Long noncoding RNA SNHG5 promotes podocyte injury via the microRNA-26a-5p/TRPC6 pathway in diabetic nephropathy

Abstract

Podocyte injury is a characteristic pathological hallmark of diabetic nephropathy (DN). However, the exact mechanism of podocyte injury in DN is incompletely understood. This study was conducted using db/db mice and immortalized mouse podocytes. High-throughput sequencing was used to identify the differentially expressed long noncoding RNAs in kidney of db/db mice. The lentiviral shRNA directed against long noncoding RNA small nucleolar RNA host gene 5 (SNHG5) or microRNA-26a-5p (miR-26a-5p) agomir was used to treat db/db mice to regulate the SNHG5/miR-26a-5p pathway. Here, we found that the expression of transient receptor potential canonical type 6 (TRPC6) was significantly increased in injured podocytes under the condition of DN, which was associated with markedly decreased miR-26a-5p. We determined that miR-26a-5p overexpression ameliorated podocyte injury in DN via binding to 3′-UTR of Trpc6, as evidenced by the markedly reduced activity of luciferase reporters by miR-26a-5p mimic. Then, the upregulated SNHG5 in podocytes and kidney in DN was identified, and it was proved to sponge to miR-26a-5p directly using luciferase activity, RNA immunoprecipitation, and RNA pull-down assay. Knockdown of SNHG5 attenuated podocyte injury in vitro, accompanied by an increased expression of miR-26a-5p and decreased expression of TRPC6, demonstrating that SNHG5 promoted podocyte injury by controlling the miR-26a-5p/TRPC6 pathway. Moreover, knockdown of SNHG5 protects against podocyte injury and progression of DN in vivo. In conclusion, SNHG5 promotes podocyte injury via the miR-26a-5p/TRPC6 pathway in DN. Our findings provide novel insights into the pathophysiology of podocyte injury and a potential new therapeutic strategy for DN. Podocyte injury is a characteristic pathological hallmark of diabetic nephropathy (DN). However, the exact mechanism of podocyte injury in DN is incompletely understood. This study was conducted using db/db mice and immortalized mouse podocytes. High-throughput sequencing was used to identify the differentially expressed long noncoding RNAs in kidney of db/db mice. The lentiviral shRNA directed against long noncoding RNA small nucleolar RNA host gene 5 (SNHG5) or microRNA-26a-5p (miR-26a-5p) agomir was used to treat db/db mice to regulate the SNHG5/miR-26a-5p pathway. Here, we found that the expression of transient receptor potential canonical type 6 (TRPC6) was significantly increased in injured podocytes under the condition of DN, which was associated with markedly decreased miR-26a-5p. We determined that miR-26a-5p overexpression ameliorated podocyte injury in DN via binding to 3′-UTR of Trpc6, as evidenced by the markedly reduced activity of luciferase reporters by miR-26a-5p mimic. Then, the upregulated SNHG5 in podocytes and kidney in DN was identified, and it was proved to sponge to miR-26a-5p directly using luciferase activity, RNA immunoprecipitation, and RNA pull-down assay. Knockdown of SNHG5 attenuated podocyte injury in vitro, accompanied by an increased expression of miR-26a-5p and decreased expression of TRPC6, demonstrating that SNHG5 promoted podocyte injury by controlling the miR-26a-5p/TRPC6 pathway. Moreover, knockdown of SNHG5 protects against podocyte injury and progression of DN in vivo. In conclusion, SNHG5 promotes podocyte injury via the miR-26a-5p/TRPC6 pathway in DN. Our findings provide novel insights into the pathophysiology of podocyte injury and a potential new therapeutic strategy for DN. Diabetes mellitus (DM) is a global public health problem (1Chan J. Lim L.L. Wareham N.J. Shaw J.E. Orchard T.J. Zhang P. et al.The Lancet Commission on Diabetes: using data to transform diabetes care and patient lives.Lancet. 2021; 396: 2019-2082Google Scholar). Diabetic nephropathy (DN) is among the most common and severe microvascular complications of DM and the primary cause of end-stage renal disease. Glomerular hypertrophy, podocyte injury, glomerular basement membrane thickening, and extracellular matrix deposition are characteristic pathological features of DN (2Umanath K. Lewis J.B. Update on diabetic nephropathy: core curriculum 2018.Am. J. Kidney Dis. 2018; 71: 884-895Google Scholar), and podocyte injury is thought to be a central link in the occurrence of DN. Nevertheless, the precise mechanisms of podocyte injury in DN are incompletely understood. Transient receptor potential canonical type 6 (TRPC6) is a nonselective receptor-operated cation channel that regulates reactive fibrosis and growth signaling (3Dietrich A. Gudermann T. TRPC6: physiological function and pathophysiological relevance.Handb. Exp. Pharmacol. 2014; 222: 157-188Google Scholar). Our previous studies found that TRPC6 is significantly upregulated in injured podocytes in the context of DN and plays a significant role in podocyte injury. Mechanistically, upregulated TRPC6 promotes podocyte injury by activating the NFAT pathway (4Ma R. Xu Y. Zhou H. Zhang D. Yao D. Song L. et al.Participation of the AngII/TRPC6/NFAT axis in the pathogenesis of podocyte injury in rats with type 2 diabetes.Mol. Med. Rep. 2019; 19: 2421-2430Google Scholar, 5Ma R. Liu L. Jiang W. Yu Y. Song H. FK506 ameliorates podocyte injury in type 2 diabetic nephropathy by down-regulating TRPC6 and NFAT expression.Int. J. Clin. Exp. Pathol. 2015; 8: 14063-14074Google Scholar). Targeting TRPC6 using pharmacological or genetic interventions could ameliorate podocyte injury and delay the progression of kidney diseases (6Ma R. Wang Y. Xu Y. Wang R. Wang X. Yu N. et al.Tacrolimus protects podocytes from apoptosis via downregulation of TRPC6 in diabetic nephropathy.J. Diabetes Res. 2021; 2021: 8832114Google Scholar, 7Spires D. Ilatovskaya D.V. Levchenko V. North P.E. Geurts A.M. Palygin O. et al.Protective role of Trpc6 knockout in the progression of diabetic kidney disease.Am. J. Physiol. Renal Physiol. 2018; 315: F1091-F1097Google Scholar). Nevertheless, the underlying mechanisms of dysregulated expression of TRPC6 in DN remain obscure. MicroRNAs (miRNAs) are a class of small noncoding RNAs that play essential regulatory roles in almost all biological processes by modulating gene expression (8Treiber T. Treiber N. Meister G. Regulation of microRNA biogenesis and its crosstalk with other cellular pathways.Nat. Rev. Mol. Cell Biol. 2019; 20: 5-20Google Scholar). Several lines of evidence suggest that miRNAs play crucial roles in the occurrence and development of kidney diseases (9Guo C. Dong G. Liang X. Dong Z. Epigenetic regulation in AKI and kidney repair: mechanisms and therapeutic implications.Nat. Rev. Nephrol. 2019; 15: 220-239Google Scholar). In the context of DN, associations between podocyte injury and miRNA expression levels in the blood, urine, and kidney tissue have been reported (10Ishii H. Kaneko S. Yanai K. Aomatsu A. Hirai K. Ookawara S. et al.MicroRNAs in podocyte injury in diabetic nephropathy.Front. Genet. 2020; 11: 993Google Scholar). Moreover, modulation of the expression of several miRNAs has been shown to protect against podocyte injury in DN (10Ishii H. Kaneko S. Yanai K. Aomatsu A. Hirai K. Ookawara S. et al.MicroRNAs in podocyte injury in diabetic nephropathy.Front. Genet. 2020; 11: 993Google Scholar). Nevertheless, it is uncertain whether specific miRNAs regulate TRPC6 in DN. Long noncoding RNAs (lncRNAs), commonly defined as RNA molecules more than 200 nucleotides in length, are significant players at almost every level of gene function and regulation (11Statello L. Guo C.J. Chen L.L. Huarte M. Gene regulation by long non-coding RNAs and its biological functions.Nat. Rev. Mol. Cell Biol. 2021; 22: 96-118Google Scholar). Several studies reported that lncRNAs combine with miRNAs such that the targeted mRNA can escape negative regulation by a mechanism known as competing endogenous RNA (12Salmena L. Poliseno L. Tay Y. Kats L. Pandolfi P.P. A ceRNA hypothesis: the Rosetta stone of a hidden RNA language.Cell. 2011; 146: 353-358Google Scholar), and lncRNA appears to participate in diabetes and DN (13Guo J. Liu Z. Gong R. Long noncoding RNA: an emerging player in diabetes and diabetic kidney disease.Clin. Sci. (Lond.). 2019; 133: 1321-1339Google Scholar). For instance, Deng et al. (14Deng Q. Wen R. Liu S. Chen X. Song S. Li X. et al.Increased long noncoding RNA maternally expressed gene 3 contributes to podocyte injury induced by high glucose through regulation of mitochondrial fission.Cell Death Dis. 2020; 11: 814Google Scholar) found that increased lncRNA Meg3 expression contributed to podocyte injury induced by high glucose, suggesting that specific lncRNA plays a critical role in the progression of DN. However, regulation of podocyte injury by specific lncRNAs via the TRPC6 pathway has not been reported. The baseline indicators of db/db mice are shown in Table 1. Histologic analysis revealed marked glomerular hypertrophy and mesangial expansion in db/db mice compared with control db/m mice (Fig. 1A). Transmission electron microscope (TEM) revealed that the foot processes were fused, and basement membranes were thickened in db/db mice (Fig. 1B). The expression of nephrin and podocin was decreased significantly (Fig. 1C), suggesting podocyte injury. Then, we found that Trpc6 mRNA and protein levels were increased significantly in the kidneys of db/db mice (Fig. 1, D and E). Immunostaining of TRPC6 indicated that the TRPC6 expression significantly increased in injured podocytes (Fig. 1F). Podocyte injury and upregulated TRPC6 expression were also observed in high glucose (HG)-treated podocytes, detected by qPCR (Fig. 1G) and Western blot (Fig. 1H), respectively. These findings suggest that TRPC6 expression increases significantly in injured podocytes in DN.Table 1Baseline indicators of mice (x¯ ± s)Indicatorsdb/m (n = 6)db/db (n = 6)t valuep valueKW/BW (mg/g)12.51 ± 2.619.31 ± 1.062.7780.020FBG (mmol/L)5.32 ± 0.4117.54 ± 2.2912.8360.000ACR (mg/mg)0.24 ± 0.121.09 ± 0.414.8860.001TG (mmol/L)1.50 ± 0.293.25 ± 0.2211.6960.000TC (mmol/L)2.91 ± 0.445.41 ± 0.866.3620.000LDL-C (mmol/L)0.77 ± 0.901.23 ± 0.156.5900.000Scr (μmol/L)13.06 ± 0.8013.40 ± 1.510.4890.636BUN (mmol/L)8.40 ± 1.038.16 ± 0.910.4350.673Abbreviations: ACR, urinary albumin to creatinine ratio; BUN, serum urea nitrogen; FBG, fasting blood glucose; KW/BW, kidney weight/body weight; LDL-C, low density lipoprotein-cholesterol; Scr, serum creatinine; TC, total cholesterol; TG, triglycerides. Open table in a new tab Abbreviations: ACR, urinary albumin to creatinine ratio; BUN, serum urea nitrogen; FBG, fasting blood glucose; KW/BW, kidney weight/body weight; LDL-C, low density lipoprotein-cholesterol; Scr, serum creatinine; TC, total cholesterol; TG, triglycerides. Given the crucial role of miRNAs in the progression of kidney diseases, we hypothesized that specific miRNAs might mediate dysregulated TRPC6 expression. We predicted the putative miRNAs containing the binding sites of Trpc6 that contribute to the podocyte injury using five online databases: TargetScan, miRDB, miRanda, DIANA-TarBase, and PicTar. Overlap analysis revealed that miR-26a-5p contains a highly conserved consequence targeting the Trpc6 3′-UTR (Fig. 2A). We also observed that levels of miR-26a-5p in kidney tissue were markedly decreased (Fig. 2B), negatively correlated with Trpc6 mRNA levels (Fig. 2C). Fluorescence in situ hybridization (FISH) analysis revealed that the decreased miR-26a-5p was located in the podocytes of glomeruli (Fig. 2D). We then measured expression levels of miR-26a-5p in podocytes exposed to HG. As expected, miR-26a-5p levels decreased significantly (Fig. 2E). Consistent with the in vivo finding, there is a negative correlation between miR-26a-5p and Trpc6 mRNA levels in HG-treated podocytes (Fig. 2F). The luciferase reporter assay showed that the activity of luciferase reporters was markedly reduced by miR-26a-5p mimic compared with mimic-NC. Furthermore, the activity of Trpc6 3′-UTR-mut luciferase reporter was not affected by the miR-26a-5p mimic, suggesting that miR-26a-5p directly interacts with 3′-UTR of Trpc6 mRNA. (Fig. 2G). These findings suggest that miR-26a-5p is involved in podocyte injury by targeting Trpc6 mRNA directly. To determine the role of miR-26a-5p in podocyte injury and progression of DN in vivo, miR-26a-5p agomir or negative control was administered via the tail vein of db/db mice. We first confirmed transfection efficiency (Fig. 3A). The level of albuminuria was markedly decreased in miR-26a-5p agomir-treated mice (Fig. 3B). Histologic analysis revealed that glomerular hypertrophy and mesangial expansion were significantly blunted in miR-26a-5p agomir-treated mice compared with agomir NC-treated mice (Fig. 3C). TEM revealed that polysaccharide aggregation and foot process fusion were partially relieved in miR-26a-5p agomir-transfected db/db mice (Fig. 3D). Podocyte injury was markedly alleviated, as demonstrated by nephrin and podocin expression levels (Fig. 3E). TRPC6 expression was decreased in this group (Fig. 3, F and G). In an in vitro experiment, podocytes were transfected with miR-26a-5p mimic to overexpress miR-26a-5p before intervention with HG (Fig. 3H). As expected, the expression of TRPC6 was markedly decreased in podocytes with miR-26a-5p mimic treatment, combined with the increased nephrin and podocin expression (Fig. 3I). These findings suggest that miR-26a-5p overexpression mitigated podocyte injury by inhibiting TRPC6. To explore the potential mechanism of the dysregulated miR-26a-5p/TRPC6 pathway, high-throughput sequencing for lncRNAs of the kidney tissue of db/db mice was used (Fig. 4, A and B). Among the top lncRNAs, the specific lncRNAs for miR-26a-5p interaction were predicted by the StarBase database. A significant increase in lncRNA small nucleolar RNA host gene 5 (SNHG5) was found, and it was selected for further study. To confirm the lncRNA profile, we measured expression levels of SNHG5 via FISH and qPCR. FISH revealed that SNHG5 expression was significantly increased, primarily in podocyte cytoplasm (Fig. 4C). We found that SNHG5 expression was upregulated in the kidney of db/db mice using qPCR (Fig. 4D), positively correlated with the albumin to creatinine ratio (ACR) (Fig. 4E). Dramatically increased SNHG5 expression was observed in HG-treated podocytes compared with normal glucose (NG)-treated and high mannitol (HM)-treated podocytes (Fig. 4F). These findings suggest that increased SNHG5 expression may be associated with podocyte injury in DN. Next, we sought to understand how SNHG5 promotes podocytes injury. Based on bioinformatics analysis, miR-26a-5p was predicted to contain appropriate binding sites for SNHG5 (Fig. 5A), a critical negative regulator of the TRPC6 signaling pathway. We performed, dual-luciferase reporter assay, RNA immunoprecipitation (RIP) and RNA pull-down to test whether there is a direct interaction between SNHG5 and miR-26a-5p in podocytes. Luciferase activity of SNHG5-wt reporter was significantly decreased with miR-26a-5p overexpression, whereas no significant change was observed in SNHG5-mut reporter expression (Fig. 5B). In RIP assays, SNHG5 levels were enriched in abundance by Ago2 antibody compared with control IgG (Fig. 5C). RNA pull-down analysis displayed a substantial enrichment of SNHG5 by miR-26a-5p compared with the control group (Fig. 5D). We found that miR-26a-5p levels were significantly increased in podocytes with SNHG5 knockdown (Fig. 5E), combined with decreased TRPC6 expression (Fig. 5F). Podocyte injury was markedly abrogated in this group (Fig. 5G). These findings suggest that SNHG5 promotes podocyte injury by targeting miR-26a-5p in DN. To confirm the functional effects of SNHG5 in vivo, we suppressed SNHG5 function in podocytes with the SNHG5 shRNA by tail vein injection (n = 6 for each group). As expected, less albuminuria was found in db/db mice with SNHG5 knockdown (Fig. 6A). Meanwhile, histologic analysis revealed that glomerular hypertrophy and mesangial expansion were significantly attenuated in SNHG5 knockdown mice (Fig. 6B). TEM analysis revealed that foot process fusion and basement membrane thickening were markedly alleviated (Fig. 6C). Expression levels of nephrin and podocin were significantly increased (Fig. 6D). Levels of miR-26a-5p in db/db mice with SNHG5 knockdown were also increased (Fig. 6E) combined with decreased TRPC6 expression in podocytes (Fig. 6, F and G). These findings suggest that SNHG5 knockdown protects podocytes by modulating the miR-26a-5p/TRPC6 axis in DN. DN is a therapeutic challenge in clinical practice, partly because of its unclear pathogenesis. Several lines of evidence demonstrated that podocyte dysfunction is central to the underlying pathophysiology of DN (15Torban E. Braun F. Wanner N. Takano T. Goodyer P.R. Lennon R. et al.From podocyte biology to novel cures for glomerular disease.Kidney Int. 2019; 96: 850-861Google Scholar). Nevertheless, the mechanism of podocyte dysfunction in DN remains poorly understood. In the present study, we found that upregulated TRPC6 in injured podocytes plays an essential role in the occurrence of DN. Meanwhile, we determined miR-26a-5p as the regulator of TRPC6 in injured podocytes. Furthermore, the lncRNA SNHG5, which sponges miR-26a-5p, regulates the TRPC6 pathway. As a terminally differentiated cell type, podocytes participate in the formation of a filtration barrier, which is critical in maintaining the physiological function of the kidney. Several lines of evidence suggest that membrane structural and signal transduction proteins in podocytes help maintain homeostasis. Among them, TRPC6, which forms a signal transduction complex with nephrin and podocin on podocytes (16Reiser J. Polu K.R. Möller C.C. Kenlan P. Altintas M.M. Wei C. et al.TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal Genet. Scholar), is critical to podocyte injury in kidney diseases A. a to novel therapeutic for kidney Pharmacol. Sci. 2019; Scholar). showed that the TRPC6 channel might be a for In the present study, we found that TRPC6 expression increased significantly in the kidney of db/db mice and HG-treated podocytes, with a previous study that TRPC6 is upregulated in podocytes of several DM L. of TRPC6 promotes and glomerular injury in Int. 2019; Scholar, D.V. G. Palygin O. Levchenko V. et pathway in podocyte regulation and renal in diabetic kidney Nephrol. 2018; Scholar, S. R. Chen L. W. Xu X. J. et promotes channel and cell injury in Exp. Med. 2021; Scholar). mechanisms have been to how TRPC6 podocyte injury. The of the channel the of which and the activity of NFAT (4Ma R. Xu Y. Zhou H. Zhang D. Yao D. Song L. et al.Participation of the AngII/TRPC6/NFAT axis in the pathogenesis of podocyte injury in rats with type 2 diabetes.Mol. Med. Rep. 2019; 19: 2421-2430Google Scholar, S. R. Chen L. W. Xu X. J. et promotes channel and cell injury in Exp. Med. 2021; Scholar, M. T. K. et of to of TRPC6 channel and its on Nephrol. 2019; Scholar). studies demonstrated that TRPC6 regulates the and of podocytes by interaction with than on K. R. M. J. J. T. et al.The TRPC6 activity to podocyte Nephrol. 2018; Scholar, R. L. A. et al.TRPC6 to and of its channel activity, and regulates podocyte cell and Nephrol. 2019; Scholar). This evidence the of how TRPC6 to podocyte injury. Nevertheless, the underlying mechanism of dysregulated TRPC6 in podocytes remains found that a class of small endogenous noncoding are of podocyte injury in DN development P. A. MicroRNAs as of glomerular function in health and Nephrol. Scholar). For low levels of were detected in blood from DM and in kidneys of with type and 2 DM Y. Li H. Liu J. P. Li X. H. et in expression the of diabetic kidney Nephrol. Scholar), was upregulated in kidneys of DM mice J. C. X. V. W. et in glomerular Nephrol. 2015; Scholar), of which play essential roles in podocytes injury. We that TRPC6 expression was by specific miRNA in DN for In the present study, we found that miR-26a-5p is a critical regulator of TRPC6 expression. The that miR-26a-5p is for cell and apoptosis has on studies of several pathophysiological C. Li Y. Y. Z. H. Y. et and its in and Rev. 2021; Scholar). Consistent with a study that miR-26a-5p and the apoptosis of podocytes Y. Q. Wang Y. Y. Chen F. X. et extracellular containing microRNA-26a-5p and protect against diabetic nephropathy.J. Biol. 2020; Scholar), we found that overexpression of miR-26a-5p podocytes from structural by the expression of TRPC6, by the of foot process and of podocyte structural and These findings suggest that miR-26a-5p is a critical regulator of podocyte injury by targeting TRPC6. Nevertheless, the precise regulatory for effects have not been in the of DN. is known that lncRNA in processes and We hypothesized that specific lncRNAs the dysregulated miR-26a-5p/TRPC6 pathway. We high-throughput sequencing to identify specific with in analysis, we an lncRNA SNHG5 that contains binding sites for miR-26a-5p. As expected, SNHG5 expression was markedly increased in podocytes under DN SNHG5 was first in by R. H. M. K. Y. S. et host gene of no potential is at the of Scholar). SNHG5 was involved in several and diseases and is associated with clinical and other and Wang Li Y. Chen a new in 2020; Scholar, Yu Liu Zhang D. J. Yu Y. et promotes by the 2019; Scholar, M. C. L.L. M.M. R. et promotes cell by mRNA Scholar, S. T. D. E. F. S. et progression by 2018; 8: Scholar). evidence indicated that upregulated SNHG5 plays a role in kidney W. L. Zhou G. W. J. Zhang C. et al.The lncRNA downregulation contributes to the progression of cell renal cell by targeting Med. 2020; Scholar, Y. Song Wei Chen G. et non-coding RNA SNHG5 the and apoptosis of renal cell by the J. Res. 2020; Scholar). kidney a study indicated that SNHG5 expression was in with accompanied with decreased cell increased and and Q. Wang C. L. C. Chen T. et non-coding RNA small nucleolar RNA host gene 5 (SNHG5) regulates renal in diabetic nephropathy via targeting Res. 2021; Scholar). Nevertheless, the potential effects of SNHG5 in podocytes remain to be In the present study, we found that SNHG5 expression was markedly increased in DN. SNHG5 levels positively correlated with of db/db mice. attenuated podocyte injury, accompanied by miR-26a-5p and decreased TRPC6 was found in HG-treated podocytes SNHG5 Meanwhile, levels of albuminuria and alleviated foot process fusion were observed in db/db mice with SNHG5 SNHG5 knockdown podocytes from injury by modulating the miR-26a-5p/TRPC6 axis in DN. These findings are the first evidence for the biological function of SNHG5 in podocytes under DN RNAs lncRNAs, are essential mechanisms of that regulate gene expression at the and levels Lim Long noncoding RNAs in Rev. 2021; Scholar). Several lines of evidence demonstrated that play critical roles in kidney diseases (9Guo C. Dong G. Liang X. Dong Z. Epigenetic regulation in AKI and kidney repair: mechanisms and therapeutic implications.Nat. Rev. Nephrol. 2019; 15: 220-239Google Scholar, M. R. and in diabetic kidney and Rev. Nephrol. 2019; 15: Scholar). is crosstalk between lncRNAs and of the most mechanisms competing for endogenous RNA X. Y. Chen J. competing endogenous RNA for 2020; Scholar). For overexpression of lncRNA protects the kidney against injury by and signaling G. M. A. H. R. et overexpression of long non-coding RNA kidney injury through of Nephrol. 2021; Scholar). the essential role of for or diseases have been and Y. Li Z. Chen X. Zhang S. Long non-coding from to 2021; 11: Scholar, at the RNA level with J. Med. 2019; Scholar, D. Chen S. P.R. et al.The of 2021; Scholar). In the present study, we also to explore the potential therapeutic for podocyte injury via targeting In conclusion, we a precise mechanism podocyte injury in DN. SNHG5 contributes to podocyte injury by targeting the miR-26a-5p/TRPC6 pathway (Fig. These findings provide insights into the mechanisms of podocyte injury and the progression of DN. this signaling a therapeutic for DN.

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