A low-protein calorie-restricted diet mitigates kidney injury in diabetic mice by modulating the gut-kidney axis

A low-protein calorie-restricted diet mitigates kidney injury in diabetic mice by modulating the gut-kidney axis | 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 A low-protein calorie-restricted diet mitigates kidney injury in diabetic mice by modulating the gut-kidney axis Ruixiang Zhang, Xiao Wei, Yijiao Xu, Chunrong Han, Xiangzeng Cai, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5440142/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Dietary interventions are a promising strategy for restoring microbial balance in chronic kidney disease. Research indicates that a low-protein calorie-restricted (LPCR) diet can reduce renal injury in diabetic rodents. However, it is unclear whether the beneficial effects of LPCR diet in mice with diabetic kidney disease (DKD) are mediated through the modulation of the gut microbiota. Methods: A mouse model of diabetes was established using high-fat diet combined with streptozotocin injection. Diabetic mice were randomly divided into four groups: normal protein (NP, 20% protein), caloric restriction (CR, 30% restriction), low protein (LP, 13% protein), and LPCR (13% protein, 30% restriction). After five weeks of intervention, blood and urine samples were collected for relevant analyses, faecal samples for gut microbiota analysis, and kidney tissues for histological and immunohistochemical assays，as well as Western blot analysis. Results: LPCR diet significantly improved fasting blood glucose levels and lipid profiles ( p < 0.01) and mitigated kidney damage in diabetic mice. Additionally, LPCR diet ameliorated gut microbiota dysbiosis, significantly suppressing the increase in Firmicutes/Bacteroidetes ratio ( p < 0.05) and decreasing serum trimethylamine oxide(TMAO) levels ( p < 0.01). Compared to the NP group, the LPCR group exhibited significant reductions in serum TNF-α levels and the expression of ASC, NLRP3, and IL-1β in kidney tissue ( p < 0.01). Conclusion: LPCR diet exerts renoprotective effects in mice with DKD, possibly by modulating the gut-kidney axis to reduce circulating TMAO levels, thereby inhibiting NLRP3 inflammasome activation in kidney tissue. low-protein caloric restriction diet gut microbiota diabetic kidney disease gut-kidney axis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Diabetic kidney disease (DKD) is a prevalent chronic complication of diabetes and a leading cause of end-stage renal disease, cardiovascular events, and mortality [1] . DKD has a complex pathogenesis, and its clinical treatment options are limited. In addition to haemodynamic abnormalities, overactivation of the renin-angiotensin system, inflammation, podocyte injury, autophagy, and gut microbiota and their metabolites play a role in DKD, presenting potential therapeutic targets [1, 2] . The pathogenic relationship between the gut microbiota and kidney diseases, known as the gut-kidney axis [3] , has gained significant attention for its potential in developing treatment strategies for DKD. A low-protein diet (LPD) is the primary clinical approach for managing DKD, offering renal protection by reducing glomerular hyperfiltration and hypertension and improving tubular interstitial injury, inflammation, and fibrosis [4] . Caloric restriction (CR) is known to extend the lifespan and mitigate age-related diseases, including type 2 diabetes [5] , and has demonstrated renoprotective effects [6] . For example, CR alleviates kidney damage in rodent models of type 2 diabetes [7, 8] and improves renal function in obese patients with type 2 diabetes [9, 10] . A previous research indicated that a low-protein, calorie-restricted (LPCR) diet reduced renal injury in rodents with type 2 diabetes [11] . Gut microbiota is vital for human health, and its composition and function are influenced by factors such as diet, disease, and antibiotic use [12] . CR can alter the gut microbiota composition with varying effects based on dietary components [13] . LPD lowers uraemic toxin levels in patients with chronic kidney disease (CKD) by modulating the gut microbiota [14] . To the best of our knowledge, no studies have examined whether LPCR diet can alleviate diabetic kidney damage through the modulation of gut microbiota. In this study, we investigated the renoprotective effects of an LPCR diet in mice with diabetes induced by a high-fat diet (HFD) combined with streptozotocin (STZ) and explored the role of the gut microbiota in this process. Methods Animals Specific pathogen-free male C57BL/6J mice, aged 6 to 8 weeks and weighing between 17 and 21 g, were obtained from SPF Biotechnology Co., Ltd. (Suzhou, China; Permit No. SYXK [SU] 2021-0025). Mice were housed in cages under controlled conditions (room temperature: 24 ± 2°C; 12-hour light/dark cycle) and had free access to water. Notably, the mice underwent a one-week acclimatization period prior to the commencement of the study. All animal experiments were approved by the Animal Experiment Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine (Ethics No. AEWC-20230531-309) and complied with the Guidelines for the Care and Use of Animals established by the Chinese Animal Management Committee. Model construction and study design After a week acclimatization period, eight mice were randomly assigned to the normal control (NC) group and fed a standard diet (STD), whereas the remaining 32 mice were fed HFD for 12 weeks, followed by four consecutive days of intraperitoneal injections of STZ (Sigma-Aldrich Co., St. Louis, MO, USA) at a dosage of 40 mg/kg to induce diabetes. After the injections, the mice were continued on HFD. Mice with fasting blood glucose (FBG) levels ≥ 16.7 mmol/L at 10 weeks after STZ injection were considered to have diabetes. Thereafter, diabetic mice were randomly assigned to four groups (n = 8 mice/group): normal protein (NP, 20% protein), CR (30% restriction), low protein (LP, 13% protein), and LPCR (13% protein, 30% restriction). All diets were purchased from Jiangsu Xietong Pharmaceutical Bioengineer Co., Ltd. (Nanjing, China) and the feed formulas are detailed in Supplementary Table 1. Daily food intake was measured, and FBG and body weight were recorded weekly, with dietary intervention lasting for a total of five weeks (Fig. 1 ). Sample collection and indicator detection After five weeks of dietary intervention, random urine samples were collected from the mice using individual metabolic cages, followed by the determination of urinary albumin and creatinine levels using enzyme-linked immunosorbent assay (ELISA). Blood samples were obtained from the retro-orbital plexus and centrifuged at 4°C to extract serum. Serum creatinine, triglycerides (TG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), glutathione (GSH), superoxide dismutase (SOD), trimethylamine oxide (TMAO), and TNF-α levels were determined using ELISA kits. ELISA kits for urinary albumin and TMAO levels were obtained from Nanjing Jin Yibai Biological Technology Co., Ltd. (Nanjing, China) and Elabscience Biotechnology Co., Ltd. (Wuhan, China), respectively. All other ELISA kits were sourced from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). At the end of the experiment, mice were anaesthetised via intraperitoneal injection with pentobarbital sodium (100 mg/kg). Intestinal faeces were collected, rapidly frozen in liquid nitrogen, and transported in dry ice to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) for gut microbiota analysis. Kidney samples were divided into two portions, and one portion was fixed in 4% paraformaldehyde for histopathological examination, while the other portion was stored at ˗80°C for Western blot analysis. Histopathology and immunohistochemistry Paraffin-embedded kidney tissue sections (4 µm thick) were prepared and stained with haematoxylin and eosin (HE), periodic acid–schiff (PAS), and Masson's trichrome stain. For immunohistochemical assay, the sections were incubated overnight at 4°C with rabbit polyclonal antibodies against fibronectin (1:200) and Collagen I (1:200). Thereafter, the sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody at room temperature for 30 min. All antibodies were obtained from Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). Gut microbiome analysis Microbial genomic DNA was extracted from faecal samples using the PF Mag-Bind Stool DNA Kit (Omega Bio-tek, Georgia, USA). DNA purity and concentration were assessed using the PF Mag-Bind Stool DNA Kit (Omega Bio-tek, Georgia, USA). Thereafter, the 16S rRNA gene was amplified using the primers 27F (5´-AGRGTTYGATYMTGGCTCAG-3´) and 1492R (5´-RGYTACCTTGTTACGACTT-3´). The PCR amplification protocol was as follows: initial denaturation at 95°C for 3 min, followed by 27 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 45 s, and final extension at 72°C for 10 min. Subsequently, a PacBio library was constructed and sequenced. PacBio data were analysed using SMRTLink 11.0, resulting in at least three complete passes with 99% sequence accuracy for high-fidelity sequences. High-fidelity sequences were processed using UPARSE 11 to classify the operational taxonomic units (OTUs) at 97% similarity. Western blot analysis Briefly, proteins were extracted frozen kidney tissues, mixed with western blotting loading buffer, separated using SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. After blocking with non-fat milk at room temperature for 2 h, the membranes were incubated overnight at 4°C with primary antibodies against ASC, NLRP3, IL-1β, and β-actin (all diluted at 1:3000). Thereafter, the membranes were incubated with the corresponding secondary antibodies at 4°C for an additional two nights. All antibodies were purchased from Cell Signalling Technology (Danvers, MA, USA). Protein bands were detected using a chemiluminescent imaging system, and the grayscale values of the bands were calculated using the ImageJ software. Statistical analysis All statistical analyses were performed using GraphPad Prism 8. Normally distributed data are expressed as mean ± standard deviation. Significant differences were determined using t-tests for two groups and one-way ANOVA was used for multiple groups. Non-normally distributed data were analysed using the Wilcoxon rank-sum test. Statistical significance was set at p < 0.05. Results LPCR diet improves glucose and lipid metabolism Table 1 summarises the changes in body weight and FBG levels across the different groups before and after the dietary intervention. Diabetic mice in the various groups showed significantly lower body weights compared to those in the NC group at the end of the experiment ( p < 0.01). Although the LPCR group showed a decreasing trend in body weight compared to the NP group, this difference was not statistically significant. In terms of FBG levels, all three dietary intervention groups experienced a reduction compared to baseline, whereas the NP group showed an increase. Notably, FBG levels were significantly lower in the LP and LPCR groups compared to the NP group at the conclusion of the intervention ( P < 0.01). Table 1 Effects of LPCR diet on body weights and fasting blood glucose levels in diabetic mice Group Body weight (g) Weight change Fasting blood glucose (mmol/L) Glucose change 22w 27w 22w 27w NC 26.18 ± 1.59 27.00 ± 1.85 0.82 ± 2.19 7.00 ± 0.76 7.05 ± 0.57 0.05 ± 1.01 NP 25.94 ± 2.80 22.04 ± 1.34** -3.90 ± 2.74** 22.50 ± 6.21** 27.89 ± 3.70** 5.39 ± 4.93 CR 25.80 ± 1.70 21.14 ± 2.45** -4.66 ± 2.36** 27.84 ± 3.34** 24.18 ± 5.17** -3.66 ± 6.68 # LP 25.88 ± 2.25 21.35 ± 1.81** -4.53 ± 2.88** 25.43 ± 4.43** 15.29 ± 6.03* ## -10.14 ± 5.98** ## LPCR 25.69 ± 1.15 19.80 ± 2.74** -5.89 ± 2.90** 20.98 ± 6.25** 15.71 ± 6.45** ## -5.27 ± 7.33 ## Differences before and after dietary intervention (22 and 27 week) in diabetic mice. Data are presented as mean ± standard deviation, n = 8 in each group. * P < 0.05 vs. NC group; ** P < 0.01 vs. NC group; # P < 0.05 vs. NP group; ## P < 0.01 vs. NP group After five weeks of dietary intervention, there was a significant increase in serum TG, T-CHO, and LDL-C levels ( p < 0.01) and a significant decrease in HDL-C levels ( p < 0.05) in the NP group compared with those in the NC group. However, both LP and LPCR diets significant improved the levels of all four lipid parameters compared with those in the NP group ( p < 0.01), with the LPCR group demonstrating particularly pronounced effects on the LDL-C and HDL-C levels (Fig. 2 ). LPCR diet mitigates kidney damage To examine the effects of LPCR on kidney function, we measured serum creatinine levels and urine albumin/creatinine ratios after five weeks of dietary intervention. Serum creatinine levels and urine albumin/creatinine ratios were significantly higher in the NP group than in the NC group ( P < 0.01). In contrast, the three dietary interventions significantly decreased serum creatinine levels and urine albumin/creatinine ratios compared with those in the NP group ( p < 0.01) (Fig. 3.1 ). HE staining revealed that mice in the NP group exhibited pathological changes characteristic of DKD, including irregular glomerular morphology, cytoplasmic vacuolation of renal tubular epithelial cells, and chronic inflammatory cell infiltration into the renal interstitium. However, the three dietary interventions ameliorated these pathological changes to varying degrees, with the LPCR group demonstrating the most significant effect (Fig. 3.2 A). PAS staining indicated enlarged areas of glycogen deposition in the kidneys of mice in the NP group, along with widening of the mesangial matrix in the glomeruli and thickening of the tubular basement membrane. However, treatment with CR, LP, and LPCR diets alleviated these pathological alterations to varying degrees, with the LPCR group showing the most significant improvement (Fig. 3.2 B). Masson’s trichrome staining demonstrated a marked increase in collagen deposition in the kidneys of mice from the NP group, indicating pronounced renal fibrosis. However, the dietary interventions effectively reduced collagen deposition to varying degrees, with the LPCR diet significantly inhibiting renal fibrosis (Fig. 3.2 C). Additionally, immunohistochemical staining showed that fibronectin expression in the glomeruli and collagen I level in the renal interstitium were significantly higher in the NP group than in the NC group. In contrast, the expression levels of both proteins were significantly lower in the LPCR group than in the NP group (Fig. 3.3 ). LPCR diet modulates gut microbiota β-diversity analysis was performed to assess the similarity of gut microbial compositions across different groups (Fig. 4 A). Notably, the NP group displayed a clear separation from the NC group, indicating significant alterations in microbial diversity. Principal coordinate analysis (PCoA) showed that the clustering of the three dietary intervention groups shifted notably towards the NC group, particularly for the LPCR group. The Gut Microbiome Health Index (GMHI), which assesses health status based on species-level taxonomic profiles from stool shotgun metagenome samples, revealed a significant disruption in the GMHI of the NP group compared to the NC group ( p < 0.01). Notably, LPCR diet led to a substantial improvement in GMHI compared to the NP group ( P < 0.01) (Fig. 4 B). At the phylum level, the abundance of Firmicutes and Firmicutes/Bacteroidetes (F/B) ratio were higher in the NP group than in the NC group. Notably, treatment with LPCR diet resulted in a significant decrease in both the abundance of Firmicutes ( p < 0.01) and F/B ratio ( p < 0.05) in mice with DKD (Fig. 4 C–D). The Wilcoxon rank-sum test revealed that the abundance of Bacteroides at the genus level and Bacteroides acidifaciens at the species level were significantly higher in the LPCR group than in the NP group ( p < 0.01) (Fig. 4 E–F). Moreover, serum TMAO levels were significantly higher in the NP group compared to the NC group ( P < 0.01). In contrast, CR, LP, and LPCR diets significantly reduced serum TMAO levels compared to those in the NP group ( p < 0.05, p < 0.01, and p < 0.01, respectively), with LPCR diet exerting the most pronounced inhibitory effect (Fig. 4 G). LPCR diet provides antioxidant and anti-inflammatory benefits Compared to the NC group, there was a significant decrease in serum GSH and SOD levels, along with a marked increase in TNF-α levels ( P < 0.01). However, treatment with LPCR diet for five weeks significantly increased GSH and SOD levels and decreased TNF-α levels compared to the NP group ( P < 0.01) (Fig. 5 A–C). Additionally, ASC, NLRP3, and IL-1β expression levels in kidney tissue were significantly higher in the NP group than in the NC group ( p < 0.01). In contrast, CR, LP, and LPCR diets significantly reduced the expression of ASC, NLRP3, and IL-1β compared to the NP group ( P < 0.01), with the LPCR group showing the most pronounced decrease in NLRP3 expression (Fig. 5 D–G). Discussion In this study, we investigated whether LPCR diet can protect the kidney by modulating the gut microbiota in a mouse model of diabetes. Our findings indicated that LPCR diet not only improved glucose and lipid metabolic disorders but also mitigated kidney damage in diabetic mice. Additionally, LPCR diet altered the gut microbiota composition, reduced circulating TMAO levels, and provided antioxidant and anti-inflammatory benefits. Overall, the renoprotective effects of LPCR diet may be mediated through the gut-kidney axis. CR, defined as reduced energy intake without malnutrition, is a recognised dietary intervention for diabetes that offers benefits, such as weight loss, improved glucose and lipid metabolism, and reduced oxidative stress and inflammation [ 15 ] . Similarly, dietary protein restriction enhances metabolic function and regulates glycaemia, lipid levels, and body weight in patients with type 2 diabetes [ 16 ] . In the present study, both the LP and LPCR diets improved glucose and lipid metabolism, whereas CR did not significantly affect FBG and serum LDL-C levels. This discrepancy may be due to severe metabolic disorders present in diabetic mice and an insufficient degree of CR. Moreover, a previous study indicated that LPCR diet was more effective than LP diet in improving glucose and lipid metabolism in diabetic rats [ 11 ] . Therefore, LPCR diet may be a superior option for enhancing metabolic function in diabetes. Based on serum creatinine levels, urine albumin/creatinine ratios, and pathological changes observed at the end of the intervention, kidney damage occurred in diabetic mice at the mid to late stages of DKD rather than in the early stage. All three dietary interventions significantly reduced serum creatinine and urinary protein/creatinine levels in DKD mice. Importantly, various pathological changes, including irregular glomerular morphology, cytoplasmic vacuolation of renal tubular epithelial cells, chronic inflammatory cell infiltration, widening of the mesangial matrix in the glomeruli, thickening of the tubular basement membrane, and renal fibrosis, showed improvement after five weeks of dietary intervention, with the LPCR group demonstrating the most pronounced improvements. Collectively, these findings suggest that LPCR diet may effectively alleviated kidney damage in diabetic mice. Dysbiosis of the gut microbiota has been implicated in the development and progression of various diseases, including type 2 diabetes and CKD [ 17 , 18 ] . CKD can alter the composition of the gut microbiota, leading to dysbiosis, which may further elevate the levels of uraemic toxins and exacerbate CKD progression [ 19 ] . Diet plays a crucial role in regulating the gut microbiota and its activity [ 20 , 21 ] , making dietary interventions a promising strategy for restoring microbial balance in CKD [ 21 ] . To explore the role of the gut microbiota in the renoprotective effects of LPCR diet, we assessed the gut microbiota composition of diabetic mice across different dietary groups. Our findings revealed significant variations in the composition of the gut microbiota based on the dietary interventions. Notably, the four dominant bacterial groups, Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, accounted for over 90% of the gut microbiota, with Firmicutes and Bacteroidetes particularly involved in regulating the host metabolism and immunity [ 22 ] . A high abundance of Firmicutes and low abundance of Bacteroidetes is strongly linked with obesity, which is often associated with diabetes [ 23 ] . Cai et al. reported a marked increase in F/B ratio in mice with DKD, which was restored by treatment with resveratrol, a CR mimetic [ 24 ] . Similarly, our findings revealed a pronounced increase in F/B ratio in DKD mice. Notably, LPCR diet significantly improved gut microbiota dysbiosis in diabetic mice. Additionally, LPCR diet induced the highest increase in Bacteroides acidificient , a species classified as Bacteroidetes . TMAO is a gut-derived toxic metabolite that is associated with increased systemic inflammation. Research findings indicate that high TMAO production is associated with elevated F/B ratios in healthy individuals [ 25 ] . Additionally, plasma TMAO levels were negatively correlated with Bacteroides abundance in mice fed high-choline diet [ 26 ] . Elevated TMAO levels in CKD patients are closely associated with decreased renal function [ 27 – 29 ] . Changes in gut microbiota contribute to the development of DKD, with TMAO and chronic inflammation playing pivotal roles in this process [ 30 ] . Furthermore, serum TMAO level has been identified as an independent risk factor for DKD [ 31 ] . In the present study, LPCR diet significantly reversed DKD-induced increase in serum TMAO levels. Overall, we speculated that LPCR diet might improve DKD by lowering F/B ratio, which subsequently reduced TMAO levels. Mechanistically, TMAO influences kidney injury primarily through the exacerbation of tubulointerstitial fibrosis and renal inflammation [ 32 – 34 ] . Recently, Stefaniash et al. demonstrated that TMAO enhances TNF-α mediated kidney inflammation by inducing the release of various cytokines, chemokines, and inflammatory mediators from renal fibroblasts [ 35 ] . Inflammation and oxidative stress are critical factors in the development and progression of CKD, and are closely interconnected and exacerbate each other [ 27 ] . The NLRP3 inflammasome has been implicated in the progression of CKD, including DKD [ 36 , 37 ] . Research indicates that TMAO can induce oxidative stress and activate the NLRP3 inflammasome, leading to the release of inflammatory cytokines [ 34 , 39 ] . Conversely, the inhibition of TMAO can reduce NLRP3 inflammasome activation and oxidative stress [ 39 ] . Our results indicated that treatment with LPCR diet for five weeks significantly increased GSH and SOD levels and downregulated TNF-α and NLRP3 inflammasome-related proteins, including ASC, NLRP3, and IL-1β, in kidney tissue. Collectively, these results suggest that the LPCR diet may have anti-inflammatory and antioxidant effects. Based on related studies, we speculated that the renoprotective effects of LPCR intervention in DKD mice may be linked to TMAO inhibition, which prevented the activation of the NLRP3 inflammasome. Despite the promising findings, the study had several limitations. For example, we set the protein ratio for the LPCR group to 13% protein with 30% caloric restriction to prevent protein-energy wastage. Previous animal studies have shown that intermittent very LPD [ 40 ] , 50% CR [ 7 ] , and intermittent CR [ 7 ] can improve DKD. Therefore, further research is needed to determine the optimal protein ratio for LPCR and explore whether intermittent approaches are more effective. Additionally, owing to the prolonged modelling period for DKD and other constraints, we did not conduct counterevidence experiments, limiting our ability to clearly define the direct effects of differential bacterial species on TMAO levels. In future studies, we plan to address this limitation by employing faecal microbiota transplantation or other counterevidence techniques to verify the regulatory effects of Firmicutes and Bacteroidetes on TMAO and DKD. Conclusively, LPCR diet mitigates kidney injury in diabetic mice, possibly by modulating the gut-kidney axis. This effect may be linked to reduced circulating TMAO levels, which in turn inhibit the activation of the NLRP3 inflammasome in kidney tissue (Fig. 6 ). Overall, this study provides the foundation for further research on the specific mechanisms by which LPCR diet regulates the gut-kidney axis, offering new insights and strategies for the clinical treatment of DKD. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Data a vailability The data that support the findings of this study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding Clinical Medicine Science and Technology Development Fund Project of Jiangsu University (JLY2021162); "Double Innovation Doctor" of Jiangsu Province (JSSCBS20211615); Special Research Startup Funding for Introduced Personnel at Nanjing Lishui People’s Hospital (KY07). Authors' contributions RXZ and XW contributed to formal analysis, investigation, draft preparation, and visualization. YJX contributed to data curation. CRH and XZC contributed to image acquisition and analysis. YLW contributed to statistical analyses. YG contributed to funding acquisition, project administration, writing and review, and editing. YG and CL contributed to conceptualization, validation, and supervision. All authors read and approved the final manuscript. Acknowledgements The skillful technical assistance of Yu Chen, Xingjia Li, Qifeng Wang, Houcai Huang, Rongling Zhong, and Zhi Xia is appreciated. References Kikuchi K, Saigusa D, Kanemitsu Y, et al. Gut microbiome-derived phenyl sulfate contributes to albuminuria in diabetic kidney disease. Nat Commun. 2019;10:1835. Mao ZH, Gao ZX, Liu DW, et al. Gut microbiota and its metabolites - molecular mechanisms and management strategies in diabetic kidney disease. Front Immunol. 2023;14:1124704. Chen YY, Chen DQ, Chen L, et al. Microbiome-metabolome reveals the contribution of gut-kidney axis on kidney disease. J Transl Med. 2019;17:5. Kitada M, Ogura Y, Monno I, et al. A Low-Protein Diet for Diabetic Kidney Disease: Its Effect and Molecular Mechanism, an Approach from Animal Studies. Nutrients. 2018;10:544. Fontana L, Partridge L, Longo VD. 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Endocr Connect. 2023;12:e220542. Zhang W, Miikeda A, Zuckerman J, et al. Inhibition of microbiota-dependent TMAO production attenuates chronic kidney disease in mice. Sci Rep. 2021;11:518. Kapetanaki S, Kumawat AK, Persson K, et al. The Fibrotic Effects of TMAO on Human Renal Fibroblasts Is Mediated by NLRP3, Caspase-1 and the PERK/Akt/mTOR Pathway. Int J Mol Sci. 2021;22:11864. Fang Q, Zheng B, Liu N, et al. Trimethylamine N-Oxide Exacerbates Renal Inflammation and Fibrosis in Rats With Diabetic Kidney Disease. Front Physiol. 2021;12:682482. Stefania K, Ashok KK, Geena PV, et al. TMAO enhances TNF-α mediated fibrosis and release of inflammatory mediators from renal fibroblasts. Sci Rep. 2024;14:9070. Ram C, Jha AK, Ghosh A, et al. Targeting NLRP3 inflammasome as a promising approach for treatment of diabetic nephropathy: Preclinical evidences with therapeutic approaches. Eur J Pharmacol. 2020;885:173503. Qiu YY, Tang LQ. Roles of the NLRP3 inflammasome in the pathogenesis of diabetic nephropathy. Pharmacol Res. 2016;114:251-64. Ke Y, Li D, Zhao M, et al. Gut flora-dependent metabolite Trimethylamine-N-oxide accelerates endothelial cell senescence and vascular aging through oxidative stress. Free Radic Biol Med. 2018;116:88-100. Boini KM, Hussain T, Li PL, et al. Trimethylamine-N-Oxide Instigates NLRP3 Inflammasome Activation and Endothelial Dysfunction. Cell Physiol Biochem. 2017;44:152-62. Kitada M, Ogura Y, Suzuki T, et al. Cyclic and intermittent very low-protein diet can have beneficial effects against advanced diabetic nephropathy in Wistar fatty (fa/fa) rats, an animal model of type 2 diabetes and obesity. Nephrology (Carlton). 2017;22:1030-4. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.docx Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5440142","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":384617110,"identity":"41cc365c-7068-43b9-aa09-7fa046adc8d5","order_by":0,"name":"Ruixiang Zhang","email":"","orcid":"","institution":"Affiliated Hospital of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ruixiang","middleName":"","lastName":"Zhang","suffix":""},{"id":384617111,"identity":"b0c44570-0e4c-4ea9-baf8-0ae7f4a26852","order_by":1,"name":"Xiao Wei","email":"","orcid":"","institution":"Affiliated Hospital of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Wei","suffix":""},{"id":384617112,"identity":"a82a9044-82c1-4227-b76a-11604bb4e2f0","order_by":2,"name":"Yijiao Xu","email":"","orcid":"","institution":"Affiliated Hospital of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yijiao","middleName":"","lastName":"Xu","suffix":""},{"id":384617113,"identity":"d59fc3e2-369b-4871-83fb-6ecdc5e24922","order_by":3,"name":"Chunrong Han","email":"","orcid":"","institution":"Nanjing Lishui People's Hospital, Zhongda Hospital Lishui Branch, Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Chunrong","middleName":"","lastName":"Han","suffix":""},{"id":384617114,"identity":"3c9aadae-3869-4670-8257-09acb484dcb6","order_by":4,"name":"Xiangzeng Cai","email":"","orcid":"","institution":"Nanjing Lishui People's Hospital, Zhongda Hospital Lishui Branch, Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Xiangzeng","middleName":"","lastName":"Cai","suffix":""},{"id":384617115,"identity":"7c06ff48-53c6-4101-9e62-c068dfe223e3","order_by":5,"name":"Yinling Wu","email":"","orcid":"","institution":"Nanjing Lishui People's Hospital, Zhongda Hospital Lishui Branch, Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Yinling","middleName":"","lastName":"Wu","suffix":""},{"id":384617117,"identity":"ccc746c1-4deb-4186-b4bd-7ad31b19474a","order_by":6,"name":"Yan Geng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYBADOTZmhsQHCRU1xGsx5mdveGzw4Mwx4rUkzuw5+EzyYQszYaXy7r2HX/PU3GHccCM5rSKxgY2Bv707Aa8WwzPn0qx5jj1jNriRlnYjcYcMg8SZsxvwa5mRY2bMw3aYzeBGDlDLGTYGA4lcAlrmvwFq+XeYx+BG/reCxDZmwlrkJXiMH/O2HZaQ7DmQxkCUFgOeHDPGuX2HDYCBnCyRcOYYD0G/yLefMf7w5tvheqD5iR9/VNTI8bf3ErDlAAObFA+SAA9OpXBbGhiYP/4gqGwUjIJRMApGNAAA45hOuDiLooEAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing Lishui People's Hospital, Zhongda Hospital Lishui Branch, Southeast University","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Geng","suffix":""},{"id":384617118,"identity":"78dd1a7c-58c1-439f-bc0e-21654c646282","order_by":7,"name":"Chao Liu","email":"","orcid":"","institution":"Affiliated Hospital of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-11-12 13:53:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5440142/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5440142/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70474995,"identity":"2ddcf1e2-b3b1-4779-bb7c-1dd64e116d20","added_by":"auto","created_at":"2024-12-03 14:06:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":487693,"visible":true,"origin":"","legend":"\u003cp\u003eDietary paradigms and study design. (A) Dietary composition. Standard diet (STD) consisted of 70% carbohydrates, 10% fat, and 20% protein. High-fat diet (HFD) contained 60% of calories from fat. Caloric restriction (CR) was set at 30% of the STD. Low-protein diet (LPD) contained 13% of calories from protein. Low-protein calorie-restricted (LPCR) diet was set at 30% CR of the LPD. (B) Study design. Eight mice in normal control (NC) group were fed STD \u003cem\u003ead libitum\u003c/em\u003e. A mouse model of diabetes was established by feeding the mice a 12-week HFD, followed by multiple intraperitoneal injections of streptozotocin (STZ), and an additional 10 weeks of HFD. The diabetic mice were randomly divided into four groups: a normal protein (NP) group fed STD, and three groups receiving dietary interventions of CR, LP, and LPCR (n = 8 mice/group). After 5 weeks of dietary intervention, blood, urine, faecal samples, and kidney tissues were collected for relevant analyses.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5440142/v1/2e652d6b1ecbb5fb708da710.png"},{"id":70474996,"identity":"59ff682e-fd70-48a6-8db7-aa9ae02e100f","added_by":"auto","created_at":"2024-12-03 14:06:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":82033,"visible":true,"origin":"","legend":"\u003cp\u003eLPCR diet improves lipid metabolism in DKD mice. (A) Mean TG level, (B) mean HDL-C level, (C) mean T-CHO level, and (D) mean LDL-C level in each group. n = 6. *\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 vs. NC group; **\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 vs. NC group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 vs. NP group\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5440142/v1/82b56f4b8402ddf9526ac648.png"},{"id":70476117,"identity":"8ce3b6f9-7dde-4117-8684-458ee32a25c6","added_by":"auto","created_at":"2024-12-03 14:14:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60122,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.1 LPCR diet improves renal function in DKD mice. (A) Mean serum creatinine level and (B) mean urinary albumin-to-creatinine ratio in each group. n = 6. **\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 vs. NC group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 vs. NP group\u003c/p\u003e","description":"","filename":"Fig.3.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5440142/v1/1898b036706d7177c5beb4ff.png"},{"id":70476118,"identity":"46b33133-b174-43b4-ab56-b63a19c0e7fb","added_by":"auto","created_at":"2024-12-03 14:14:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1970828,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.2 LPCR diet alleviates partial pathological abnormalities of the kidneys in DKD mice. (A) Representative photomicrographs of HE-stained kidney sections, (B) representative photomicrographs of PAS-stained kidney sections, and (C) representative photomicrographs of Masson’s trichrome-stained kidney sections in each group. Magnification = 200×.\u003c/p\u003e","description":"","filename":"Fig.3.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5440142/v1/89dda1109a985b197f5cd14f.png"},{"id":70476119,"identity":"2e4ae1b2-7dcb-4a7a-b7c0-646806535ccf","added_by":"auto","created_at":"2024-12-03 14:14:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2100888,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3.3 LPCR diet alleviates renal fibrosis in DKD mice. (A) Representative immunohistochemical images of fibronectin and (B) representative immunohistochemical images of Collagen I in each group. Magnification = 200×.\u003c/p\u003e","description":"","filename":"Fig.3.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5440142/v1/f45247251abce801b5de7125.png"},{"id":70474999,"identity":"56f31005-edb7-4708-995d-89a27a6bc4e6","added_by":"auto","created_at":"2024-12-03 14:06:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":525014,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 4 LPCR diet modulates gut microbiota in DKD mice. (A) Principal coordinate analysis (PCoA) of each group. (B) Comparison of Gut Microbiome Health Index (GMHI) among different groups. (C–D) Mean Firmicutes abundance (C) and mean Firmicutes/Bacteroidetes ratio (D) in each group. (E–F) Comparison of gut microbiota at the genus level (E) and species level (F) between NP and LPCR groups. (G) Mean serum trimethylamine oxide (TMAO) level in each group. n = 6–8. *\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 vs. NC group; **\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 vs. NC group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs. NP group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 vs. NP group\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5440142/v1/1f3d712c44a35f402c900e44.png"},{"id":70475003,"identity":"6c75d346-2b0c-4ae2-821a-d5f9ed207eb5","added_by":"auto","created_at":"2024-12-03 14:06:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":288896,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 5 LPCR diet provides antioxidant and anti-inflammatory benefits in DKD mice. (A–C) Mean GSH level (A), mean SOD level (B), and mean TNF-α level (C) in each group；(D) Representative Western blot images of NLRP3 inflammasome-related protein in the kidney tissue of DKD mice; (E–G) Western blot densitometric analysis of ACS (E), NLRP3 (F), and IL-1β (G) in each group. n = 3–6. *\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 vs. NC group; **\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 vs. NC group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 vs. NP group\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-5440142/v1/ad19856c21e28c9ed07fcf87.png"},{"id":70475001,"identity":"1dad46f4-907f-45b1-bc65-8cbfe2714979","added_by":"auto","created_at":"2024-12-03 14:06:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":454377,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 6 Graphical abstract. LPCR diet mitigates kidney injury in diabetic mice by modulating the gut-kidney axis.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-5440142/v1/9138c9838f1f85b111464547.png"},{"id":70477883,"identity":"9c396df0-c798-4caf-8917-20a48563b995","added_by":"auto","created_at":"2024-12-03 14:30:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6726384,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5440142/v1/5cfce370-ad5c-47ad-aa75-e136a12fffd6.pdf"},{"id":70476120,"identity":"b372e0bd-8370-4182-9ea7-8e60063d119b","added_by":"auto","created_at":"2024-12-03 14:14:48","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":16333,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5440142/v1/e0bdfe0f029b9b4c16da2bee.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A low-protein calorie-restricted diet mitigates kidney injury in diabetic mice by modulating the gut-kidney axis","fulltext":[{"header":"Background","content":"\u003cp\u003eDiabetic kidney disease (DKD) is a prevalent chronic complication of diabetes and a leading cause of end-stage renal disease, cardiovascular events, and mortality\u003csup\u003e[1]\u003c/sup\u003e. DKD has a complex pathogenesis, and its clinical treatment options are limited. In addition to haemodynamic abnormalities, overactivation of the renin-angiotensin system, inflammation, podocyte injury, autophagy, and gut microbiota and their metabolites play a role in DKD, presenting potential therapeutic targets\u003csup\u003e[1, 2]\u003c/sup\u003e. The pathogenic relationship between the gut microbiota and kidney diseases, known as the gut-kidney axis\u003csup\u003e[3]\u003c/sup\u003e, has gained significant attention for its potential in developing treatment strategies for DKD.\u003c/p\u003e\n\u003cp\u003eA low-protein diet (LPD) is\u0026nbsp;the\u0026nbsp;primary clinical approach for managing DKD, offering renal protection by reducing glomerular hyperfiltration and hypertension\u0026nbsp;and\u0026nbsp;improving tubular interstitial injury, inflammation, and fibrosis\u003csup\u003e[4]\u003c/sup\u003e. Caloric restriction (CR) is known to extend\u0026nbsp;the\u0026nbsp;lifespan and mitigate age-related diseases, including type 2 diabetes\u003csup\u003e[5]\u003c/sup\u003e, and has demonstrated renoprotective effects\u003csup\u003e[6]\u003c/sup\u003e.\u0026nbsp;For example, CR alleviates kidney damage in\u0026nbsp;rodent models of type 2 diabetes\u003csup\u003e[7, 8]\u003c/sup\u003e and\u0026nbsp;improves\u0026nbsp;renal function in obese patients with type 2 diabetes\u003csup\u003e[9, 10]\u003c/sup\u003e.\u0026nbsp;A\u0026nbsp;previous research indicated that a low-protein,\u0026nbsp;calorie-restricted (LPCR) diet\u0026nbsp;reduced\u0026nbsp;renal injury in\u0026nbsp;rodents with\u0026nbsp;type 2\u0026nbsp;diabetes\u003csup\u003e[11]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eGut\u0026nbsp;microbiota is vital for human health,\u0026nbsp;and\u0026nbsp;its composition and function\u0026nbsp;are\u0026nbsp;influenced by factors such as diet, disease, and antibiotic use\u003csup\u003e[12]\u003c/sup\u003e. CR can alter\u0026nbsp;the\u0026nbsp;gut microbiota composition with varying effects based on dietary components\u003csup\u003e[13]\u003c/sup\u003e. LPD\u0026nbsp;lowers uraemic\u0026nbsp;toxin levels in patients with chronic kidney disease (CKD) by modulating\u0026nbsp;the\u0026nbsp;gut microbiota\u003csup\u003e[14]\u003c/sup\u003e. To\u0026nbsp;the best of our knowledge, no studies\u0026nbsp;have examined\u0026nbsp;whether LPCR diet can alleviate diabetic kidney damage through\u0026nbsp;the modulation of\u0026nbsp;gut microbiota.\u003c/p\u003e\n\u003cp\u003eIn this study, we investigated the renoprotective effects of an LPCR diet in mice with diabetes induced by a high-fat diet (HFD) combined with streptozotocin (STZ) and explored the role of the gut microbiota in this process.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eSpecific pathogen-free male C57BL/6J mice, aged 6 to 8 weeks and weighing between 17 and 21 g, were obtained from SPF Biotechnology Co., Ltd. (Suzhou, China; Permit No. SYXK [SU] 2021-0025). Mice were housed in cages under controlled conditions (room temperature: 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C; 12-hour light/dark cycle) and had free access to water. Notably, the mice underwent a one-week acclimatization period prior to the commencement of the study. All animal experiments were approved by the Animal Experiment Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine (Ethics No. AEWC-20230531-309) and complied with the Guidelines for the Care and Use of Animals established by the Chinese Animal Management Committee.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eModel construction and study design\u003c/h3\u003e\n\u003cp\u003eAfter a week acclimatization period, eight mice were randomly assigned to the normal control (NC) group and fed a standard diet (STD), whereas the remaining 32 mice were fed HFD for 12 weeks, followed by four consecutive days of intraperitoneal injections of STZ (Sigma-Aldrich Co., St. Louis, MO, USA) at a dosage of 40 mg/kg to induce diabetes. After the injections, the mice were continued on HFD. Mice with fasting blood glucose (FBG) levels\u0026thinsp;\u0026ge;\u0026thinsp;16.7 mmol/L at 10 weeks after STZ injection were considered to have diabetes. Thereafter, diabetic mice were randomly assigned to four groups (n\u0026thinsp;=\u0026thinsp;8 mice/group): normal protein (NP, 20% protein), CR (30% restriction), low protein (LP, 13% protein), and LPCR (13% protein, 30% restriction). All diets were purchased from Jiangsu Xietong Pharmaceutical Bioengineer Co., Ltd. (Nanjing, China) and the feed formulas are detailed in Supplementary Table\u0026nbsp;1. Daily food intake was measured, and FBG and body weight were recorded weekly, with dietary intervention lasting for a total of five weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSample collection and indicator detection\u003c/h3\u003e\n\u003cp\u003eAfter five weeks of dietary intervention, random urine samples were collected from the mice using individual metabolic cages, followed by the determination of urinary albumin and creatinine levels using enzyme-linked immunosorbent assay (ELISA). Blood samples were obtained from the retro-orbital plexus and centrifuged at 4\u0026deg;C to extract serum. Serum creatinine, triglycerides (TG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), glutathione (GSH), superoxide dismutase (SOD), trimethylamine oxide (TMAO), and TNF-α levels were determined using ELISA kits. ELISA kits for urinary albumin and TMAO levels were obtained from Nanjing Jin Yibai Biological Technology Co., Ltd. (Nanjing, China) and Elabscience Biotechnology Co., Ltd. (Wuhan, China), respectively. All other ELISA kits were sourced from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).\u003c/p\u003e \u003cp\u003eAt the end of the experiment, mice were anaesthetised via intraperitoneal injection with pentobarbital sodium (100 mg/kg). Intestinal faeces were collected, rapidly frozen in liquid nitrogen, and transported in dry ice to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) for gut microbiota analysis. Kidney samples were divided into two portions, and one portion was fixed in 4% paraformaldehyde for histopathological examination, while the other portion was stored at ˗80\u0026deg;C for Western blot analysis.\u003c/p\u003e\n\u003ch3\u003eHistopathology and immunohistochemistry\u003c/h3\u003e\n\u003cp\u003eParaffin-embedded kidney tissue sections (4 \u0026micro;m thick) were prepared and stained with haematoxylin and eosin (HE), periodic acid\u0026ndash;schiff (PAS), and Masson's trichrome stain. For immunohistochemical assay, the sections were incubated overnight at 4\u0026deg;C with rabbit polyclonal antibodies against fibronectin (1:200) and Collagen I (1:200). Thereafter, the sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody at room temperature for 30 min. All antibodies were obtained from Wuhan Servicebio Technology Co., Ltd. (Wuhan, China).\u003c/p\u003e\n\u003ch3\u003eGut microbiome analysis\u003c/h3\u003e\n\u003cp\u003eMicrobial genomic DNA was extracted from faecal samples using the PF Mag-Bind Stool DNA Kit (Omega Bio-tek, Georgia, USA). DNA purity and concentration were assessed using the PF Mag-Bind Stool DNA Kit (Omega Bio-tek, Georgia, USA). Thereafter, the 16S rRNA gene was amplified using the primers 27F (5\u0026acute;-AGRGTTYGATYMTGGCTCAG-3\u0026acute;) and 1492R (5\u0026acute;-RGYTACCTTGTTACGACTT-3\u0026acute;). The PCR amplification protocol was as follows: initial denaturation at 95\u0026deg;C for 3 min, followed by 27 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 60\u0026deg;C for 30 s, extension at 72\u0026deg;C for 45 s, and final extension at 72\u0026deg;C for 10 min. Subsequently, a PacBio library was constructed and sequenced. PacBio data were analysed using SMRTLink 11.0, resulting in at least three complete passes with 99% sequence accuracy for high-fidelity sequences. High-fidelity sequences were processed using UPARSE 11 to classify the operational taxonomic units (OTUs) at 97% similarity.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eBriefly, proteins were extracted frozen kidney tissues, mixed with western blotting loading buffer, separated using SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. After blocking with non-fat milk at room temperature for 2 h, the membranes were incubated overnight at 4\u0026deg;C with primary antibodies against ASC, NLRP3, IL-1β, and β-actin (all diluted at 1:3000). Thereafter, the membranes were incubated with the corresponding secondary antibodies at 4\u0026deg;C for an additional two nights. All antibodies were purchased from Cell Signalling Technology (Danvers, MA, USA). Protein bands were detected using a chemiluminescent imaging system, and the grayscale values of the bands were calculated using the ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism 8. Normally distributed data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Significant differences were determined using t-tests for two groups and one-way ANOVA was used for multiple groups. Non-normally distributed data were analysed using the Wilcoxon rank-sum test. 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=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLPCR diet improves glucose and lipid metabolism\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarises the changes in body weight and FBG levels across the different groups before and after the dietary intervention. Diabetic mice in the various groups showed significantly lower body weights compared to those in the NC group at the end of the experiment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Although the LPCR group showed a decreasing trend in body weight compared to the NP group, this difference was not statistically significant. In terms of FBG levels, all three dietary intervention groups experienced a reduction compared to baseline, whereas the NP group showed an increase. Notably, FBG levels were significantly lower in the LP and LPCR groups compared to the NP group at the conclusion of the intervention (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of LPCR diet on body weights and fasting blood glucose levels in diabetic mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eBody weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eWeight change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eFasting blood glucose (mmol/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003cp\u003echange\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e22w 27w\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e22w 27w\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e7.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e22.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.74**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e22.50\u0026thinsp;\u0026plusmn;\u0026thinsp;6.21**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e27.89\u0026thinsp;\u0026plusmn;\u0026thinsp;3.70**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e5.39\u0026thinsp;\u0026plusmn;\u0026thinsp;4.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-4.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e27.84\u0026thinsp;\u0026plusmn;\u0026thinsp;3.34**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e24.18\u0026thinsp;\u0026plusmn;\u0026thinsp;5.17**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e-3.66\u0026thinsp;\u0026plusmn;\u0026thinsp;6.68\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-4.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.88**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e25.43\u0026thinsp;\u0026plusmn;\u0026thinsp;4.43**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e15.29\u0026thinsp;\u0026plusmn;\u0026thinsp;6.03*\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e-10.14\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98**\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLPCR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e19.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.74**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.90**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e20.98\u0026thinsp;\u0026plusmn;\u0026thinsp;6.25**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e15.71\u0026thinsp;\u0026plusmn;\u0026thinsp;6.45**\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e-5.27\u0026thinsp;\u0026plusmn;\u0026thinsp;7.33\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eDifferences before and after dietary intervention (22 and 27 week) in diabetic mice. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, n\u0026thinsp;=\u0026thinsp;8 in each group. *\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 vs. NC group; **\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01 vs. NC group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 vs. NP group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01 vs. NP group\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter five weeks of dietary intervention, there was a significant increase in serum TG, T-CHO, and LDL-C levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and a significant decrease in HDL-C levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the NP group compared with those in the NC group. However, both LP and LPCR diets significant improved the levels of all four lipid parameters compared with those in the NP group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with the LPCR group demonstrating particularly pronounced effects on the LDL-C and HDL-C levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLPCR diet mitigates kidney damage\u003c/h2\u003e \u003cp\u003eTo examine the effects of LPCR on kidney function, we measured serum creatinine levels and urine albumin/creatinine ratios after five weeks of dietary intervention. Serum creatinine levels and urine albumin/creatinine ratios were significantly higher in the NP group than in the NC group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, the three dietary interventions significantly decreased serum creatinine levels and urine albumin/creatinine ratios compared with those in the NP group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3.1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHE staining revealed that mice in the NP group exhibited pathological changes characteristic of DKD, including irregular glomerular morphology, cytoplasmic vacuolation of renal tubular epithelial cells, and chronic inflammatory cell infiltration into the renal interstitium. However, the three dietary interventions ameliorated these pathological changes to varying degrees, with the LPCR group demonstrating the most significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e A).\u003c/p\u003e \u003cp\u003ePAS staining indicated enlarged areas of glycogen deposition in the kidneys of mice in the NP group, along with widening of the mesangial matrix in the glomeruli and thickening of the tubular basement membrane. However, treatment with CR, LP, and LPCR diets alleviated these pathological alterations to varying degrees, with the LPCR group showing the most significant improvement (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e B).\u003c/p\u003e \u003cp\u003eMasson\u0026rsquo;s trichrome staining demonstrated a marked increase in collagen deposition in the kidneys of mice from the NP group, indicating pronounced renal fibrosis. However, the dietary interventions effectively reduced collagen deposition to varying degrees, with the LPCR diet significantly inhibiting renal fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e C). Additionally, immunohistochemical staining showed that fibronectin expression in the glomeruli and collagen I level in the renal interstitium were significantly higher in the NP group than in the NC group. In contrast, the expression levels of both proteins were significantly lower in the LPCR group than in the NP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3.3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLPCR diet modulates gut microbiota\u003c/h2\u003e \u003cp\u003eβ-diversity analysis was performed to assess the similarity of gut microbial compositions across different groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Notably, the NP group displayed a clear separation from the NC group, indicating significant alterations in microbial diversity. Principal coordinate analysis (PCoA) showed that the clustering of the three dietary intervention groups shifted notably towards the NC group, particularly for the LPCR group.\u003c/p\u003e \u003cp\u003eThe Gut Microbiome Health Index (GMHI), which assesses health status based on species-level taxonomic profiles from stool shotgun metagenome samples, revealed a significant disruption in the GMHI of the NP group compared to the NC group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Notably, LPCR diet led to a substantial improvement in GMHI compared to the NP group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eAt the phylum level, the abundance of Firmicutes and Firmicutes/Bacteroidetes (F/B) ratio were higher in the NP group than in the NC group. Notably, treatment with LPCR diet resulted in a significant decrease in both the abundance of Firmicutes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and F/B ratio (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in mice with DKD (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;D). The Wilcoxon rank-sum test revealed that the abundance of \u003cem\u003eBacteroides\u003c/em\u003e at the genus level and \u003cem\u003eBacteroides acidifaciens\u003c/em\u003e at the species level were significantly higher in the LPCR group than in the NP group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;F).\u003c/p\u003e \u003cp\u003eMoreover, serum TMAO levels were significantly higher in the NP group compared to the NC group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, CR, LP, and LPCR diets significantly reduced serum TMAO levels compared to those in the NP group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively), with LPCR diet exerting the most pronounced inhibitory effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLPCR diet provides antioxidant and anti-inflammatory benefits\u003c/h2\u003e \u003cp\u003eCompared to the NC group, there was a significant decrease in serum GSH and SOD levels, along with a marked increase in TNF-α levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, treatment with LPCR diet for five weeks significantly increased GSH and SOD levels and decreased TNF-α levels compared to the NP group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;C).\u003c/p\u003e \u003cp\u003eAdditionally, ASC, NLRP3, and IL-1β expression levels in kidney tissue were significantly higher in the NP group than in the NC group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, CR, LP, and LPCR diets significantly reduced the expression of ASC, NLRP3, and IL-1β compared to the NP group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with the LPCR group showing the most pronounced decrease in NLRP3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;G).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated whether LPCR diet can protect the kidney by modulating the gut microbiota in a mouse model of diabetes. Our findings indicated that LPCR diet not only improved glucose and lipid metabolic disorders but also mitigated kidney damage in diabetic mice. Additionally, LPCR diet altered the gut microbiota composition, reduced circulating TMAO levels, and provided antioxidant and anti-inflammatory benefits. Overall, the renoprotective effects of LPCR diet may be mediated through the gut-kidney axis.\u003c/p\u003e \u003cp\u003eCR, defined as reduced energy intake without malnutrition, is a recognised dietary intervention for diabetes that offers benefits, such as weight loss, improved glucose and lipid metabolism, and reduced oxidative stress and inflammation\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Similarly, dietary protein restriction enhances metabolic function and regulates glycaemia, lipid levels, and body weight in patients with type 2 diabetes\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In the present study, both the LP and LPCR diets improved glucose and lipid metabolism, whereas CR did not significantly affect FBG and serum LDL-C levels. This discrepancy may be due to severe metabolic disorders present in diabetic mice and an insufficient degree of CR. Moreover, a previous study indicated that LPCR diet was more effective than LP diet in improving glucose and lipid metabolism in diabetic rats\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Therefore, LPCR diet may be a superior option for enhancing metabolic function in diabetes.\u003c/p\u003e \u003cp\u003eBased on serum creatinine levels, urine albumin/creatinine ratios, and pathological changes observed at the end of the intervention, kidney damage occurred in diabetic mice at the mid to late stages of DKD rather than in the early stage. All three dietary interventions significantly reduced serum creatinine and urinary protein/creatinine levels in DKD mice. Importantly, various pathological changes, including irregular glomerular morphology, cytoplasmic vacuolation of renal tubular epithelial cells, chronic inflammatory cell infiltration, widening of the mesangial matrix in the glomeruli, thickening of the tubular basement membrane, and renal fibrosis, showed improvement after five weeks of dietary intervention, with the LPCR group demonstrating the most pronounced improvements. Collectively, these findings suggest that LPCR diet may effectively alleviated kidney damage in diabetic mice.\u003c/p\u003e \u003cp\u003eDysbiosis of the gut microbiota has been implicated in the development and progression of various diseases, including type 2 diabetes and CKD\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. CKD can alter the composition of the gut microbiota, leading to dysbiosis, which may further elevate the levels of uraemic toxins and exacerbate CKD progression\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Diet plays a crucial role in regulating the gut microbiota and its activity\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, making dietary interventions a promising strategy for restoring microbial balance in CKD\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. To explore the role of the gut microbiota in the renoprotective effects of LPCR diet, we assessed the gut microbiota composition of diabetic mice across different dietary groups. Our findings revealed significant variations in the composition of the gut microbiota based on the dietary interventions. Notably, the four dominant bacterial groups, Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, accounted for over 90% of the gut microbiota, with Firmicutes and Bacteroidetes particularly involved in regulating the host metabolism and immunity\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. A high abundance of Firmicutes and low abundance of Bacteroidetes is strongly linked with obesity, which is often associated with diabetes\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Cai et al. reported a marked increase in F/B ratio in mice with DKD, which was restored by treatment with resveratrol, a CR mimetic\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Similarly, our findings revealed a pronounced increase in F/B ratio in DKD mice. Notably, LPCR diet significantly improved gut microbiota dysbiosis in diabetic mice. Additionally, LPCR diet induced the highest increase in \u003cem\u003eBacteroides acidificient\u003c/em\u003e, a species classified as \u003cem\u003eBacteroidetes\u003c/em\u003e. TMAO is a gut-derived toxic metabolite that is associated with increased systemic inflammation. Research findings indicate that high TMAO production is associated with elevated F/B ratios in healthy individuals\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Additionally, plasma TMAO levels were negatively correlated with \u003cem\u003eBacteroides\u003c/em\u003e abundance in mice fed high-choline diet\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Elevated TMAO levels in CKD patients are closely associated with decreased renal function\u003csup\u003e[\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Changes in gut microbiota contribute to the development of DKD, with TMAO and chronic inflammation playing pivotal roles in this process\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Furthermore, serum TMAO level has been identified as an independent risk factor for DKD\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In the present study, LPCR diet significantly reversed DKD-induced increase in serum TMAO levels. Overall, we speculated that LPCR diet might improve DKD by lowering F/B ratio, which subsequently reduced TMAO levels.\u003c/p\u003e \u003cp\u003eMechanistically, TMAO influences kidney injury primarily through the exacerbation of tubulointerstitial fibrosis and renal inflammation\u003csup\u003e[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Recently, Stefaniash et al. demonstrated that TMAO enhances TNF-α mediated kidney inflammation by inducing the release of various cytokines, chemokines, and inflammatory mediators from renal fibroblasts\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Inflammation and oxidative stress are critical factors in the development and progression of CKD, and are closely interconnected and exacerbate each other\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. The NLRP3 inflammasome has been implicated in the progression of CKD, including DKD\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Research indicates that TMAO can induce oxidative stress and activate the NLRP3 inflammasome, leading to the release of inflammatory cytokines\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Conversely, the inhibition of TMAO can reduce NLRP3 inflammasome activation and oxidative stress\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Our results indicated that treatment with LPCR diet for five weeks significantly increased GSH and SOD levels and downregulated TNF-α and NLRP3 inflammasome-related proteins, including ASC, NLRP3, and IL-1β, in kidney tissue. Collectively, these results suggest that the LPCR diet may have anti-inflammatory and antioxidant effects. Based on related studies, we speculated that the renoprotective effects of LPCR intervention in DKD mice may be linked to TMAO inhibition, which prevented the activation of the NLRP3 inflammasome.\u003c/p\u003e \u003cp\u003eDespite the promising findings, the study had several limitations. For example, we set the protein ratio for the LPCR group to 13% protein with 30% caloric restriction to prevent protein-energy wastage. Previous animal studies have shown that intermittent very LPD\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, 50% CR\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, and intermittent CR\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e can improve DKD. Therefore, further research is needed to determine the optimal protein ratio for LPCR and explore whether intermittent approaches are more effective. Additionally, owing to the prolonged modelling period for DKD and other constraints, we did not conduct counterevidence experiments, limiting our ability to clearly define the direct effects of differential bacterial species on TMAO levels. In future studies, we plan to address this limitation by employing faecal microbiota transplantation or other counterevidence techniques to verify the regulatory effects of Firmicutes and Bacteroidetes on TMAO and DKD.\u003c/p\u003e \u003cp\u003eConclusively, LPCR diet mitigates kidney injury in diabetic mice, possibly by modulating the gut-kidney axis. This effect may be linked to reduced circulating TMAO levels, which in turn inhibit the activation of the NLRP3 inflammasome in kidney tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Overall, this study provides the foundation for further research on the specific mechanisms by which LPCR diet regulates the gut-kidney axis, offering new insights and strategies for the clinical treatment of DKD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData a\u003c/strong\u003e\u003cstrong\u003evailability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical\u0026nbsp;Medicine\u0026nbsp;Science\u0026nbsp;and\u0026nbsp;Technology\u0026nbsp;Development\u0026nbsp;Fund\u0026nbsp;Project of\u0026nbsp;Jiangsu\u0026nbsp;University (JLY2021162); \u0026quot;Double\u0026nbsp;Innovation\u0026nbsp;Doctor\u0026quot; of\u0026nbsp;Jiangsu\u0026nbsp;Province (JSSCBS20211615); Special Research Startup Funding for Introduced Personnel at Nanjing Lishui People\u0026rsquo;s Hospital (KY07).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRXZ and XW contributed to formal analysis, investigation, draft preparation, and visualization. YJX contributed to data curation. CRH and XZC contributed to image acquisition and analysis. YLW contributed to statistical analyses. YG contributed to funding acquisition, project administration, writing and review, and editing. YG and CL contributed to conceptualization, validation, and supervision. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe skillful technical assistance of Yu Chen, Xingjia Li, Qifeng Wang, Houcai Huang, Rongling Zhong, and Zhi Xia is appreciated.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKikuchi K, Saigusa D, Kanemitsu Y, et al. Gut microbiome-derived phenyl sulfate contributes to albuminuria in diabetic kidney disease. Nat Commun. 2019;10:1835.\u003c/li\u003e\n\u003cli\u003eMao ZH, Gao ZX, Liu DW, et al. 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Mol Nutr Food Res. 2017;61.\u003c/li\u003e\n\u003cli\u003eWang X, Li X, Dong Y. Vitamin D Decreases Plasma Trimethylamine-N-oxide Level in Mice by Regulating Gut Microbiota. Biomed Res Int. 2020;2020:9896743.\u003c/li\u003e\n\u003cli\u003eEl-Deeb OS, Atef MM, Hafez YM. The interplay between microbiota-dependent metabolite trimethylamine N-oxide, Transforming growth factor \u0026beta;/SMAD signaling and inflammasome activation in chronic kidney disease patients: A new mechanistic perspective. J Cell Biochem. 2019;120:14476-85.\u003c/li\u003e\n\u003cli\u003eTang WH, Wang Z, Kennedy DJ, et al. Gut microbiota-dependent trimethylamine N-oxide (TMAO) pathway contributes to both development of renal insufficiency and mortality risk in chronic kidney disease. Circ Res. 2015;116:448-55.\u003c/li\u003e\n\u003cli\u003eMissailidis C, H\u0026auml;llqvist J, Qureshi AR, et al. Serum Trimethylamine-N-Oxide Is Strongly Related to Renal Function and Predicts Outcome in Chronic Kidney Disease. 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Free Radic Biol Med. 2018;116:88-100.\u003c/li\u003e\n\u003cli\u003eBoini KM, Hussain T, Li PL, et al. Trimethylamine-N-Oxide Instigates NLRP3 Inflammasome Activation and Endothelial Dysfunction. Cell Physiol Biochem. 2017;44:152-62.\u003c/li\u003e\n\u003cli\u003eKitada M, Ogura Y, Suzuki T, et al. Cyclic and intermittent very low-protein diet can have beneficial effects against advanced diabetic nephropathy in Wistar fatty (fa/fa) rats, an animal model of type 2 diabetes and obesity. Nephrology (Carlton). 2017;22:1030-4.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"low-protein, caloric restriction, diet, gut microbiota, diabetic kidney disease, gut-kidney axis","lastPublishedDoi":"10.21203/rs.3.rs-5440142/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5440142/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eDietary interventions are a promising strategy for restoring microbial balance in chronic kidney disease. Research indicates that a low-protein calorie-restricted (LPCR) diet can reduce renal injury in diabetic rodents. However, it is unclear whether the beneficial effects of LPCR diet in mice with diabetic kidney disease (DKD) are mediated through the modulation of the gut microbiota.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA mouse model of diabetes was established using high-fat diet combined with streptozotocin injection. Diabetic mice were randomly divided into four groups: normal protein (NP, 20% protein), caloric restriction (CR, 30% restriction), low protein (LP, 13% protein), and LPCR (13% protein, 30% restriction). After five weeks of intervention, blood and urine samples were collected for relevant analyses, faecal samples for gut microbiota analysis, and kidney tissues for histological and immunohistochemical assays，as well as Western blot analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eLPCR diet significantly improved fasting blood glucose levels and lipid profiles (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) and mitigated kidney damage in diabetic mice. Additionally, LPCR diet ameliorated gut microbiota dysbiosis, significantly suppressing the increase in Firmicutes/Bacteroidetes ratio (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) and decreasing serum trimethylamine oxide(TMAO) levels (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Compared to the NP group, the LPCR group exhibited significant reductions in serum TNF-α levels and the expression of ASC, NLRP3, and IL-1β in kidney tissue (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eLPCR diet exerts renoprotective effects in mice with DKD, possibly by modulating the gut-kidney axis to reduce circulating TMAO levels, thereby inhibiting NLRP3 inflammasome activation in kidney tissue.\u003c/p\u003e","manuscriptTitle":"A low-protein calorie-restricted diet mitigates kidney injury in diabetic mice by modulating the gut-kidney axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-03 14:06:43","doi":"10.21203/rs.3.rs-5440142/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57331ad3-1058-435c-b911-4b819d5346da","owner":[],"postedDate":"December 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-03T14:06:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-03 14:06:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5440142","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5440142","identity":"rs-5440142","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}