SGLT2 inhibitor empagliflozin ameliorates tubulointerstitial fibrosis in DKD by downregulating renal tubular PKM2

SGLT2 inhibitor empagliflozin ameliorates tubulointerstitial fibrosis in DKD by downregulating renal tubular PKM2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article SGLT2 inhibitor empagliflozin ameliorates tubulointerstitial fibrosis in DKD by downregulating renal tubular PKM2 Xiang Cai, Huanyi Cao, Meijun Wang, Piaojian Yu, Xiaoqi Liang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5563608/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Apr, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Background and Objective Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to prevent the progression of diabetic kidney disease (DKD). However, their impact on renal fibrosis remains largely uninvestigated. This study aimed to explore the effect of SGLT2 inhibitor empagliflozin on renal fibrosis in DKD patients and DKD models, and the molecular mechanisms involved. Methods Kidney samples of DKD patients and DKD models were used in this study. DKD mouse models included STZ-treated CD-1 mice and HFD-fed C57BL/6 mice were all treated with empagliflozin for 6 to 12 weeks. Kidney pathological changes were analysed and fibrotic factors were detected. HK-2 cells were treated with normal glucose (NG), high glucose (HG), or HG with empagliflozin. RNA sequencing was employed to identify the differentially expressed genes. Epithelial–mesenchymal transition (EMT) markers were detected. Binding of transcription factor and target gene was determined using a dual-luciferase reporter assay. Results Empagliflozin significantly ameliorated kidney fibrosis in DKD patients and DKD models. This was evidenced by tubulointerstitial fibrosis reduction observed through PAS and Masson staining, along with fibrotic factors downregulation. RNA sequencing and the subsequent in vitro and in vivo validation identified PKM2 as the most significantly upregulated glycolytic enzyme in DKD patients and models. Empagliflozin downregulated PKM2 and alleviated EMT and renal fibrosis. Importantly, empagliflozin improves fibrosis by downregulating PKM2. The downregulation of PKM2 by empagliflozin was achieved by inhibiting the binding of estrogen-related receptor α at the promoter. Conclusions Empagliflozin ameliorates kidney fibrosis via downregulating PKM2 in DKD. Diabetic kidney disease Epithelial-to-mesenchymal transition Pyruvate kinase M2 Sodium-glucose cotransporter 2 inhibition Tubulointerstitial fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Diabetic kidney disease (DKD), one of the most prevalent complications of diabetes mellitus, is a leading cause for end-stage renal disease and increasingly aggravate the significant global burden [ 1 ]. Renal fibrosis is a key characteristic of DKD. The development of renal fibrosis has been shown to cause the progress of renal function decline and serves as a common result to renal failure. SGLT2 inhibitors have been recommended in the American Diabetes Association guidelines for chronic kidney disease treatment in diabetic patients[ 2 , 3 ]. Large clinical trials have shown that SGLT2 inhibitors significantly slow the progression to renal failure in patients with DKD [ 4 – 8 ]. However, these trials primarily focus on parameters such as blood creatinine levels, estimated glomerular filtration rate (eGFR), and urine albumin-to-creatinine ratio (UACR), which are more indicative of glomerular injury rather than renal fibrosis, an important cause of renal failure. Moreover, in both clinical and preclinical studies, SGLT2 inhibitors have been reported to modulate multiple pathways implicated in renal fibrosis among patients with diabetic kidney disease (DKD). Canagliflozin has been reported to downregulate plasma levels of inflammatory markers, including TNF receptor 1, IL-6, matrix metalloproteinase 7, and fibronectin 1, in patients with diabetic kidney disease[ 9 ]. SGLT2 inhibitors also reduce DNA oxidation and activate antioxidant mechanisms in diabetic patients, indicating their role in anti-oxidation and the maintenance of cellular redox homeostasis in diabetic kidney disease (DKD) patients [ 10 – 12 ]. Additionally, SGLT2 inhibitors have been observed to reduce renal macrophage infiltration in DKD mouse models and in type 2 diabetic patients at high cardiovascular risk[ 13 , 14 ]. Given the significant effects of SGLT2 inhibitors on improving DKD and the critical role of renal fibrosis in driving the progression of DKD, it is essential to continue exploring the specific molecular mechanisms through which these drugs ameliorate renal fibrosis, which will aid in identifying additional therapeutic targets for DKD. Consequently, extensive pre-clinical and clinical studies are still required to investigate the anti-fibrotic effects of SGLT2 inhibitors. SGLT2 inhibitors directly target renal tubular proximal cells (RTPCs) in DKD. Importantly, during the development of renal fibrosis, proximal renal tubule is the epicenter [ 15 , 16 ]. RTPCs are not only affected members of the injury, but also active promoters. Renal tubular epithelial cells can promote renal fibrosis through multiple mechanisms. Epithelial-to-mesenchymal transition (EMT) is critical for the development of renal interstitial fibrosis [ 17 ]. EMT is characterized by a loss of epithelial phenotype and a gain of profibrotic features [ 18 , 19 ]. Upon activation of EMT, RTPCs undergo a transition where they lose their epithelial markers, leading to collagen deposition and renal fibrosis. In addition to EMT, damaged tubules produce and release bioactive molecules that recruit inflammatory cells, thereby activating myofibroblast differentiation, proliferation, and matrix secretion[ 20 ]. Moreover, G2/M phase cell cycle arrest and apoptosis of tubular epithelial cells can promote renal fibrosis by activating the JNK signaling pathway, which leads to the upregulation of profibrotic factors, stimulation of fibroblast proliferation, and accumulation of extracellular matrix[ 21 , 22 ]. Therefore, it is crucial to explore the impact of SGLT2 inhibitors on RTPCs in DKD-related renal fibrosis, as well as the underlying molecular mechanisms involved. In the present study, we examined the effect of empagliflozin, an SGLT2 inhibitor on ameliorating kidney fibrosis in patients with DKD and in DKD models, while also investigating the underlying molecular mechanism. Materials and Methods Human samples Diabetic patients were diagnosed with DKD by kidney biopsy. The human biopsy samples from DKD patients were obtained from puncture specimens. The patients were divided into two groups: one group did not receive SGLT2 inhibitors (SGLT2is), while the other group were treated with SGLT2is. All patients included in this study received the maximum tolerated dose of angiotensin-converting enzyme inhibitor/angiotensin‐receptor blocker. The exclusion criteria were as follows: age < 18 years; the presence of other types of kidney disease; pregnancy; infection; genetic disease; taking GLP-1 receptor agonists, finerenone, and other medications that have demonstrated a significant kidney protective effect; taking SGLT2 inhibitors less than 3 months before the renal biopsy. Patient information, including gender, age, diabetes duration, blood pressure, urinary albumin-to-creatinine ratio (ACR), estimate glomerular filtration rate (eGFR), glycated hemoglobin A1c (HbA1c), plasma lipid profile, medication history, images of PAS staining, Masson staining and transmission election microscopy results were retrospectively obtained from the hospital medical record system. The present study was conducted in accordance with the 1964 Helsinki ethical declaration and its subsequent amendments. The experimental design was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (approval number: II2024-056). Animal models All experiments were approved by the Animal Ethics Committee of Sun Yat-sen University (approval number: IACUC-F3–22–0415, SYSU-IACUC-2020000006) and conducted in accordance with the standard protocols approved by the National Research Council's Guide for the Care and Use of Laboratory Animals and the Animal Ethics Committee of Sun Yat-sen University. All mice had free access to water and food, and were housed under a 12-hour light/12-hour dark cycle. Seven-week-old male CD-1 mice were purchased from Guangdong Zhiyuan Biomedical Technology Co (Guangzhou, China). CD-1 mice were kept in a barrier environment at the Animal Experimental Center of the Third Affiliated Hospital of Sun Yat-sen University. After one week of adaptive feeding, CD-1 mice were randomly and blindly divided into two groups (n = 6 per group). The STZ-induced diabetic mouse model was constructed according to previously published study[ 23 , 24 ]. In brief, the streptozotocin (STZ) group received an intraperitoneal injection of a single dose of STZ at 150 mg/kg in a citrate buffer (10 mM) to induce diabetes, while an equal volume of citrate buffer (10mM) was injected in control group. Two weeks after STZ injection, mice with diabetes were confirmed by fasting blood glucose level greater than 16 mM. Eight weeks after diabetes induction, empagliflozin (10 mg/kg/day) dissolved in 0.5% hydroxypropyl methylcellulose (HPMC) or the same volume of 0.5% HPMC (control) was administered by oral gavage until ACR was significantly decreased (12 weeks) (n = 6 per group). Fasting blood glucose and body weight were measured every two weeks. The ACR was quantified by the Department of Laboratory Medicine of the Third Affiliated Hospital at Sun Yat-sen University. Seven-week-old male C57BL/6 mice were obtained from GemPharmatech (Nanjing, China). After one week of adaptive feeding, C57BL/6 mice were randomly and blindly divided into two groups (n = 6 per group). The chow diet (CD) group were fed a diet containing 11% fat (Guangdong Medical Laboratory Animal Center, Guangzhou, China). The high-fat diet (HFD) group were fed a diet containing 58% fat (D12331, Research Diets, New Brunswick, NJ, USA)[ 24 , 25 ]. DKD of C57BL/6 mice was confirmed by significantly elevated FBG, impaired glucose tolerance and increased ACR after 14 weeks of HFD-feeding. After 14 weeks of feeding, empagliflozin (10 mg/kg/day) or the same volume of 0.5% HPMC were administered via oral gavage until ACR was significantly decreased (6 weeks) (n = 6 per group). Fasting blood glucose and body weight were measured every two weeks. The plasma insulin level was detected by ELISA kits from Elabscience (Wuhan, China). The urinary albumin-to-creatinine ratio (ACR) was calculated by urinary microalbumin /urinary creatinine. The urinary albumin level and urinary creatinine level was determined using the creatinine companion and the Albuwell M kits purchased from Guangzhou Ruishu Biotechnology Co., Ltd (Guangzhou, China). Seven-week-old B6·V-Lep ob /J ( ob/ob ) mice were purchased from GemPharmatech (Nanjing, China) [ 26 ]. After one week of adaptive feeding, ob/ob mice were randomly divided into two groups (n = 6 per group) and fed a methionine- and choline-deficient (MCD) diet for 8 weeks. Mice were treated with empagliflozin (10 mg/kg/day) or an equal volume of 0.5% HPMC via oral gavage until ACR was significantly decreased (8 weeks). After fasted overnight, the blood samples were collected from DKD mice anesthetized using isoflurane, followed by administration of more than 100 mg/kg of pentobarbital for euthanasia. Subsequently, mice were sacrificed through cervical vertebra dislocation, followed by tissue collection. The left kidneys from five mice in each group were cut in half longitudinally, and the half were then fixed in 4% formaldehyde for at least 24 h, embedded in paraffin and sectioned at 4–6 µm thickness for renal histological analysis, immunofluorescence, and immunochemistry. Glucose and insulin tolerance test The methods of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were similar to those described in our previous study[ 27 , 28 ]. In brief, after fasting for 6 hours, mice were intraperitoneally injected with glucose (2 g/kg) solved in saline to perform an IPGTT. Blood samples were collected from the tail tip at 0, 30, 60 and 120 minutes after injection. Blood glucose was measured by a glucometer (ONETOUCH UltraVue, Johnson & Johnson, USA). An IPITT was performed by intraperitoneal injection of human insulin (0.65 IU/kg) (Novolin R, Novo Nordisk A/S, Copenhagen, Denmark) after 4 hours of fasting. Cell culture and treatment Mycoplasma-free human immortalized RTPC (HK-2) (GNHu47, Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) and Ham’s F12 medium (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) with 10% (v/v) fetal bovine serum (Procell, Wuhan, China). After starving in a serum-free medium for 12 h, HK-2 cells were synchronized and then treated with normal concentration of glucose (NG, 5.5 mM), high concentration of glucose (HG, 30 mM), or HG with empagliflozin (1 mM) for 72 hours. For construction of non-diabetic renal fibrosis model, HK-2 cells were treated with TGF-β (5ng/ml) for 24 hours, and then treated with empagliflozin (1mM) for another 72 hours. Knockdown and overexpression of PKM2 The PKM2 shRNA and PKM2-overexpression vector (pcDNA3-PKM2, OE) were purchased from GenePharma (Shanghai GenePharma Co., Ltd., China) [ 29 , 30 ]. HK-2 cells were transfected at 60–65% confluence with PKM2 shRNA or overexpression vector by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions[ 31 ]. For PKM2 knockdown, the sequences of shRNA were cloned into pGPU6 vector. shPKM2: “5′-TTATTTGAGGAACTCCGCCGC-3”. For transfection, cells were seeded into a 6-well plate, cultured overnight to 60–70% confluence, and then transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific, 11668019) supplemented with 2500 ng DNA or 50 nM (final concentration) siRNA[ 32 ]. Renal histopathology Periodic acid-Schiff (PAS) staining and Masson trichrome staining were conducted according to previous published studies[ 33 ]. For PAS staining, the paraffin-embedded kidney slides were stained with 0.5% periodic acid for 10 min, washed, and stained again with Schiff reagent for 15 min. For Masson trichrome staining, the paraffin-embedded kidney sections were stained using Weigert’s iron hematoxylin, azophloxine staining solution, phosphotungstic acid orange G, and light-green SF solution using a step-by-step method. After dehydration and xylene clearing, stained sections were observed under a light microscope (Olympus BX63; Olympus, Tokyo, Japan). Three photographs of each stained slide were taken. Tubular injury was defined as tubular dilation, tubular atrophy, formation of cylindrical tubules, detachment of tubular epithelial cells or loss of brush border and thickening of the tubular basement membrane. The scoring system used was as follows: 0 points, no tubular damage; 1 point, 75% renal tubular injury damage[ 34 ]. Immunohistochemical analysis Kidney sections from DKD patients, CD-1 mice and C57BL/6 mice were used for immunohistochemical staining. Immunohistochemical was carried out according to standard procedures reported in previous published studies[ 35 , 36 ]. In brief, kidney tissues were fixed in 4% paraformaldehyde at 4°C for 24 hours, followed by dehydration by an ascending series of ethanol baths. Then the tissues were cleared with xylene and embedded in paraffin. The paraffin-embedded kidney tissues were cut into 4-µm sections. The kidney sections were then dewaxed with xylene and rehydrated with gradient ethanol. Specimens were incubated with 1% bovine serum albumin in PBS for 1 hour and then incubated with the primary antibodies anti-neutrophil gelatinase associated lipocalin (NGAL) (1:500), anti-pyruvate kinase M2 (PKM2) (1:200), anti-E-cadherin (1:200), anti-Vimentin (1:200), anti-collagen type III α1 (COL3A1) (1:200), anti-α smooth muscle actin (αSMA) (1:200) primary antibodies at 4ºC overnight. Then the kidney sections were incubated with an enzyme-conjugated secondary antibody (1:2000) for 50 min at 37ºC. The detailed antibody information is shown in Table 1 of the extra supplementary material (ESM). Immunofluorescence staining The initial immune-staining steps were performed as previously described [ 37 , 38 ]. For kidney immunofluorescence staining, tissues were paraffin-embedded, de-paraffinized and rehydrated. Samples were incubated with primary antibodies for COL3A1 (1:200), PKM2 (1:200), and Lotus tetragonolobus lectin (LTL; 1:200) at 4 ºC overnight, followed by incubation with fluorophore-conjugated secondary antibody (1:200) for 1 h at 37℃. Subsequently, 4,6-diamino-2-phenyl indole (DAPI) was applied for 5 min. For HK-2 cells immunofluorescence staining, cells were fixed in 4% paraformaldehyde for 30 minutes, and permeabilized with 0.5% Triton X-100 for 15 min. After that, the cells were blocked in 1% bovine serum albumin (BSA) for 1 hour and incubated with the primary antibody for estrogen-related receptor α (ESRRA) (1:200) overnight at 4 ºC. Next, they were incubated with fluorophore-conjugated secondary antibody (1:10000) for 1 h, followed by DAPI solution for 5 min at 37ºC. Images were obtained using a Leica fluorescence microscope (Leica Microsystems, Wetzlar, Germany) at 200× magnification. The detailed antibody information is shown in ESM Table 2. RNA sequencing HK-2 cells were seeded in T25 culture flask and treated with NG, HG, or HG with empagliflozin for 72 h. Total RNA was isolated using TRIzol (#15596018, Invitrogen, Carlsbad, CA, USA). Each sample in NG, HG, and HG with empagliflozin groups were resultant mix of nine RNA extraction. According to the TruSeq™ RNA Sample Preparation Guide, paired-end libraries were synthesized used the TruSeq™ RNA Sample Prep Kit (Illumina, San Diego, CA, USA). Briefly, poly-A-containing mRNA molecules were purified using poly-T oligo-linked magnetic beads and then fragmented into small pieces using divalent cations at 94ºC for 8 min. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. DNA Polymerase I and RNase H were used to synthesize second strand cDNA. These cDNA fragments went through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final cDNA library. Purified libraries were quantified with a Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and validated using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to confirm the insert size and calculate the mole concentration. Clusters were generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina NovaSeq 6000 (Illumina, San Diego, USA). The library construction and sequencing were performed by the Guangzhou Promegene Biotechnology Co., Ltd (Guangzhou, China). FPKM values were normalized per gene to obtain relative expression values. Nuclear-cytosolic protein extraction HK-2 cells were collected in ice-cold PBS (0.01 M) and then centrifuged at 800 RCF for 5 min. Tissue samples were cut into pieces and homogenized in the presence of phenylmethyl sulfonyl fluoride (PMSF). The samples were stored at -80°C for subsequent western blot analysis. The cytoplasmic and nuclear protein fractions were separated using corresponding extraction reagents using a Nuclear and Cytoplasmic Protein Extraction Kit (#p0028; Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s protocol [ 39 , 40 ]. Cytoplasmic protein and nuclear protein were separately collected and stored at -80°C for western blot analysis. Western blot Western blot was performed according to previous published paper[ 26 ]. Kidney tissues and HK-2 cells were each homogenized in a lysis buffer containing protease and phosphatase inhibitors. Protein lysates were subjected to 8–10% polyacrylamide dodecyl sulfate gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. PVDF membranes were then incubated in primary antibodies against COL3A1 (1:1000), alpha smooth muscle actin (α-SMA;1:1000), PKM2 (1:1000), hexokinase 2 (HK2) (1:1000), phosphofructokinase (PFKP) (1:1000), ESRRA (1:1000), beta-actin (β-ACTIN; 1:1000), and Lamin B1 (1:1000) at 4°C overnight. The membranes were then incubated with corresponding secondary antibodies (1:10000) at 37°C for 1 h. Band intensities were quantified using Image-Pro Plus v6.0 (Media Cybernetics, Washington DC, USA). The detailed antibody information is shown in ESM Table 3. Quantitative reverse transcription-PCR Total RNA extracted from mice kidneys was converted to cDNA using a cDNA reverse transcription kit (Cat# RR047A, TAKARA, Kyoto, Japan) according to the manufacturer’s instructions. Real-time quantitative PCR was performed using TB Green Premix Ex TaqII (Cat# RR820A, TAKARA, Kyoto, Japan) and conducted using a Light Cycler 480II Real-Time PCR System (Roche Diagnostics, Mannheim, Germany). β-actin was used as a housekeeping gene. The sequences of primers were listed in ESM Table 4. Luciferase reporter assay The JASPAR database ( https://jaspar.genereg.net/ ) was used to identify the predicted ESSRA binding sites 2,000 base pairs (bp) upstream and 100 bp downstream of the transcription start site. The luciferase reporter assay was performed according to the previous published paper [ 37 , 41 ]. The wild-type or mutant promoters of human PKM2 were transduced into pGL3-basic vectors. The ESRRA expression plasmid, and wild-type or mutant pGL3-basic vectors were co-transfected into HEK 293T cells (Dongze Biotech Co., Ltd, Guangdong, China). Subsequently, luciferase assays were performed on HEK 293T cells at 70–80% confluence using a dual-luciferase reporter system (#E2940; Promega, Madison, WI, USA) according to the manufacturer’s protocol. Chromatin immunoprecipitation (ChIP)-qPCR Chromatin immunoprecipitation (ChIP) assays were performed using a Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the instructions provided by the manufacturer[ 42 , 43 ]. In brief, ESRRA antibody (Cell signaling technology, #13826) was adopted for immunoprecipitation of chromatin, and IgG was negative control. Isolated RNA was assayed by RT-qPCR. Primers designed for the predicted binding site 1 of ESRRA at PKM2 promoter regions were as follows: forward primer: 5’- CGGCGGAGGGATTGCG-3’, reverse primer: 5’- GCTACGCTGCAAAGACGAAGA-3’. Primers designed for the predicted binding site 2 of ESRRA at PKM2 promoter regions were as follows: forward primer:5’- ACCGAAAGGGCAACCTGC-3’, reverse primer: 5’- GGGCCGCCGCAATCC-3’. Statistical analysis All quantitative experiments were repeated at least 3 times independently. GraphPad Prism 8.0.1 was used for statistical analysis and creating graphs (GraphPad Software, San Diego, CA, USA). Data are expressed as the mean ± standard error of the mean (SEM). Comparisons between two groups were performed using the unpaired Student t-test. Comparisons between multiple groups were performed using one-way ANOVA, followed by Tukey's multiple comparisons test. Correlations were assessed using Spearman's rank correlation. Statistical significance was set at P < 0.05. Results SGLT2i ameliorate kidney fibrosis in DKD patients and DKD models Patients without SGLT2i treatment were recruited into group 1, patients treated with SGLT2i were recruited into group 2. Clinical information on the patients is summarized in Table 1. The male to female ratios were both 3 to 2 in the two groups. There was no statistical difference in mean age, diabetes duration, systolic blood pressure, diastolic blood pressure, urinary creatine, eGFR, and glycated hemoglobin between the two patient groups (Table 1). The medication duration of group 1 was longer than in group 2 (Table 1). As expected, the urinary ACR was significantly lower in group 2 (Table 1). Extracellular matrix accumulation (PAS staining) and tubulointerstitial fibrosis (MASSON staining) were ameliorated in group 2 compared with group 1, indicating that SGLT2i treatment alleviated in DKD (Fig. 1 A). The tubular injury marker NGAL were downregulated by empagliflozin (Fig. 1 A). Fibrosis marker COL3A1 and αSMA were downregulated by empagliflozin in DKD patients (Fig. 1 B, ESM Fig. 1 A, B). Transmission electron microscopy (TEM) also showed the tubulointerstitial collagen deposition, basement thickening and foot process fusion were ameliorated by empagliflozin treatment (Fig. 1 C). After that, in vivo and in vitro experiments were carried out to confirmed the anti-fibrotic effect of empagliflozin. FBG and ACR were elevated in both STZ-treated CD-1 mice and HFD-treated C57BL/6 mice (ESM Fig. 1 C-D, F-G). Body weight was reduced in STZ-treated CD1 mice (ESM Fig. 1 E) and elevated in HFD-treated C57BL/6 mice (ESM Fig. 1 H). Tubulointerstitial fibrosis were observed by PAS and MASSON staining in both of the two DKD mouse models (ESM Fig. 1 I-J). Tubular injury score and NGAL were upregulated under STZ and HFD treatment (ESM Fig. 1 K-N)). Empagliflozin lowered FBG, improved body weight, and reduced ACR in both STZ-treated and HFD-treated mice (ESM Fig. 2 A-H). Empagliflozin also lowered plasma insulin level, improved glucose tolerance (tested by IPGTT and IPITT) in HFD-treated mice (ESM Fig. 2 I-K). Notably, empagliflozin significantly ameliorated tubular dilation, extracellular matrix accumulation (PAS staining), and tubulointerstitial fibrosis (MASSON staining) in both STZ-treated and HFD-treated mice (Fig. 1 D, F). Tubular injury score and NGAL were also downregulated by empagliflozin (Fig. 1 D, F, ESM Fig. 2 L-O). Immunofluorescence and western blot were then used to detect fibrosis markers. Empagliflozin significantly downregulated COL3A1 and αSMA in both STZ-induced and HFD-induced DKD (Fig. 1 E, G, H-I). In vitro experiments were carried out in HK-2 cells. Under HG condition, COL3A1 and αSMA were upregulated in HK-2 cells, while empagliflozin significantly downregulated these fibrosis makers (Fig. 1 J). Empagliflozin downregulated mRNA level of PKM2 in DKD To investigate the mechanisms underlying the anti-fibrotic effect of empagliflozin, RNA sequencing was conducted in HK-2 cells (Fig. 2 A). Both KEGG and GO analyses revealed that pathways associated with fibrosis were upregulated under HG conditions (Fig. 2 B-C). Additionally, KEGG analysis was performed to identify the top ten enriched pathways that were upregulated in the HG group and simultaneously downregulated by empagliflozin. Differentially expressed genes were involved in cytokine-cytokine receptor interaction, calcium signaling, chemokine signaling, JAK-STAT signaling, TGF-beta signaling, glucagon signaling, glycolysis, starch and sucrose metabolism, renin-angiotensin system, and the pentose phosphate pathway (Fig. 3 D). We focused our attention on differentially expressed genes involved in glycolysis, since the mechanisms by which empagliflozin reduces glycolysis have not yet been fully elucidated. RNA sequencing result showed that some of the genes involved in glycolytic pathway including PKM2, GAPDHS, PGAM2, FBP1, ADH1 were upregulated by HG and downregulated by empagliflozin. While other glycolytic genes including PFKP and HK2 showed no significant difference between NG, HG, and HG with empagliflozin group (Fig. 2 E). The above glycolic genes in RNA sequencing were validated by RT-qPCR both in vitro . PKM2, PFKP , and HK2 were upregulated by HG in HK-2 cells. Only PKM2 was downregulated by empagliflozin (Fig. 2 F). The results were further validated in DKD mouse models. PKM2 was upregulated by STZ and HFD, and was downregulated by empagliflozin (Fig. 2 G, H). Empagliflozin downregulated protein level of PKM2 in DKD Genes fulfill their functions by being translated into functional proteins. In this study, we examined the protein expression levels of three key glycolytic enzymes: PKM2, HK2, and PFKP, in the context of diabetic kidney disease (DKD). Notably, PKM2 showed the highest level of upregulation under DKD conditions, while it was the most significantly downregulated enzyme in response to empagliflozin treatment (Fig. 3 A-C). Immunofluorescence analysis revealed that in both the STZ and HFD groups, PKM2 was highly expressed and co-localized with LTL, a marker indicative of kidney proximal tubule cells. Notably, empagliflozin significantly downregulated PKM2 expression in both STZ-treated and HFD-treated mice (Fig. 3 D-E). These findings were further corroborated by immunohistochemical analysis (Fig. 3 F, G ESM Fig. 2 P, Q). Importantly, PKM2 expression was found to be upregulated in the renal tubules of DKD patients, while it was downregulated in the empagliflozin-treated group (Fig. 3 H, ESM Fig. 2 R). Furthermore, a positive correlation was observed between PKM2 levels and the expression of fibrosis markers COL3A1 and αSMA in both DKD models and DKD patients (Fig. 3 I-K). Empagliflozin ameliorated renal fibrosis by downregulating PKM2 in vitro To further elucidate whether empagliflozin ameliorates renal fibrosis via the downregulation of PKM2, we conducted experiments to overexpress and knock down PKM2 in HK-2 cells separately. The effects of overexpression and knockdown were validated through Western blot analysis (Fig. 4 A-D). The PKM2 overexpression resulted in an elevation of fibrosis markers, including COL3A1 and αSMA (Fig. 4 E-G). Importantly, the overexpression of PKM2 diminished the ameliorative effects of empagliflozin on renal fibrosis (Fig. 4 H-K). Furthermore, PKM2 knockdown attenuated the downregulatory effects of empagliflozin on COL3A1 and αSMA, as evidenced by the comparison between the HG + shPKM2 and HG + shPKM2 + Empagliflozin groups (Fig. 4 L-O). Empagliflozin ameliorated EMT in DKD Glycolysis plays a crucial role in the process of EMT. In this study, we examined the expression of the epithelial cell marker E-cadherin and the mesenchymal cell marker Vimentin in human kidney specimens as well as in kidney sections from DKD mouse models. Our findings revealed that E-cadherin was downregulated while Vimentin was upregulated in the renal tubular cells of DKD patients, STZ-treated mice, and HFD-treated mice (Fig. 5 A-C). Notably, empagliflozin significantly ameliorated EMT in both DKD patients and DKD mouse models (Fig. 5 A-C). Furthermore, the positive areas of E-cadherin were negatively correlated with the positive areas of PKM2. The positive areas of Vimentin were positively correlated with the areas of PKM2 (Fig. 5 D-F). Empagliflozin downregulated PKM2 by blocking the binding of ESRRA to the promoter ESRRA, as a transcription factor, transferred into nucleus to activate downstream target genes. The nuclear translocation of ESRRA was increased in STZ-treated mice, HFD-fed mice, and HG-treated HK-2 cells, and was decreased under empagliflozin treatment (Fig. 6 A-D). The two predicted binding sites were further verified by dual-luciferase reporter gene assays. The mutation of either binding site 1 or binding site 2 significantly decreased the transcriptional activity of ESRRA (Fig. 6 E). ChIP-qPCR analysis further indicated the enrichment of ESRRA at the PKM2 promoter (Fig. 6 F). The results of ChIP-qPCR analysis also showed that the binding of ESRRA was upregulated by HG and was downregulated by empagliflozin at the two predicted binding sites (Fig. 6 G). Empagliflozin downregulated fibrosis markers in TGF-β-treated HK-2 cells After stimulation of TGF-β, COL3A1 and αSMA were upregulated in HK-2 cells. Empagliflozin downregulated the fibrosis markers, indicating a direct amelioration effect of empagliflozin on renal fibrosis (ESM Fig. 2 S). Empagliflozin ameliorated kidney fibrosis and downregulated PKM2-PKM2 in MCD treated ob/ob mice In MCD diet-treated ob/ob (ob/MCD) mice, empagliflozin ameliorated renal tubular injury, as evidenced by PAS staining, and reduced tubulointerstitial fibrosis, as shown by Masson staining. It also suppressed the accumulation of NGAL and decreased the tubular injury score (ESM Fig. 3 A-C). Additionally, COL3A1 and αSMA expression were downregulated by empagliflozin (ESM Fig. 3 D, E). Pkm2 was significantly downregulated by empagliflozin (ESM Fig. 3 F). Western blot validated that PKM2 was the downregulated by empagliflozin, while empagliflozin showed no effect on HK2 and PFKP (ESM Fig. 3 G). Immunofluorescence and immunochemistry results indicated that PKM2 was downregulated after empagliflozin treatment. Furthermore, E-cadherin was upregulated and Vimentin was downregulated in empagliflozin-treated group (ESM Fig. 3 H-J). Empagliflozin decreased the nuclear translocation of ESRRA in ob/MCD mice (ESM Fig. 3 K). Discussion The present study demonstrated that SGLT2 inhibitor empagliflozin can effectively alleviate EMT-related tubulointerstitial fibrosis by downregulating PKM2 in DKD. The study showed that empagliflozin ameliorated kidney fibrosis in DKD patients and DKD models. PKM2 was significantly upregulated in diabetic kidneys and HG-treated HK-2 cells. Empagliflozin downregulated PKM2 and PKM2 in DKD models both in vivo and in vitro . Empagliflozin ameliorated renal fibrosis by reducing the recruitment of ESRRA to the PKM2 promoter, thereby suppressing the transcription of PKM2 . The renal benefits of SGLT2 inhibitors have been validated in clinical trials [ 4 – 7 ]. RTPCs are the direct targets of SGLT2 inhibitors. Increasing evidence suggests that renal tubules play a critical role in the progression of DKD[ 44 – 48 ]. Large clinical trials have shown that SGLT2 inhibitors significantly reduce the risk of end-stage kidney disease in DKD patients. However, the outcomes of the above clinical trials mainly indicate the function of glomerulus[ 4 – 8 ]. The anti-fibrotic effect of SGLT2 inhibitors has been confirmed in several in vivo studies[ 9 , 47 , 49 , 50 ]. Also, SGLT2 inhibitors have been reported to modulate multiple pathways implicated in renal fibrosis among DKD patients, potentially uncovering the mechanisms through which SGLT2 inhibition may ameliorate renal fibrosis[ 9 – 14 ]. Direct evidence that SGLT2 inhibitors improve renal fibrosis in DKD patients is yet to be fully established. In this study, we retrospectively analyzed renal biopsy results from DKD patients who were treated with SGLT2 inhibitors compared to those who were not. Staining techniques, including PAS staining and MASSON staining revealed that SGLT2 inhibitors reduced tubulointerstitial fibrosis in DKD patients. Transmission electron microscopy results show that empagliflozin improves renal interstitial collagen fiber proliferation, basement membrane thickening and foot process fusion in DKD patients. Furthermore, the anti-fibrotic effects of empagliflozin were validated in various DKD mouse models, including STZ-treated CD-1 mice, HFD-treated C57BL/6 mice, and MCD-treated ob/ob mice. We then investigated the molecular mechanisms underlying empagliflozin-ameliorated-fibrosis. Although glucose metabolism is not the main energy supply method of renal tubules, key enzymes of glycolysis and gluconeogenesis still play some roles in renal tubular cells. Hexokinase, a key glycolytic enzyme, reduced mitochondrial membrane injury after metabolic stress[ 51 ]. Gluconeogenic enzymes including phosphoenolpyruvatecarboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) are highly expressed in the proximal tubules, making proximal tubules important in maintaining systemic glucose homeostasis[ 52 – 54 ]. In the present study, RNA sequencing analysis revealed that glycolysis pathway was significantly upregulated under HG condition and was downregulated by empagliflozin. SGLT2 inhibitors are reported to ameliorate glycolysis in metabolic disorders, including non-alcoholic fatty liver disease and DKD[ 41 ]. The progression of glycolysis depends on key glycolytic enzymes. However, the relationship between various glycolytic enzymes and the anti-fibrotic effects of SGLT2 inhibitors remains to be thoroughly investigated. Herein, PKM2 exhibited the most significant elevation among glycolytic genes in HG-treated HK-2 cells. The upregulation of Pkm2 was further confirmed in DKD mouse models. The expression of PKM2 was positively corelated with fibrosis markers COL3A1 and αSMA. Notably, empagliflozin downregulated Pkm2 in both in vivo and in vitro DKD models. These findings suggest that PKM2 is the key glycolytic gene involved in kidney fibrosis and the anti-fibrotic effect of empagliflozin in DKD. PKM2 is translated from PKM2 , serving as the rate-limiting enzyme of glycolysis [ 55 ]. In this study, we observed significant upregulation of PKM2 across various DKD models. This upregulation was predominantly observed in RPTCs. Furthermore, the expression levels of PKM2 were also positively correlated with fibrosis markers, suggesting a critical role for PKM2 of RTPCs in kidney fibrosis. Empagliflozin effectively downregulated renal tubular PKM2. These findings further substantiate the role of PKM2 in inducing renal fibrosis, underscoring its significance in empagliflozin-ameliorated renal fibrosis. Under normal condition, fatty acid oxidation is the main metabolic way in RTPCs. When glycolysis enhanced, the metabolic shift alters RTPCs identity and cell fate[ 56 ]. Transition to glycolysis state suppresses the normal epithelial phenotype and promotes a mesenchymal phenotype, leading to EMT[ 57 ]. This transition further stimulates the secretion of extracellular matrix components, including collagen and fibronectin, ultimately resulting in renal fibrosis [ 17 , 58 , 59 ]. Suppressing EMT in RTPCs may contribute to the anti-fibrotic effect of SGLT2 inhibitors. Thus, we then detected the EMT markers in the kidneys of DKD patients and DKD models. As expected, EMT was observed in the renal tubular cells in various DKD mouse models. Notably, empagliflozin effectively alleviated EMT. These findings underscore the anti-fibrotic effect of empagliflozin through the suppression of EMT in renal tubular cells. Finally, we investigated the transcription factors responsible for PKM2 upregulation. The transcription factors of PKM2 were predicted by UCSC ( https://genome.ucsc.edu/index.html ) and hTFtarget database ( https://guolab.wchscu.cn/hTFtarget/ ). Among the predicted transcription factors, ESRRA has been rarely reported in DKD. As an orphan nuclear receptor, ESRRA regulates cell proliferation and cell metabolism by targeting various downstream genes. It is associated with various metabolic disorders, including obesity and non-alcoholic fatty liver disease [ 60 – 62 ]. Moreover, ESRRA is closely related with renal fibrosis induced by folic acid or unilateral ureteral obstruction [ 63 ]. However, there is little research focusing on the role of ESRRA in DKD. Herein, we found that ESRRA nuclear translocation was increased in DKD models both in vivo and in vitro and was significantly decreased by empagliflozin. Next, the binding sites of ESRRA at the PKM2 promoter were predicted and confirmed. Empagliflozin downregulated PKM2 via inhibiting the binding of ESRRA to PKM2 promoter. These findings have further clarified the down-regulatory effect of SGLT2 inhibitors on PKM2 by elucidating the molecular mechanism. To further validate the sufficient of PKM2 downregulation in the renal fibrosis amelioration mediated by empagliflozin, PKM2 was overexpressed and knocked down separately in HK-2 cells. Both overexpression and knockdown of PKM2 diminished the effect of empagliflozin on downregulation the fibrosis markers in HK-2 cells. These findings highlight the importance of PKM2 as a therapeutic target for DKD-related renal fibrosis. In parallel to ongoing epidemics of obesity, The incidence of metabolic dysfunction-associated fatty liver disease (MAFLD) is increasing globally. Clinical trials indicate that the presence of MAFLD significantly increases the risk of end-stage kidney disease and renal fibrosis[ 64 , 65 ]. However, the effect of SGLT2 inhibitors on renal fibrosis in MAFLD remains largely unclear. The MAFLD of ob/ob mice treated with methionine- and choline-deficient (MCD) diet used in the present study was confirmed in our previous published study[ 26 ]. Herein, renal fibrosis was observed in MCD-treated ob/ob mice. Furthermore, empagliflozin ameliorated renal fibrosis and EMT, downregulated PKM2 as well as reduced the nuclear translocation of ESRRA in the MAFLD mouse model. To our knowledge, our study is the first to report the upregulation of renal tubular PKM2 and its downregulation induced by empagliflozin in patients with DKD. Other studies have analyzed the expression of PKM2 in podocytes from DKD patients. PKM2 was found to be downregulated in podocytes of DKD patients[ 66 ]. This disparity may arise from differing metabolic dependencies: tubular epithelial cells (TECs) rely predominantly on fatty acid β-oxidation, whereas podocytes are primarily dependent on glycolysis [ 46 , 67 ]. In the context of DKD, glycolysis is observed to increase in TECs while decreasing in podocytes, which may explain the different trends of PKM2 in TECs and podocytes under diabetic condition[ 41 , 68 ]. This study had a few limitations that must be considered. Firstly, the small number of patients included in our study could be considered a potential limitation. Many patients are reluctant to undergo renal biopsy due to its invasive nature and the associated risks of complications, including bleeding and infection. Secondly, the clinical information and pathology data of DKD patients were retrospectively collected, resulting in an inherent risk of recall bias and confounders (wash-out period absence). Thirdly, the PKM2 tubules-specific overexpression or knockdown mouse model is needed in the future study to further validate that the downregulation of PKM2 is sufficient to the attenuation of renal fibrosis by empagliflozin. In conclusion, empagliflozin improves kidney fibrosis in DKD patients and DKD models. It ameliorates EMT of renal tubular cells through downregulating PKM2. At the molecular level, empagliflozin inhibits the transcription of PKM2 by reducing the nuclear translocation of ESRRA and inhibiting its binding to the PKM2 promoter. Our findings emphasize the significant role of PKM2 in both DKD-related kidney fibrosis and the amelioration of tubulointerstitial fibrosis mediated by empagliflozin. Declarations Fundings This work was supported by the National Natural Science Foundation of China (81670762 to Mengyin Cai, 82270942 to Fen Xu), Natural Science Foundation of Guangdong Province (2020A1515011245 and 2016A030313258 to Mengyin Cai), the Guangzhou Municipal Science and Technology Project (201707010118 to Mengyin Cai). Competing interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Xiang Cai, Huanyi Cao, and Piaojian Yu carried out experiments, and wrote the manuscript. Meijun Wang and Hua liang contributed to data analysis. Xiaoqi Liang provided technical support with animal care. Fen Xu and Mengyin Cai contributed to the study design, data interpretation and revising the manuscript. Data availability Original data are available upon reasonable request from the corresponding author. Ethical approval The retrospective study was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (approval number: II2024-056). Informed consent was obtained from all individual participants included in the study. All animal experiments were approved by the Animal Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (approval number: IACUC-F3–22–0415, SYSU-IACUC-2020000006). Consent to participate Informed consent was obtained from all individual participants included in the study. References Webster, A.C., et al., Chronic Kidney Disease. Lancet, 2017. 389 (10075): p. 1238-1252. ElSayed, N.A., et al., 11. Chronic Kidney Disease and Risk Management: Standards of Care in Diabetes-2023. Diabetes Care, 2023. 46 (Suppl 1): p. S191-s202. KDIGO 2022 Clinical Practice Guideline for Diabetes Management in Chronic Kidney Disease. Kidney Int, 2022. 102 (5s): p. S1-s127. Cherney, D.Z.I., et al., Effects of empagliflozin on the urinary albumin-to-creatinine ratio in patients with type 2 diabetes and established cardiovascular disease: an exploratory analysis from the EMPA-REG OUTCOME randomised, placebo-controlled trial. Lancet Diabetes Endocrinol, 2017. 5 (8): p. 610-621. Wanner, C., et al., Empagliflozin and Progression of Kidney Disease in Type 2 Diabetes. N Engl J Med, 2016. 375 (4): p. 323-34. Perkovic, V., et al., Canagliflozin and Renal Outcomes in Type 2 Diabetes and Nephropathy. N Engl J Med, 2019. 380 (24): p. 2295-2306. Wiviott, S.D., et al., Dapagliflozin and Cardiovascular Outcomes in Type 2 Diabetes. N Engl J Med, 2019. 380 (4): p. 347-357. Herrington, W.G., et al., Empagliflozin in Patients with Chronic Kidney Disease. N Engl J Med, 2023. 388 (2): p. 117-127. Heerspink, H.J.L., et al., Canagliflozin reduces inflammation and fibrosis biomarkers: a potential mechanism of action for beneficial effects of SGLT2 inhibitors in diabetic kidney disease. Diabetologia, 2019. 62 (7): p. 1154-1166. Llorens-Cebrià, C., et al., Antioxidant Roles of SGLT2 Inhibitors in the Kidney. Biomolecules, 2022. 12 (1). Nabrdalik-Leśniak, D., et al., Influence of SGLT2 Inhibitor Treatment on Urine Antioxidant Status in Type 2 Diabetic Patients: A Pilot Study. Oxid Med Cell Longev, 2021. 2021 : p. 5593589. van Bommel, E.J.M., et al., The renal hemodynamic effects of the SGLT2 inhibitor dapagliflozin are caused by post-glomerular vasodilatation rather than pre-glomerular vasoconstriction in metformin-treated patients with type 2 diabetes in the randomized, double-blind RED trial. Kidney Int, 2020. 97 (1): p. 202-212. Kim, S.R., et al., SGLT2 inhibition modulates NLRP3 inflammasome activity via ketones and insulin in diabetes with cardiovascular disease. Nat Commun, 2020. 11 (1): p. 2127. Sunilkumar, S., et al., REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy. Cell Death Dis, 2025. 16 (1): p. 79. Liu, X., et al., Tubule-derived exosomes play a central role in fibroblast activation and kidney fibrosis. Kidney Int, 2020. 97 (6): p. 1181-1195. Liu, B.C., et al., Renal tubule injury: a driving force toward chronic kidney disease. Kidney Int, 2018. 93 (3): p. 568-579. Lovisa, S., et al., Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis. Nat Med, 2015. 21 (9): p. 998-1009. Thiery, J.P. and J.P. Sleeman, Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol, 2006. 7 (2): p. 131-42. Thiery, J.P., et al., Epithelial-mesenchymal transitions in development and disease. Cell, 2009. 139 (5): p. 871-90. Huang, R., P. Fu, and L. Ma, Kidney fibrosis: from mechanisms to therapeutic medicines. Signal Transduct Target Ther, 2023. 8 (1): p. 129. Yang, L., et al., Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury. Nat Med, 2010. 16 (5): p. 535-43, 1p following 143. Thomas, K., et al., Glutamine prevents acute kidney injury by modulating oxidative stress and apoptosis in tubular epithelial cells. JCI Insight, 2022. 7 (21). Xu, Z., et al., METTL14-regulated PI3K/Akt signaling pathway via PTEN affects HDAC5-mediated epithelial-mesenchymal transition of renal tubular cells in diabetic kidney disease. Cell Death Dis, 2021. 12 (1): p. 32. Urabe, H., et al., Ablation of a small subpopulation of diabetes-specific bone marrow-derived cells in mice protects against diabetic neuropathy. Am J Physiol Endocrinol Metab, 2016. 310 (4): p. E269-75. Müller, T.D., et al., p62 links β-adrenergic input to mitochondrial function and thermogenesis. J Clin Invest, 2013. 123 (1): p. 469-78. Shen, Y., et al., SGLT2 inhibitor empagliflozin downregulates miRNA-34a-5p and targets GREM2 to inactivate hepatic stellate cells and ameliorate non-alcoholic fatty liver disease-associated fibrosis. Metabolism, 2023. 146 : p. 155657. Cao, H., et al., Malonylation of Acetyl-CoA carboxylase 1 promotes hepatic steatosis and is attenuated by ketogenic diet in NAFLD. Cell Rep, 2023. 42 (4): p. 112319. Zheng, X., et al., SIRT1/HSF1/HSP pathway is essential for exenatide-alleviated, lipid-induced hepatic endoplasmic reticulum stress. Hepatology, 2017. 66 (3): p. 809-824. Wang, Q., et al., Suppression of osteoclast multinucleation via a posttranscriptional regulation-based spatiotemporally selective delivery system. Sci Adv, 2022. 8 (26): p. eabn3333. Hu, Y., et al., Demethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification. Mol Cancer, 2022. 21 (1): p. 34. Bai, Y., et al., Marrow mesenchymal stem cell mediates diabetic nephropathy progression via modulation of Smad2/3/WTAP/m6A/ENO1 axis. Faseb j, 2024. 38 (11): p. e23729. Wu, J., et al., APOL1 risk variants in individuals of African genetic ancestry drive endothelial cell defects that exacerbate sepsis. Immunity, 2021. 54 (11): p. 2632-2649.e6. Chen, J.H., et al., The down-regulation of XBP1, an unfolded protein response effector, promotes acute kidney injury to chronic kidney disease transition. J Biomed Sci, 2022. 29 (1): p. 46. Xu, S., et al., Bone marrow mesenchymal stem cell-derived exosomal miR-21a-5p alleviates renal fibrosis by attenuating glycolysis by targeting PFKM. Cell Death Dis, 2022. 13 (10): p. 876. Ju, B., et al., Co-activation of hedgehog and AKT pathways promote tumorigenesis in zebrafish. Mol Cancer, 2009. 8 : p. 40. Liu, Y., et al., Nrf2 deficiency deteriorates diabetic kidney disease in Akita model mice. Redox Biol, 2022. 58 : p. 102525. Wang, M.J., et al., SIRT1-dependent deacetylation of Txnip H3K9ac is critical for exenatide-improved diabetic kidney disease. Biomed Pharmacother, 2023. 167 : p. 115515. Lu, Y.H., et al., Empagliflozin Attenuates Hyperuricemia by Upregulation of ABCG2 via AMPK/AKT/CREB Signaling Pathway in Type 2 Diabetic Mice. Int J Biol Sci, 2020. 16 (3): p. 529-542. Huang, X., et al., Targeting Epigenetic Crosstalk as a Therapeutic Strategy for EZH2-Aberrant Solid Tumors. Cell, 2018. 175 (1): p. 186-199.e19. Lou, F., et al., Excessive Polyamine Generation in Keratinocytes Promotes Self-RNA Sensing by Dendritic Cells in Psoriasis. Immunity, 2020. 53 (1): p. 204-216.e10. Cai, T., et al., Sodium-glucose cotransporter 2 inhibition suppresses HIF-1 α-mediated metabolic switch from lipid oxidation to glycolysis in kidney tubule cells of diabetic mice. Cell Death Dis, 2020. 11 (5): p. 390. Xing, S., et al., Hypoxia downregulated miR-4521 suppresses gastric carcinoma progression through regulation of IGF2 and FOXM1. Mol Cancer, 2021. 20 (1): p. 9. Hong, S., G. Zheng, and J.W. Wiley, Epigenetic regulation of genes that modulate chronic stress-induced visceral pain in the peripheral nervous system. Gastroenterology, 2015. 148 (1): p. 148-157.e7. Zeni, L., et al., A more tubulocentric view of diabetic kidney disease. J Nephrol, 2017. 30 (6): p. 701-717. Di Vincenzo, A., et al., Renal structure in type 2 diabetes: facts and misconceptions. J Nephrol, 2020. 33 (5): p. 901-907. Kang, H.M., et al., Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development. Nat Med, 2015. 21 (1): p. 37-46. Vallon, V. and S.C. Thomson, The tubular hypothesis of nephron filtration and diabetic kidney disease. Nat Rev Nephrol, 2020. 16 (6): p. 317-336. Doke, T. and K. Susztak, The multifaceted role of kidney tubule mitochondrial dysfunction in kidney disease development. Trends Cell Biol, 2022. 32 (10): p. 841-853. Kogot-Levin, A., et al., Proximal Tubule mTORC1 Is a Central Player in the Pathophysiology of Diabetic Nephropathy and Its Correction by SGLT2 Inhibitors. Cell Rep, 2020. 32 (4): p. 107954. Zhang, Y., et al., A sodium-glucose cotransporter 2 inhibitor attenuates renal capillary injury and fibrosis by a vascular endothelial growth factor-dependent pathway after renal injury in mice. Kidney Int, 2018. 94 (3): p. 524-535. Gall, J.M., et al., Hexokinase regulates Bax-mediated mitochondrial membrane injury following ischemic stress. Kidney Int, 2011. 79 (11): p. 1207-16. Pollock, A.S., Induction of renal phosphoenolpyruvate carboxykinase mRNA: suppressive effect of glucose. Am J Physiol, 1989. 257 (1 Pt 2): p. F145-51. Mithieux, G., F. Rajas, and A. Gautier-Stein, A novel role for glucose 6-phosphatase in the small intestine in the control of glucose homeostasis. J Biol Chem, 2004. 279 (43): p. 44231-4. Sasaki, M., et al., Dual Regulation of Gluconeogenesis by Insulin and Glucose in the Proximal Tubules of the Kidney. Diabetes, 2017. 66 (9): p. 2339-2350. Christofk, H.R., et al., The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature, 2008. 452 (7184): p. 230-3. Zhu, Z., et al., Transition of acute kidney injury to chronic kidney disease: role of metabolic reprogramming. Metabolism, 2022. 131 : p. 155194. Srivastava, S.P., et al., SIRT3 deficiency leads to induction of abnormal glycolysis in diabetic kidney with fibrosis. Cell Death Dis, 2018. 9 (10): p. 997. Zhou, D. and Y. Liu, Renal fibrosis in 2015: Understanding the mechanisms of kidney fibrosis. Nat Rev Nephrol, 2016. 12 (2): p. 68-70. Djudjaj, S. and P. Boor, Cellular and molecular mechanisms of kidney fibrosis. Mol Aspects Med, 2019. 65 : p. 16-36. B'Chir, W., et al., Divergent Role of Estrogen-Related Receptor α in Lipid- and Fasting-Induced Hepatic Steatosis in Mice. Endocrinology, 2018. 159 (5): p. 2153-2164. Chen, C.Y., et al., Inhibition of Estrogen-Related Receptor α Blocks Liver Steatosis and Steatohepatitis and Attenuates Triglyceride Biosynthesis. Am J Pathol, 2021. 191 (7): p. 1240-1254. Yang, M., et al., Dysfunction of estrogen-related receptor alpha-dependent hepatic VLDL secretion contributes to sex disparity in NAFLD/NASH development. Theranostics, 2020. 10 (24): p. 10874-10891. Dhillon, P., et al., The Nuclear Receptor ESRRA Protects from Kidney Disease by Coupling Metabolism and Differentiation. Cell Metab, 2021. 33 (2): p. 379-394.e8. Zhao, L., et al., Impact of non-alcoholic fatty liver disease and fibrosis on mortality and kidney outcomes in patients with type 2 diabetes and chronic kidney disease: A multi-cohort longitudinal study. Diabetes Obes Metab, 2024. 26 (10): p. 4241-4250. Musso, G., et al., Association of non-alcoholic fatty liver disease with chronic kidney disease: a systematic review and meta-analysis. PLoS Med, 2014. 11 (7): p. e1001680. Chen, Z., et al., Reduction of anaerobic glycolysis contributes to angiotensin II-induced podocyte injury with foot process effacement. Kidney Int, 2023. 103 (4): p. 735-748. Ozawa, S., et al., Glycolysis, but not Mitochondria, responsible for intracellular ATP distribution in cortical area of podocytes. Sci Rep, 2015. 5 : p. 18575. Yuan, Q., et al., Role of pyruvate kinase M2-mediated metabolic reprogramming during podocyte differentiation. Cell Death Dis, 2020. 11 (5): p. 355. Table 1 Table 1 is available in the Supplementary Files section. Supplementary Files Table1.tif ESMmaterials250328.docx Cite Share Download PDF Status: Published Journal Publication published 16 Apr, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Accept as is 31 Mar, 2025 Reviewers agreed at journal 29 Mar, 2025 Reviewers invited by journal 29 Mar, 2025 Editor assigned by journal 29 Mar, 2025 First submitted to journal 27 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5563608","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":435665401,"identity":"92e09710-7cef-4d69-a31e-17862b6deccc","order_by":0,"name":"Xiang Cai","email":"","orcid":"","institution":"Third Affiliated Hospital of Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Cai","suffix":""},{"id":435665402,"identity":"88512347-6955-4b29-bd71-265ad78bbb30","order_by":1,"name":"Huanyi Cao","email":"","orcid":"","institution":"Guangdong Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huanyi","middleName":"","lastName":"Cao","suffix":""},{"id":435665403,"identity":"846407b7-9395-4a04-8d88-986446f78431","order_by":2,"name":"Meijun Wang","email":"","orcid":"","institution":"Xunfei Healthcare Techonology Co.Ltd","correspondingAuthor":false,"prefix":"","firstName":"Meijun","middleName":"","lastName":"Wang","suffix":""},{"id":435665404,"identity":"593ca7e1-ad29-4bf8-bd40-5bf5e145c4b8","order_by":3,"name":"Piaojian Yu","email":"","orcid":"","institution":"Third Affiliated Hospital of Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Piaojian","middleName":"","lastName":"Yu","suffix":""},{"id":435665405,"identity":"5359475f-c8a5-4b50-ad73-2c5cc1a294aa","order_by":4,"name":"Xiaoqi Liang","email":"","orcid":"","institution":"Third Affiliated Hospital of Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqi","middleName":"","lastName":"Liang","suffix":""},{"id":435665406,"identity":"d7df0542-7e90-4e73-9ef2-409fbe4ffb82","order_by":5,"name":"Hua Liang","email":"","orcid":"","institution":"Shunde Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Liang","suffix":""},{"id":435665407,"identity":"2d085f3a-93aa-484f-a190-9fc42bad85c9","order_by":6,"name":"Fen Xu","email":"","orcid":"","institution":"Third Affiliated Hospital of Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Fen","middleName":"","lastName":"Xu","suffix":""},{"id":435665408,"identity":"3cabf7da-efd3-4d1a-8842-f1820823f668","order_by":7,"name":"Mengyin Cai","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-4329-1162","institution":"Third Affiliated Hospital of Sun Yat-Sen University","correspondingAuthor":true,"prefix":"","firstName":"Mengyin","middleName":"","lastName":"Cai","suffix":""}],"badges":[],"createdAt":"2024-12-02 10:20:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5563608/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5563608/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-05688-8","type":"published","date":"2025-04-16T15:57:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79745824,"identity":"dbe02fc6-c61b-47b0-9716-f0719f38a38b","added_by":"auto","created_at":"2025-04-02 08:49:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6290042,"visible":true,"origin":"","legend":"\u003cp\u003eEmpagliflozin ameliorate kidney fibrosis in DKD patients and DKD models (A) Representative images of PAS staining, Masson staining, and immunochemistry staining of NGAL from kidney sections of DKD patients. Images were obtained under a light microscope at 100X lens and 200X lens. (B) Representative images of immunochemistry staining of COL3A1 and αSMA from kidney sections of DKD patients. Images were obtained under a light microscope at 100X lens. (C) Representative kidney transmission electron microscopy (TEM) images of DKD patients. Thickened glomerular basement membrane was indicated by red arrow. Foot process effacement was indicated by black arrow. Scale bar, 2μM. (D) Representative images of PAS staining, Masson staining, and immunochemistry staining of NGAL from kidney sections of CD-1 mice. Images were obtained under a light microscope at 100X lens. (E) Representative images of immunofluorescence staining for COL3A1 and Lotus tetragonolobus lectin (LTL) in CD-1 mice. Scale bar, 100 μm. (F) Representative images of PAS staining, Masson staining, and immunochemistry staining of NGAL from kidney sections of C57BL/6 mice. Images were obtained under a light microscope at 100X lens. (G) Representative images of immunofluorescence staining for COL3A1 and Lotus tetragonolobus lectin (LTL) in C57BL/6 mice. Scale bar, 100 μm.(H) Western blot analysis for collagenIII α1 chain (COL3A1) and αSMA in the kidneys of CD-1 mice. For quantification, the band intensities of COL3A1 and αSMA protein were normalized to respective band intensities of β-actin. (I) Western blot analysis for COL3A1 and αSMA in the kidneys of C57BL/6 mice. For quantification, the band intensities of COL3A1 and αSMA protein were normalized to respective band intensities of β-actin. (J) Western blot analysis for COL3A1 and αSMA in the kidneys of HK-2 mice. For quantification, the band intensities of COL3A1 and αSMA protein were normalized to respective band intensities of β-actin. For all panels, *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05. Abbreviations: NG, normal glucose; HG, high glucose; HFD, high-fat diet; EMPA, empagliflozin.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5563608/v1/7255b1b5e6cfdc40083ebf7d.png"},{"id":79745825,"identity":"0d7db516-fe70-4333-9179-9d3d7a479ce9","added_by":"auto","created_at":"2025-04-02 08:49:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1184297,"visible":true,"origin":"","legend":"\u003cp\u003eEmpagliflozin downregulated the mRNA level of \u003cem\u003ePKM2\u003c/em\u003e in DKD (A) Heatmap and volcano map of the identified differentially expressed genes (DEGs) (B) Bubble chart of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment for fibrosis-related pathways in Normal glucose (NG)-treated and high glucose (HG)-treated HK-2 cells. (C) \u0026nbsp;Bubble chart of Gene Ontology (GO) enrichment for fibrosis-related pathways in Normal glucose (NG)-treated and high glucose (HG)-treated HK-2 cells. (D) KEGG analyses of enriched pathways upregulated in HG group and meanwhile downregulated by empagliflozin according to RNA-sequencing. (E) FPKM levels of glycolytic genes expression of glycolytic genes in HK-2 cells treated NG, HG or HG with empagliflozin according to RNA-sequencing. (F) Expression of glycolytic genes in HK-2 cells treated NG, HG or HG with empagliflozin analysed by RT-qPCR. (G) mRNA expression of \u003cem\u003ePKM2\u003c/em\u003e, \u003cem\u003ePFKP\u003c/em\u003e, and \u003cem\u003eHK2\u003c/em\u003e in each CD-1 mouse group. (H) mRNA expression of \u003cem\u003ePKM2\u003c/em\u003e, \u003cem\u003ePFKP\u003c/em\u003e, and \u003cem\u003eHK2\u003c/em\u003e in each C57BL/6 mouse group.For all panels, *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05. Abbreviations: NG, normal glucose; HG, high glucose; EMPA, empagliflozin; CD, chow diet; HFD, high fat diet.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5563608/v1/993d26c7e97817f5466cad7b.png"},{"id":79747191,"identity":"c8c29243-0378-4d7d-8f02-ad9383788f8e","added_by":"auto","created_at":"2025-04-02 08:57:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3524931,"visible":true,"origin":"","legend":"\u003cp\u003eEmpagliflozin downregulated the protein level of PKM2in DKD (A) Western blot analysis for PKM2 in HK-2 cells treated with NG, HG, or HG with empagliflozin. For quantification, the band intensities of PKM2 proteins were normalized to respective band intensities of β-ACTIN. (B) Western blot analysis forPKM2 in each group of CD-1 mice. For quantification, the band intensities of PKM2 proteins were normalized to respective band intensities of β-ACTIN. (C) Western blot analysis forPKM2 in each group of C57BL/6 mice. For quantification, the band intensities of PKM2 proteins were normalized to respective band intensities of β-ACTIN. (D-E) Representative images of immunofluorescence staining for PKM2 and Lotus tetragonolobus lectin (LTL) in CD-1 and C57BL/6 mice. Scale bar, 100 μm. (F) Representative images of immunochemistry staining of PKM2 from kidney sections of CD-1 mice. Images were obtained under a light microscope at 100X lens. (G) Representative images of immunochemistry staining of PKM2 from kidney sections of C57BL/6 mice. Images were obtained under a light microscope at 100X lens. (H) Representative images of immunochemistry staining of PKM2 from kidney sections of DKD patients. Images were obtained under a light microscope at 100X lens. (I-K) Correlation analysis of the expression of PKM2\u003cem\u003e \u003c/em\u003eprotein levels with COL3A1 and αSMA in kidneys of DKD patients, each group of CD-1 mice and each group or C57BL/6 mice. For all panels, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Abbreviations: NG, normal glucose; HG, high glucose; Ctrl, control; CD, chow diet; HFD, high-fat diet; EMPA, empagliflozin.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5563608/v1/cb05d17d6b71d7070646d29b.png"},{"id":79745827,"identity":"a1dcd272-1928-43a7-8c7f-b9bcf40bbed2","added_by":"auto","created_at":"2025-04-02 08:49:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":769893,"visible":true,"origin":"","legend":"\u003cp\u003eEmpagliflozin ameliorated renal fibrosis by downregulating PKM2 in vitro\u003cstrong\u003e \u003c/strong\u003e(A-B) Western blot analysis for PKM2 in HK-2 cells treated with empty vector or PKM2-overexpression plasmid (pcDNA3-PKM2). (C-D) Western blot analysis of PKM2 in HK-2 cells treated with shRNA-NC or shRNA-PKM2. (E-G) Western blot analysis of COL3A1 and αSMA in HK-2 cells treated with empty vector or pcDNA3-PKM2. (H-K) Western blot analysis of COL3A1, αSMA, and PKM2 in each group of HK-2 cells. (L-O) Western blot analysis of COL3A1, αSMA, and PKM2 in each group of HK-2 cells. For quantification, the band intensities of PKM2, COL3A1, and αSMA were normalized to respective band intensities of β-ACTIN.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5563608/v1/8d082c3cf51646c89599c6e2.png"},{"id":79745830,"identity":"db5588b1-2b5e-47f3-bc24-ef95d2164742","added_by":"auto","created_at":"2025-04-02 08:49:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2888204,"visible":true,"origin":"","legend":"\u003cp\u003eEmpagliflozin ameliorated EMT in DKD (A) Representative images of immunochemistry staining and the quantitative analysis of E-cadherin and Vimentin from kidney sections of DKD patients. Images were obtained under a light microscope at 100X lens. (B) Representative images of immunochemistry staining and the quantitative analysis of E-cadherin and Vimentin from kidney sections of each group of CD-1 mice. Images were obtained under a light microscope at 100X lens. (C) Representative images of immunochemistry staining and the quantitative analysis of E-cadherin and Vimentin from kidney sections of each group of C57BL/6 mice. Images were obtained under a light microscope at 100X lens. (E-F) Correlation analysis of the positive area of PKM2 with E-cadherin and Vimentin in kidneys of DKD patients, each group of CD-1 mice and C57BL/6 mice. For all panels, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Abbreviations: Ctrl, control; CD, chow diet; HFD, high-fat diet; NG, normal glucose; HG, high glucose; EMPA, empagliflozin; ECAD, E-cadherin; VIM, vimentin.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5563608/v1/10cb4981a8a8c268054faf2a.png"},{"id":79745832,"identity":"78f2a965-e8ff-41d6-ad0b-998a81e472f1","added_by":"auto","created_at":"2025-04-02 08:49:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1398311,"visible":true,"origin":"","legend":"\u003cp\u003eEmpagliflozin downregulated \u003cem\u003ePKM2\u003c/em\u003e by blocking the binding of estrogen-related receptor alpha (ESRRA) to the promoter (A) Western blot analysis for cytoplasmic and nuclear ESRRA in kidneys of each CD-1 mouse group. For quantification, the band intensities of cytoplasmic ESRRA were normalized to respective band intensities of β-ACTIN. The band intensities of nuclear ESRRA were normalized to respective band intensities of LAMIN-B. (B) Western blot analysis for cytoplasmic and nuclear ESRRA in kidneys of each C57BL/6 mouse group. For quantification, the band intensities of cytoplasmic ESRRA were normalized to respective band intensities of β-ACTIN. The band intensities of nuclear ESRRA were normalized to respective band intensities of LAMIN-B. (C) Western blot analysis for cytoplasmic and nuclear ESRRA in HK-2 cells treated with NG, HG, or HG with empagliflozin. For quantification, the band intensities of cytoplasmic ESRRA were normalized to respective band intensities of β-ACTIN. The band intensities of nuclear ESRRA were normalized to respective band intensities of PCNA. \u0026nbsp;\u0026nbsp;(D) Representative images of intracellular immunofluorescence staining for ESRRA in HK-2 cells treated with NG, HG, or HG with empagliflozin. Scale bar, 100 μm. (E) Luciferase reporter assays for the \u003cem\u003ePKM2\u003c/em\u003e promoter were performed in HK-2 cells co-transfected with ESRRA expression plasmids and luciferase reporter plasmids containing wild-type or mutant mouse \u003cem\u003ePKM2\u003c/em\u003e promoters. (F) ChIP analysis for ESRRA binding to the promoter of \u003cem\u003ePKM2\u003c/em\u003e gene in HK-2 cells at different binding sites. (G) ChIP analysis for ESRRA binding to the promoter of \u003cem\u003ePKM2\u003c/em\u003egene in HK-2 cells treated with NG, HG, or HG with Empagliflozin at different binding sites. For all panels, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Abbreviations: STZ, streptozotocin; CD, control diet; HFD, high fat diet; NG, normal glucose; HG, high glucose; EMPA, empagliflozin, DBS, distant binding site, BS, binding site.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5563608/v1/1f2e3463262b0d4a75603884.png"},{"id":81050972,"identity":"9f7a4424-3404-4229-b91e-3ed61060041d","added_by":"auto","created_at":"2025-04-21 16:09:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19725677,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5563608/v1/08c13bfb-d300-42a4-89fb-869083d57e4c.pdf"},{"id":79745834,"identity":"b2e550d6-b852-4a37-9fec-7708cf939c29","added_by":"auto","created_at":"2025-04-02 08:49:12","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":784600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Table1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5563608/v1/75f319e94a29b93c572dcd34.tif"},{"id":79745835,"identity":"aa2dc37f-459d-4ce9-9d85-3fc57cdadf91","added_by":"auto","created_at":"2025-04-02 08:49:12","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4828815,"visible":true,"origin":"","legend":"","description":"","filename":"ESMmaterials250328.docx","url":"https://assets-eu.researchsquare.com/files/rs-5563608/v1/1887c81c3e7a0305febd0dbe.docx"}],"financialInterests":"","formattedTitle":"SGLT2 inhibitor empagliflozin ameliorates tubulointerstitial fibrosis in DKD by downregulating renal tubular PKM2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic kidney disease (DKD), one of the most prevalent complications of diabetes mellitus, is a leading cause for end-stage renal disease and increasingly aggravate the significant global burden [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Renal fibrosis is a key characteristic of DKD. The development of renal fibrosis has been shown to cause the progress of renal function decline and serves as a common result to renal failure.\u003c/p\u003e \u003cp\u003eSGLT2 inhibitors have been recommended in the American Diabetes Association guidelines for chronic kidney disease treatment in diabetic patients[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Large clinical trials have shown that SGLT2 inhibitors significantly slow the progression to renal failure in patients with DKD [\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, these trials primarily focus on parameters such as blood creatinine levels, estimated glomerular filtration rate (eGFR), and urine albumin-to-creatinine ratio (UACR), which are more indicative of glomerular injury rather than renal fibrosis, an important cause of renal failure. Moreover, in both clinical and preclinical studies, SGLT2 inhibitors have been reported to modulate multiple pathways implicated in renal fibrosis among patients with diabetic kidney disease (DKD). Canagliflozin has been reported to downregulate plasma levels of inflammatory markers, including TNF receptor 1, IL-6, matrix metalloproteinase 7, and fibronectin 1, in patients with diabetic kidney disease[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. SGLT2 inhibitors also reduce DNA oxidation and activate antioxidant mechanisms in diabetic patients, indicating their role in anti-oxidation and the maintenance of cellular redox homeostasis in diabetic kidney disease (DKD) patients [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Additionally, SGLT2 inhibitors have been observed to reduce renal macrophage infiltration in DKD mouse models and in type 2 diabetic patients at high cardiovascular risk[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Given the significant effects of SGLT2 inhibitors on improving DKD and the critical role of renal fibrosis in driving the progression of DKD, it is essential to continue exploring the specific molecular mechanisms through which these drugs ameliorate renal fibrosis, which will aid in identifying additional therapeutic targets for DKD. Consequently, extensive pre-clinical and clinical studies are still required to investigate the anti-fibrotic effects of SGLT2 inhibitors.\u003c/p\u003e \u003cp\u003eSGLT2 inhibitors directly target renal tubular proximal cells (RTPCs) in DKD. Importantly, during the development of renal fibrosis, proximal renal tubule is the epicenter [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. RTPCs are not only affected members of the injury, but also active promoters. Renal tubular epithelial cells can promote renal fibrosis through multiple mechanisms. Epithelial-to-mesenchymal transition (EMT) is critical for the development of renal interstitial fibrosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. EMT is characterized by a loss of epithelial phenotype and a gain of profibrotic features [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Upon activation of EMT, RTPCs undergo a transition where they lose their epithelial markers, leading to collagen deposition and renal fibrosis. In addition to EMT, damaged tubules produce and release bioactive molecules that recruit inflammatory cells, thereby activating myofibroblast differentiation, proliferation, and matrix secretion[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Moreover, G2/M phase cell cycle arrest and apoptosis of tubular epithelial cells can promote renal fibrosis by activating the JNK signaling pathway, which leads to the upregulation of profibrotic factors, stimulation of fibroblast proliferation, and accumulation of extracellular matrix[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, it is crucial to explore the impact of SGLT2 inhibitors on RTPCs in DKD-related renal fibrosis, as well as the underlying molecular mechanisms involved.\u003c/p\u003e \u003cp\u003eIn the present study, we examined the effect of empagliflozin, an SGLT2 inhibitor on ameliorating kidney fibrosis in patients with DKD and in DKD models, while also investigating the underlying molecular mechanism.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman samples\u003c/h2\u003e \u003cp\u003eDiabetic patients were diagnosed with DKD by kidney biopsy. The human biopsy samples from DKD patients were obtained from puncture specimens. The patients were divided into two groups: one group did not receive SGLT2 inhibitors (SGLT2is), while the other group were treated with SGLT2is. All patients included in this study received the maximum tolerated dose of angiotensin-converting enzyme inhibitor/angiotensin‐receptor blocker. The exclusion criteria were as follows: age\u0026thinsp;\u0026lt;\u0026thinsp;18 years; the presence of other types of kidney disease; pregnancy; infection; genetic disease; taking GLP-1 receptor agonists, finerenone, and other medications that have demonstrated a significant kidney protective effect; taking SGLT2 inhibitors less than 3 months before the renal biopsy. Patient information, including gender, age, diabetes duration, blood pressure, urinary albumin-to-creatinine ratio (ACR), estimate glomerular filtration rate (eGFR), glycated hemoglobin A1c (HbA1c), plasma lipid profile, medication history, images of PAS staining, Masson staining and transmission election microscopy results were retrospectively obtained from the hospital medical record system. The present study was conducted in accordance with the 1964 Helsinki ethical declaration and its subsequent amendments. The experimental design was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (approval number: II2024-056).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal models\u003c/h3\u003e\n\u003cp\u003e All experiments were approved by the Animal Ethics Committee of Sun Yat-sen University (approval number: IACUC-F3\u0026ndash;22\u0026ndash;0415, SYSU-IACUC-2020000006) and conducted in accordance with the standard protocols approved by the National Research Council's Guide for the Care and Use of Laboratory Animals and the Animal Ethics Committee of Sun Yat-sen University. All mice had free access to water and food, and were housed under a 12-hour light/12-hour dark cycle.\u003c/p\u003e \u003cp\u003eSeven-week-old male CD-1 mice were purchased from Guangdong Zhiyuan Biomedical Technology Co (Guangzhou, China). CD-1 mice were kept in a barrier environment at the Animal Experimental Center of the Third Affiliated Hospital of Sun Yat-sen University. After one week of adaptive feeding, CD-1 mice were randomly and blindly divided into two groups (n\u0026thinsp;=\u0026thinsp;6 per group). The STZ-induced diabetic mouse model was constructed according to previously published study[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In brief, the streptozotocin (STZ) group received an intraperitoneal injection of a single dose of STZ at 150 mg/kg in a citrate buffer (10 mM) to induce diabetes, while an equal volume of citrate buffer (10mM) was injected in control group. Two weeks after STZ injection, mice with diabetes were confirmed by fasting blood glucose level greater than 16 mM. Eight weeks after diabetes induction, empagliflozin (10 mg/kg/day) dissolved in 0.5% hydroxypropyl methylcellulose (HPMC) or the same volume of 0.5% HPMC (control) was administered by oral gavage until ACR was significantly decreased (12 weeks) (n\u0026thinsp;=\u0026thinsp;6 per group). Fasting blood glucose and body weight were measured every two weeks. The ACR was quantified by the Department of Laboratory Medicine of the Third Affiliated Hospital at Sun Yat-sen University.\u003c/p\u003e \u003cp\u003eSeven-week-old male C57BL/6 mice were obtained from GemPharmatech (Nanjing, China). After one week of adaptive feeding, C57BL/6 mice were randomly and blindly divided into two groups (n\u0026thinsp;=\u0026thinsp;6 per group). The chow diet (CD) group were fed a diet containing 11% fat (Guangdong Medical Laboratory Animal Center, Guangzhou, China). The high-fat diet (HFD) group were fed a diet containing 58% fat (D12331, Research Diets, New Brunswick, NJ, USA)[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. DKD of C57BL/6 mice was confirmed by significantly elevated FBG, impaired glucose tolerance and increased ACR after 14 weeks of HFD-feeding. After 14 weeks of feeding, empagliflozin (10 mg/kg/day) or the same volume of 0.5% HPMC were administered via oral gavage until ACR was significantly decreased (6 weeks) (n\u0026thinsp;=\u0026thinsp;6 per group). Fasting blood glucose and body weight were measured every two weeks. The plasma insulin level was detected by ELISA kits from Elabscience (Wuhan, China). The urinary albumin-to-creatinine ratio (ACR) was calculated by urinary microalbumin /urinary creatinine. The urinary albumin level and urinary creatinine level was determined using the creatinine companion and the Albuwell M kits purchased from Guangzhou Ruishu Biotechnology Co., Ltd (Guangzhou, China).\u003c/p\u003e \u003cp\u003eSeven-week-old B6\u0026middot;V-Lep\u003csup\u003e\u003cem\u003eob\u003c/em\u003e\u003c/sup\u003e/J (\u003cem\u003eob/ob\u003c/em\u003e) mice were purchased from GemPharmatech (Nanjing, China) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. After one week of adaptive feeding, \u003cem\u003eob/ob\u003c/em\u003e mice were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;6 per group) and fed a methionine- and choline-deficient (MCD) diet for 8 weeks. Mice were treated with empagliflozin (10 mg/kg/day) or an equal volume of 0.5% HPMC via oral gavage until ACR was significantly decreased (8 weeks).\u003c/p\u003e \u003cp\u003eAfter fasted overnight, the blood samples were collected from DKD mice anesthetized using isoflurane, followed by administration of more than 100 mg/kg of pentobarbital for euthanasia. Subsequently, mice were sacrificed through cervical vertebra dislocation, followed by tissue collection. The left kidneys from five mice in each group were cut in half longitudinally, and the half were then fixed in 4% formaldehyde for at least 24 h, embedded in paraffin and sectioned at 4\u0026ndash;6 \u0026micro;m thickness for renal histological analysis, immunofluorescence, and immunochemistry.\u003c/p\u003e\n\u003ch3\u003eGlucose and insulin tolerance test\u003c/h3\u003e\n\u003cp\u003eThe methods of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were similar to those described in our previous study[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In brief, after fasting for 6 hours, mice were intraperitoneally injected with glucose (2 g/kg) solved in saline to perform an IPGTT. Blood samples were collected from the tail tip at 0, 30, 60 and 120 minutes after injection. Blood glucose was measured by a glucometer (ONETOUCH UltraVue, Johnson \u0026amp; Johnson, USA). An IPITT was performed by intraperitoneal injection of human insulin (0.65 IU/kg) (Novolin R, Novo Nordisk A/S, Copenhagen, Denmark) after 4 hours of fasting.\u003c/p\u003e\n\u003ch3\u003eCell culture and treatment\u003c/h3\u003e\n\u003cp\u003eMycoplasma-free human immortalized RTPC (HK-2) (GNHu47, Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in a 1:1 mixture of Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) and Ham\u0026rsquo;s F12 medium (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) with 10% (v/v) fetal bovine serum (Procell, Wuhan, China). After starving in a serum-free medium for 12 h, HK-2 cells were synchronized and then treated with normal concentration of glucose (NG, 5.5 mM), high concentration of glucose (HG, 30 mM), or HG with empagliflozin (1 mM) for 72 hours. For construction of non-diabetic renal fibrosis model, HK-2 cells were treated with TGF-β (5ng/ml) for 24 hours, and then treated with empagliflozin (1mM) for another 72 hours.\u003c/p\u003e\n\u003ch3\u003eKnockdown and overexpression of PKM2\u003c/h3\u003e\n\u003cp\u003eThe PKM2 shRNA and PKM2-overexpression vector (pcDNA3-PKM2, OE) were purchased from GenePharma (Shanghai GenePharma Co., Ltd., China) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. HK-2 cells were transfected at 60\u0026ndash;65% confluence with PKM2 shRNA or overexpression vector by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. For \u003cem\u003ePKM2\u003c/em\u003e knockdown, the sequences of shRNA were cloned into pGPU6 vector. shPKM2: \u0026ldquo;5\u0026prime;-TTATTTGAGGAACTCCGCCGC-3\u0026rdquo;. For transfection, cells were seeded into a 6-well plate, cultured overnight to 60\u0026ndash;70% confluence, and then transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific, 11668019) supplemented with 2500 ng DNA or 50 nM (final concentration) siRNA[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRenal histopathology\u003c/h2\u003e \u003cp\u003ePeriodic acid-Schiff (PAS) staining and Masson trichrome staining were conducted according to previous published studies[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. For PAS staining, the paraffin-embedded kidney slides were stained with 0.5% periodic acid for 10 min, washed, and stained again with Schiff reagent for 15 min. For Masson trichrome staining, the paraffin-embedded kidney sections were stained using Weigert\u0026rsquo;s iron hematoxylin, azophloxine staining solution, phosphotungstic acid orange G, and light-green SF solution using a step-by-step method. After dehydration and xylene clearing, stained sections were observed under a light microscope (Olympus BX63; Olympus, Tokyo, Japan). Three photographs of each stained slide were taken.\u003c/p\u003e \u003cp\u003eTubular injury was defined as tubular dilation, tubular atrophy, formation of cylindrical tubules, detachment of tubular epithelial cells or loss of brush border and thickening of the tubular basement membrane. The scoring system used was as follows: 0 points, no tubular damage; 1 point, \u0026lt;\u0026thinsp;10% renal tubular damage; 2 points; 10\u0026ndash;25% renal tubular damage; 3 points, 25\u0026ndash;50% renal tubular damage; 4 points, 50\u0026ndash;74% renal tubular injury; 5 points, \u0026gt;\u0026thinsp;75% renal tubular injury damage[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemical analysis\u003c/h3\u003e\n\u003cp\u003eKidney sections from DKD patients, CD-1 mice and C57BL/6 mice were used for immunohistochemical staining. Immunohistochemical was carried out according to standard procedures reported in previous published studies[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In brief, kidney tissues were fixed in 4% paraformaldehyde at 4\u0026deg;C for 24 hours, followed by dehydration by an ascending series of ethanol baths. Then the tissues were cleared with xylene and embedded in paraffin. The paraffin-embedded kidney tissues were cut into 4-\u0026micro;m sections. The kidney sections were then dewaxed with xylene and rehydrated with gradient ethanol. Specimens were incubated with 1% bovine serum albumin in PBS for 1 hour and then incubated with the primary antibodies anti-neutrophil gelatinase associated lipocalin (NGAL) (1:500), anti-pyruvate kinase M2 (PKM2) (1:200), anti-E-cadherin (1:200), anti-Vimentin (1:200), anti-collagen type III α1 (COL3A1) (1:200), anti-α smooth muscle actin (αSMA) (1:200) primary antibodies at 4\u0026ordm;C overnight. Then the kidney sections were incubated with an enzyme-conjugated secondary antibody (1:2000) for 50 min at 37\u0026ordm;C. The detailed antibody information is shown in Table\u0026nbsp;1 of the extra supplementary material (ESM).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eThe initial immune-staining steps were performed as previously described [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. For kidney immunofluorescence staining, tissues were paraffin-embedded, de-paraffinized and rehydrated. Samples were incubated with primary antibodies for COL3A1 (1:200), PKM2 (1:200), and Lotus tetragonolobus lectin (LTL; 1:200) at 4 \u0026ordm;C overnight, followed by incubation with fluorophore-conjugated secondary antibody (1:200) for 1 h at 37℃. Subsequently, 4,6-diamino-2-phenyl indole (DAPI) was applied for 5 min. For HK-2 cells immunofluorescence staining, cells were fixed in 4% paraformaldehyde for 30 minutes, and permeabilized with 0.5% Triton X-100 for 15 min. After that, the cells were blocked in 1% bovine serum albumin (BSA) for 1 hour and incubated with the primary antibody for estrogen-related receptor α (ESRRA) (1:200) overnight at 4 \u0026ordm;C. Next, they were incubated with fluorophore-conjugated secondary antibody (1:10000) for 1 h, followed by DAPI solution for 5 min at 37\u0026ordm;C. Images were obtained using a Leica fluorescence microscope (Leica Microsystems, Wetzlar, Germany) at 200\u0026times; magnification. The detailed antibody information is shown in ESM Table\u0026nbsp;2.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing\u003c/h2\u003e \u003cp\u003eHK-2 cells were seeded in T25 culture flask and treated with NG, HG, or HG with empagliflozin for 72 h. Total RNA was isolated using TRIzol (#15596018, Invitrogen, Carlsbad, CA, USA). Each sample in NG, HG, and HG with empagliflozin groups were resultant mix of nine RNA extraction. According to the TruSeq\u0026trade; RNA Sample Preparation Guide, paired-end libraries were synthesized used the TruSeq\u0026trade; RNA Sample Prep Kit (Illumina, San Diego, CA, USA). Briefly, poly-A-containing mRNA molecules were purified using poly-T oligo-linked magnetic beads and then fragmented into small pieces using divalent cations at 94\u0026ordm;C for 8 min. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. DNA Polymerase I and RNase H were used to synthesize second strand cDNA. These cDNA fragments went through an end repair process, the addition of a single \u0026lsquo;A\u0026rsquo; base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final cDNA library. Purified libraries were quantified with a Qubit\u0026reg; 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and validated using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to confirm the insert size and calculate the mole concentration. Clusters were generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina NovaSeq 6000 (Illumina, San Diego, USA). The library construction and sequencing were performed by the Guangzhou Promegene Biotechnology Co., Ltd (Guangzhou, China). FPKM values were normalized per gene to obtain relative expression values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eNuclear-cytosolic protein extraction\u003c/h2\u003e \u003cp\u003eHK-2 cells were collected in ice-cold PBS (0.01 M) and then centrifuged at 800 RCF for 5 min. Tissue samples were cut into pieces and homogenized in the presence of phenylmethyl sulfonyl fluoride (PMSF). The samples were stored at -80\u0026deg;C for subsequent western blot analysis. The cytoplasmic and nuclear protein fractions were separated using corresponding extraction reagents using a Nuclear and Cytoplasmic Protein Extraction Kit (#p0028; Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer\u0026rsquo;s protocol [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Cytoplasmic protein and nuclear protein were separately collected and stored at -80\u0026deg;C for western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eWestern blot was performed according to previous published paper[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Kidney tissues and HK-2 cells were each homogenized in a lysis buffer containing protease and phosphatase inhibitors. Protein lysates were subjected to 8\u0026ndash;10% polyacrylamide dodecyl sulfate gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. PVDF membranes were then incubated in primary antibodies against COL3A1 (1:1000), alpha smooth muscle actin (α-SMA;1:1000), PKM2 (1:1000), hexokinase 2 (HK2) (1:1000), phosphofructokinase (PFKP) (1:1000), ESRRA (1:1000), beta-actin (β-ACTIN; 1:1000), and Lamin B1 (1:1000) at 4\u0026deg;C overnight. The membranes were then incubated with corresponding secondary antibodies (1:10000) at 37\u0026deg;C for 1 h. Band intensities were quantified using Image-Pro Plus v6.0 (Media Cybernetics, Washington DC, USA). The detailed antibody information is shown in ESM Table\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcription-PCR\u003c/h2\u003e \u003cp\u003e Total RNA extracted from mice kidneys was converted to cDNA using a cDNA reverse transcription kit (Cat# RR047A, TAKARA, Kyoto, Japan) according to the manufacturer\u0026rsquo;s instructions. Real-time quantitative PCR was performed using TB Green Premix Ex TaqII (Cat# RR820A, TAKARA, Kyoto, Japan) and conducted using a Light Cycler 480II Real-Time PCR System (Roche Diagnostics, Mannheim, Germany). β-actin was used as a housekeeping gene. The sequences of primers were listed in ESM Table\u0026nbsp;4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003eThe JASPAR database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jaspar.genereg.net/\u003c/span\u003e\u003cspan address=\"https://jaspar.genereg.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to identify the predicted ESSRA binding sites 2,000 base pairs (bp) upstream and 100 bp downstream of the transcription start site. The luciferase reporter assay was performed according to the previous published paper [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The wild-type or mutant promoters of human \u003cem\u003ePKM2\u003c/em\u003e were transduced into pGL3-basic vectors. The ESRRA expression plasmid, and wild-type or mutant pGL3-basic vectors were co-transfected into HEK 293T cells (Dongze Biotech Co., Ltd, Guangdong, China). Subsequently, luciferase assays were performed on HEK 293T cells at 70\u0026ndash;80% confluence using a dual-luciferase reporter system (#E2940; Promega, Madison, WI, USA) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003cp\u003eChromatin immunoprecipitation (ChIP)-qPCR\u003c/p\u003e \u003cp\u003eChromatin immunoprecipitation (ChIP) assays were performed using a Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the instructions provided by the manufacturer[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In brief, ESRRA antibody (Cell signaling technology, #13826) was adopted for immunoprecipitation of chromatin, and IgG was negative control. Isolated RNA was assayed by RT-qPCR. Primers designed for the predicted binding site 1 of ESRRA at \u003cem\u003ePKM2\u003c/em\u003e promoter regions were as follows: forward primer: 5\u0026rsquo;- CGGCGGAGGGATTGCG-3\u0026rsquo;, reverse primer: 5\u0026rsquo;- GCTACGCTGCAAAGACGAAGA-3\u0026rsquo;. Primers designed for the predicted binding site 2 of ESRRA at \u003cem\u003ePKM2\u003c/em\u003e promoter regions were as follows: forward primer:5\u0026rsquo;- ACCGAAAGGGCAACCTGC-3\u0026rsquo;, reverse primer: 5\u0026rsquo;- GGGCCGCCGCAATCC-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll quantitative experiments were repeated at least 3 times independently. GraphPad Prism 8.0.1 was used for statistical analysis and creating graphs (GraphPad Software, San Diego, CA, USA). Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Comparisons between two groups were performed using the unpaired Student t-test. Comparisons between multiple groups were performed using one-way ANOVA, followed by Tukey's multiple comparisons test. Correlations were assessed using Spearman's rank correlation. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSGLT2i ameliorate kidney fibrosis in DKD patients and DKD models\u003c/h2\u003e \u003cp\u003ePatients without SGLT2i treatment were recruited into group 1, patients treated with SGLT2i were recruited into group 2. Clinical information on the patients is summarized in Table\u0026nbsp;1. The male to female ratios were both 3 to 2 in the two groups. There was no statistical difference in mean age, diabetes duration, systolic blood pressure, diastolic blood pressure, urinary creatine, eGFR, and glycated hemoglobin between the two patient groups (Table\u0026nbsp;1). The medication duration of group 1 was longer than in group 2 (Table\u0026nbsp;1). As expected, the urinary ACR was significantly lower in group 2 (Table\u0026nbsp;1). Extracellular matrix accumulation (PAS staining) and tubulointerstitial fibrosis (MASSON staining) were ameliorated in group 2 compared with group 1, indicating that SGLT2i treatment alleviated in DKD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The tubular injury marker NGAL were downregulated by empagliflozin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Fibrosis marker COL3A1 and αSMA were downregulated by empagliflozin in DKD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Transmission electron microscopy (TEM) also showed the tubulointerstitial collagen deposition, basement thickening and foot process fusion were ameliorated by empagliflozin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter that, \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments were carried out to confirmed the anti-fibrotic effect of empagliflozin. FBG and ACR were elevated in both STZ-treated CD-1 mice and HFD-treated C57BL/6 mice (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D, F-G). Body weight was reduced in STZ-treated CD1 mice (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) and elevated in HFD-treated C57BL/6 mice (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Tubulointerstitial fibrosis were observed by PAS and MASSON staining in both of the two DKD mouse models (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J). Tubular injury score and NGAL were upregulated under STZ and HFD treatment (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-N)). Empagliflozin lowered FBG, improved body weight, and reduced ACR in both STZ-treated and HFD-treated mice (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-H). Empagliflozin also lowered plasma insulin level, improved glucose tolerance (tested by IPGTT and IPITT) in HFD-treated mice (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-K). Notably, empagliflozin significantly ameliorated tubular dilation, extracellular matrix accumulation (PAS staining), and tubulointerstitial fibrosis (MASSON staining) in both STZ-treated and HFD-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, F). Tubular injury score and NGAL were also downregulated by empagliflozin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, F, ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL-O). Immunofluorescence and western blot were then used to detect fibrosis markers. Empagliflozin significantly downregulated COL3A1 and αSMA in both STZ-induced and HFD-induced DKD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, G, H-I). \u003cem\u003eIn vitro\u003c/em\u003e experiments were carried out in HK-2 cells. Under HG condition, COL3A1 and αSMA were upregulated in HK-2 cells, while empagliflozin significantly downregulated these fibrosis makers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEmpagliflozin downregulated mRNA level of PKM2 in DKD\u003c/h2\u003e \u003cp\u003eTo investigate the mechanisms underlying the anti-fibrotic effect of empagliflozin, RNA sequencing was conducted in HK-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Both KEGG and GO analyses revealed that pathways associated with fibrosis were upregulated under HG conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Additionally, KEGG analysis was performed to identify the top ten enriched pathways that were upregulated in the HG group and simultaneously downregulated by empagliflozin. Differentially expressed genes were involved in cytokine-cytokine receptor interaction, calcium signaling, chemokine signaling, JAK-STAT signaling, TGF-beta signaling, glucagon signaling, glycolysis, starch and sucrose metabolism, renin-angiotensin system, and the pentose phosphate pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). We focused our attention on differentially expressed genes involved in glycolysis, since the mechanisms by which empagliflozin reduces glycolysis have not yet been fully elucidated. RNA sequencing result showed that some of the genes involved in glycolytic pathway including PKM2, GAPDHS, PGAM2, FBP1, ADH1 were upregulated by HG and downregulated by empagliflozin. While other glycolytic genes including PFKP and HK2 showed no significant difference between NG, HG, and HG with empagliflozin group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The above glycolic genes in RNA sequencing were validated by RT-qPCR both \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003ePKM2, PFKP\u003c/em\u003e, and \u003cem\u003eHK2\u003c/em\u003e were upregulated by HG in HK-2 cells. Only PKM2 was downregulated by empagliflozin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The results were further validated in DKD mouse models. \u003cem\u003ePKM2\u003c/em\u003e was upregulated by STZ and HFD, and was downregulated by empagliflozin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEmpagliflozin downregulated protein level of PKM2 in DKD\u003c/h2\u003e \u003cp\u003eGenes fulfill their functions by being translated into functional proteins. In this study, we examined the protein expression levels of three key glycolytic enzymes: PKM2, HK2, and PFKP, in the context of diabetic kidney disease (DKD). Notably, PKM2 showed the highest level of upregulation under DKD conditions, while it was the most significantly downregulated enzyme in response to empagliflozin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Immunofluorescence analysis revealed that in both the STZ and HFD groups, PKM2 was highly expressed and co-localized with LTL, a marker indicative of kidney proximal tubule cells. Notably, empagliflozin significantly downregulated PKM2 expression in both STZ-treated and HFD-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). These findings were further corroborated by immunohistochemical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eP, Q). Importantly, PKM2 expression was found to be upregulated in the renal tubules of DKD patients, while it was downregulated in the empagliflozin-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eR). Furthermore, a positive correlation was observed between PKM2 levels and the expression of fibrosis markers COL3A1 and αSMA in both DKD models and DKD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-K).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEmpagliflozin ameliorated renal fibrosis by downregulating PKM2 in vitro\u003c/h2\u003e \u003cp\u003eTo further elucidate whether empagliflozin ameliorates renal fibrosis via the downregulation of PKM2, we conducted experiments to overexpress and knock down PKM2 in HK-2 cells separately. The effects of overexpression and knockdown were validated through Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). The PKM2 overexpression resulted in an elevation of fibrosis markers, including COL3A1 and αSMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G). Importantly, the overexpression of PKM2 diminished the ameliorative effects of empagliflozin on renal fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-K). Furthermore, PKM2 knockdown attenuated the downregulatory effects of empagliflozin on COL3A1 and αSMA, as evidenced by the comparison between the HG\u0026thinsp;+\u0026thinsp;shPKM2 and HG\u0026thinsp;+\u0026thinsp;shPKM2\u0026thinsp;+\u0026thinsp;Empagliflozin groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL-O).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEmpagliflozin ameliorated EMT in DKD\u003c/h2\u003e \u003cp\u003eGlycolysis plays a crucial role in the process of EMT. In this study, we examined the expression of the epithelial cell marker E-cadherin and the mesenchymal cell marker Vimentin in human kidney specimens as well as in kidney sections from DKD mouse models. Our findings revealed that E-cadherin was downregulated while Vimentin was upregulated in the renal tubular cells of DKD patients, STZ-treated mice, and HFD-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Notably, empagliflozin significantly ameliorated EMT in both DKD patients and DKD mouse models (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Furthermore, the positive areas of E-cadherin were negatively correlated with the positive areas of PKM2. The positive areas of Vimentin were positively correlated with the areas of PKM2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEmpagliflozin downregulated PKM2 by blocking the binding of ESRRA to the promoter\u003c/h2\u003e \u003cp\u003eESRRA, as a transcription factor, transferred into nucleus to activate downstream target genes. The nuclear translocation of ESRRA was increased in STZ-treated mice, HFD-fed mice, and HG-treated HK-2 cells, and was decreased under empagliflozin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). The two predicted binding sites were further verified by dual-luciferase reporter gene assays. The mutation of either binding site 1 or binding site 2 significantly decreased the transcriptional activity of ESRRA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). ChIP-qPCR analysis further indicated the enrichment of ESRRA at the PKM2 promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). The results of ChIP-qPCR analysis also showed that the binding of ESRRA was upregulated by HG and was downregulated by empagliflozin at the two predicted binding sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eEmpagliflozin downregulated fibrosis markers in TGF-β-treated HK-2 cells\u003c/h2\u003e \u003cp\u003eAfter stimulation of TGF-β, COL3A1 and αSMA were upregulated in HK-2 cells. Empagliflozin downregulated the fibrosis markers, indicating a direct amelioration effect of empagliflozin on renal fibrosis (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eS).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eEmpagliflozin ameliorated kidney fibrosis and downregulated PKM2-PKM2 in MCD treated ob/ob mice\u003c/h2\u003e \u003cp\u003eIn MCD diet-treated ob/ob (ob/MCD) mice, empagliflozin ameliorated renal tubular injury, as evidenced by PAS staining, and reduced tubulointerstitial fibrosis, as shown by Masson staining. It also suppressed the accumulation of NGAL and decreased the tubular injury score (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Additionally, COL3A1 and αSMA expression were downregulated by empagliflozin (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). \u003cem\u003ePkm2\u003c/em\u003e was significantly downregulated by empagliflozin (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Western blot validated that PKM2 was the downregulated by empagliflozin, while empagliflozin showed no effect on HK2 and PFKP (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Immunofluorescence and immunochemistry results indicated that PKM2 was downregulated after empagliflozin treatment. Furthermore, E-cadherin was upregulated and Vimentin was downregulated in empagliflozin-treated group (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-J). Empagliflozin decreased the nuclear translocation of ESRRA in ob/MCD mice (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study demonstrated that SGLT2 inhibitor empagliflozin can effectively alleviate EMT-related tubulointerstitial fibrosis by downregulating \u003cem\u003ePKM2\u003c/em\u003e in DKD. The study showed that empagliflozin ameliorated kidney fibrosis in DKD patients and DKD models. \u003cem\u003ePKM2\u003c/em\u003e was significantly upregulated in diabetic kidneys and HG-treated HK-2 cells. Empagliflozin downregulated \u003cem\u003ePKM2\u003c/em\u003e and PKM2 in DKD models both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Empagliflozin ameliorated renal fibrosis by reducing the recruitment of ESRRA to the \u003cem\u003ePKM2\u003c/em\u003e promoter, thereby suppressing the transcription of \u003cem\u003ePKM2\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe renal benefits of SGLT2 inhibitors have been validated in clinical trials [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. RTPCs are the direct targets of SGLT2 inhibitors. Increasing evidence suggests that renal tubules play a critical role in the progression of DKD[\u003cspan additionalcitationids=\"CR45 CR46 CR47\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Large clinical trials have shown that SGLT2 inhibitors significantly reduce the risk of end-stage kidney disease in DKD patients. However, the outcomes of the above clinical trials mainly indicate the function of glomerulus[\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The anti-fibrotic effect of SGLT2 inhibitors has been confirmed in several \u003cem\u003ein vivo\u003c/em\u003e studies[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Also, SGLT2 inhibitors have been reported to modulate multiple pathways implicated in renal fibrosis among DKD patients, potentially uncovering the mechanisms through which SGLT2 inhibition may ameliorate renal fibrosis[\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Direct evidence that SGLT2 inhibitors improve renal fibrosis in DKD patients is yet to be fully established. In this study, we retrospectively analyzed renal biopsy results from DKD patients who were treated with SGLT2 inhibitors compared to those who were not. Staining techniques, including PAS staining and MASSON staining revealed that SGLT2 inhibitors reduced tubulointerstitial fibrosis in DKD patients. Transmission electron microscopy results show that empagliflozin improves renal interstitial collagen fiber proliferation, basement membrane thickening and foot process fusion in DKD patients. Furthermore, the anti-fibrotic effects of empagliflozin were validated in various DKD mouse models, including STZ-treated CD-1 mice, HFD-treated C57BL/6 mice, and MCD-treated \u003cem\u003eob/ob\u003c/em\u003e mice.\u003c/p\u003e \u003cp\u003eWe then investigated the molecular mechanisms underlying empagliflozin-ameliorated-fibrosis. Although glucose metabolism is not the main energy supply method of renal tubules, key enzymes of glycolysis and gluconeogenesis still play some roles in renal tubular cells. Hexokinase, a key glycolytic enzyme, reduced mitochondrial membrane injury after metabolic stress[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Gluconeogenic enzymes including phosphoenolpyruvatecarboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) are highly expressed in the proximal tubules, making proximal tubules important in maintaining systemic glucose homeostasis[\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In the present study, RNA sequencing analysis revealed that glycolysis pathway was significantly upregulated under HG condition and was downregulated by empagliflozin. SGLT2 inhibitors are reported to ameliorate glycolysis in metabolic disorders, including non-alcoholic fatty liver disease and DKD[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The progression of glycolysis depends on key glycolytic enzymes. However, the relationship between various glycolytic enzymes and the anti-fibrotic effects of SGLT2 inhibitors remains to be thoroughly investigated. Herein, \u003cem\u003ePKM2\u003c/em\u003e exhibited the most significant elevation among glycolytic genes in HG-treated HK-2 cells. The upregulation of \u003cem\u003ePkm2\u003c/em\u003e was further confirmed in DKD mouse models. The expression of \u003cem\u003ePKM2\u003c/em\u003e was positively corelated with fibrosis markers COL3A1 and αSMA. Notably, empagliflozin downregulated \u003cem\u003ePkm2\u003c/em\u003e in both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e DKD models. These findings suggest that \u003cem\u003ePKM2\u003c/em\u003e is the key glycolytic gene involved in kidney fibrosis and the anti-fibrotic effect of empagliflozin in DKD.\u003c/p\u003e \u003cp\u003ePKM2 is translated from \u003cem\u003ePKM2\u003c/em\u003e, serving as the rate-limiting enzyme of glycolysis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In this study, we observed significant upregulation of PKM2 across various DKD models. This upregulation was predominantly observed in RPTCs. Furthermore, the expression levels of PKM2 were also positively correlated with fibrosis markers, suggesting a critical role for PKM2 of RTPCs in kidney fibrosis. Empagliflozin effectively downregulated renal tubular PKM2. These findings further substantiate the role of PKM2 in inducing renal fibrosis, underscoring its significance in empagliflozin-ameliorated renal fibrosis.\u003c/p\u003e \u003cp\u003eUnder normal condition, fatty acid oxidation is the main metabolic way in RTPCs. When glycolysis enhanced, the metabolic shift alters RTPCs identity and cell fate[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Transition to glycolysis state suppresses the normal epithelial phenotype and promotes a mesenchymal phenotype, leading to EMT[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. This transition further stimulates the secretion of extracellular matrix components, including collagen and fibronectin, ultimately resulting in renal fibrosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Suppressing EMT in RTPCs may contribute to the anti-fibrotic effect of SGLT2 inhibitors. Thus, we then detected the EMT markers in the kidneys of DKD patients and DKD models. As expected, EMT was observed in the renal tubular cells in various DKD mouse models. Notably, empagliflozin effectively alleviated EMT. These findings underscore the anti-fibrotic effect of empagliflozin through the suppression of EMT in renal tubular cells.\u003c/p\u003e \u003cp\u003eFinally, we investigated the transcription factors responsible for \u003cem\u003ePKM2\u003c/em\u003e upregulation. The transcription factors of PKM2 were predicted by UCSC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.ucsc.edu/index.html\u003c/span\u003e\u003cspan address=\"https://genome.ucsc.edu/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and hTFtarget database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://guolab.wchscu.cn/hTFtarget/\u003c/span\u003e\u003cspan address=\"https://guolab.wchscu.cn/hTFtarget/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Among the predicted transcription factors, ESRRA has been rarely reported in DKD. As an orphan nuclear receptor, ESRRA regulates cell proliferation and cell metabolism by targeting various downstream genes. It is associated with various metabolic disorders, including obesity and non-alcoholic fatty liver disease [\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Moreover, ESRRA is closely related with renal fibrosis induced by folic acid or unilateral ureteral obstruction [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. However, there is little research focusing on the role of ESRRA in DKD. Herein, we found that ESRRA nuclear translocation was increased in DKD models both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e and was significantly decreased by empagliflozin. Next, the binding sites of ESRRA at the \u003cem\u003ePKM2\u003c/em\u003e promoter were predicted and confirmed. Empagliflozin downregulated \u003cem\u003ePKM2\u003c/em\u003e via inhibiting the binding of ESRRA to \u003cem\u003ePKM2\u003c/em\u003e promoter. These findings have further clarified the down-regulatory effect of SGLT2 inhibitors on PKM2 by elucidating the molecular mechanism.\u003c/p\u003e \u003cp\u003eTo further validate the sufficient of PKM2 downregulation in the renal fibrosis amelioration mediated by empagliflozin, PKM2 was overexpressed and knocked down separately in HK-2 cells. Both overexpression and knockdown of PKM2 diminished the effect of empagliflozin on downregulation the fibrosis markers in HK-2 cells. These findings highlight the importance of PKM2 as a therapeutic target for DKD-related renal fibrosis.\u003c/p\u003e \u003cp\u003eIn parallel to ongoing epidemics of obesity, The incidence of metabolic dysfunction-associated fatty liver disease (MAFLD) is increasing globally. Clinical trials indicate that the presence of MAFLD significantly increases the risk of end-stage kidney disease and renal fibrosis[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. However, the effect of SGLT2 inhibitors on renal fibrosis in MAFLD remains largely unclear. The MAFLD of ob/ob mice treated with methionine- and choline-deficient (MCD) diet used in the present study was confirmed in our previous published study[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Herein, renal fibrosis was observed in MCD-treated ob/ob mice. Furthermore, empagliflozin ameliorated renal fibrosis and EMT, downregulated PKM2 as well as reduced the nuclear translocation of ESRRA in the MAFLD mouse model.\u003c/p\u003e \u003cp\u003eTo our knowledge, our study is the first to report the upregulation of renal tubular PKM2 and its downregulation induced by empagliflozin in patients with DKD. Other studies have analyzed the expression of PKM2 in podocytes from DKD patients. PKM2 was found to be downregulated in podocytes of DKD patients[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. This disparity may arise from differing metabolic dependencies: tubular epithelial cells (TECs) rely predominantly on fatty acid β-oxidation, whereas podocytes are primarily dependent on glycolysis [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. In the context of DKD, glycolysis is observed to increase in TECs while decreasing in podocytes, which may explain the different trends of PKM2 in TECs and podocytes under diabetic condition[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. This study had a few limitations that must be considered. Firstly, the small number of patients included in our study could be considered a potential limitation. Many patients are reluctant to undergo renal biopsy due to its invasive nature and the associated risks of complications, including bleeding and infection. Secondly, the clinical information and pathology data of DKD patients were retrospectively collected, resulting in an inherent risk of recall bias and confounders (wash-out period absence). Thirdly, the \u003cem\u003ePKM2\u003c/em\u003e tubules-specific overexpression or knockdown mouse model is needed in the future study to further validate that the downregulation of PKM2 is sufficient to the attenuation of renal fibrosis by empagliflozin.\u003c/p\u003e \u003cp\u003eIn conclusion, empagliflozin improves kidney fibrosis in DKD patients and DKD models. It ameliorates EMT of renal tubular cells through downregulating PKM2. At the molecular level, empagliflozin inhibits the transcription of \u003cem\u003ePKM2\u003c/em\u003e by reducing the nuclear translocation of ESRRA and inhibiting its binding to the \u003cem\u003ePKM2\u003c/em\u003e promoter. Our findings emphasize the significant role of PKM2 in both DKD-related kidney fibrosis and the amelioration of tubulointerstitial fibrosis mediated by empagliflozin.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (81670762 to Mengyin Cai, 82270942 to Fen Xu), Natural Science Foundation of Guangdong Province (2020A1515011245 and 2016A030313258 to Mengyin Cai), the Guangzhou Municipal Science and Technology Project (201707010118 to Mengyin Cai).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiang Cai, Huanyi Cao, and Piaojian Yu carried out experiments, and wrote the manuscript. Meijun Wang and Hua liang contributed to data analysis. Xiaoqi Liang provided technical support with animal care. Fen Xu and Mengyin Cai contributed to the study design, data interpretation and revising the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOriginal data are available upon reasonable request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe retrospective study was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (approval number: II2024-056). Informed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Animal Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (approval number: IACUC-F3\u0026ndash;22\u0026ndash;0415, SYSU-IACUC-2020000006).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the\u003c/p\u003e\n\u003cp\u003estudy.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWebster, A.C., et al., \u003cem\u003eChronic Kidney Disease.\u003c/em\u003e Lancet, 2017. \u003cstrong\u003e389\u003c/strong\u003e(10075): p. 1238-1252.\u003c/li\u003e\n\u003cli\u003eElSayed, N.A., et al., \u003cem\u003e11. Chronic Kidney Disease and Risk Management: Standards of Care in Diabetes-2023.\u003c/em\u003e Diabetes Care, 2023. \u003cstrong\u003e46\u003c/strong\u003e(Suppl 1): p. S191-s202.\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eKDIGO 2022 Clinical Practice Guideline for Diabetes Management in Chronic Kidney Disease.\u003c/em\u003e Kidney Int, 2022. \u003cstrong\u003e102\u003c/strong\u003e(5s): p. S1-s127.\u003c/li\u003e\n\u003cli\u003eCherney, D.Z.I., et al., \u003cem\u003eEffects of empagliflozin on the urinary albumin-to-creatinine ratio in patients with type 2 diabetes and established cardiovascular disease: an exploratory analysis from the EMPA-REG OUTCOME randomised, placebo-controlled trial.\u003c/em\u003e Lancet Diabetes Endocrinol, 2017. \u003cstrong\u003e5\u003c/strong\u003e(8): p. 610-621.\u003c/li\u003e\n\u003cli\u003eWanner, C., et al., \u003cem\u003eEmpagliflozin and Progression of Kidney Disease in Type 2 Diabetes.\u003c/em\u003e N Engl J Med, 2016. \u003cstrong\u003e375\u003c/strong\u003e(4): p. 323-34.\u003c/li\u003e\n\u003cli\u003ePerkovic, V., et al., \u003cem\u003eCanagliflozin and Renal Outcomes in Type 2 Diabetes and Nephropathy.\u003c/em\u003e N Engl J Med, 2019. \u003cstrong\u003e380\u003c/strong\u003e(24): p. 2295-2306.\u003c/li\u003e\n\u003cli\u003eWiviott, S.D., et al., \u003cem\u003eDapagliflozin and Cardiovascular Outcomes in Type 2 Diabetes.\u003c/em\u003e N Engl J Med, 2019. \u003cstrong\u003e380\u003c/strong\u003e(4): p. 347-357.\u003c/li\u003e\n\u003cli\u003eHerrington, W.G., et al., \u003cem\u003eEmpagliflozin in Patients with Chronic Kidney Disease.\u003c/em\u003e N Engl J Med, 2023. \u003cstrong\u003e388\u003c/strong\u003e(2): p. 117-127.\u003c/li\u003e\n\u003cli\u003eHeerspink, H.J.L., et al., \u003cem\u003eCanagliflozin reduces inflammation and fibrosis biomarkers: a potential mechanism of action for beneficial effects of SGLT2 inhibitors in diabetic kidney disease.\u003c/em\u003e Diabetologia, 2019. \u003cstrong\u003e62\u003c/strong\u003e(7): p. 1154-1166.\u003c/li\u003e\n\u003cli\u003eLlorens-Cebri\u0026agrave;, C., et al., \u003cem\u003eAntioxidant Roles of SGLT2 Inhibitors in the Kidney.\u003c/em\u003e Biomolecules, 2022. \u003cstrong\u003e12\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eNabrdalik-Leśniak, D., et al., \u003cem\u003eInfluence of SGLT2 Inhibitor Treatment on Urine Antioxidant Status in Type 2 Diabetic Patients: A Pilot Study.\u003c/em\u003e Oxid Med Cell Longev, 2021. \u003cstrong\u003e2021\u003c/strong\u003e: p. 5593589.\u003c/li\u003e\n\u003cli\u003evan Bommel, E.J.M., et al., \u003cem\u003eThe renal hemodynamic effects of the SGLT2 inhibitor dapagliflozin are caused by post-glomerular vasodilatation rather than pre-glomerular vasoconstriction in metformin-treated patients with type 2 diabetes in the randomized, double-blind RED trial.\u003c/em\u003e Kidney Int, 2020. \u003cstrong\u003e97\u003c/strong\u003e(1): p. 202-212.\u003c/li\u003e\n\u003cli\u003eKim, S.R., et al., \u003cem\u003eSGLT2 inhibition modulates NLRP3 inflammasome activity via ketones and insulin in diabetes with cardiovascular disease.\u003c/em\u003e Nat Commun, 2020. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 2127.\u003c/li\u003e\n\u003cli\u003eSunilkumar, S., et al., \u003cem\u003eREDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy.\u003c/em\u003e Cell Death Dis, 2025. \u003cstrong\u003e16\u003c/strong\u003e(1): p. 79.\u003c/li\u003e\n\u003cli\u003eLiu, X., et al., \u003cem\u003eTubule-derived exosomes play a central role in fibroblast activation and kidney fibrosis.\u003c/em\u003e Kidney Int, 2020. \u003cstrong\u003e97\u003c/strong\u003e(6): p. 1181-1195.\u003c/li\u003e\n\u003cli\u003eLiu, B.C., et al., \u003cem\u003eRenal tubule injury: a driving force toward chronic kidney disease.\u003c/em\u003e Kidney Int, 2018. \u003cstrong\u003e93\u003c/strong\u003e(3): p. 568-579.\u003c/li\u003e\n\u003cli\u003eLovisa, S., et al., \u003cem\u003eEpithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis.\u003c/em\u003e Nat Med, 2015. \u003cstrong\u003e21\u003c/strong\u003e(9): p. 998-1009.\u003c/li\u003e\n\u003cli\u003eThiery, J.P. and J.P. Sleeman, \u003cem\u003eComplex networks orchestrate epithelial-mesenchymal transitions.\u003c/em\u003e Nat Rev Mol Cell Biol, 2006. \u003cstrong\u003e7\u003c/strong\u003e(2): p. 131-42.\u003c/li\u003e\n\u003cli\u003eThiery, J.P., et al., \u003cem\u003eEpithelial-mesenchymal transitions in development and disease.\u003c/em\u003e Cell, 2009. \u003cstrong\u003e139\u003c/strong\u003e(5): p. 871-90.\u003c/li\u003e\n\u003cli\u003eHuang, R., P. Fu, and L. Ma, \u003cem\u003eKidney fibrosis: from mechanisms to therapeutic medicines.\u003c/em\u003e Signal Transduct Target Ther, 2023. \u003cstrong\u003e8\u003c/strong\u003e(1): p. 129.\u003c/li\u003e\n\u003cli\u003eYang, L., et al., \u003cem\u003eEpithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.\u003c/em\u003e Nat Med, 2010. \u003cstrong\u003e16\u003c/strong\u003e(5): p. 535-43, 1p following 143.\u003c/li\u003e\n\u003cli\u003eThomas, K., et al., \u003cem\u003eGlutamine prevents acute kidney injury by modulating oxidative stress and apoptosis in tubular epithelial cells.\u003c/em\u003e JCI Insight, 2022. \u003cstrong\u003e7\u003c/strong\u003e(21).\u003c/li\u003e\n\u003cli\u003eXu, Z., et al., \u003cem\u003eMETTL14-regulated PI3K/Akt signaling pathway via PTEN affects HDAC5-mediated epithelial-mesenchymal transition of renal tubular cells in diabetic kidney disease.\u003c/em\u003e Cell Death Dis, 2021. \u003cstrong\u003e12\u003c/strong\u003e(1): p. 32.\u003c/li\u003e\n\u003cli\u003eUrabe, H., et al., \u003cem\u003eAblation of a small subpopulation of diabetes-specific bone marrow-derived cells in mice protects against diabetic neuropathy.\u003c/em\u003e Am J Physiol Endocrinol Metab, 2016. \u003cstrong\u003e310\u003c/strong\u003e(4): p. E269-75.\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller, T.D., et al., \u003cem\u003ep62 links \u003c/em\u003e\u003cem\u003e\u0026beta;-adrenergic input to mitochondrial function and thermogenesis.\u003c/em\u003e J Clin Invest, 2013. \u003cstrong\u003e123\u003c/strong\u003e(1): p. 469-78.\u003c/li\u003e\n\u003cli\u003eShen, Y., et al., \u003cem\u003eSGLT2 inhibitor empagliflozin downregulates miRNA-34a-5p and targets GREM2 to inactivate hepatic stellate cells and ameliorate non-alcoholic fatty liver disease-associated fibrosis.\u003c/em\u003e Metabolism, 2023. \u003cstrong\u003e146\u003c/strong\u003e: p. 155657.\u003c/li\u003e\n\u003cli\u003eCao, H., et al., \u003cem\u003eMalonylation of Acetyl-CoA carboxylase 1 promotes hepatic steatosis and is attenuated by ketogenic diet in NAFLD.\u003c/em\u003e Cell Rep, 2023. \u003cstrong\u003e42\u003c/strong\u003e(4): p. 112319.\u003c/li\u003e\n\u003cli\u003eZheng, X., et al., \u003cem\u003eSIRT1/HSF1/HSP pathway is essential for exenatide-alleviated, lipid-induced hepatic endoplasmic reticulum stress.\u003c/em\u003e Hepatology, 2017. \u003cstrong\u003e66\u003c/strong\u003e(3): p. 809-824.\u003c/li\u003e\n\u003cli\u003eWang, Q., et al., \u003cem\u003eSuppression of osteoclast multinucleation via a posttranscriptional regulation-based spatiotemporally selective delivery system.\u003c/em\u003e Sci Adv, 2022. \u003cstrong\u003e8\u003c/strong\u003e(26): p. eabn3333.\u003c/li\u003e\n\u003cli\u003eHu, Y., et al., \u003cem\u003eDemethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification.\u003c/em\u003e Mol Cancer, 2022. \u003cstrong\u003e21\u003c/strong\u003e(1): p. 34.\u003c/li\u003e\n\u003cli\u003eBai, Y., et al., \u003cem\u003eMarrow mesenchymal stem cell mediates diabetic nephropathy progression via modulation of Smad2/3/WTAP/m6A/ENO1 axis.\u003c/em\u003e Faseb j, 2024. \u003cstrong\u003e38\u003c/strong\u003e(11): p. e23729.\u003c/li\u003e\n\u003cli\u003eWu, J., et al., \u003cem\u003eAPOL1 risk variants in individuals of African genetic ancestry drive endothelial cell defects that exacerbate sepsis.\u003c/em\u003e Immunity, 2021. \u003cstrong\u003e54\u003c/strong\u003e(11): p. 2632-2649.e6.\u003c/li\u003e\n\u003cli\u003eChen, J.H., et al., \u003cem\u003eThe down-regulation of XBP1, an unfolded protein response effector, promotes acute kidney injury to chronic kidney disease transition.\u003c/em\u003e J Biomed Sci, 2022. \u003cstrong\u003e29\u003c/strong\u003e(1): p. 46.\u003c/li\u003e\n\u003cli\u003eXu, S., et al., \u003cem\u003eBone marrow mesenchymal stem cell-derived exosomal miR-21a-5p alleviates renal fibrosis by attenuating glycolysis by targeting PFKM.\u003c/em\u003e Cell Death Dis, 2022. \u003cstrong\u003e13\u003c/strong\u003e(10): p. 876.\u003c/li\u003e\n\u003cli\u003eJu, B., et al., \u003cem\u003eCo-activation of hedgehog and AKT pathways promote tumorigenesis in zebrafish.\u003c/em\u003e Mol Cancer, 2009. \u003cstrong\u003e8\u003c/strong\u003e: p. 40.\u003c/li\u003e\n\u003cli\u003eLiu, Y., et al., \u003cem\u003eNrf2 deficiency deteriorates diabetic kidney disease in Akita model mice.\u003c/em\u003e Redox Biol, 2022. \u003cstrong\u003e58\u003c/strong\u003e: p. 102525.\u003c/li\u003e\n\u003cli\u003eWang, M.J., et al., \u003cem\u003eSIRT1-dependent deacetylation of Txnip H3K9ac is critical for exenatide-improved diabetic kidney disease.\u003c/em\u003e Biomed Pharmacother, 2023. \u003cstrong\u003e167\u003c/strong\u003e: p. 115515.\u003c/li\u003e\n\u003cli\u003eLu, Y.H., et al., \u003cem\u003eEmpagliflozin Attenuates Hyperuricemia by Upregulation of ABCG2 via AMPK/AKT/CREB Signaling Pathway in Type 2 Diabetic Mice.\u003c/em\u003e Int J Biol Sci, 2020. \u003cstrong\u003e16\u003c/strong\u003e(3): p. 529-542.\u003c/li\u003e\n\u003cli\u003eHuang, X., et al., \u003cem\u003eTargeting Epigenetic Crosstalk as a Therapeutic Strategy for EZH2-Aberrant Solid Tumors.\u003c/em\u003e Cell, 2018. \u003cstrong\u003e175\u003c/strong\u003e(1): p. 186-199.e19.\u003c/li\u003e\n\u003cli\u003eLou, F., et al., \u003cem\u003eExcessive Polyamine Generation in Keratinocytes Promotes Self-RNA Sensing by Dendritic Cells in Psoriasis.\u003c/em\u003e Immunity, 2020. \u003cstrong\u003e53\u003c/strong\u003e(1): p. 204-216.e10.\u003c/li\u003e\n\u003cli\u003eCai, T., et al., \u003cem\u003eSodium-glucose cotransporter 2 inhibition suppresses HIF-1\u003c/em\u003e\u003cem\u003e\u0026alpha;-mediated metabolic switch from lipid oxidation to glycolysis in kidney tubule cells of diabetic mice.\u003c/em\u003e Cell Death Dis, 2020. \u003cstrong\u003e11\u003c/strong\u003e(5): p. 390.\u003c/li\u003e\n\u003cli\u003eXing, S., et al., \u003cem\u003eHypoxia downregulated miR-4521 suppresses gastric carcinoma progression through regulation of IGF2 and FOXM1.\u003c/em\u003e Mol Cancer, 2021. \u003cstrong\u003e20\u003c/strong\u003e(1): p. 9.\u003c/li\u003e\n\u003cli\u003eHong, S., G. Zheng, and J.W. Wiley, \u003cem\u003eEpigenetic regulation of genes that modulate chronic stress-induced visceral pain in the peripheral nervous system.\u003c/em\u003e Gastroenterology, 2015. \u003cstrong\u003e148\u003c/strong\u003e(1): p. 148-157.e7.\u003c/li\u003e\n\u003cli\u003eZeni, L., et al., \u003cem\u003eA more tubulocentric view of diabetic kidney disease.\u003c/em\u003e J Nephrol, 2017. \u003cstrong\u003e30\u003c/strong\u003e(6): p. 701-717.\u003c/li\u003e\n\u003cli\u003eDi Vincenzo, A., et al., \u003cem\u003eRenal structure in type 2 diabetes: facts and misconceptions.\u003c/em\u003e J Nephrol, 2020. \u003cstrong\u003e33\u003c/strong\u003e(5): p. 901-907.\u003c/li\u003e\n\u003cli\u003eKang, H.M., et al., \u003cem\u003eDefective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development.\u003c/em\u003e Nat Med, 2015. \u003cstrong\u003e21\u003c/strong\u003e(1): p. 37-46.\u003c/li\u003e\n\u003cli\u003eVallon, V. and S.C. Thomson, \u003cem\u003eThe tubular hypothesis of nephron filtration and diabetic kidney disease.\u003c/em\u003e Nat Rev Nephrol, 2020. \u003cstrong\u003e16\u003c/strong\u003e(6): p. 317-336.\u003c/li\u003e\n\u003cli\u003eDoke, T. and K. Susztak, \u003cem\u003eThe multifaceted role of kidney tubule mitochondrial dysfunction in kidney disease development.\u003c/em\u003e Trends Cell Biol, 2022. \u003cstrong\u003e32\u003c/strong\u003e(10): p. 841-853.\u003c/li\u003e\n\u003cli\u003eKogot-Levin, A., et al., \u003cem\u003eProximal Tubule mTORC1 Is a Central Player in the Pathophysiology of Diabetic Nephropathy and Its Correction by SGLT2 Inhibitors.\u003c/em\u003e Cell Rep, 2020. \u003cstrong\u003e32\u003c/strong\u003e(4): p. 107954.\u003c/li\u003e\n\u003cli\u003eZhang, Y., et al., \u003cem\u003eA sodium-glucose cotransporter 2 inhibitor attenuates renal capillary injury and fibrosis by a vascular endothelial growth factor-dependent pathway after renal injury in mice.\u003c/em\u003e Kidney Int, 2018. \u003cstrong\u003e94\u003c/strong\u003e(3): p. 524-535.\u003c/li\u003e\n\u003cli\u003eGall, J.M., et al., \u003cem\u003eHexokinase regulates Bax-mediated mitochondrial membrane injury following ischemic stress.\u003c/em\u003e Kidney Int, 2011. \u003cstrong\u003e79\u003c/strong\u003e(11): p. 1207-16.\u003c/li\u003e\n\u003cli\u003ePollock, A.S., \u003cem\u003eInduction of renal phosphoenolpyruvate carboxykinase mRNA: suppressive effect of glucose.\u003c/em\u003e Am J Physiol, 1989. \u003cstrong\u003e257\u003c/strong\u003e(1 Pt 2): p. F145-51.\u003c/li\u003e\n\u003cli\u003eMithieux, G., F. Rajas, and A. Gautier-Stein, \u003cem\u003eA novel role for glucose 6-phosphatase in the small intestine in the control of glucose homeostasis.\u003c/em\u003e J Biol Chem, 2004. \u003cstrong\u003e279\u003c/strong\u003e(43): p. 44231-4.\u003c/li\u003e\n\u003cli\u003eSasaki, M., et al., \u003cem\u003eDual Regulation of Gluconeogenesis by Insulin and Glucose in the Proximal Tubules of the Kidney.\u003c/em\u003e Diabetes, 2017. \u003cstrong\u003e66\u003c/strong\u003e(9): p. 2339-2350.\u003c/li\u003e\n\u003cli\u003eChristofk, H.R., et al., \u003cem\u003eThe M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth.\u003c/em\u003e Nature, 2008. \u003cstrong\u003e452\u003c/strong\u003e(7184): p. 230-3.\u003c/li\u003e\n\u003cli\u003eZhu, Z., et al., \u003cem\u003eTransition of acute kidney injury to chronic kidney disease: role of metabolic reprogramming.\u003c/em\u003e Metabolism, 2022. \u003cstrong\u003e131\u003c/strong\u003e: p. 155194.\u003c/li\u003e\n\u003cli\u003eSrivastava, S.P., et al., \u003cem\u003eSIRT3 deficiency leads to induction of abnormal glycolysis in diabetic kidney with fibrosis.\u003c/em\u003e Cell Death Dis, 2018. \u003cstrong\u003e9\u003c/strong\u003e(10): p. 997.\u003c/li\u003e\n\u003cli\u003eZhou, D. and Y. Liu, \u003cem\u003eRenal fibrosis in 2015: Understanding the mechanisms of kidney fibrosis.\u003c/em\u003e Nat Rev Nephrol, 2016. \u003cstrong\u003e12\u003c/strong\u003e(2): p. 68-70.\u003c/li\u003e\n\u003cli\u003eDjudjaj, S. and P. Boor, \u003cem\u003eCellular and molecular mechanisms of kidney fibrosis.\u003c/em\u003e Mol Aspects Med, 2019. \u003cstrong\u003e65\u003c/strong\u003e: p. 16-36.\u003c/li\u003e\n\u003cli\u003eB\u0026apos;Chir, W., et al., \u003cem\u003eDivergent Role of Estrogen-Related Receptor \u003c/em\u003e\u003cem\u003e\u0026alpha; in Lipid- and Fasting-Induced Hepatic Steatosis in Mice.\u003c/em\u003e Endocrinology, 2018. \u003cstrong\u003e159\u003c/strong\u003e(5): p. 2153-2164.\u003c/li\u003e\n\u003cli\u003eChen, C.Y., et al., \u003cem\u003eInhibition of Estrogen-Related Receptor \u003c/em\u003e\u003cem\u003e\u0026alpha; Blocks Liver Steatosis and Steatohepatitis and Attenuates Triglyceride Biosynthesis.\u003c/em\u003e Am J Pathol, 2021. \u003cstrong\u003e191\u003c/strong\u003e(7): p. 1240-1254.\u003c/li\u003e\n\u003cli\u003eYang, M., et al., \u003cem\u003eDysfunction of estrogen-related receptor alpha-dependent hepatic VLDL secretion contributes to sex disparity in NAFLD/NASH development.\u003c/em\u003e Theranostics, 2020. \u003cstrong\u003e10\u003c/strong\u003e(24): p. 10874-10891.\u003c/li\u003e\n\u003cli\u003eDhillon, P., et al., \u003cem\u003eThe Nuclear Receptor ESRRA Protects from Kidney Disease by Coupling Metabolism and Differentiation.\u003c/em\u003e Cell Metab, 2021. \u003cstrong\u003e33\u003c/strong\u003e(2): p. 379-394.e8.\u003c/li\u003e\n\u003cli\u003eZhao, L., et al., \u003cem\u003eImpact of non-alcoholic fatty liver disease and fibrosis on mortality and kidney outcomes in patients with type 2 diabetes and chronic kidney disease: A multi-cohort longitudinal study.\u003c/em\u003e Diabetes Obes Metab, 2024. \u003cstrong\u003e26\u003c/strong\u003e(10): p. 4241-4250.\u003c/li\u003e\n\u003cli\u003eMusso, G., et al., \u003cem\u003eAssociation of non-alcoholic fatty liver disease with chronic kidney disease: a systematic review and meta-analysis.\u003c/em\u003e PLoS Med, 2014. \u003cstrong\u003e11\u003c/strong\u003e(7): p. e1001680.\u003c/li\u003e\n\u003cli\u003eChen, Z., et al., \u003cem\u003eReduction of anaerobic glycolysis contributes to angiotensin II-induced podocyte injury with foot process effacement.\u003c/em\u003e Kidney Int, 2023. \u003cstrong\u003e103\u003c/strong\u003e(4): p. 735-748.\u003c/li\u003e\n\u003cli\u003eOzawa, S., et al., \u003cem\u003eGlycolysis, but not Mitochondria, responsible for intracellular ATP distribution in cortical area of podocytes.\u003c/em\u003e Sci Rep, 2015. \u003cstrong\u003e5\u003c/strong\u003e: p. 18575.\u003c/li\u003e\n\u003cli\u003eYuan, Q., et al., \u003cem\u003eRole of pyruvate kinase M2-mediated metabolic reprogramming during podocyte differentiation.\u003c/em\u003e Cell Death Dis, 2020. \u003cstrong\u003e11\u003c/strong\u003e(5): p. 355.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Diabetic kidney disease, Epithelial-to-mesenchymal transition, Pyruvate kinase M2, Sodium-glucose cotransporter 2 inhibition, Tubulointerstitial fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-5563608/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5563608/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and Objective\u003c/h2\u003e \u003cp\u003eSodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to prevent the progression of diabetic kidney disease (DKD). However, their impact on renal fibrosis remains largely uninvestigated. This study aimed to explore the effect of SGLT2 inhibitor empagliflozin on renal fibrosis in DKD patients and DKD models, and the molecular mechanisms involved.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eKidney samples of DKD patients and DKD models were used in this study. DKD mouse models included STZ-treated CD-1 mice and HFD-fed C57BL/6 mice were all treated with empagliflozin for 6 to 12 weeks. Kidney pathological changes were analysed and fibrotic factors were detected. HK-2 cells were treated with normal glucose (NG), high glucose (HG), or HG with empagliflozin. RNA sequencing was employed to identify the differentially expressed genes. Epithelial\u0026ndash;mesenchymal transition (EMT) markers were detected. Binding of transcription factor and target gene was determined using a dual-luciferase reporter assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eEmpagliflozin significantly ameliorated kidney fibrosis in DKD patients and DKD models. This was evidenced by tubulointerstitial fibrosis reduction observed through PAS and Masson staining, along with fibrotic factors downregulation. RNA sequencing and the subsequent \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e validation identified PKM2 as the most significantly upregulated glycolytic enzyme in DKD patients and models. Empagliflozin downregulated PKM2 and alleviated EMT and renal fibrosis. Importantly, empagliflozin improves fibrosis by downregulating PKM2. The downregulation of PKM2 by empagliflozin was achieved by inhibiting the binding of estrogen-related receptor α at the promoter.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eEmpagliflozin ameliorates kidney fibrosis via downregulating PKM2 in DKD.\u003c/p\u003e","manuscriptTitle":"SGLT2 inhibitor empagliflozin ameliorates tubulointerstitial fibrosis in DKD by downregulating renal tubular PKM2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-02 08:49:07","doi":"10.21203/rs.3.rs-5563608/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept as is","date":"2025-04-01T00:03:09+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-30T00:12:37+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-29T09:23:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-29T05:05:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-03-28T03:12:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12c5172c-3657-4df3-8244-e6203efe2db1","owner":[],"postedDate":"April 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T16:03:06+00:00","versionOfRecord":{"articleIdentity":"rs-5563608","link":"https://doi.org/10.1007/s00018-025-05688-8","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2025-04-16 15:57:22","publishedOnDateReadable":"April 16th, 2025"},"versionCreatedAt":"2025-04-02 08:49:07","video":"","vorDoi":"10.1007/s00018-025-05688-8","vorDoiUrl":"https://doi.org/10.1007/s00018-025-05688-8","workflowStages":[]},"version":"v1","identity":"rs-5563608","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5563608","identity":"rs-5563608","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}