Study On Renoprotective Effect Of SGLT2 Inhibitor On Diabetic Nephropathy By Regulating Complement Factor B

Objective:Diabetic nephropathy(DN)has become one of the leading causes of end-stage renal disease(ESRD)worldwide.Among the DN renal pathological injuries,glomerulopathy was thought to be the most dominant indicator affecting the clinical characteristics and prognosis of patients,and tubulointerstitial fibrosis was the prevalent pathological feature in the progression of DN to advanced stages.However,a close relationship between the early damage of renal tubules and the prognosis of patients’ kidneys and the decline of renal function has been discovered in recent years.Epithelial-mesenchymal transition(EMT)plays an important role in the early progression of renal interstitial fibrosis.Abnormal activation of the complement system has been confirmed to be involved in the pathogenesis of DN.Inhibition of the complement common pathway can obviously alleviate the fibrosis of DN kidneys.Complement factor B(CFB)is the initiating factor of the alternative pathway of complement.Increased CFB has been found in the kidney and urine of patients with DN.Sodium-glucose cotransporter 2(SGLT2)inhibitors are a new class of glucose lowering drugs discovered in recent years.Numerous clinical studies have shown significant renoprotective effects independent of glucose lowering effect.Intervention with SGLT2 inhibitors can significantly improve renal fibrosis in diabetic model mice.At present,whether complement alternative pathway actually plays a promoting role in the pathogenesis of DN and whether the renoprotective effect of SGLT2 inhibitors are related to the regulation of complement activation have not been reported.This study aimed to explore the role of complement alternative pathway initiator,CFB,in renal tubular epithelial-mesenchymal transition in DN,and elucidate whether SGLT2 inhibitors could exert renoprotective effect against renal fibrosis by downregulating CFB and subsequent activation of complement factors.Materials and Methods:1.The association between serum CFB and clinicopathological and renal prognosis of DN patients:Diabetic patients who underwent renal biopsy and were diagnosed with DN at the Department of Nephrology,West China Hospital of Sichuan University from January 2010 to April 2019 were screened.The clinical and pathological characteristics of the included patients were collected at the time of renal biopsy.Patients were grouped by median level of serum CFB,and the differences of clinicopathological characteristics between the two groups were compared;the correlation between serum CFB and clinicopathological parameters was analyzed;Cox regression analysis of patient outcome was performed to explore the possible role of CFB in patient renal outcomes;the renal tissue of patients with DN and para-carcinoma renal tissue were stained with CFB using immunohistochemical staining,while the para-carcinoma renal tissue was taken as normal control group.The correlation between the expression level of CFB in renal tissues and clinical indicators was analyzed.2.Effects of CFB on EMT of tubule epithelial cells in type 2 diabetic model:(1)For in-vivo experiments,a mouse model of type 2 diabetes was constructed by high-glucose and high-fat diet combined with continuous low-dose intraperitoneal injection of streptozocin(STZ).The mice were divided into control group,diabetes group,and LNP023 intervention group.The control group was fed with a normal chow diet,and other groups were fed with a high-glucose and high-fat diet for 4 weeks before the administration of 50 mg/kg STZ by continuous intraperitoneal injection for 6 days.After successful modeling,the control group and the diabetes group were given double distilled water by gavage,and the LNP023 intervention group was given 20 mg/kg LNP023 by gavage every 12 hours.The mice were sacrificed after 10 weeks.Lipid parameters,serum CFB,C3,C4 and renal function indexes were determined.The renal tissues were subjected to HE staining to observe the histopathological morphological changes,and Masson staining to observe the collagen fiber deposition.Immunohistochemical staining was performed to detect expression levels of CFB,C3,C5b-9,E-cadherin,vimentin and α-SMA.The protein expression changes in CFB,C3,E-cadherin,vimentin,α-SMA and TGF-β were determined by Western blot.(2)In vitro,human tubular epithelial cells(HK-2)were used and divided into three groups: low glucose group(LG Group);high glucose group(HG group);high glucose + LNP023 intervention group(HG + LNP023 group).High glucose of 30mmol/L was administered to stimulate epithelial-mesenchymal transition of HK-2cells.The CCK-8 assay was used to determine the appropriate concentration of CFB inhibitor,LNP023.The effects of LNP023 on EMT of HK-2 cells were evaluated by scratch assay,as well as the expression changes of related m RNA and proteins including CFB,E-cadherin and vimentin by cell immunofluorescence staining,RT-q PCR and Western blot.HK-2 cells overexpressing CFB were further constructed and cultured under normal glucose conditions.The effects of CFB overexpression on EMT of HK-2 cells were validated and examined.3.Effects of SGLT2 inhibitors on CFB expression and EMT of tubular epithelial cells in diabetes mellitus models:(1)In-vivo experiments were conducted by dividing experimental mouse into three groups,which were control group,diabetes group and empagliflozin intervention group.The type 2 diabetes mouse model was constructed following the method described previously.Empagliflozin suspension was administered by gavage at the concentration of 10 mg/kg,while the remaining groups were administered blank suspension by gavage once a day.The mice were sacrificed after 10 weeks.Lipid parameters,serum CFB,C3,C4 and renal function indexes were detected.Similarly,the renal tissues were subjected to HE staining to observe the histopathological morphological changes,and Masson staining to observe the collagen fiber deposition.The expression changes in CFB,C3,C5b-9,E-cadherin,vimentin,α-SMA and TGF-β were determined by immunohistochemical staining and Western blot.(2)In vitro,HK-2 cells were divided into three groups: low glucose group(LG Group);high glucose group(HG group);high glucose + empagliflozin intervention group(HG + empagliflozin group).Empagliflozin intervention group was treated with 500 nmol/L empagliflozin under high glucose condition.The expression changes of CFB,C3 and EMT related indicators were detected by RT-q PCR and Western blot,respectively.Effects of empagliflozin on EMT of HK-2 cells and expression levels of complement components including CFB were observed.Results:1.The association between serum CFB and clinicopathological and renal prognosis of DN patients:After screening exclusion and inclusion criteria,246 DN patients were finally included and divided into two groups according to the median level of serum CFB.A smaller proportion of patients with higher serum CFB level had a diagnosis of DR at the time of renal biopsy than patients in the low CFB group(P=0.001).Higher levels of fasting glucose(P = 0.003),hemoglobin(P = 0.001),serum albumin(P = 0.02),e GFR(P < 0.001),C3(P < 0.001)and C4(P < 0.001)were observed in patients in high CFB group than those in the low CFB group.However,the 24-hour urinary protein excretion level and the scores of every pathological index were not significantly different between the two groups.In the cohort with e GFR < 60ml/min/1.73 m2,patients in the high CFB group had higher C3 and C4 levels(P <0.001),while the other clinical parameters were not significantly different from those in the low CFB group.Among the pathological indexes,patients in the high CFB group had more severe IFTA scores than those in the low CFB group(P = 0.044),while the remaining pathological indexes were not statistically different.CFB was a statistically significant protective factor for renal outcomes in DN patients in univariate Cox regression analysis.However,after multivariate adjustment,CFB had no predictive role for renal outcomes in DN.In the para-carcinoma renal tissue,CFB was rarely detected.In the renal tissues of DN patients,CFB is mainly expressed in the tubular epithelial cells,with a small amount in the glomeruli.The results of semi-quantitative analysis of CFB immunohistochemical staining showed that there was no significant difference in the deposition of CFB between the IFTA 0+1 group and the IFTA 2+3 group,and the deposition of CFB in these two groups was significantly higher than that in the normal control group(P < 0.05).According to e GFR,patients were divided into two groups.In patients with e GFR < 60 ml/min/1.73 m2,the deposition of CFB in the IFTA 2+3 group was significantly more severe than that in the IFTA 0+1 group(P <0.05),and the deposition of CFB in the two groups was significantly more severe than that in the normal control group(P < 0.05);In patients with e GFR ≥ 60 ml/min/1.73 m2,the expression of CFB protein in renal tissue of the IFTA 2+3 group was significantly higher than that of the control group and the IFTA 0+1 group(P <0.05).Meanwhile,there was no significant difference in the expression of CFB protein between the IFTA 0+1 group and the control group.The CFB level in renal tissue of DN patients was negatively correlated with serum CFB(r=-0.608,P =0.036),e GFR(r =-0.636,P = 0.026),fasting blood glucose(r =-0.601,P = 0.039),and hemoglobin(r =-0.753,P = 0.005).2.Effects of CFB on EMT of tubule epithelial cells in type 2 diabetic model:LDL,HDL,total cholesterol,triglycerides,serum creatinine and urinary albumin to creatinine ratio were higher in diabetic mice than in normal controls.The intervention of LNP023 decreased LDL level,down-regulated serum CFB and C3 levels,and decreased serum creatinine and urinary albumin-to-creatinine ratio.HE staining and Masson staining showed that LNP023 ameliorated histopathological changes and improved collagen deposition in the kidneys of diabetic mice.The levels of CFB,C3,C5b-9,vimentin and α-SMA were increased in the kidneys of diabetic mice compared with normal mice by immunohistochemical staining and Western blot.The expression level of E-cadherin was decreased in the diabetic model group.The intervention of LNP023 could inhibit the changes of the above indicators.Results of CCK-8 assay showed that the HK-2 cells activity was the best at intervention concentrations of 1 μmol/L and 10 μmol/L of LNP023 with high glucose stimulation.The differences between these two concentrations and the other concentrations were statistically significant(P<0.05).Scratch assay showed that high glucose stimulation at 30 mmol/L increased the migration ability of HK-2 cells,and the intervention of LNP023 could inhibit this change to some extent.The results of cell immunofluorescence staining,RT-qPCR and Western blot showed that the m RNA and proteins of CFB,C3,vimentin and α-SMA were up-regulated after high glucose stimulation.E-cadherin was down-regulated under high glucose.The intervention of LNP023 could inhibit the above changes and alleviate HK-2 cell EMT stimulated by high glucose.In addition to upregulating subsequent complement activation,overexpression of CFB can also up-regulate the expression level of vimentin and α-SMA,and down-regulate E-cadherin in HK-2 cells.3.Effects of SGLT2 inhibitors on CFB expression and EMT of tubular epithelial cells in diabetes mellitus models:Triglyceride level,serum CFB,C3,serum creatinine and urinary albumin-to-creatinine ratio were lower in the empagliflozin intervention group than in the diabetes model group.HE staining and Masson staining showed that empagliflozin ameliorated histopathological changes and improved collagen deposition in the kidneys of diabetic mice.Immunohistochemical staining and Western blot showed that treatment with empagliflozin down regulated the levels of CFB,C3,C5b-9,vimentin and α-SMA,which are highly expressed in the kidneys of diabetic mice.E-cadherin levels in the kidneys of diabetic mice were also restored by empagliflozin intervention.After the intervention with empagliflozin,the m RNA and proteins of CFB and C3,vimentin and α-SMA,which were highly expressed in HK-2 cells under high glucose conditions,were downregulated.E-cadherin,which were downregulated by high glucose stimulation,was also restored to some extent after empagliflozin intervention.Conclusions:(1)Compared with patients with lower serum CFB level,patients with higher serum CFB level had higher baseline e GFR,C3,and C4 level,whereas the 24-hour proteinuria level and pathological indexes were not significantly different between the two groups.Serum CFB is associated with renal prognosis but fails to be an independent predictor.(2)The expression of complement alternative pathway initiator,CFB,in renal tissue of patients with diabetic nephropathy was high in renal tubules,which was negatively correlated with serum CFB level and e GFR.CFB may be involved in the tubulointerstitial injury of diabetic nephropathy;(3)CFB can induce epithelial-mesenchymal transition of HK-2 cells and promote renal fibrosis;(4)An SGLT2 inhibitor(empagliflozin)may exert antifibrotic effects by downregulating CFB expression in the diabetic kidney and delaying the progression of kidney disease.