The Mechanisms Of IL-37 In Renal Fibrosis Of Diabetic Kidney Disease

Background:Diabetic kidney disease(DKD)is the complications of diabetes mellitus related microvascular damage and is the leading cause of chronic kidney disease and end-stage renal disease.While glomerular injury is often considered an important cause of DKD progression,but a growing body of studies confirms that tubulointerstitial also play an important role in the pathogenesis of DKD.Renal interstitial fibrosis can occur in the early stage of DKD,and is the main and irreversible factor for renal dysfunction.Except for proper blood pressure and glycemic control,therapeutic options to revert or deter the progression of fibrosis are very limited.Therefore,it is interesting to elucidate the importance of tubulointerstitial in the pathogenesis of DKD.Interleukin-37(IL-37)is the seventh member of the IL-1 family.Function as a natural inhibitor of immune responses and inflammation.Previous study has showed that IL-37 can inhibit the release of inflammatory cytokines in renal tubular epithelial cells during ischemia refusion injury,suggestion that IL-37 may plays a key role in kidney disease.Moreover,a few studies also revealed that IL-37 was relevant to fibrotic disease.For example,intranasal administration of IL-37 prevented bleomycin-induced lung fibrosis and inflammation.In addition,IL-37 transgenic(IL-37tg)mice relieved autoimmune liver diseases related fibrosis and immune cell infiltrate.Of note,current studies have also showed that IL-37 tg mice could protect against high fat diet-induced obesity and insulin resistance.These studies suggest that IL-37 may be associated with renal fibrosis in DKD.However,the role of IL-37 in DKD are rarely explored.Therefore,we first observed the expression of IL-37 in DKD patient’s kidney biopsy,and analyzed the correlation between the expression of IL-37 in renal and clinical features of renal impairment.Then we established streptozotocin plus high fat diet(STZ/HFD)-induced DKD mice model with wild type(WT)or IL-37 tg mice.Then observed the renal injury and renal fibrosis in different groups.In addition,RNA-sequencing was applied to explore the potential mechanisms of IL-37 effect on renal fibrosis.In vitro,human proximal tubular(HK-2)cells were cultured to elucidated the possible mechanism of IL-37 protect from DKD renal fibrosis.Eventually,verified the mechanism of IL-37 on DKD renal interstitial fibrosis in WT or IL-37 tg mice.Methods and results:Part one: The expression of IL-37 in DKD patient’s renalThe expression of IL-37 in tubular epithelial cells of patients with DKD decreasedThe renal section of tissue away from renal cell carcinoma as control group(n=10)and DKD group(n=10)of patients diagnosed by renal biopsy.All of them were from Department of Nephrology of the Second Affiliated Hospital of Army Medical University from January2018 to December 2019.Clinical features of renal impairment: serum creatinine(SCr),24 h urinary protein excretion,estimated glomerular filtration rate(eGFR)were recorded in DKD patients.HE and PAS performed the pathological changes of renal glomerulus.Masson and IL-37 immunohistochemical staining observe renal interstitial fibrosis and the expression of IL-37.Analyze the correlation between the expression level of IL-37 in the kidney and clinical features of renal impairment in DKD patients.The results showed that the expanding of glomerular volume and mesangial in DKD patients.Meanwhile,DKD patients appear renal fibrosis and the expression of IL-37 significantly decreased in tubular epithelial cells.The expression level of IL-37 in renal was negatively correlated with renal interstitial fibrosis,SCr and proteinuria,but positively correlated with eGFR.These data indicate that the low expression of IL-37 in renal tubules is closely related to the degree of renal interstitial fibrosis and renal injury in DKD,and IL-37 may be involved in the progression of DKD.Part two: The role of IL-37 in DKD1.The expression of IL-37 alleviated renal injury in DKDThe expression of IL-37 was confirmed by western blot and IHC staining in IL-37 tg mice.The results showed that WT mice did not express IL-37,but IL-37 tg mice express IL-37 significantly.It means the establish of IL-37 tg mice was successful.We established STZ/HFD-induced DKD mice model with WT or IL-37 tg mice.24 hours urine were collected by metabolic cages in different groups and monitor the plasma glucose.Sixteen weeks after modeling,these mice were sacrificed.The weight and kidney weight of mice were weighed.Urine albumin,urine creatinine and SCr were detected.And the expression of KIM1 and NGAL in kidney were detected by quantitative real-time polymerase chain reaction(RT-qPCR).The results showed that the mice of STZ/HFD appear the significantly increased of plasma glucose,SCr and urine albumin to creatinine ratio(URCA),and the expanding of glomerular volume and mesangial,that means the successful of DKD model.IL-37 tg DKD mice showed significantly decreased UACR and SCr compared with WT DKD mice,but plasma glucose.WT DKD mice showed significantly decreased of body weight and the increased of the kidney to body weight ratio,the KIM-1 and NGAL expression compared with WT control mice.However,these were reversed by IL-37 expression.These data indicate that IL-37 could protect from renal injury.2.The expression of IL-37 attenuated renal fibrosis in DKDWT DKD mice showed significantly collagen deposition in renal interstitial compared with WT control mice.It means renal interstitial fibrosis.However,the area of fibrosis decreased in IL-37 tg DKD mice detected by Masson stain.The immunochemistry staining and Western blot performed that the expression of fibrosis marker of α-SMA and Fibronectin in IL-37 tg mice of DKD were signified decreased than WT DKD mice.These data show that the expression of IL-37 could alleviate DKD related-renal fibrosis.3.IL-37 reversed high glucose-induced HK-2 cell fibrosis markers expressionIn vitro,HK-2 cells were treated by 30 mmol/L high glucose,and with or without or 300ng/m L human recombinant IL-37(rec IL-37).Western blot was used to observe the expression of α-SMA and fibronectin in HK-2 cells.The results show that high glucose could induce the protein expression of α-SMA and fibronectin in HK-2 cells,while treatment with 300 ng/m L rec IL-37 would reduce these proteins expression.These data indicated that IL-37 could suppression the expression of fibrosis markers in renal tubular epithelial cell induced by high glucose in vitro.Part three: The mechanism of IL-37 in renal fibrosis of DKD1.The transcriptional landscape of DKD highlighted the enrichment in fatty acid metabolismRNA-sequencing was performed to profile the whole transcriptome in the primary renal tubular epithelial cells from WT or IL-37 tg mice with DKD.Processing GO、KEGG、Reactom and GSEA enrichment in different expression genes.The results showed that compared with WT DKD mice,IL-37 tg DKD mice exhibited significantly different transcriptome.Bioinformatics analysis from three methods(GO,KEGG pathway and Reactcome enrichment analysis)all emphasized specific changes in fatty acid metabolism consistently.Gene set enrichment analysis(GSEA)also determined a strong enrichment of fatty acid β-oxidation(FAO)among the fatty acid metabolism.These data indicate that IL-37 may alleviate renal fibrosis by modulating FAO in renal tubular epithelial cells.In vitro,HK-2 cells were cultured,treated by 30 mmol/L high glucose,and with or without 300 ng/m L rec IL-37.Detecting adenosine triphosphate(ATP)content,RT-qPCR were used to performed the expression of FAO related genes as MCAD、CROT、HADHB,and oil red O staining observed the lipid accumulation.Results showed that IL-37 could markedly reverse HK-2 cells ATP content and FAO related gens expression decreased,and lipid accumulation induce by high glucose.These data indicate that IL-37 could alleviate FAO impairment and lipid accumulation in HK-2 cell induced by high glucose.2.IL-37 ameliorate the expression of fibrosis markers by restoring carnitine palmitoyl-transferase 1A(CPT1A)mediated FAO in HK-2 cells under high glucose2.1 IL-37 upregulate CPT1A expression in HK-2 cells.Further analyzing the data of RNA-sequencing relative FAO different transcriptome.The result showed that the expression of CPT1A was significantly upregulated in IL-37 tg DKD mice,compared with that in WT DKD mice.Therefore,detected the orle of IL-37 in HK-2 cell about CPT1A expression was significant.Western blot performed that CPT1A was decreased in HK-2 cell induced by 30mmol/L high glucose.But recovered by co-cultured rec IL-37.2.2 Overexpression of CPT1A alleviated fibrosis markers expression and FAO impairment in HK-2 cell under high glucose.After HK-2 cells transfected with expression plasmid of CPT1A,treated with 30 mmol/L high glucose then detecting ATP content,oil red O stain observe lipid accumulation,and Western blot performed the expression of α-SMA and Fibronectin.These results showed that the ATP generation,lipid accumulation,and the increased of fibrosis marker expression induced by high glucose were significantly reversed by CPT1A overexpression.These data indicate that overexpression of CPT1A could ameliorate high glucose-induced FAO impaired and fibrosis markers expression.2.3 The effect of IL-37 in HK-2 cells was abolished by CPT1A gene silenceThe gene of CPT1A was knockdown by siRNA.Then HK-2 cells treated by 30 mmol/L high glucose,and with or without 300 ng/m L rec IL-37.Detecting ATP content,oil red O stain observe lipid accumulation,and Western blot performed the expression of α-SMA and Fibronectin.These results showed that IL-37 could alleviate ATP content,lipid accumulation and fibrosis marker high expression,but these alterations were abolished by CPT1A knockdown.These data indicate that IL-37 effect on the expression of fibrosis markers and FAO by restoring CPT1A expression in HK-2 cells.3.The expression of IL-37 restored renal CPT1A and FAO related genes expression level,and lipid accumulation in renal tubular epithelial cells in mice with DKDWT and IL-37 tg DKD mice as studied.Western blot and immunohistochemical staining were used to detected the expression of CPT1A.RT-qPCR was used to performed FAO related gene expression and ATP contend was determined.The result showed the expression of CPT1A and FAO related gens,ATP content were decreased in WT DKD mice compared as WT control mice.However,these were reversed by IL-37 expression.Moreover,the adipocyte differentiation-related protein(adipophilin,ADRP)and the marker of proximal tubular epithelial cell(Lotus tetragolonobus lectin,LTL)immunofluorescence stain and oil red O stain indicated the increased of ADRP expression and decreased of LTL expression,and more lipid accumulation in WT DKD mice compared with WT control mice.But alleviated in IL-37 tg DKD mice compared with WT DKD mice.These results demonstrate that IL-37 could ameliorate renal interstitial fibrosis in DKD by restoring renal tubular epithelial cells FAO impairment and lipid accumulation.Conclusion:In summary,in this work,we verified the decreased expression of IL-37 in renal tubular epithelial cells in DKD patients.The protein level of IL-37 in kidney was negatively correlated with renal interstitial fibrosis,SCr and proteinuria,but positively correlated with eGFR.IL-37 expression markedly attenuated kidney injury and renal interstitial fibrosis in DKD mice.Further confirmed that IL-37 ameliorates renal fibrosis by restoring CPT1A mediated FAO in DKD.These results indicated that IL-37 may be a potential therapeutic avenue to treat renal fibrosis in DKD...