PTPN2 improved renal injury and fibrosis by suppressing STAT-induced inflammation in early diabetic nephropathy

Abstract

Diabetic nephropathy (DN) is a chronic inflammatory disease triggered by disordered metabolism. Recent studies suggested that protein tyrosine phosphatase non-receptor type 2 (PTPN2) could ameliorate metabolic disorders and suppress inflammatory responses. This study investigated PTPN2's role in modulating DN and the possible cellular mechanisms involved. In a mouse model combining hyperglycaemia and hypercholesterolaemia (streptozotocin diabetic, ApoE^-/- mice), mice showed severe insulin resistance, renal dysfunction, micro-inflammation, subsequent extracellular matrix expansion and decreased expression of PTPN2. We found that mice treated with PTPN2 displayed reduced serum creatinine, serum BUN and proteinuria. PTPN2 gene therapy markedly attenuated metabolic disorders and hyperglycaemia. In addition, PTPN2 gene transfer significantly suppressed renal activation of signal transducers and activators of transcription (STAT), STAT-dependent pro-inflammatory and pro-fibrotic genes expression, and influx of lymphocytes in DN, indicating anti-inflammatory effects of PTPN2 by inhibiting the activation of STAT signalling pathway in vivo. Furthermore, PTPN2 overexpression inhibited the high-glucose induced phosphorylation of STAT, target genes expression and proliferation in mouse mesangial and tubuloepithelial cells, suggesting that the roles of PTPN2 on STAT activation was independent of glycaemic changes. Our results demonstrated that PTPN2 gene therapy could exert protective effects on DN via ameliorating metabolic disorders and inhibiting renal STAT-dependent micro-inflammation, suggesting its potential role for treatment of human DN.

Keywords: PTPN2; STAT1/3; diabetes nephropathy; fibrosis; inflammation.

Figure 1

Renal PTPN2 expression decreased after diabetes induction in mice. A, ApoE^‐/‐ mice were fed on chow diet (Chow) or high‐fat diet (DM) and analysed by intraperitoneal glucose tolerance test (IPGTT) at 4, 10 and 12 weeks of feeding. B, AUC (area under curve) in 4, 10 and 12‐week‐old ApoE^‐/‐ mice. C, Body weight at the ages of 4, 10, 12, 20, 22 and 24 weeks. D, Serum creatinine, serum BUN (blood urea nitrogen) and proteinuria levels (urine albumin‐to‐creatinine ratio) in ApoE^‐/‐ mice. E, Representative images of periodic acid‐Schiff (PAS)‐stained kidney sections. F, Glomerular area quantification and PAS + mesangial area analysis in ApoE^‐/‐ mice. G, Representative micrographs showing positive PTPN2, P‐STAT1, and PSTAT3 immunostaining in glomerular and tubulointerstitium of diabetic mice. H, Quantification of PTPN2, PSTAT1, and P‐STAT3 immunostaining in glomerular and tubulointerstitial compartments. I, Western blot analyses of PTPN2, P‐STAT1 and P‐STAT3 expression in renal cortical lysates from diabetic mice. N: normal; DM: diabetes mellitus; IOD: integrated optical density. Data are mean ± SEM of seven to eight animals per group. *P < 0.05 vs. N. Original magnification, ×200 in E and G

Figure 2

PTPN2 gene therapy protected from diabetes‐associated renal injury in ApoE^‐/‐ mice. A, Serum creatinine levels in ApoE^‐/‐ mice with established nephropathy after PTPN2 gene therapy. B, Serum BUN levels in ApoE^‐/‐ mice with established nephropathy after PTPN2 gene therapy. C, Proteinuria levels in ApoE^‐/‐ mice with established nephropathy after PTPN2 gene therapy. D, Representative images of HE and PAS staining in renal sections. E, Glomerular area quantification and PAS + mesangial area analysis in the experimental groups. N: normal; DM: diabetes mellitus. Data are mean ± SEM of seven to eight animals per group. *P < 0.05 vs. N + Vehicle; ^# P < 0.05 vs. DM + Vehicle. Original magnification, ×200 in D

Figure 3

PTPN2 gene therapy improved insulin resistance and metabolic disorder in diabetic mice. A, Glucose tolerance tests and the area under blood glucose concentration curve (AUC) at the age of 24 weeks. B, Fast blood glucose levels at the age of 24 weeks. C, Serum levels of ALT (alanine transaminase) and AST (aspartate transaminase) after PTPN2 gene therapy. D, Liver index of mice with established nephropathy after PTPN2 gene therapy. Liver index = Liver weight (mg)/Body weight (g). E, Representative micrographs of HE and Sirius Red‐sensitive collagen staining to detect liver histopathological alterations. F, Quantification of liver pathological alteration and fibrosis in diabetic mice after PTPN2 gene therapy. N: normal; DM: diabetes mellitus. Data are mean ± SEM of seven to eight animals per group. *P < 0.05 vs. N + Vehicle; ^# P < 0.05 vs. DM + Vehicle. Original magnification, ×200 in E

Figure 4

PTPN2 gene therapy reduced diabetes‐induced renal inflammation. A, Representative micrographs showing positive CD3, F4/80, Arg I (Arginase I), Arg II (Arginase II), CD11c and CD206 immunostaining in glomerular and tubulointerstitium. B, Quantification of CD3, F4/80, Arg I, Arg II, CD11c and CD206 immunostaining in glomerular and tubulointerstitial compartments. C, Representative micrographs showing positive TNF‐α (tumor necrosis factor‐α), IL‐6 (interleukin‐6), ICAM‐1 (intercellular cell adhesion molecule‐1) and MCP‐1 (monocyte chemotactic protein 1) immunostaining in glomerular and tubulointerstitium. D, Quantification of TNF‐α, IL‐6, ICAM‐1, and MCP‐1 immunostaining in glomerular and tubulointerstitial compartments. E, Representative Western blot analyses of Arg I, Arg II, TNF‐α, IL‐6, ICAM‐1, and MCP‐1 expression in renal cortical lysates. F, Quantitative analysis of the results in E. IOD: integrated optical density; N: normal; DM: diabetes mellitus; IOD: integrated optical density. Data are mean ± SEM of seven to eight animals per group. *P < 0.05 vs. N + Vehicle; ^# P < 0.05 vs. DM + Vehicle. Original magnification, ×200 in A and C

Figure 5

PTPN2 gene therapy decreased diabetes‐induced renal fibrosis. A, Representative images of MASSON, and Sirius Red‐sensitive collagen staining in renal sections. B, Representative micrographs showing positive Col I (collagen I), Col IV (Collagen IV), Fn (fibronectin), PAI‐1 (plasminogen activator inhibitor‐1), and TGF‐β (transforming growth factor‐β) and α‐SMA (α‐smooth muscle actin) immunostaining in glomerular and tubulointerstitium. C, Quantification of fibrosis (% Sirius Red area), Col I, Col IV, Fn, PAI‐1, TGF‐β and α‐SMA immunostaining in glomerular and tubulointerstitial compartments. D, Representative Western blot analyses of Col I, Col IV, Fn, PAI‐1 and TGF‐β expression in renal cortical lysates. E, Quantitative analysis of the results in D. N: normal; DM: diabetes mellitus; IOD: integrated optical density. Data are mean ± SEM of seven to eight animals per group. *P < 0.05 vs. N + Vehicle; ^# P < 0.05 vs. DM + Vehicle. Original magnification, ×200 in A and B

Figure 6

PTPN2 gene therapy inhibited STAT activation in vivo. A, Representative micrographs showing positive PTPN2, P‐STAT1 and P‐STAT3 immunostaining in glomerular and tubulointerstitium. B, Quantification of PTPN2 immunostaining in glomerular and tubulointerstitium. C, Quantification of P‐STAT1 immunostaining in glomerular and tubulointerstitium. D, Quantification of P‐STAT3 immunostaining in glomerular and tubulointerstitium. E, Representative Western blot analyses for PTPN2, P‐STAT1, and P‐STAT3 in renal cortical lysates. N: normal; DM: diabetes mellitus; IOD: integrated optical density. Data are mean ± SEM of seven to eight animals per group. *P < 0.05 vs. N + Vehicle; ^# P < 0.05 vs. DM + Vehicle. Original magnification, ×200 in A

Figure 7

PTPN2 gene therapy prevented diabetes‐induced renal angiogenesis. A, Representative immunofluorescence microscopy of vascular endothelial growth factor (VEGF) and CD31 in peritubular and glomerular regions. B, Quantitative analysis of glomerular VEGF expression. C, Quantitative analysis of glomerular CD31 expression. D, Representative Western blot analyses for VEGF expression in renal cortical lysates. E, Quantitative analysis of the results in D. N: normal; DM: diabetes mellitus. Data are mean ± SEM of seven to eight animals per group. *P < 0.05 vs. N + Vehicle; ^# P < 0.05 vs. DM + Vehicle. Original magnification, ×400 in A

Figure 8

HG caused PTPN2 down‐regulation and STAT activation in cultured renal cells. A, Immunofluorescence microscopy of MC (murine mesangial cells) showed PTPN2 staining in green and nuclear staining in blue. Cells were either left untreated or incubated with high glucose (HG, 30 mmol/L Dglucose) for 24 h. B, Immunofluorescence microscopy of MCT (murine tubuloepithelial cells) showed PTPN2 staining in green and nuclear staining in blue. Cells were either left untreated or incubated with HG for 24 h. C, Representative Western blot analyses showing time course of P‐STAT1, P‐STAT3, and PTPN2 induction by low glucose (LG, 5.5 mmol/L D‐glucose) in MC. D, Quantitative analysis of the results in C. E, Representative Western blot analyses showing time course of P‐STAT1, P‐STAT3, and PTPN2 induction by LG in MCT. F, Quantitative analysis of the results in E. G, Representative Western blot analyses showing time course of P‐STAT1, P‐STAT3 and PTPN2 induction by HG in MC. H, Quantitative analysis of the results in G. I, Representative Western blot analyses showing time course of P‐STAT1, P‐STAT3, and PTPN2 induction by HG in MCT. J, Quantitative analysis of the results in I. LG: low glucose; HG: high glucose. Data are mean ± SEM of three experiments in duplicate. *P < 0.05 vs. Control (0 h)

Figure 9

PTPN2 inhibited HG‐induced STAT activation, STAT‐dependent genes, and cell proliferation in vitro. MC and MCT were infected with PTPN2‐expressing adenovirus or control adenovirus. After 24 h, cells were stimulated for an additional 4 h with LG or HG. A, Representative Western blot analyses for P‐STAT1, P‐STAT3 and PTPN2 proteins in total cell extracts from MC. B, Quantitative analysis of the results in A. C, Representative Western blot analyses for P‐STAT1, P‐STAT3 and PTPN2 proteins in total cell extracts from MCT. D, Quantitative analysis of the results in C. E, Representative Western blot analyses for intercellular cell adhesion molecule‐1 (ICAM‐1), tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), collagen I (Col I), collagen IV (Col IV), fibronectin (Fn), plasminogen activator inhibitor‐1 (PAI‐1), and transforming growth factor‐β (TGF‐β) proteins in total cell extracts from MC. F, Western blot analyses of ICAM‐1, TNF‐α, and IL‐6 expression in total cell extracts from MC. G, Western blot analyses of Col I, Col IV, Fn, PAI‐1, and TGF‐β expression in total cell extracts from MC. H, Representative Western blot analyses for ICAM‐1, TNF‐α, IL‐6, Col I, Col IV, Fn, PAI‐1 and TGF‐β proteins in total cell extracts from MCT. I, Western blot analyses of ICAM‐1, TNF‐α and IL‐6 expression in total cell extracts from MCT. J, Western blot analyses of Col I, Col IV, Fn, PAI‐1 and TGF‐β expression in total cell extracts from MCT. K, Monocyte chemotactic protein‐1 (MCP‐1) concentration in MC supernatants measured by ELISA. L, Cell proliferation assay in cells transfected with PTPN2‐expressing adenovirus or control adenovirus after 48 h of incubation in LG or HG. LG: low glucose; HG: high glucose. Data are mean ± SEM of three experiments in duplicate. *P < 0.05 vs. LG + Vehicle; ^# P < 0.05 vs. HG + Vehicle