miR-299a-5p is a novel mediator of fibrosis in diabetic kidney disease through its regulation of antifibrotic proteins follistatin and cripto-1

miR-299a-5p is a novel mediator of fibrosis in diabetic kidney disease through its regulation of antifibrotic proteins follistatin and cripto-1 | 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 Article miR-299a-5p is a novel mediator of fibrosis in diabetic kidney disease through its regulation of antifibrotic proteins follistatin and cripto-1 Joan Krepinsky, Ifeanyi Nmecha, Gaolin Bo, Melissa McDonald, Dan Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5419387/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Dec, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Glomerular extracellular matrix protein accumulation, mediated largely by mesangial cells, is a defining feature of early diabetic kidney disease. Previously we showed that TGFβ1, a profibrotic cytokine with a well-defined pathogenic role in kidney fibrosis, inhibits expression of the antifibrotic follistatin through induction of microRNA-299a-5p. Whether this microRNA contributes to diabetic kidney disease is unknown. We show that microRNA-299a-5p is increased in mouse and human diabetic kidneys, and by high glucose in primary mesangial cells. Overexpression of microRNA-299a-5p in mesangial cells increased basal extracellular matrix protein production. Conversely, microRNA-299a-5p inhibition prevented the glucose-induced profibrotic response. Bioinformatics screening revealed that cripto-1 is also a target of microRNA-299a-5p. It is known that follistatin and cripto-1 inhibit activin A and TGFβ1 respectively. Induction of microRNA-299a-5p by high glucose mediated the mesangial cell fibrotic response by inhibiting expression of both follistatin and cripto-1 which led to increased activin A and TGFβ1 signaling. In vivo , microRNA-299a-5p inhibition reduced albuminuria, glomerular hypertrophy, loss of podocyte nephrin and extracellular matrix production, and this was associated with increased expression of follistatin and cripto-1. Thus, microRNA-299a-5p is an important mediator of glucose-induced profibrotic responses in mesangial cells and diabetic kidneys. Its inhibition may be a potential novel therapy. Biological sciences/Molecular biology/RNAi Biological sciences/Molecular biology/Non-coding RNAs/miRNAs Biological sciences/Biological techniques/Gene delivery/Transfection/Bacterial transformation diabetic kidney disease microRNA-299a-5p cripto-1 activins transforming growth factor beta 1 (TGFβ1) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 4 Figure 5 Figure 5 Figure 6 Figure 6 Figure 7 Introduction Diabetic kidney disease (DKD) is a major complication of diabetes mellitus, developing in up to 40% of patients. It is the leading cause of end-stage kidney disease, associated with reduced quality of life and increased mortality. 1 , 2 The current standard of care for DKD includes control of blood glucose and blood pressure, and use of inhibitors of the renin-angiotensin-aldosterone system, and for type 2 diabetics sodium glucose co-transporter-2 inhibitors. However, current therapies, even in combination, are unable to halt DKD progression. The identification of novel therapeutic targets aimed at preventing disease progression is thus a major clinical need. Initial pathologic changes of the diabetic kidney occur in the glomerulus, characterized by basement membrane thickening and mesangial expansion from accumulation of extracellular matrix proteins. The profibrotic cytokine transforming growth factor β1 (TGFβ1) is well known to be a major pathologic mediator of these changes. 3 , 4 However, the pleiotropic roles of TGFβ1 make its direct therapeutic targeting challenging. Previously, our lab identified that TGFβ1 inhibits the production of an antifibrotic protein, follistatin (FST) by mesangial cells (MC) through upregulation of miR-299a-5p. 5 FST is a potent inhibitor of activins, members of the TGFβ superfamily, in particular activins A and B. The importance of both activins to fibrosis in DKD has been shown. 6 – 8 We had also shown that FST attenuated high glucose (HG)-induced matrix production by MC and reduced fibrosis in a model of DKD. 9 However, effects are attenuated at higher doses, indicating that administration may have a narrow therapeutic window. Novel strategies to increase endogenous FST may thus be more effective and better clinically tolerated. miR-299a-5p is a member of the miR-154 family, the second largest miRNA cluster in the human genome. Containing over 40 members, it is highly conserved between species. 10 Members of this family are implicated in fibrosis in other organs. In idiopathic pulmonary fibrosis (IPF), several members are increased including miR-299a-5p and miR-154, with the latter shown to heighten IPF fibroblast response to TGFβ1. 11 miR-154 expression alone also increased cardiac fibroblast collagen production, 12 and its LNA inhibition protected against cardiac fibrosis and dysfunction in a pressure overload model. 13 Increased miR-299a-5p was also found in fibrotic liver from patients with primary biliary cirrhosis. 14 Our previous data show its increase in a hypertensive model of chronic kidney disease, 5 but whether miR-299a-5p contributes to the progression of fibrosis in DKD is unknown. Here, we show that miR-299a-5p expression is significantly upregulated in glomeruli and tubules in both animal models of type 1 diabetes and in kidneys of type 2 DKD patients. HG increases the expression of miR-299a-5p by MC to inhibit FST production. Bioinformatics screening identified cripto-1 as an additional miR-299a-5p target. Unlike FST which has no neutralizing activity against TGFβ1, 15 cripto-1 inhibits the actions of both activin A (actA) and TGFβ1. 16 , 17 We hypothesize that inhibition of miR-299a-5p in vivo with anti-miR administration ameliorated DKD, in association with elevated expression of FST and cripto-1. Our study highlights a potential therapeutic role for miR-299a-5p inhibition in restoring endogenous antifibrotic protein expression to slow the progression of DKD. Methods 2.1 Cell Culture Primary MC were grown from glomeruli of male C57BL/6J mice isolated using Dynabeads (Invitrogen, Carlsbad, CA) as described previously. 5 MC were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 20% fetal bovine serum (FBS), streptomycin (100µg/ml) and penicillin (100µg/ml) at 37˚C in 95% O 2 , 5% CO 2 . Cells were serum deprived in DMEM with 0.5% FBS for 24h following transfection and prior to treatment with high glucose (30mM) for 72 hrs, drugs or recombinant proteins ( Supplementary Table 1). Passage 9–16 MCs were used for experiments. 2.2 mRNA and miRNA Extraction and qPCR RNA was extracted using TRIzol (Life Technologies, Carlsbad, CA) and 1µg was reverse transcribed using qScript cDNA SuperMix Reagent (Quanta Biosciences). miRNA-enriched cDNA was generated using the qScript microRNA Quantification System (Quanta Biosciences). Real-time polymerase chain reaction (PCR) was performed using a SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA) on the ViiA 7 Sequence Detector (Life Technologies). Amplification of cripto-1 or miRNA expression, relative to 18S or U6 respectively, was measured using the ΔΔCT method. Primers are listed in Supplementary Table 2 . miRNeasy serum advanced kit (Qiagen) was used to extract RNA from the serum of diabetic mice after which miRNA-enriched cDNA was generated as above. 2.3 miRNA in-situ Hybridization (ISH) Formalin-fixed paraffin-embedded kidney sections (4µm) were deparaffinized, dehydrated, and treated with proteinase K. After incubation in hybridization buffer, sections were incubated with DIG-labeled miRCURY LNA anti-miR detection probes ( Supplementary Table 3 ) targeting miR-299a or U6 (18h). After further washes 18 , sections were blocked in 1×Casein Solution (Vector labs) and incubated with anti-digoxigenin-AP Fab fragment. Chromogenic reaction was carried out using NBT/BCIP (Vector Labs). Slides were then mounted with Vectamount (Vector labs). Images were taken at 40x using a BX41 Olympus microscope and quantified using ImageJ. For human studies, kidney biopsy samples with a diagnosis of DKD were obtained. Normal kidney tissues surrounding resected renal cancers were used as controls (Research Ethics Board approval number 2010 − 159). 2.4 Transient Transfections For luciferase experiments, MC were plated in triplicate at 60–70% confluence and transfected with 0.5µg of luciferase construct (pGL3-CAGA12-luc or pGL3-COL1α1-luc) with 0.05µg pCMV β-galactosidase (Clontech) using Effectene (Qiagen). At harvest, MC were lysed with 1X Reporter Lysis Buffer (Promega, Madison, WI) and frozen at -80ºC overnight. Luciferase and β-gal activities were measured on clarified lysate using specific kits (Promega) with a Spectramax Plus 384 plate reader set to luminescence and 420nm, respectively. β-gal activity was used to adjust for transfection efficiency. miR299a-5p regulatory element (MRE) luciferase was generated in order to measure miR299a-5p activity as previously described, 5 and MCs were transfected as above. For transient expression, MC resuspended in electroporation buffer containing 10µg expression plasmid were electroporated using a single square pulse set at 200V for 35ms (ECM830, Harvard Bioscience). After 18h, media was exchanged. To assess transfection efficiency of miR plasmids ( Supplementary Table 4 ), mCherry (ex 550nm/em 620nm) and GFP (ex 490nm/em 525nm) immunofluorescence were confirmed by imaging (EVOS FL Cell Imaging System, Thermo Fisher Scientific). Luciferase constructs and pCMV β-galactosidase were transfected 4h later in some experiments. 2.5 Protein Extraction and Immunoblotting MC were washed 3x with cold phosphate buffered saline and lysed in buffer with protease inhibitors (10µg/ml PMSF, 2µg/ml leupeptin, 2µg/ml aprotinin). After clarification, proteins were separated by SDS-PAGE and analyzed by immunoblotting. Antibodies used are listed in Supplementary Table 5 . 2.6 Animal Studies Animal studies were approved by the McMaster University Animal Research Ethics Board and carried out in accordance with the Canadian Council on Animal Care guidelines and ARRIVE guidelines and checklist. Animals were housed under standard conditions with free access to chow and water. Two animal models were assessed by IHC and ISH: (1) Male type 1 diabetic Akita (C57BL/6-Ins2 Akita /J) or wild-type mice (C57BL/6J) (Jackson Laboratory, Bar Harbor, ME, USA) at age 40 weeks (ethics #18-07-30). (2) Uninephrectomized male CD1 mice aged 9 weeks were injected with 200µg streptozotocin and euthanized after 12 weeks of diabetes as described previously 19 , 20 (#14-11-48). C57BL/6 HH Akita mice expressing hypermorphic (gain in function) alleles for TGFβ1 (denoted HH-A), resulting in ~ 300% increase in transcript expression, were described previously. 28 Controls were non-diabetic HH mice. Male mice were enrolled at age 12 weeks, randomly assigned and treated with 2mg/kg of miR-LNA inhibitor (LNAi) or negative control “B” (LNAc, Qiagen) intraperitoneally once weekly for 12 weeks. There were four groups: HH-LNAc (n = 8), HH-LNAi (n = 8), HH-A-LNAc (n = 12) and HH-A-LNAi (n = 12). Mice with glucometer measure of tail vein blood glucose > 17mM were enrolled as diabetics. Those that developed ketonuria (dipstick, Bayer Multistix) or progressive weight loss were administered ¼ insulin pellet (LinShin Canada) to maintain body weight while also maintaining hyperglycemia. Blood pressure was measured in the morning, at 4, 8, and 12 weeks using tail-cuff plethysmography (Coda non-invasive blood pressure monitoring system, Kent Scientific). After 12 weeks, urine was collected (6h, Nalgene metabolic cage 650 − 0210). Urine albumin and creatinine were measured using Albuwell M (Exocell) and Mouse Creatinine Assay kits (Crystal Chem) respectively to determine the albumin to creatinine ratio (ACR). Glomerular filtration rate (GFR) was measured using fluorescein isothiocyanate (FITC)-labeled sinistrin (Fresenius Kabi Linz) as previously described. 9 Mice were then anesthetized, perfused with saline and kidneys harvested for analysis. ELISA kits (R&D Systems) were used to measure total TGFβ1 or actA in urine, normalized to urine creatinine. TGFβ1 samples underwent acid activation. 2.7 Imaging Formalin-fixed paraffin-embedded kidney sections (4µm) were deparaffinized and stained using Trichrome (Sigma), PAS or Picrosirius red (PSR) (Polysciences Inc.) or using antibodies ( Supplementary Table 5 ). Images were captured using the Olympus BX41 microscope at 20x (or Olympus IX81 fluorescence microscope for PSR) and quantified as percentage of positive area using ImageJ. Glomerular hypertrophy was assessed by glomerular cross-sectional area measures on Periodic acid-Schiff-stained sections as described previously. 15 For immunofluorescence, 10µm OCT-embedded frozen kidney sections were fixed in 4% paraformaldehyde prior to blocking and nephrin antibody incubation. Nuclei were stained with DAPI. Images were taken at 40x for quantification. 2.8 Statistical Analysis GraphPad Prism 10 was used to analyze differences between groups using a one-way ANOVA with Tukey’s post-hoc testing, or two-tail unpaired t-test. Repeated measures two-way ANOVA was used to analyze blood pressure. Statistical significance was set at P ≤ 0.05, with data presented as mean ± SEM. Results 3.1 miR-299a-5p expression is increased by HG in MC and in mouse and human DKD. We first investigated the effects of HG on miR-299a-5p expression. HG, but not the osmotic control mannitol, increased the expression of miR-299a-5p in MC (Fig. 1 a ). We next used a construct in which the miR-299a-5p regulatory element (MRE) is placed downstream of the luciferase gene. 5 Fig. 1 b shows decreased luciferase activity in HG, indicating increased miR-299a-5p binding. We next assessed miR-299a-5p expression in vivo. By PCR, miR-299a-5p was increased in kidney cortex of CD1 streptozotocin-induced diabetic mice (Fig. 1 c), confirmed by ISH (Fig. 1 d,e). Increased expression was also seen in type 1 diabetic Akita mice (Fig. 1 f,g), and in human DKD kidney biopsies (Fig. 1 h,i). Expression was observed in both glomeruli and tubules, suggesting a role for this miR across kidney cell types. 3.2 miR-299a-5p promotes HG-induced profibrotic responses in MC. Previously, we showed that FST is a target of miR-299a-5p, 5 and that FST regulates both basal and HG-induced matrix production through its potent inhibition of actA. 9 We thus assessed whether miR-299a-5p regulates HG-induced fibrotic responses. We used plasmids to either inhibit or overexpress this miR, 5 with transfection efficiency measured by mCherry or GFP immunofluorescence respectively (Fig. 2 a,e). MiR-299a-5p inhibition significantly attenuated HG (72hr) profibrotic effects, measured as collagen Iα1 luciferase activity (Fig. 2 b) and matrix protein (fibronectin, collagen Iα1) and profibrotic cytokine (connective tissue growth factor (CTGF)) expression (Fig. 2 c,d). Conversely, miR-299a-5p overexpression augmented basal collagen Iα1 promoter activity (Fig. 2 f) and profibrotic protein synthesis ( Fig. 2 g,h) to levels seen with HG. Profibrotic responses to HG in MC are known to require Smad3, 21 which mediates both actA and TGFβ1 signaling. 3 , 22 , 23 We thus assessed whether miR-299a-5p mediates HG (72hr)-induced Smad3 activation. Figure 2 i,j shows that miR-299a-5p inhibition prevented HG-induced Smad3 C-terminal phosphorylation, required for its activation. Smad3 transcriptional activity, assessed using its reporter CAGA 12 -luciferase, was also inhibited (Fig. 2 k). Conversely, miR-299a-5p overexpression alone increased Smad3 phosphorylation and transcriptional activity to levels seen with HG (Fig. 2 l-n). Interestingly, as for matrix protein synthesis, this was not augmented by HG, suggesting a major role for miR-299a-5p in Smad3 activation by HG. 3.3 miR-299a-5p inhibits expression of anti-fibrotic proteins cripto-1 and FST. We next assessed whether, in addition to FST, other potentially anti-fibrotic genes were regulated by miR-299a-5p. Bioinformatics screening of potential targets using TargetScan8.0, miRDB, miRWalk and Fireplex discovery engine revealed cripto-1, a known antagonist of both TGFβ1 and actA, as a target. As seen in Fig. 3 a, the miR-299a-5p response element is conserved in the mouse and human cripto-1 3’UTR. 16 , 17 To confirm regulation by miR-299a-5p, we tested its effects on a cripto-1 3’UTR-luciferase reporter. Figure 3 b shows that HG (72hr) reduced cripto-1 3’UTR luciferase activity. This was reversed by miR-299a-5p inhibition (Fig. 3 c). As seen in Fig. 3 d-f, both cripto-1 mRNA and protein expression were decreased by HG, with reduced FST expression also confirmed. Decreased cripto-1 and FST expression were also observed in kidney cortex immunoblots of type 1 DKD (Akita) mice (Fig. 3 g,h), with cripto-1 reduction confirmed by immunohistochemistry (Fig. 3 i,j). Reduction in cripto-1 and FST were confirmed in a second DKD mouse model (streptozotocin-treated, uninephrectomized CD1 mice) (Fig. 3 k-n), 19 and cripto-1 reduction also confirmed in human DKD biopsies (Fig. 3 o, p). We next sought to confirm that the regulation of cripto-1 by HG is mediated by miR-299a-5p. As seen in Fig. 3 q,r, miR-299a-5p inhibition attenuated HG-induced repression of cripto-1. Conversely, miR-299a-5p overexpression reduced basal expression of cripto-1 similarly to HG-mediated reduction (Fig. 3 s,t). FST was similarly affected by miR inhibition and overexpression. Together, these results indicate that HG-induced miR-299a-5p expression reduces cripto-1 and FST synthesis. 3.4 Cripto-1 inhibits HG-induced profibrotic responses in MC. Cripto-1 is known to antagonize both TGFβ1 and actA signaling. 16 , 17 We first confirmed this in MC. Supplementary Fig. 1 shows that Smad3 transcriptional activation by TGFβ1 or actA was inhibited by cripto-1, although ActA inhibition was greater. We next determined whether cripto-1 could prevent HG-induced Smad3 activation. Figure 4 a, b shows that the increased Smad3 C-terminal phosphorylation induced by HG (72hr) was inhibited by cripto-1, as was Smad3 transcriptional activity (Fig. 4 c). Figure 4 d-f show that cripto-1 also inhibited HG-induced collagen Iα1 luciferase activation and the increase in expression of fibrotic proteins. Since both FST and cripto-1 are decreased by miR-299a-5p in HG and both inhibit actA, but only cripto-1 inhibits TGFβ1, we tested if their combination would have an additive effect. Figure 4 g shows that each individually suppressed HG (72hr)-induced Smad3 transcriptional activity, with a small additive effect. In response to miR-299a-5p overexpression, cripto-1 and FST alone reduced Smad3 reporter activity, with this effect augmented by their combination (Fig. 4 h). Similar augmented effects were seen for collagen Iα1 luciferase (Fig. 4 i). Finally, using neutralizing antibodies we explored effects of individual inhibition of TGFβ1 or the two activins primarily neutralized by FST, actA and activin B (actB). Interestingly, neutralization of either actA or TGFβ1, but not actB, inhibited Smad3 transcriptional activation by miR-299a-5p overexpression (Fig. 4 j). This suggests that cripto-1 and FST prevent Smad3-dependent fibrotic signaling through their inhibition of actA and TGFβ1 with no significant contribution from actB. Together, these data also suggest that direct inhibition of miR-299a-5p may have greater therapeutic efficacy than use of FST or cripto-1 alone. 3.5 miR-299a-5p is increased in caveolin-1 knockout MC. Previously, we showed a reduction in both basal and HG-induced matrix protein expression in MC that lack caveolin-1 and thus caveolae, membrane microdomains which regulate profibrotic signaling. 24 – 26 Furthermore, FST was significantly upregulated in MC derived from caveolin-1 knockout (KO) mice, 9 , 27 mediated by suppressed miR-299a-5p expression and activity in these cells. 5 This translated to protection from DKD in caveolin-1 KO mice. 9 We thus hypothesized that cripto-1 would also be increased in KO MC. To do this, primary mouse MCs were isolated from caveolin-1 wild-type (WT) and caveolin-1 KO B6129SF1/J mice as described previously. 5 Indeed, Supplementary Fig. 2a, b show that cripto-1 expression was higher in caveolin-1 KO compared to WT cells, and this expression was reduced by miR-299a-5p overexpression ( Supplementary Fig. 2c, d ). Furthermore, miR-299a-5p inhibition in WT cells increased cripto-1 and FST expression ( Supplementary Fig. 2e, f ). Interestingly, bioinformatics screening showed that another target of miR-299a-5p is the transcription factor SP1, which we previously showed regulates FST promoter activation in MC. 27 Supplementary Fig. 3 shows that miR-299a-5p overexpression significantly decreased SP1 expression. 3.6 miR-299a-5p inhibition improves DKD. We next assessed the therapeutic potential of miR-299a-5p inhibition in DKD. We used Akita mice with genetically increased TGFβ1 expression which was previously shown to exacerbate DKD. 28 MiR-299a-5p was competitively inhibited from binding to its targets using a Locked Nucleic Acid (LNA) anti-miR. Mice were treated for 12 weeks (12–24 weeks of age, Fig. 4 a). We first confirmed that miR-299a-5p inhibition could rescue reduced cripto-1 and FST expression in diabetic kidneys. Figure 4 b,c shows that both proteins were significantly increased by miR-299a-5p inhibition, confirmed for FST by IHC (Fig. 4 d,e). Supplementary table 6 shows that endpoint fasting blood glucose and weight were not affected by miR-299a-5p inhibition. Blood pressure was measured at enrollment and every 4 weeks ( Supplementary Table 7 ). By study endpoint both systolic and diastolic pressures were increased in diabetic mice, with reduction by miR-299a-5p inhibition. No effect on blood pressure was seen in non-diabetic mice. Diabetic mice showed the expected hyperfiltration (increased GFR) and kidney hypertrophy characteristic of early DKD. These were unaffected by miR-299a-5p inhibition (Fig. 4 f,g). However, miR inhibition significantly decreased albuminuria (Fig. 4 h) and glomerular hypertrophy (Fig. 4 i), two key features of early DKD 29 . MiR inhibition also significantly rescued loss of nephrin, a marker of podocyte injury which has been correlated with albuminuria in diabetic mice 30 , 31 (Fig. 4 j, k). We next assessed fibrosis. Picrosirius red (PSR) showed the expected increase in collagen I/III expression in diabetic kidneys, which was reduced by miR inhibition (Fig. 5 a,b). Similarly, IHC showed that fibronectin, collagen Iα1 and CTGF expression were also decreased by miR-299a-5p inhibition in diabetic mice (Fig. 5 a,c-e). This was confirmed by immunoblotting of kidney cortex (Figs. 5 f-i). MiR-299a-5p inhibition also reduced activation of Smad3, assessed by its phosphorylation (Fig. 6 a,b). Furthermore, the expected increase in actA expression in diabetic kidneys 9 was markedly reduced in both glomeruli and tubules by miR-299a-5p inhibition, as shown by IHC (Fig. 6 c,d). Urinary actA, significantly elevated in diabetic mice, was also decreased by the inhibitor (Fig. 6 e). Similarly, urinary TGFβ1 was reduced by miR inhibition (Fig. 6 f). Interestingly, serum miR-299a-5p was increased in diabetic mice, highlighting its potential role as a biomarker for DKD ( Fig. 6 g). These data support potential therapeutic value of miR-299a-5p inhibition as an antifibrotic agent in DKD through its restoration of cripto-1 and FST, endogenous antagonists of profibrotic TGFβ1 and actA. Discussion miRs play various regulatory roles in cellular processes of healthy kidneys. However, disease-specific alteration of particular miRs may enable their therapeutic targeting and/or use as diagnostic markers. We identified a novel role for miR-299a-5p in the pathogenesis of fibrosis in DKD. It not only downregulates expression of FST, which inhibits fibrosis through its potent neutralization of activins, but it also downregulates the TGFβ1 antagonist cripto-1. By restoring endogenous FST and cripto-1, miR-299a-5p inhibition ameliorates DKD, as summarized in Fig. 6 h. We previously identified FST as a miR-299a-5p target. 5 Its greatest efficacy is against actA 32 , known to be increased in rodent and human DKD. 9 , 33 While FST reduced fibrosis and protected against podocyte loss in Akita mice, higher dosing did not increase benefit, and in a non-diabetic model showed reduced efficacy 5 . These data suggested a therapeutic window for FST, possibly related to inhibition of other TGFβ family members. Restoring reduced endogenous FST levels in DKD may thus be a better treatment option. Our identification of increased miR-299a-5p expression in both glomeruli and tubules of mouse and human DKD suggested the potential therapeutic value of targeting miR-299a-5p, supported by our preclinical study. As expected, this was associated with reduced actA in kidneys and urine of diabetic mice. Given that miRNAs have several targets, we further performed bioinformatics analysis to identify potential additional mechanisms by which miR-299a-5p may contribute to fibrosis. Interestingly, we found that the cripto-1 3’UTR is also a target of miR-299a-5p. Cripto-1 is a glycosylphosphatidylinositol-linked membrane protein essential in human embryonic development and also implicated in tumor progression 34 . While it functions as a co-receptor for TGFβ family members nodal, growth differentiation factor (GDF)1 and GDF3, 35 it was also shown to block signaling of actA, actB and TGFβ1 itself. 11 , 12 , 36 Through binding these ligands and their type I/II receptors, cripto-1 inhibits ligand-receptor interaction to prevent signaling. Cripto-1 can also be shed from the membrane, 35 likely further enabling interaction with these cytokines. Indeed, our data showed that recombinant cripto-1 effectively inhibits signaling by actA and TGFβ1 in MC. Little is known of the role of cripto-1 in the kidney. One study found no significant kidney expression of cripto-1 by immunohistochemistry, but this was in comparison to renal cell carcinoma in which expression may be highly elevated. 37 We now show that cripto-1 is expressed in normal kidney in both glomeruli and tubules, likely at much lower levels than seen in cancer cells, and that this is attenuated in DKD. In agreement, miR-299a-5p-regulated inhibition of cripto-1 expression by HG was also seen in MC. We confirmed that recombinant cripto-1 reduces signaling by both actA and TGFβ1 and now show that it also attenuates the profibrotic effects of HG. Interestingly, specific neutralization studies suggested that the contribution of actA to these effects was greater than that of actB. Of importance therapeutically, we observed synergy between cripto-1 and FST in attenuating the profibrotic effects of either miR-299a-5p overexpression or of HG. Together, the targeting of both actA and TGFβ1 should theoretically provide more effective inhibition of fibrosis in DKD than inhibition of either ligand separately, although this would need to be demonstrated using target-specific miR blockers. Previously, we showed that caveolae and their structural membrane protein caveolin-1 are important regulators of FST expression. Caveolin-1 deletion in MC significantly reduced miR-299a-5p, leading to increased expression of FST and reduced expression of fibrotic proteins. 9 Caveolin-1 knockout in mice also protected against glomerular fibrosis in DKD. 9 Our data now extend these findings to show the negative regulation of cripto-1 expression by caveolin-1, mediated by miR-299a-5p. The protective effects of cav-1 deletion on DKD can thus likely be attributed to additional antifibrotic effects of cripto-1. Interestingly, Bianco et al. showed that caveolin-1 interaction with cripto-1 in mammary epithelial cells inhibits cripto-1 biologic activity 13 , suggesting that caveolin-1 can regulate both cripto-1 expression and activity. A negative feedback mechanism was also suggested, with cripto-1 reducing caveolin-1 expression in these cells 13 . The reduction in cripto-1 seen in diabetic kidneys may thus serve to augment caveolin-1 expression, further reducing FST and cripto-1. Additional studies are needed to test whether this feedback mechanism exists. Our data support a novel role for miR-299a-5p in DKD. Increased by HG in MC and in diabetic kidneys, its inhibition protects against DKD. Overexpression of miR-299a-5p alone in the absence of HG promotes matrix synthesis, highlighting the important role it plays in the cellular fibrotic response. This is not surprising given its inhibition of at least two potent antifibrotic targets. Further studies are needed to determine whether additional miR-299a-5p targets which could contribute to kidney fibrosis exist. Interestingly, miR-299a-5p was found to suppress Atg5 expression and thereby autophagy in neurons. 38 As autophagy dysfunction is also found in DKD, 39 miR-299a-5p may affect multiple pathogenic processes important to disease. Additional miR-154 family members may also contribute to DKD. Increased miR-377 in type 2 diabetic db/db mice and with HG in MC led to increased profibrotic PAI-1 and TGFβ1 expression through PPARγ inhibition. 40 Sensitive biomarkers for diagnosis and prediction of DKD progression are needed. Measures of urinary and/or serum miR levels represent a potential non-invasive and affordable means to do this. Several urinary miRNAs (95-3p, 185-5p, 1246, 631) were shown to distinguish various forms of nephropathy in diabetic patients, and two of these reflected DKD severity. 41 Here we show a significant increase in serum miR-299a-5p in diabetic mice (Fig. 6 g), suggesting that its measure could be explored further in patient cohorts to determine its clinical utility in the context of kidney disease progression. Two other miR-154 family members were also found increased in serum in DKD patients. Circulating miR-154-5p correlated with serum TGFβ1 and albuminuria, and inversely correlated with kidney function, 42 and serum miR-377 was also suggested to be a potential early biomarker of DKD. 43 Larger studies, however, are needed to confirm these findings. In conclusion, we identify a novel role for miR-299a-5p in promoting fibrosis in DKD. This study supports further assessment of miR-299a-5p inhibition in the treatment of DKD. The restoration of endogenous antifibrotic proteins and inhibition of pathologic TGFβ1 signaling in the kidney may be better tolerated than systemic inhibition of these ligands. Indeed, targeting TGFβ1 has proven challenging due to its homeostatic role. 44 , 45 Further work should also address some limitations of this study. Given that miRs are not specific for single targets, confirmation that beneficial effects of miR-299a-5p targeting occur via increased cripto-1 and FST expression could be ascertained using specific target site blockers to restrict effects to each of these RNAs. Declarations Acknowledgments The authors recognize the support of The Research Institute at St. Joe’s Hamilton for nephrology research. Joan Krepinsky is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Author Contributions I.K.N, B.G, M.M, D.Z, U.B, and J.C performed experiments; I.K.N and J.C.K conceived experimental design; I.K.N, D.Z and J.T analyzed the data; I.K.N and J.C.K wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Data Sharing/Availability Statement This study does not involve large data sets, new software or custom codes. The original data obtained and presented in this article are available from the corresponding author upon reasonable request. Disclosure The authors have nothing to disclose and declare no competing interests. Funding This work was supported by the Canadian Institutes of Health Research (CIHR) (JCK, PJT-148628). IKN is a recipient of an Ontario Graduate Scholarship award and the 2023 Canadian Graduate Student (CIHR) award (189316). References Kato M, Natarajan R. Epigenetics and epigenomics in diabetic kidney disease and metabolic memory. 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Inhibition of SREBP with Fatostatin Does Not Attenuate Early Diabetic Nephropathy in Male Mice. Endocrinology . 2018;159(3):1479-1495. doi:10.1210/EN.2018-00093 Zhu QJ, Zhu M, Xu XX, Meng XM, Wu YG. Exosomes from high glucose-treated macrophages activate glomerular mesangial cells via TGF-β1/Smad3 pathway in vivo and in vitro. FASEB J . 2019;33(8):9279-9290. doi:10.1096/FJ.201802427RRR Mehta N, Krepinsky JC. The emerging role of activins in renal disease. Curr Opin Nephrol Hypertens . 2020;29(1):136-144. doi:10.1097/MNH.0000000000000560 Soomro A, Khajehei M, Li R, et al. A therapeutic target for CKD: activin A facilitates TGFβ1 profibrotic signaling. Cell Mol Biol Lett . 2023;28(1):10. doi:10.1186/S11658-023-00424-1 Peng F, Zhang B, Wu D, Ingram AJ, Gao B, Krepinsky JC. TGFβ-induced RhoA activation and fibronectin production in mesangial cells require caveolae. Am J Physiol Renal Physiol . 2008;295(1):F153. doi:10.1152/AJPRENAL.00419.2007 Guan TH, Chen G, Gao B, et al. Caveolin-1 deficiency protects against mesangial matrix expansion in a mouse model of type 1 diabetic nephropathy. Diabetologia . 2013;56(9):2068-2077. doi:10.1007/S00125-013-2968-Z Guan T, Gao B, Chen G, et al. Colchicine attenuates renal injury in a model of hypertensive chronic kidney disease. Am J Physiol Renal Physiol . 2013;305(10). doi:10.1152/AJPRENAL.00057.2013 Mehta N, Zhang D, Li R, et al. Caveolin-1 regulation of Sp1 controls production of the antifibrotic protein follistatin in kidney mesangial cells. Cell Commun Signal . 2019;17(1). doi:10.1186/S12964-019-0351-5 Hathaway CK, Gasim AMH, Grant R, et al. Low TGFβ1 expression prevents and high expression exacerbates diabetic nephropathy in mice. Proc Natl Acad Sci U S A . 2015;112(18):5815-5820. doi:10.1073/PNAS.1504777112/-/DCSUPPLEMENTAL Kolset SO, Reinholt FP, Jenssen T. Diabetic Nephropathy and Extracellular Matrix. J Histochem Cytochem . 2012;60(12):976. doi:10.1369/0022155412465073 Kandasamy Y, Smith R, Lumbers ER, Rudd D. Nephrin - a biomarker of early glomerular injury. Biomark Res . 2014;2(1). doi:10.1186/2050-7771-2-21 Welsh GI, Saleem MA. Nephrin - Signature molecule of the glomerular podocyte? J Pathol . 2010;220(3):328-337. doi:10.1002/PATH.2661 Thompson TB, Lerch TF, Cook RW, Woodruff TK, Jardetzky TS. The Structure of the Follistatin:Activin Complex Reveals Antagonism of Both Type I and Type II Receptor Binding. Dev Cell . 2005;9(4):535-543. doi:10.1016/J.DEVCEL.2005.09.008 Bian X, Griffin TP, Zhu X, et al. Senescence marker activin A is increased in human diabetic kidney disease: association with kidney function and potential implications for therapy. BMJ Open Diabetes Res Care . 2019;7(1):720. doi:10.1136/BMJDRC-2019-000720 Sousa ER, Zoni E, Karkampouna S, et al. A Multidisciplinary Review of the Roles of Cripto in the Scientific Literature Through a Bibliometric Analysis of its Biological Roles. Cancers 2020, Vol 12, Page 1480 . 2020;12(6):1480. doi:10.3390/CANCERS12061480 Gray PC, Vale W. Cripto/GRP78 modulation of the TGF-β pathway in development and oncogenesis. FEBS Lett . 2012;586(14):1836. doi:10.1016/J.FEBSLET.2012.01.051 Shukla A, Ho Y, Liu X, Ryscavage A, Glick AB. Cripto-1 alters keratinocyte differentiation via blockade of transforming growth factor-beta1 signaling: role in skin carcinogenesis. Mol Cancer Res . 2008;6(3):509-516. doi:10.1158/1541-7786.MCR-07-0396 Xue YJ, Chen SN, Chen WG, et al. Cripto-1 expression in patients with clear cell renal cell carcinoma is associated with poor disease outcome. J Exp Clin Cancer Res . 2019;38(1). doi:10.1186/S13046-019-1386-6 Zhang Y, Liu C, Wang J, et al. MiR-299-5p regulates apoptosis through autophagy in neurons and ameliorates cognitive capacity in APPswe/PS1dE9 mice. Sci Reports 2016 61 . 2016;6(1):1-14. doi:10.1038/srep24566 Gonzalez CD, Carro Negueruela MP, Santamarina CN, Resnik R, Vaccaro MI. Autophagy Dysregulation in Diabetic Kidney Disease: From Pathophysiology to Pharmacological Interventions. Cells . 2021;10(9). doi:10.3390/CELLS10092497 Duan LJ, Ding M, Hou LJ, Cui YT, Li CJ, Yu DM. Long noncoding RNA TUG1 alleviates extracellular matrix accumulation via mediating microRNA-377 targeting of PPARγ in diabetic nephropathy. Biochem Biophys Res Commun . 2017;484(3):598-604. doi:10.1016/J.BBRC.2017.01.145 Han Q, Zhang Y, Jiao T, et al. Urinary sediment microRNAs can be used as potential noninvasive biomarkers for diagnosis, reflecting the severity and prognosis of diabetic nephropathy. Nutr Diabetes . 2021;11(1). doi:10.1038/S41387-021-00166-Z Ren H, Wu C, Shao Y, Liu S, Zhou Y, Wang Q. Correlation between serum miR-154-5p and urinary albumin excretion rates in patients with type 2 diabetes mellitus: a cross-sectional cohort study. Front Med . 2020;14(5):642-650. doi:10.1007/S11684-019-0719-3/METRICS Xing C, Huo L, Tang H, et al. The predictive value of miR-377 and phospholipase A2 in the early diagnosis of diabetic kidney disease and their relationship with inflammatory factors. Immunobiology . 2024;229(2):152792. doi:10.1016/J.IMBIO.2024.152792 Kulkarni AB, Ward JM, Yaswen L, et al. Transforming growth factor-beta 1 null mice. An animal model for inflammatory disorders. Am J Pathol . 1995;146(1):264. Accessed July 22, 2024. /pmc/articles/PMC1870760/?report=abstract Voelker J, Berg PH, Sheetz M, et al. Anti-TGF-b1 antibody therapy in patients with diabetic nephropathy. J Am Soc Nephrol . 2017;28(3):953-962. doi:10.1681/ASN.2015111230/-/DCSUPPLEMENTAL Additional Declarations There is NO Competing Interest. Supplementary Files OriginalBlotsforreviewers.pdf Original blots for reviewers only SupplementalTablesApril29th2025.pdf Supplemental Tables (Table 1-7) SupplementaryFigure1.pdf Supplemental Figure 1 SupplementaryFigure2.pdf Supplemental Figure 2 SupplementaryFigure3.pdf Supplemental Figure 3 Cite Share Download PDF Status: Published Journal Publication published 11 Dec, 2025 Read the published version in Communications Biology → 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. 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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-5419387","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":456630398,"identity":"62abba5b-6e24-4821-b185-f8c8e7c3eb59","order_by":0,"name":"Joan Krepinsky","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYBACCRCRwMAmR7oWYwg3gVgtQJDYQLQWydmHjz14uIMvfW376cSPP3/Y5DOwH36AV4s0X1q6QeIZttxtZ3I3S/MkpFk28KQZ4NUix8NjJpHYBtRyg3eDNEPCYQOgWwlp4f8G0pJudoN3888fCf+BWtg/4HcYDw8bSEsCUMs2CZ6EA0AtPPhtkexhMzcAajEE+mWbNU9asgEbT04BXi0SZ5ifPfzZdkze7PjZzTd/2NgZ8LMf34BXCxCwAfExVC4xWmqIUDcKRsEoGAUjFgAAvSs/dYu+vfoAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6761-909X","institution":"McMaster University","correspondingAuthor":true,"prefix":"","firstName":"Joan","middleName":"","lastName":"Krepinsky","suffix":""},{"id":456630399,"identity":"14be7850-e1fa-4d66-a350-80d471888689","order_by":1,"name":"Ifeanyi Nmecha","email":"","orcid":"","institution":"McMaster University","correspondingAuthor":false,"prefix":"","firstName":"Ifeanyi","middleName":"","lastName":"Nmecha","suffix":""},{"id":456630400,"identity":"373147b8-5888-49aa-9431-0b42bf67a5a2","order_by":2,"name":"Gaolin Bo","email":"","orcid":"","institution":"McMaster University","correspondingAuthor":false,"prefix":"","firstName":"Gaolin","middleName":"","lastName":"Bo","suffix":""},{"id":456630401,"identity":"f5275beb-3405-4e4c-8b55-0c6e02dabcfe","order_by":3,"name":"Melissa McDonald","email":"","orcid":"","institution":"McMaster University","correspondingAuthor":false,"prefix":"","firstName":"Melissa","middleName":"","lastName":"McDonald","suffix":""},{"id":456630402,"identity":"109ff756-aecd-4ec8-a1d1-3707ec8a0a08","order_by":4,"name":"Dan Zhang","email":"","orcid":"","institution":"McMaster University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Zhang","suffix":""},{"id":456630403,"identity":"81ca2c15-df13-4ef3-bdc8-fe4afde0a7b9","order_by":5,"name":"Jason Choi","email":"","orcid":"","institution":"McMaster University","correspondingAuthor":false,"prefix":"","firstName":"Jason","middleName":"","lastName":"Choi","suffix":""},{"id":456630404,"identity":"75c0a0c6-c467-4a7f-be9e-8128249656bc","order_by":6,"name":"Jackie Trink","email":"","orcid":"","institution":"McMaster University","correspondingAuthor":false,"prefix":"","firstName":"Jackie","middleName":"","lastName":"Trink","suffix":""},{"id":456630405,"identity":"9da7dd4c-0e9e-4de8-b38b-48a2504659ca","order_by":7,"name":"Urooj Bajwa","email":"","orcid":"","institution":"McMaster University","correspondingAuthor":false,"prefix":"","firstName":"Urooj","middleName":"","lastName":"Bajwa","suffix":""}],"badges":[],"createdAt":"2024-11-09 02:45:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5419387/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5419387/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-09271-6","type":"published","date":"2025-12-11T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82979673,"identity":"77e2e4ee-213c-40e1-b64b-83e2944cacec","added_by":"auto","created_at":"2025-05-18 10:02:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":663913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-299a-5p expression is increased by HG in MC and in mouse and human DKD. A)\u003c/strong\u003e miR-299a-5p expression was increased, as assessed by qPCR, after HG but not mannitol treatment of MC for 72h. \u003cstrong\u003eB)\u003c/strong\u003e miR-299a-5p MRE-Luc activity was assessed in MCs after HG (72h), showing increased miR-299a-5p binding. \u003cstrong\u003eC)\u003c/strong\u003e miR-299a-5p was increased in kidney cortex of streptozotocin-induced type 1 diabetic CD1 mice, as assessed by qPCR. ISH showed an increase in miR-299a-5p expression in: \u003cstrong\u003eD-E)\u003c/strong\u003estreptozotocin-induced type-1 diabetic CD1 mice, with blue staining indicating a positive signal; \u003cstrong\u003eF-G)\u003c/strong\u003e in 40-week type 1 diabetic Akita mice; and in \u003cstrong\u003eH-I)\u003c/strong\u003ekidney biopsies of type 2 diabetic patients (*p\u0026lt;0.05, ** p\u0026lt;0.01, ***p\u0026lt;0.001, **** p\u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/efee6d0018e0a8e142b42bc7.jpg"},{"id":82979672,"identity":"3fd48716-b79c-44cd-a8d3-1bee58af1309","added_by":"auto","created_at":"2025-05-18 10:02:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":802654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-299a-5p promotes HG-induced profibrotic responses in MC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e MC were transfected with miR-299a-5p inhibitor or its control. Effective transfection was confirmed with mCherry immunofluorescence. \u003cstrong\u003eB) \u003c/strong\u003emiR-299a-5p inhibition decreased collagen 1α1 promoter activation by HG (72h). \u003cstrong\u003eC-D) \u003c/strong\u003emiR-299a-5p inhibition decreased basal and HG-induced matrix protein and cytokine (CTGF) production. \u003cstrong\u003eE) \u003c/strong\u003emiR-299a-5p was overexpressed in MC.\u0026nbsp; Effective transfection was confirmed with GFP immunofluorescence. \u003cstrong\u003eF) \u003c/strong\u003emiR-299a-5p overexpression increased collagen 1α1 promoter activation, with no additive effect by HG.\u003cstrong\u003e G-H) \u003c/strong\u003emiR-299a-5p overexpression increased basal matrix protein and CTGF expression to levels seen with HG. \u003cstrong\u003eI-J) \u003c/strong\u003emiR-299a-5p inhibition prevented HG-induced Smad3 activation, assessed by its C-terminus phosphorylation. \u003cstrong\u003eK) \u003c/strong\u003emiR-299a-5p inhibition prevented HG-induced Smad3 transcriptional activation, assessed using the Smad3-responsive CAGA\u003csub\u003e12\u003c/sub\u003e luciferase. \u003cstrong\u003eL-M) \u003c/strong\u003emiR-299a-5p overexpression induced Smad3 activation, assessed by phosphorylation, to levels seen with HG, as well as\u003cstrong\u003e N) \u003c/strong\u003eSmad3\u003cstrong\u003e \u003c/strong\u003etranscriptional activation (*p\u0026lt;0.05, ** p\u0026lt;0.01, ***p\u0026lt;0.001, **** p\u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/fd9a6f3968fcafb68f2977d8.jpg"},{"id":82980064,"identity":"cbb50315-bcfc-4729-8d4e-acf67c5ff827","added_by":"auto","created_at":"2025-05-18 10:10:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":870447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-299a-5p inhibits expression of anti-fibrotic proteins cripto-1 and FST.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eThe miRNA regulatory element (MRE) for miR-299a-5p is conserved in the 3′UTR of mouse and human cripto-1.\u003cstrong\u003e B) \u003c/strong\u003eCripto-1 3’UTR-luciferase activity was attenuated by HG (72h), and \u003cstrong\u003eC) \u003c/strong\u003ethis was prevented by miR-299a-5p inhibition. \u003cstrong\u003eD) \u003c/strong\u003eCripto-1 mRNA expression was reduced by HG (72h). \u003cstrong\u003eE-F) \u003c/strong\u003eCripto-1 and FST protein expression were significantly reduced by HG (72h) in MC, and \u003cstrong\u003eG-H)\u003c/strong\u003e in type 1 diabetic Akita mice.\u003cstrong\u003e \u003c/strong\u003eIHC showed that expression of cripto-1 is reduced in type 1 diabetic Akita mice (\u003cstrong\u003eI-J\u003c/strong\u003e) and in CD1 streptozotocin-induced CD1 diabetic mice (\u003cstrong\u003eK-L\u003c/strong\u003e). (\u003cstrong\u003eM-N\u003c/strong\u003e) IHC for FST confirms its reduction in streptozotocin-induced diabetes. \u003cstrong\u003eO-P) \u003c/strong\u003emiR-299a-5p inhibition prevented HG (72h)-induced reduction of cripto-1 and FST protein expression in MC. \u003cstrong\u003eQ-R) \u003c/strong\u003emiR-299a-5p overexpression reduced cripto-1 and FST protein expression (*p \u0026lt;0.05, **p \u0026lt;0.01, ***p \u0026lt;0.001).\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/1afb07a66ccec68f18b9d807.jpg"},{"id":82980128,"identity":"90d7c1b4-28a9-40e7-9e75-b0450168d108","added_by":"auto","created_at":"2025-05-18 10:18:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":589076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCripto-1 inhibits HG-induced profibrotic responses in MC.\u003c/strong\u003e \u003cstrong\u003eA-B)\u003c/strong\u003e Cripto-1 (1µg/ml) decreased HG (72h)-induced Smad3 phosphorylation in MC, as well as \u003cstrong\u003e(C) \u003c/strong\u003eits transcriptional activation assessed using CAGA\u003csub\u003e12\u003c/sub\u003e-luciferase. \u003cstrong\u003eD) \u003c/strong\u003eCollagen 1α1 promoter activation by HG (72h) was inhibited by cripto-1. \u003cstrong\u003eE-F) \u003c/strong\u003eHG (72h)-induced\u003cstrong\u003e \u003c/strong\u003ematrix protein\u003cstrong\u003e \u003c/strong\u003eand CTGF upregulation were inhibited by cripto-1. \u003cstrong\u003eG) \u003c/strong\u003eHG-induced Smad3 transcriptional activation was inhibited by cripto-1 (1µg/ml) and FST (500 ng/ml), with a synergistic effect of both together. \u003cstrong\u003eH)\u003c/strong\u003e Overexpression of miR-299a-5p increased Smad3 transcriptional activity. This was inhibited by cripto-1 and FST, with a synergistic effect seen with both. \u003cstrong\u003eI) \u003c/strong\u003eMiR-299a-5p overexpression increased collagen 1α1 promoter activation, which was inhibited by cripto-1 or FST alone, and a greater effect seen when used together. \u003cstrong\u003eJ) \u003c/strong\u003eActA or TGFβ1 inhibition using specific neutralizing antibodies inhibited Smad3 transcriptional activation induced by miR-299a-5p overexpression, similarly to FST. However, actB neutralization had no effect (*p \u0026lt;0.05, **p \u0026lt;0.01, ***p \u0026lt;0.001, ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/52ca8ecfe3125efcebd4e60b.jpg"},{"id":82980068,"identity":"6250c87a-bccf-4f75-98cd-5c792e00a221","added_by":"auto","created_at":"2025-05-18 10:10:42","extension":"pdf","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1119078,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/54fd1fd26848739b9ad68b60.pdf"},{"id":82979687,"identity":"36dc412d-18bb-4112-9023-76d4717f46e9","added_by":"auto","created_at":"2025-05-18 10:02:42","extension":"pdf","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":515312,"visible":true,"origin":"","legend":" Cripto-1 (1\u0026micro;g/ml) decreased HG (72h)-induced Smad3 phosphorylation in MC, as well as its transcriptional activation assessed using CAGA-luciferase. Collagen 1α1 promoter activation by HG (72h) was inhibited by cripto-1. HG (72h)-induced matrix protein and CTGF upregulation were inhibited by cripto-1. HG-induced Smad3 transcriptional activation was inhibited by cripto-1 (1\u0026micro;g/ml) and FST (500 ng/ml), with a synergistic effect of both together. Overexpression of miR-299a-5p increased Smad3 transcriptional activity. This was inhibited by cripto-1 and FST, with a synergistic effect seen with both. MiR-299a-5p overexpression increased collagen 1α1 promoter activation, which was inhibited by cripto-1 or FST alone, and a greater effect seen when used together. ActA or TGFβ1 inhibition using specific neutralizing antibodies inhibited Smad3 transcriptional activation induced by miR-299a-5p overexpression, similarly to FST. However, actB neutralization had no effect (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).","description":"","filename":"Figure4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/dd5e4e530cbe0fab30741c3e.pdf"},{"id":82979678,"identity":"6198ce9f-1464-41ba-9c69-ba1f87739e3b","added_by":"auto","created_at":"2025-05-18 10:02:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":809475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-299a-5p inhibition improves DKD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eExperimental timeline for miR-299a-5p inhibition studies. \u003cstrong\u003eB-C) \u003c/strong\u003eMiR-299a-5p inhibition restored the reduced cripto-1 and FST protein expression seen in DKD. \u003cstrong\u003eD-E) \u003c/strong\u003eFST reduction in DKD, seen by IHC, was restored by miR-299a-5p inhibition. \u003cstrong\u003eF)\u003c/strong\u003e Glomerular filtration rate (GFR) was increased in DKD, with miR-299a-5p inhibition having no effect. \u003cstrong\u003eG) \u003c/strong\u003eKidney hypertrophy was increased in DKD. This was unaffected by miR-299a-5p inhibition. \u003cstrong\u003eH) \u003c/strong\u003eThe increased albuminuria seen in DKD was attenuated by anti-miR-299a-5p inhibition. \u003cstrong\u003eI) \u003c/strong\u003eGlomerular volume was increased in DKD, and this was reduced by miR-299a-5p inhibition.\u003cstrong\u003e J-K) \u003c/strong\u003eThe decreased expression of the podocyte marker nephrin in DKD was restored by miR-299a-5p inhibition (*p \u0026lt;0.05, **p \u0026lt;0.01, ***p \u0026lt;0.001, ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/425982b5c5902adfb1ad5d3b.jpg"},{"id":82980067,"identity":"de2019d3-001e-425b-b69e-64e56f6ec017","added_by":"auto","created_at":"2025-05-18 10:10:42","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":871796,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-299a-5p inhibition reduces fibrosis in DKD. \u003c/strong\u003e(\u003cstrong\u003eA-E\u003c/strong\u003e) Increased fibrosis was seen in DKD, assessed by PSR (\u003cstrong\u003eA-B\u003c/strong\u003e) and IHC for fibronectin, collagen IVα1 and CTGF (\u003cstrong\u003eC-E\u003c/strong\u003e). All were reduced by miR-299a-5p inhibition. (\u003cstrong\u003eF-I\u003c/strong\u003e) Immunoblotting for fibronectin, collagen 1α1 and CTGF showed similar results (*p \u0026lt;0.05, **p \u0026lt;0.01, ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/b4e3a0e34379ac9c733221e9.jpg"},{"id":82979691,"identity":"3aee1db8-8049-4c3c-927a-a9d9c0fd859b","added_by":"auto","created_at":"2025-05-18 10:02:42","extension":"pdf","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":919061,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/e1885bf1b86419d34938296b.pdf"},{"id":82979684,"identity":"f67f98af-b1fc-475b-936f-eb115ce0ca45","added_by":"auto","created_at":"2025-05-18 10:02:42","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":579633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-299a-5p inhibition reduces actA, TGFβ1 and active Smad3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B) \u003c/strong\u003eElevated Smad3 phosphorylation in DKD was reduced by miR-299a-5p inhibition (*p\u0026lt;0.05, **p\u0026lt;0.01). ActA protein expression in the kidney cortex and excretion in the urine was assessed by \u003cstrong\u003e(C-D)\u003c/strong\u003e IHC and \u003cstrong\u003e(E) \u003c/strong\u003eELISA. Both were increased in DKD and reduced by miR-299a-5p inhibition (*p\u0026lt;0.05, **p\u0026lt;0.01, ****p\u0026lt;0.0001). \u003cstrong\u003e(F) \u003c/strong\u003eUrinary TGFβ1 was increased in diabetic mice, and this was reduced by miR-299a-5p inhibition (*p\u0026lt;0.05, **p\u0026lt;0.01). \u003cstrong\u003e(G)\u003c/strong\u003emiR-299a-5p expression was increased in the serum of diabetic Akita mice (*p\u0026lt;0.05). \u003cstrong\u003e(H)\u003c/strong\u003e Schematic diagram of study findings, showing the contribution of actA and TGFβ1 to DKD, and the role of FST and cripto-1 in their inhibition and improvement of DKD as shown by these studies.\u003c/p\u003e","description":"","filename":"fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/6c760d0b07c7ce524b11d117.jpg"},{"id":99588673,"identity":"c0c7f18e-5670-4548-919c-467e82152fed","added_by":"auto","created_at":"2026-01-06 08:23:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6193126,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/add9f8b2-864e-477f-a7de-66491af305be.pdf"},{"id":82979677,"identity":"dd8a6de1-3c7a-4714-9767-364eb738b62b","added_by":"auto","created_at":"2025-05-18 10:02:42","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1143736,"visible":true,"origin":"","legend":"Original blots for reviewers only","description":"","filename":"OriginalBlotsforreviewers.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/e35fa96970be24521185db4c.pdf"},{"id":82980065,"identity":"8843724f-bf92-429c-8d2c-57db3b632430","added_by":"auto","created_at":"2025-05-18 10:10:42","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":157596,"visible":true,"origin":"","legend":"Supplemental Tables (Table 1-7)","description":"","filename":"SupplementalTablesApril29th2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/e9baffde7e8f652fc94d3085.pdf"},{"id":82979681,"identity":"a5f15532-67e8-448a-a4d0-739d340fa0fd","added_by":"auto","created_at":"2025-05-18 10:02:42","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":238761,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure 1\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/22bb5696642eeeb6ddf94a56.pdf"},{"id":82979682,"identity":"cf8df0e3-4842-44c1-901a-42bafe759047","added_by":"auto","created_at":"2025-05-18 10:02:42","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":426973,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure 2\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/bb9ab28141295cbdb3bc4939.pdf"},{"id":82980069,"identity":"2386fc3b-5900-4e40-8e17-466281f141a9","added_by":"auto","created_at":"2025-05-18 10:10:42","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":285336,"visible":true,"origin":"","legend":"Supplemental Figure 3","description":"","filename":"SupplementaryFigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5419387/v1/19f896e738ddb9a720cfa1be.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"miR-299a-5p is a novel mediator of fibrosis in diabetic kidney disease through its regulation of antifibrotic proteins follistatin and cripto-1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic kidney disease (DKD) is a major complication of diabetes mellitus, developing in up to 40% of patients. It is the leading cause of end-stage kidney disease, associated with reduced quality of life and increased mortality.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The current standard of care for DKD includes control of blood glucose and blood pressure, and use of inhibitors of the renin-angiotensin-aldosterone system, and for type 2 diabetics sodium glucose co-transporter-2 inhibitors. However, current therapies, even in combination, are unable to halt DKD progression. The identification of novel therapeutic targets aimed at preventing disease progression is thus a major clinical need.\u003c/p\u003e \u003cp\u003eInitial pathologic changes of the diabetic kidney occur in the glomerulus, characterized by basement membrane thickening and mesangial expansion from accumulation of extracellular matrix proteins. The profibrotic cytokine transforming growth factor β1 (TGFβ1) is well known to be a major pathologic mediator of these changes.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e However, the pleiotropic roles of TGFβ1 make its direct therapeutic targeting challenging. Previously, our lab identified that TGFβ1 inhibits the production of an antifibrotic protein, follistatin (FST) by mesangial cells (MC) through upregulation of miR-299a-5p.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFST is a potent inhibitor of activins, members of the TGFβ superfamily, in particular activins A and B. The importance of both activins to fibrosis in DKD has been shown.\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e We had also shown that FST attenuated high glucose (HG)-induced matrix production by MC and reduced fibrosis in a model of DKD.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e However, effects are attenuated at higher doses, indicating that administration may have a narrow therapeutic window. Novel strategies to increase endogenous FST may thus be more effective and better clinically tolerated.\u003c/p\u003e \u003cp\u003emiR-299a-5p is a member of the miR-154 family, the second largest miRNA cluster in the human genome. Containing over 40 members, it is highly conserved between species.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Members of this family are implicated in fibrosis in other organs. In idiopathic pulmonary fibrosis (IPF), several members are increased including miR-299a-5p and miR-154, with the latter shown to heighten IPF fibroblast response to TGFβ1.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e miR-154 expression alone also increased cardiac fibroblast collagen production,\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and its LNA inhibition protected against cardiac fibrosis and dysfunction in a pressure overload model.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Increased miR-299a-5p was also found in fibrotic liver from patients with primary biliary cirrhosis.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Our previous data show its increase in a hypertensive model of chronic kidney disease,\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e but whether miR-299a-5p contributes to the progression of fibrosis in DKD is unknown.\u003c/p\u003e \u003cp\u003eHere, we show that miR-299a-5p expression is significantly upregulated in glomeruli and tubules in both animal models of type 1 diabetes and in kidneys of type 2 DKD patients. HG increases the expression of miR-299a-5p by MC to inhibit FST production. Bioinformatics screening identified cripto-1 as an additional miR-299a-5p target. Unlike FST which has no neutralizing activity against TGFβ1,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e cripto-1 inhibits the actions of both activin A (actA) and TGFβ1.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e We hypothesize that inhibition of miR-299a-5p \u003cem\u003ein vivo\u003c/em\u003e with anti-miR administration ameliorated DKD, in association with elevated expression of FST and cripto-1. Our study highlights a potential therapeutic role for miR-299a-5p inhibition in restoring endogenous antifibrotic protein expression to slow the progression of DKD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell Culture\u003c/h2\u003e \u003cp\u003ePrimary MC were grown from glomeruli of male C57BL/6J mice isolated using Dynabeads (Invitrogen, Carlsbad, CA) as described previously.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e MC were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) with 20% fetal bovine serum (FBS), streptomycin (100\u0026micro;g/ml) and penicillin (100\u0026micro;g/ml) at 37˚C in 95% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were serum deprived in DMEM with 0.5% FBS for 24h following transfection and prior to treatment with high glucose (30mM) for 72 hrs, drugs or recombinant proteins (\u003cb\u003eSupplementary Table\u0026nbsp;1).\u003c/b\u003e Passage 9\u0026ndash;16 MCs were used for experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.2 mRNA and miRNA Extraction and qPCR\u003c/h3\u003e\n\u003cp\u003eRNA was extracted using TRIzol (Life Technologies, Carlsbad, CA) and 1\u0026micro;g was reverse transcribed using qScript cDNA SuperMix Reagent (Quanta Biosciences). miRNA-enriched cDNA was generated using the qScript microRNA Quantification System (Quanta Biosciences). Real-time polymerase chain reaction (PCR) was performed using a SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA) on the ViiA 7 Sequence Detector (Life Technologies). Amplification of cripto-1 or miRNA expression, relative to 18S or U6 respectively, was measured using the ΔΔCT method. Primers are listed in \u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e. miRNeasy serum advanced kit (Qiagen) was used to extract RNA from the serum of diabetic mice after which miRNA-enriched cDNA was generated as above.\u003c/p\u003e\n\u003ch3\u003e2.3 miRNA in-situ Hybridization (ISH)\u003c/h3\u003e\n\u003cp\u003eFormalin-fixed paraffin-embedded kidney sections (4\u0026micro;m) were deparaffinized, dehydrated, and treated with proteinase K. After incubation in hybridization buffer, sections were incubated with DIG-labeled miRCURY LNA anti-miR detection probes (\u003cb\u003eSupplementary Table\u0026nbsp;3\u003c/b\u003e) targeting miR-299a or U6 (18h). After further washes\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, sections were blocked in 1\u0026times;Casein Solution (Vector labs) and incubated with anti-digoxigenin-AP Fab fragment. Chromogenic reaction was carried out using NBT/BCIP (Vector Labs). Slides were then mounted with Vectamount (Vector labs). Images were taken at 40x using a BX41 Olympus microscope and quantified using ImageJ.\u003c/p\u003e \u003cp\u003eFor human studies, kidney biopsy samples with a diagnosis of DKD were obtained. Normal kidney tissues surrounding resected renal cancers were used as controls (Research Ethics Board approval number 2010\u0026thinsp;\u0026minus;\u0026thinsp;159).\u003c/p\u003e\n\u003ch3\u003e2.4 Transient Transfections\u003c/h3\u003e\n\u003cp\u003eFor luciferase experiments, MC were plated in triplicate at 60\u0026ndash;70% confluence and transfected with 0.5\u0026micro;g of luciferase construct (pGL3-CAGA12-luc or pGL3-COL1α1-luc) with 0.05\u0026micro;g pCMV β-galactosidase (Clontech) using Effectene (Qiagen). At harvest, MC were lysed with 1X Reporter Lysis Buffer (Promega, Madison, WI) and frozen at -80\u0026ordm;C overnight. Luciferase and β-gal activities were measured on clarified lysate using specific kits (Promega) with a Spectramax Plus 384 plate reader set to luminescence and 420nm, respectively. β-gal activity was used to adjust for transfection efficiency.\u003c/p\u003e \u003cp\u003emiR299a-5p regulatory element (MRE) luciferase was generated in order to measure miR299a-5p activity as previously described,\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and MCs were transfected as above.\u003c/p\u003e \u003cp\u003eFor transient expression, MC resuspended in electroporation buffer containing 10\u0026micro;g expression plasmid were electroporated using a single square pulse set at 200V for 35ms (ECM830, Harvard Bioscience). After 18h, media was exchanged. To assess transfection efficiency of miR plasmids (\u003cb\u003eSupplementary Table\u0026nbsp;4\u003c/b\u003e), mCherry (ex 550nm/em 620nm) and GFP (ex 490nm/em 525nm) immunofluorescence were confirmed by imaging (EVOS FL Cell Imaging System, Thermo Fisher Scientific). Luciferase constructs and pCMV β-galactosidase were transfected 4h later in some experiments.\u003c/p\u003e\n\u003ch3\u003e2.5 Protein Extraction and Immunoblotting\u003c/h3\u003e\n\u003cp\u003eMC were washed 3x with cold phosphate buffered saline and lysed in buffer with protease inhibitors (10\u0026micro;g/ml PMSF, 2\u0026micro;g/ml leupeptin, 2\u0026micro;g/ml aprotinin). After clarification, proteins were separated by SDS-PAGE and analyzed by immunoblotting. Antibodies used are listed in \u003cb\u003eSupplementary Table\u0026nbsp;5\u003c/b\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Animal Studies\u003c/h2\u003e \u003cp\u003e Animal studies were approved by the McMaster University Animal Research Ethics Board and carried out in accordance with the Canadian Council on Animal Care guidelines and ARRIVE guidelines and checklist. Animals were housed under standard conditions with free access to chow and water. Two animal models were assessed by IHC and ISH: (1) Male type 1 diabetic Akita (C57BL/6-Ins2\u003csup\u003eAkita\u003c/sup\u003e/J) or wild-type mice (C57BL/6J) (Jackson Laboratory, Bar Harbor, ME, USA) at age 40 weeks (ethics #18-07-30). (2) Uninephrectomized male CD1 mice aged 9 weeks were injected with 200\u0026micro;g streptozotocin and euthanized after 12 weeks of diabetes as described previously\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e (#14-11-48).\u003c/p\u003e \u003cp\u003eC57BL/6\u003csup\u003eHH\u003c/sup\u003e Akita mice expressing hypermorphic (gain in function) alleles for TGFβ1 (denoted HH-A), resulting in ~\u0026thinsp;300% increase in transcript expression, were described previously.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Controls were non-diabetic HH mice. Male mice were enrolled at age 12 weeks, randomly assigned and treated with 2mg/kg of miR-LNA inhibitor (LNAi) or negative control \u0026ldquo;B\u0026rdquo; (LNAc, Qiagen) intraperitoneally once weekly for 12 weeks. There were four groups: HH-LNAc (n\u0026thinsp;=\u0026thinsp;8), HH-LNAi (n\u0026thinsp;=\u0026thinsp;8), HH-A-LNAc (n\u0026thinsp;=\u0026thinsp;12) and HH-A-LNAi (n\u0026thinsp;=\u0026thinsp;12). Mice with glucometer measure of tail vein blood glucose\u0026thinsp;\u0026gt;\u0026thinsp;17mM were enrolled as diabetics. Those that developed ketonuria (dipstick, Bayer Multistix) or progressive weight loss were administered \u0026frac14; insulin pellet (LinShin Canada) to maintain body weight while also maintaining hyperglycemia. Blood pressure was measured in the morning, at 4, 8, and 12 weeks using tail-cuff plethysmography (Coda non-invasive blood pressure monitoring system, Kent Scientific).\u003c/p\u003e \u003cp\u003eAfter 12 weeks, urine was collected (6h, Nalgene metabolic cage 650\u0026thinsp;\u0026minus;\u0026thinsp;0210). Urine albumin and creatinine were measured using Albuwell M (Exocell) and Mouse Creatinine Assay kits (Crystal Chem) respectively to determine the albumin to creatinine ratio (ACR). Glomerular filtration rate (GFR) was measured using fluorescein isothiocyanate (FITC)-labeled sinistrin (Fresenius Kabi Linz) as previously described.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Mice were then anesthetized, perfused with saline and kidneys harvested for analysis.\u003c/p\u003e \u003cp\u003eELISA kits (R\u0026amp;D Systems) were used to measure total TGFβ1 or actA in urine, normalized to urine creatinine. TGFβ1 samples underwent acid activation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.7 Imaging\u003c/h3\u003e\n\u003cp\u003eFormalin-fixed paraffin-embedded kidney sections (4\u0026micro;m) were deparaffinized and stained using Trichrome (Sigma), PAS or Picrosirius red (PSR) (Polysciences Inc.) or using antibodies (\u003cb\u003eSupplementary Table\u0026nbsp;5\u003c/b\u003e). Images were captured using the Olympus BX41 microscope at 20x (or Olympus IX81 fluorescence microscope for PSR) and quantified as percentage of positive area using ImageJ. Glomerular hypertrophy was assessed by glomerular cross-sectional area measures on Periodic acid-Schiff-stained sections as described previously.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFor immunofluorescence, 10\u0026micro;m OCT-embedded frozen kidney sections were fixed in 4% paraformaldehyde prior to blocking and nephrin antibody incubation. Nuclei were stained with DAPI. Images were taken at 40x for quantification.\u003c/p\u003e\n\u003ch3\u003e2.8 Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eGraphPad Prism 10 was used to analyze differences between groups using a one-way ANOVA with Tukey\u0026rsquo;s post-hoc testing, or two-tail unpaired t-test. Repeated measures two-way ANOVA was used to analyze blood pressure. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, with data presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003e3.1 miR-299a-5p expression is increased by HG in MC and in mouse and human DKD.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe first investigated the effects of HG on miR-299a-5p expression. HG, but not the osmotic control mannitol, increased the expression of miR-299a-5p in MC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e).\u003c/b\u003e We next used a construct in which the miR-299a-5p regulatory element (MRE) is placed downstream of the luciferase gene.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb shows decreased luciferase activity in HG, indicating increased miR-299a-5p binding. We next assessed miR-299a-5p expression \u003cem\u003ein vivo.\u003c/em\u003e By PCR, miR-299a-5p was increased in kidney cortex of CD1 streptozotocin-induced diabetic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), confirmed by ISH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed,e). Increased expression was also seen in type 1 diabetic Akita mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef,g), and in human DKD kidney biopsies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh,i). Expression was observed in both glomeruli and tubules, suggesting a role for this miR across kidney cell types.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003e3.2 miR-299a-5p promotes HG-induced profibrotic responses in MC.\u003c/em\u003e \u003c/p\u003e \u003cp\u003ePreviously, we showed that FST is a target of miR-299a-5p,\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and that FST regulates both basal and HG-induced matrix production through its potent inhibition of actA.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e We thus assessed whether miR-299a-5p regulates HG-induced fibrotic responses. We used plasmids to either inhibit or overexpress this miR,\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e with transfection efficiency measured by mCherry or GFP immunofluorescence respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,e). MiR-299a-5p inhibition significantly attenuated HG (72hr) profibrotic effects, measured as collagen Iα1 luciferase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and matrix protein (fibronectin, collagen Iα1) and profibrotic cytokine (connective tissue growth factor (CTGF)) expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,d). Conversely, miR-299a-5p overexpression augmented basal collagen Iα1 promoter activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) and profibrotic protein synthesis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg,h) to levels seen with HG.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProfibrotic responses to HG in MC are known to require Smad3,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e which mediates both actA and TGFβ1 signaling.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e We thus assessed whether miR-299a-5p mediates HG (72hr)-induced Smad3 activation. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei,j shows that miR-299a-5p inhibition prevented HG-induced Smad3 C-terminal phosphorylation, required for its activation. Smad3 transcriptional activity, assessed using its reporter CAGA\u003csub\u003e12\u003c/sub\u003e-luciferase, was also inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek). Conversely, miR-299a-5p overexpression alone increased Smad3 phosphorylation and transcriptional activity to levels seen with HG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el-n). Interestingly, as for matrix protein synthesis, this was not augmented by HG, suggesting a major role for miR-299a-5p in Smad3 activation by HG.\u003c/p\u003e \u003cp\u003e \u003cem\u003e3.3 miR-299a-5p inhibits expression of anti-fibrotic proteins cripto-1 and FST.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe next assessed whether, in addition to FST, other potentially anti-fibrotic genes were regulated by miR-299a-5p. Bioinformatics screening of potential targets using TargetScan8.0, miRDB, miRWalk and Fireplex discovery engine revealed cripto-1, a known antagonist of both TGFβ1 and actA, as a target. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, the miR-299a-5p response element is conserved in the mouse and human cripto-1 3\u0026rsquo;UTR.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e To confirm regulation by miR-299a-5p, we tested its effects on a cripto-1 3\u0026rsquo;UTR-luciferase reporter. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb shows that HG (72hr) reduced cripto-1 3\u0026rsquo;UTR luciferase activity. This was reversed by miR-299a-5p inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f, both cripto-1 mRNA and protein expression were decreased by HG, with reduced FST expression also confirmed. Decreased cripto-1 and FST expression were also observed in kidney cortex immunoblots of type 1 DKD (Akita) mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg,h), with cripto-1 reduction confirmed by immunohistochemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei,j). Reduction in cripto-1 and FST were confirmed in a second DKD mouse model (streptozotocin-treated, uninephrectomized CD1 mice) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek-n),\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and cripto-1 reduction also confirmed in human DKD biopsies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eo, p).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next sought to confirm that the regulation of cripto-1 by HG is mediated by miR-299a-5p. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eq,r, miR-299a-5p inhibition attenuated HG-induced repression of cripto-1. Conversely, miR-299a-5p overexpression reduced basal expression of cripto-1 similarly to HG-mediated reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003es,t). FST was similarly affected by miR inhibition and overexpression. Together, these results indicate that HG-induced miR-299a-5p expression reduces cripto-1 and FST synthesis.\u003c/p\u003e \u003cp\u003e \u003cem\u003e3.4 Cripto-1 inhibits HG-induced profibrotic responses in MC.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCripto-1 is known to antagonize both TGFβ1 and actA signaling.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e We first confirmed this in MC. \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e shows that Smad3 transcriptional activation by TGFβ1 or actA was inhibited by cripto-1, although ActA inhibition was greater. We next determined whether cripto-1 could prevent HG-induced Smad3 activation. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b shows that the increased Smad3 C-terminal phosphorylation induced by HG (72hr) was inhibited by cripto-1, as was Smad3 transcriptional activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f show that cripto-1 also inhibited HG-induced collagen Iα1 luciferase activation and the increase in expression of fibrotic proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince both FST and cripto-1 are decreased by miR-299a-5p in HG and both inhibit actA, but only cripto-1 inhibits TGFβ1, we tested if their combination would have an additive effect. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg shows that each individually suppressed HG (72hr)-induced Smad3 transcriptional activity, with a small additive effect. In response to miR-299a-5p overexpression, cripto-1 and FST alone reduced Smad3 reporter activity, with this effect augmented by their combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Similar augmented effects were seen for collagen Iα1 luciferase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003eFinally, using neutralizing antibodies we explored effects of individual inhibition of TGFβ1 or the two activins primarily neutralized by FST, actA and activin B (actB). Interestingly, neutralization of either actA or TGFβ1, but not actB, inhibited Smad3 transcriptional activation by miR-299a-5p overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). This suggests that cripto-1 and FST prevent Smad3-dependent fibrotic signaling through their inhibition of actA and TGFβ1 with no significant contribution from actB. Together, these data also suggest that direct inhibition of miR-299a-5p may have greater therapeutic efficacy than use of FST or cripto-1 alone.\u003c/p\u003e \u003cp\u003e \u003cem\u003e3.5 miR-299a-5p is increased in caveolin-1 knockout MC.\u003c/em\u003e \u003c/p\u003e \u003cp\u003ePreviously, we showed a reduction in both basal and HG-induced matrix protein expression in MC that lack caveolin-1 and thus caveolae, membrane microdomains which regulate profibrotic signaling.\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Furthermore, FST was significantly upregulated in MC derived from caveolin-1 knockout (KO) mice,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e mediated by suppressed miR-299a-5p expression and activity in these cells.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e This translated to protection from DKD in caveolin-1 KO mice.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e We thus hypothesized that cripto-1 would also be increased in KO MC. To do this, primary mouse MCs were isolated from caveolin-1 wild-type (WT) and caveolin-1 KO B6129SF1/J mice as described previously.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Indeed, \u003cb\u003eSupplementary Fig.\u0026nbsp;2a, b\u003c/b\u003e show that cripto-1 expression was higher in caveolin-1 KO compared to WT cells, and this expression was reduced by miR-299a-5p overexpression (\u003cb\u003eSupplementary Fig.\u0026nbsp;2c, d\u003c/b\u003e). Furthermore, miR-299a-5p inhibition in WT cells increased cripto-1 and FST expression (\u003cb\u003eSupplementary Fig.\u0026nbsp;2e, f\u003c/b\u003e). Interestingly, bioinformatics screening showed that another target of miR-299a-5p is the transcription factor SP1, which we previously showed regulates FST promoter activation in MC.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e \u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e shows that miR-299a-5p overexpression significantly decreased SP1 expression.\u003c/p\u003e \u003cp\u003e \u003cem\u003e3.6 miR-299a-5p inhibition improves DKD.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe next assessed the therapeutic potential of miR-299a-5p inhibition in DKD. We used Akita mice with genetically increased TGFβ1 expression which was previously shown to exacerbate DKD.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e MiR-299a-5p was competitively inhibited from binding to its targets using a Locked Nucleic Acid (LNA) anti-miR. Mice were treated for 12 weeks (12\u0026ndash;24 weeks of age, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eWe first confirmed that miR-299a-5p inhibition could rescue reduced cripto-1 and FST expression in diabetic kidneys. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c shows that both proteins were significantly increased by miR-299a-5p inhibition, confirmed for FST by IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed,e). \u003cb\u003eSupplementary table 6\u003c/b\u003e shows that endpoint fasting blood glucose and weight were not affected by miR-299a-5p inhibition. Blood pressure was measured at enrollment and every 4 weeks (\u003cb\u003eSupplementary Table\u0026nbsp;7\u003c/b\u003e). By study endpoint both systolic and diastolic pressures were increased in diabetic mice, with reduction by miR-299a-5p inhibition. No effect on blood pressure was seen in non-diabetic mice. Diabetic mice showed the expected hyperfiltration (increased GFR) and kidney hypertrophy characteristic of early DKD. These were unaffected by miR-299a-5p inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef,g). However, miR inhibition significantly decreased albuminuria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh) and glomerular hypertrophy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei), two key features of early DKD\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. MiR inhibition also significantly rescued loss of nephrin, a marker of podocyte injury which has been correlated with albuminuria in diabetic mice\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej, k).\u003c/p\u003e \u003cp\u003eWe next assessed fibrosis. Picrosirius red (PSR) showed the expected increase in collagen I/III expression in diabetic kidneys, which was reduced by miR inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b). Similarly, IHC showed that fibronectin, collagen Iα1 and CTGF expression were also decreased by miR-299a-5p inhibition in diabetic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,c-e). This was confirmed by immunoblotting of kidney cortex (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-i). MiR-299a-5p inhibition also reduced activation of Smad3, assessed by its phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,b). Furthermore, the expected increase in actA expression in diabetic kidneys\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e was markedly reduced in both glomeruli and tubules by miR-299a-5p inhibition, as shown by IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec,d). Urinary actA, significantly elevated in diabetic mice, was also decreased by the inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Similarly, urinary TGFβ1 was reduced by miR inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Interestingly, serum miR-299a-5p was increased in diabetic mice, highlighting its potential role as a biomarker for DKD \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). These data support potential therapeutic value of miR-299a-5p inhibition as an antifibrotic agent in DKD through its restoration of cripto-1 and FST, endogenous antagonists of profibrotic TGFβ1 and actA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003emiRs play various regulatory roles in cellular processes of healthy kidneys. However, disease-specific alteration of particular miRs may enable their therapeutic targeting and/or use as diagnostic markers. We identified a novel role for miR-299a-5p in the pathogenesis of fibrosis in DKD. It not only downregulates expression of FST, which inhibits fibrosis through its potent neutralization of activins, but it also downregulates the TGFβ1 antagonist cripto-1. By restoring endogenous FST and cripto-1, miR-299a-5p inhibition ameliorates DKD, as summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh.\u003c/p\u003e \u003cp\u003eWe previously identified FST as a miR-299a-5p target.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Its greatest efficacy is against actA\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, known to be increased in rodent and human DKD.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e While FST reduced fibrosis and protected against podocyte loss in Akita mice, higher dosing did not increase benefit, and in a non-diabetic model showed reduced efficacy\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These data suggested a therapeutic window for FST, possibly related to inhibition of other TGFβ family members. Restoring reduced endogenous FST levels in DKD may thus be a better treatment option. Our identification of increased miR-299a-5p expression in both glomeruli and tubules of mouse and human DKD suggested the potential therapeutic value of targeting miR-299a-5p, supported by our preclinical study. As expected, this was associated with reduced actA in kidneys and urine of diabetic mice.\u003c/p\u003e \u003cp\u003eGiven that miRNAs have several targets, we further performed bioinformatics analysis to identify potential additional mechanisms by which miR-299a-5p may contribute to fibrosis. Interestingly, we found that the cripto-1 3\u0026rsquo;UTR is also a target of miR-299a-5p. Cripto-1 is a glycosylphosphatidylinositol-linked membrane protein essential in human embryonic development and also implicated in tumor progression\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. While it functions as a co-receptor for TGFβ family members nodal, growth differentiation factor (GDF)1 and GDF3,\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e it was also shown to block signaling of actA, actB and TGFβ1 itself.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Through binding these ligands and their type I/II receptors, cripto-1 inhibits ligand-receptor interaction to prevent signaling. Cripto-1 can also be shed from the membrane, \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e likely further enabling interaction with these cytokines. Indeed, our data showed that recombinant cripto-1 effectively inhibits signaling by actA and TGFβ1 in MC.\u003c/p\u003e \u003cp\u003eLittle is known of the role of cripto-1 in the kidney. One study found no significant kidney expression of cripto-1 by immunohistochemistry, but this was in comparison to renal cell carcinoma in which expression may be highly elevated.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e We now show that cripto-1 is expressed in normal kidney in both glomeruli and tubules, likely at much lower levels than seen in cancer cells, and that this is attenuated in DKD. In agreement, miR-299a-5p-regulated inhibition of cripto-1 expression by HG was also seen in MC. We confirmed that recombinant cripto-1 reduces signaling by both actA and TGFβ1 and now show that it also attenuates the profibrotic effects of HG. Interestingly, specific neutralization studies suggested that the contribution of actA to these effects was greater than that of actB. Of importance therapeutically, we observed synergy between cripto-1 and FST in attenuating the profibrotic effects of either miR-299a-5p overexpression or of HG. Together, the targeting of both actA and TGFβ1 should theoretically provide more effective inhibition of fibrosis in DKD than inhibition of either ligand separately, although this would need to be demonstrated using target-specific miR blockers.\u003c/p\u003e \u003cp\u003ePreviously, we showed that caveolae and their structural membrane protein caveolin-1 are important regulators of FST expression. Caveolin-1 deletion in MC significantly reduced miR-299a-5p, leading to increased expression of FST and reduced expression of fibrotic proteins.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Caveolin-1 knockout in mice also protected against glomerular fibrosis in DKD.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Our data now extend these findings to show the negative regulation of cripto-1 expression by caveolin-1, mediated by miR-299a-5p. The protective effects of cav-1 deletion on DKD can thus likely be attributed to additional antifibrotic effects of cripto-1. Interestingly, Bianco \u003cem\u003eet al.\u003c/em\u003e showed that caveolin-1 interaction with cripto-1 in mammary epithelial cells inhibits cripto-1 biologic activity\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, suggesting that caveolin-1 can regulate both cripto-1 expression and activity. A negative feedback mechanism was also suggested, with cripto-1 reducing caveolin-1 expression in these cells\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The reduction in cripto-1 seen in diabetic kidneys may thus serve to augment caveolin-1 expression, further reducing FST and cripto-1. Additional studies are needed to test whether this feedback mechanism exists.\u003c/p\u003e \u003cp\u003eOur data support a novel role for miR-299a-5p in DKD. Increased by HG in MC and in diabetic kidneys, its inhibition protects against DKD. Overexpression of miR-299a-5p alone in the absence of HG promotes matrix synthesis, highlighting the important role it plays in the cellular fibrotic response. This is not surprising given its inhibition of at least two potent antifibrotic targets. Further studies are needed to determine whether additional miR-299a-5p targets which could contribute to kidney fibrosis exist. Interestingly, miR-299a-5p was found to suppress Atg5 expression and thereby autophagy in neurons.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e As autophagy dysfunction is also found in DKD,\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e miR-299a-5p may affect multiple pathogenic processes important to disease. Additional miR-154 family members may also contribute to DKD. Increased miR-377 in type 2 diabetic db/db mice and with HG in MC led to increased profibrotic PAI-1 and TGFβ1 expression through PPARγ inhibition.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSensitive biomarkers for diagnosis and prediction of DKD progression are needed. Measures of urinary and/or serum miR levels represent a potential non-invasive and affordable means to do this. Several urinary miRNAs (95-3p, 185-5p, 1246, 631) were shown to distinguish various forms of nephropathy in diabetic patients, and two of these reflected DKD severity.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Here we show a significant increase in serum miR-299a-5p in diabetic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg), suggesting that its measure could be explored further in patient cohorts to determine its clinical utility in the context of kidney disease progression. Two other miR-154 family members were also found increased in serum in DKD patients. Circulating miR-154-5p correlated with serum TGFβ1 and albuminuria, and inversely correlated with kidney function,\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and serum miR-377 was also suggested to be a potential early biomarker of DKD.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Larger studies, however, are needed to confirm these findings.\u003c/p\u003e \u003cp\u003eIn conclusion, we identify a novel role for miR-299a-5p in promoting fibrosis in DKD. This study supports further assessment of miR-299a-5p inhibition in the treatment of DKD. The restoration of endogenous antifibrotic proteins and inhibition of pathologic TGFβ1 signaling in the kidney may be better tolerated than systemic inhibition of these ligands. Indeed, targeting TGFβ1 has proven challenging due to its homeostatic role.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e Further work should also address some limitations of this study. Given that miRs are not specific for single targets, confirmation that beneficial effects of miR-299a-5p targeting occur via increased cripto-1 and FST expression could be ascertained using specific target site blockers to restrict effects to each of these RNAs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors recognize the support of The Research Institute at St. Joe’s Hamilton for nephrology research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJoan Krepinsky is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI.K.N, B.G, M.M, D.Z, U.B, and J.C performed experiments; I.K.N and J.C.K conceived experimental design; I.K.N, D.Z and J.T analyzed the data; I.K.N and J.C.K wrote the manuscript. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Sharing/Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not involve large data sets, new software or custom codes. The original data obtained and presented in this article are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have nothing to disclose and declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Canadian Institutes of Health Research (CIHR) (JCK, PJT-148628). IKN is a recipient of an Ontario Graduate Scholarship award and the 2023 Canadian Graduate Student (CIHR) award (189316).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKato M, Natarajan R. Epigenetics and epigenomics in diabetic kidney disease and metabolic memory. \u003cem\u003eNat Rev Nephrol\u003c/em\u003e. 2019;15(6):327. doi:10.1038/S41581-019-0135-6\u003c/li\u003e\n\u003cli\u003eVarghese RT, Jialal I. Diabetic Nephropathy. \u003cem\u003eStatPearls\u003c/em\u003e. Published online July 24, 2023. Accessed October 25, 2023. https://www.ncbi.nlm.nih.gov/books/NBK534200/\u003c/li\u003e\n\u003cli\u003eGewin LS. TGF-\u0026beta; and Diabetic Nephropathy: Lessons Learned Over the Past 20 Years. \u003cem\u003eAm J Med Sci\u003c/em\u003e. 2020;359(2):70-72. doi:10.1016/J.AMJMS.2019.11.010\u003c/li\u003e\n\u003cli\u003eWang L, Wang HL, Liu TT, Lan HY. 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The predictive value of miR-377 and phospholipase A2 in the early diagnosis of diabetic kidney disease and their relationship with inflammatory factors. \u003cem\u003eImmunobiology\u003c/em\u003e. 2024;229(2):152792. doi:10.1016/J.IMBIO.2024.152792\u003c/li\u003e\n\u003cli\u003eKulkarni AB, Ward JM, Yaswen L, et al. Transforming growth factor-beta 1 null mice. An animal model for inflammatory disorders. \u003cem\u003eAm J Pathol\u003c/em\u003e. 1995;146(1):264. Accessed July 22, 2024. /pmc/articles/PMC1870760/?report=abstract\u003c/li\u003e\n\u003cli\u003eVoelker J, Berg PH, Sheetz M, et al. Anti-TGF-b1 antibody therapy in patients with diabetic nephropathy. \u003cem\u003eJ Am Soc Nephrol\u003c/em\u003e. 2017;28(3):953-962. doi:10.1681/ASN.2015111230/-/DCSUPPLEMENTAL\u003c/li\u003e\n\u003c/ol\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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"diabetic kidney disease, microRNA-299a-5p, cripto-1, activins, transforming growth factor beta 1 (TGFβ1)","lastPublishedDoi":"10.21203/rs.3.rs-5419387/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5419387/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlomerular extracellular matrix protein accumulation, mediated largely by mesangial cells, is a defining feature of early diabetic kidney disease. Previously we showed that TGFβ1, a profibrotic cytokine with a well-defined pathogenic role in kidney fibrosis, inhibits expression of the antifibrotic follistatin through induction of microRNA-299a-5p. Whether this microRNA contributes to diabetic kidney disease is unknown.\u003c/p\u003e \u003cp\u003eWe show that microRNA-299a-5p is increased in mouse and human diabetic kidneys, and by high glucose in primary mesangial cells. Overexpression of microRNA-299a-5p in mesangial cells increased basal extracellular matrix protein production. Conversely, microRNA-299a-5p inhibition prevented the glucose-induced profibrotic response. Bioinformatics screening revealed that cripto-1 is also a target of microRNA-299a-5p. It is known that follistatin and cripto-1 inhibit activin A and TGFβ1 respectively. Induction of microRNA-299a-5p by high glucose mediated the mesangial cell fibrotic response by inhibiting expression of both follistatin and cripto-1 which led to increased activin A and TGFβ1 signaling. \u003cem\u003eIn vivo\u003c/em\u003e, microRNA-299a-5p inhibition reduced albuminuria, glomerular hypertrophy, loss of podocyte nephrin and extracellular matrix production, and this was associated with increased expression of follistatin and cripto-1.\u003c/p\u003e \u003cp\u003eThus, microRNA-299a-5p is an important mediator of glucose-induced profibrotic responses in mesangial cells and diabetic kidneys. Its inhibition may be a potential novel therapy.\u003c/p\u003e","manuscriptTitle":"miR-299a-5p is a novel mediator of fibrosis in diabetic kidney disease through its regulation of antifibrotic proteins follistatin and cripto-1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-18 10:02:37","doi":"10.21203/rs.3.rs-5419387/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4a5f07d1-8d0f-4f1b-a76b-62d2b76e4514","owner":[],"postedDate":"May 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48534316,"name":"Biological sciences/Molecular biology/RNAi"},{"id":48534317,"name":"Biological sciences/Molecular biology/Non-coding RNAs/miRNAs"},{"id":48534318,"name":"Biological sciences/Biological techniques/Gene delivery/Transfection/Bacterial transformation"}],"tags":[],"updatedAt":"2026-01-06T08:23:11+00:00","versionOfRecord":{"articleIdentity":"rs-5419387","link":"https://doi.org/10.1038/s42003-025-09271-6","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-12-11 05:00:00","publishedOnDateReadable":"December 11th, 2025"},"versionCreatedAt":"2025-05-18 10:02:37","video":"","vorDoi":"10.1038/s42003-025-09271-6","vorDoiUrl":"https://doi.org/10.1038/s42003-025-09271-6","workflowStages":[]},"version":"v1","identity":"rs-5419387","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5419387","identity":"rs-5419387","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}