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.

pressure and lipids can relieve DN symptoms, current treatments are insufficient to efficiently prevent the progression of DN from stage 3 to stage 4. 5 Hence, there is an urgent need for new strategies that interrupt mechanisms underlying the progression of stage 3 DN induced by hyperglycaemia.
Hyperglycaemia-induced inflammation pathways play central roles in the progression of stage 3 DN. [6][7][8] The major clinical and recognized hallmark of stage 3 DN is elevated albuminuria secretion (20 μg/min < urine albumin excretion rate < 200 μg/min). 9 The classical histological features of stage 3 DN include mesangial expansion, glomerular basement membrane thickening, tubule-interstitial fibrosis and lymphocytes influx. 10 Recent findings suggest that the basic underlying mechanisms of DN involve high-glucose (HG) induced production of inflammatory mediators in glomerular and tubular cells, which triggers lymphocytes infiltration, renal cell proliferation and extracellular matrix expansion. 11,12 Thus, there is a obvious need for new strategies that simultaneously ameliorate systemic metabolism disorder and renal micro-inflammation to slow the decline of renal function in DN.
Protein tyrosine phosphatase non-receptor type 2 (PTPN2) might be the key regulator that controls metabolism and micro-inflammation. [13][14][15] PTPN2, one of 17 intracellular and non-receptor PTPs, was originally cloned from a human T-cell cDNA library. 16 It is ubiquitously expressed (eg intestinal and renal epithelium, fibroblasts, hepatocytes). 17 It has been indicated that PTPN2 is indispensable for maintaining metabolic homeostasis, 13 and liver-specific PTPN2 deficiency promotes hepatic steatosis, obesity and insulin resistance (IR). 15 Evidence is emerging for the involvement of PTPN2 in the onset and progression of inflammatory diseases, such as Crohn's disease, T1DM and rheumatoid arthritis. 14,16,18,19 Although these results suggest the important role of PTPN2 in metabolic diseases and inflammation, it remains unknown whether PTPN2 contributes to the progression of DN. HG-induced STAT activation contributes to the expression of pro-inflammatory and pro-fibrotic factors in glomerular and tubular cells and infiltration by circulating inflammatory cells, which amplifies and perpetuates the inflammatory process in the kidney, finally resulting in the development and progression of DN. [20][21][22] PTPN2 is the key negative regulator that controls the magnitude and duration of STAT signalling through several mechanisms, including kinase inhibition and STAT binding. 23,24 Thus, PTPN2 may suppress the expression of pro-inflammatory cytokines via inhibiting STAT signalling pathway and thus ameliorate renal injury and fibrosis in DN.
In the present study, we constructed an early DN ApoE -/mouse model. We aimed to explore whether PTPN2 gene therapy could improve DN by regulating systemic metabolic disorders and inhibiting local inflammation in kidney.

| Diabetic model and in vivo experiments
Four-week-old male ApoE -/mice were randomly assigned to a control group and diabetic group. The control group was fed a normal diet; the diabetic groups received a high-fat (HF) diet (

| Intraperitoneal glucose tolerance test
Intraperitoneal glucose tolerance test (IPGTT) was performed to access glucose tolerance after mice fasted for 12 hours. A bolus of glucose (2 g/kg) was injected intraperitoneally. Blood glucose levels were obtained from the tail vein and measured with the OneTouch Glucometer (LifeScan, Milpitas, CA) at 0, 15, 30, 60 and 120 minutes after injection.

| Production of adenoviral vector
The recombinant pAdxsi adenovirus constitutively expressing PTPN2 was constructed using the pAdxsi Adenoviral System (Hanbio Biotechnology Co., Ltd., Shanghai, China). The PTPN2 cDNAs from mouse were inserted into pShuttle-CMV-EGFP vector. The pAdxsi vector adenovirus was used as the control vehicle virus. After amplification, viruses were purified, titered and stored at −80°C until used.

| Blood and urine examination
At the end of the experiment, the mice were fasted overnight and killed by an overdose of pentobarbital. Fasting blood glucose, total cholesterol (Chol), LDL-cholesterol (LDL-chol), HDL-cholesterol (HDL-chol), triglyceride (TG) and non-estesterified fatty acid (NEFA) were measured. Different serum enzymes were estimated following standard methods namely aspartate transaminase (AST) and alanine transaminase (ALT). 26 Blood urea nitrogen (BUN) and creatinine in the plasma and urinary albumin were estimated using standard kits (Nanjing Jiancheng, Nanjing, China). Red staining for fibrosis analysis. The vacuolized hepatic cells were quantified in at least 10 sections per mice by the Image J plugins cell counter and expressed as percentage of total cells to analyse liver pathological alteration. Image acquisition was performed on a confocal FV 1000 SPD laser scanning microscope (Olympus, Japan).

| Immunofluorescence staining and microscopy
The cryosections were blocked with 5% BSA blocking buffer and stained with antibodies against PTPN2, VEGF and CD31 at 4°C

| Statistical analyses
Values are presented as mean ± SEM. Results were compared by one-way ANOVA. P < 0.05 was considered statistically significant.

| Generation of mouse DN model
At the age of 4 weeks, the levels of blood glucose between the control and diabetic group were similar ( Figure 1A,B). At the age of 10 weeks, IR was induced after a 6-week HF diet, confirmed by IPGTT ( Figure 1A,B). At the age of 12 weeks, the HF/STZ-treatment resulted in frank hyperglycaemia, glucose tolerance and IR ( Figure 1A,B). The mean bodyweight was markedly higher for the diabetic mice than normal diet mice at the age of 4, 10, 20, 22 and 24 weeks ( Figure 1C).
Diabetes was associated with renal decline, as demonstrated by an increased serum creatinine, serum BUN and urine albumin-tocreatinine ratio ( Figure 1D). Histologic assessment of periodic acid-Schiff (PAS)-stained kidney samples revealed diabetes increased glomerular size and PAS + -mesangial area ( Figure 1E,F).
As shown in Figure 1G,

| PTPN2 gene therapy protected from diabetesassociated renal injury in ApoE -/mice
We then investigated the effects of PTPN2 gene therapy on renal dysfunctions in mice with established experimental DN. As shown in Figure 2A,B, PTPN2 gene therapy markedly reduced serum levels of creatinine and BUN in diabetic mice, indicating largely improved renal lesions after PTPN2 gene transfer. Moreover, the proteinuria level, a clinical predictor of renal injury in DN, was significantly ameliorated in diabetic mice after PTPN2 overexpression ( Figure 2C). In addition, PTPN2 gene therapy resulted in significant decrease of kidney/body weight ratio (Table 1).
Histological examination of HE and PAS-stained kidney samples showed that PTPN2 improved several morphologic changes within the glomerulus (hypercellularity, mesangial matrix expansion and capillary dilation), tubules (atrophy and degeneration) and interstitium (fibrosis and inflammatory infiltrate) of diabetic mice ( Figure 2D, Table 2). Digital quantification further indicated that PTPN2 gene therapy reduced glomerular size and PAS + -mesangial area ( Figure 2E). Collectively, these data suggested that PTPN2 overexpression effectively attenuated renal injury in experimental DN.

| PTPN2 gene therapy improved insulin resistance and metabolic disorders in diabetic mice
It has been well acknowledged that DN is an inflammatory disease triggered by disordered metabolism. Thus, we investigated whether PTPN2 could regulate metabolism in mice with established experimental DN. As shown in Figure 3A,B, PTPN2 gene therapy markedly improved frank hyperglycaemia, glucose tolerance and IR, indicating that better blood glucose control in diabetic mice by PTPN2 overexpression ( Figure 3A,B). It has been universally recognized that liver plays a central role in metabolic balance with numerous functions.
Thus, we then explored whether liver lesions induced by diabetes were improved after PTPN2 overexpression. As shown in Figure 3C (Table 1). These results indicated that PTPN2 gene transfer ameliorated hyperglycaemia and disordered metabolism, which exert a protective effect on DN.

| PTPN2 gene therapy reduced diabetesinduced renal inflammation
The induction of diabetes was associated with the recruitment, retention and activation of leucocytes in the mouse kidney, as indicated by increased expression of leucocyte markers and pro-inflammatory genes ( Figure 4A,C). PTPN2-treated mice showed a significant decrease in the number of infiltrating CD3 + T lymphocytes and F4/80 + macrophages ( Figure 4A,B). Moreover, PTPN2 gene therapy (CD206 and Arg I) the predominant macrophage marker in the PTPN2 overexpression group (Figure 4A,E). In summary, these results indicated that PTPN2 gene therapy effectively prevented diabetes-induced renal inflammation in DN.

| PTPN2 gene therapy decreased fibrosis in diabetic mice
Since overproduction of extracellular matrix is a hallmark of DN and results in glomerular sclerosis and interstitial fibrosis, we next explored whether PTPN2 could ameliorate renal lesions by preventing renal fibrosis. Diabetic mice showed progressive renal fibrosis with increased total collagen deposition ( Figure 5A,C).
PTPN2 gene therapy significantly improved renal pathological alterations and collagen accumulation, as assessed by Sirius Red staining and Masson staining ( Figure 5A,C, Table 2). In addition, the overexpression of Col I, Col IV, Fn, PAI-1, TGF-β and α-SMA in renal tissue of diabetic mice was markedly attenuated after PTPN2 gene therapy ( Figure 5B,C). Quantitative analysis by Western blotting also showed significant decreased protein expressions of Col I, Col IV, Fn, PAI-1, TGF-β and α-SMA in the kidney from diabetic mice after PTPN2 gene therapy ( Figure 5D,E). Thus, these results suggested that renal fibrosis was markedly improved after PTPN2 overexpression in DN.

| PTPN2 gene therapy inhibited STAT activation in diabetic kidneys
Mounting evidence has demonstrated that the STAT signalling pathway modulates a broad range of mediators participated in pro-inflammatory and pro-fibrotic factors and is an important mechanism through which hyperglycaemia contribute to DN. Thus, we explored whether PTPN2 could modulate STAT activation in DN. As shown in Figure 6A,E, the protein expression of PTPN2 in kidney was markedly increased in the PTPN2 overexpression group. Furthermore, immunohistochemistry to detect the activation status of STAT proteins in the kidney revealed an intense nuclear staining of phosphorylated STAT1 (P-STAT1) and P-STAT3 in glomeruli and tubule-interstitium of diabetic mice receiving vehicle, a significant reduction in PTPN2-treated mice ( Figure 6C,D).

Western blot analysis further confirmed diabetic mice treated with
Ad-PTPN2 showed a decrease in the tyrosine phosphorylation of STAT1 and STAT3 ( Figure 6E). Our results suggested PTPN2 overexpression inhibited activation of STAT signalling pathway, through which PTPN2 exerted anti-inflammatory effects in established experimental DN. In summary, these data indicated that PTPN2 gene therapy could exert comprehensive therapeutic effects on DN via improving disordered metabolism and abolishing renal STAT activation.

| PTPN2 gene therapy prevented diabetesinduced renal angiogenesis
Abnormal angiogenesis contributes to the formation of new vessels that exert pathological effects on DN. Immunofluorescence staining of CD31 on renal tissue showed increased peritubular capillary formation in diabetic mice ( Figure 7A,C). Meanwhile, glomerular capillary formation was also increased in diabetic mice ( Figure 7A,C). PTPN2 inhibited the increase of glomerular CD31 expression in diabetic mice ( Figure 7A,C). As shown in Figure 7B,D, diabetic mice exhibited increased VEGF expression in renal tissue.
PTPN2 overexpression inhibited VEGF expression in established experimental DN ( Figure 7B,D). These data indicated that PTPN2 gene therapy could improve DN through abolishing diabetes-induced renal angiogenesis.

| Hyperglycaemia caused PTPN2 downregulation and STAT activation in renal cells
To corroborate the experimental model we assessed, in vitro, the effect of PTPN2 on murine MC and MCT stimulated with high-glucose concentrations (HG, medium containing 30 mmol/L D-glucose) in an attempt to mimic the diabetic milieu. Therefore, we treated the cells with HG for 24 hours and performed immunofluorescence studies. As shown in Figure 8A

| PTPN2 inhibited HG-induced STAT activation, STAT-dependent genes and cell proliferation
To investigate the modulation of the STAT pathway by PTPN2 in renal cells, overexpression of PTPN2 protein was induced by adenovirus infection. As shown in Figure 9A To evaluate the functional consequences of inflammatory gene reduction, we next examined the effect of PTPN2 on cell proliferation-important processes involved in renal damage during DN. As shown in Figure 9L, the proliferative effect of HG on MC and MCT was prevented by PTPN2 overexpression.
These data suggested that PTPN2 could inhibit HG-induced STAT activation, STAT-dependent genes and cell proliferation in murine mesangial cells and tubuloepithelial cells.

| D ISCUSS I ON
In this study, we found that diabetic mice developed albuminuria, PTPN2 stands at the crossroad of multiple signalling mechanisms and has emerged as an interesting therapeutic target with regulation of metabolism and micro-inflammation. 13  Consistent with these results, we found that angiogenesis using CD31 as marker was significantly increased in kidney in the diabetic group. PTPN2 gene therapy significantly suppressed VEGF expression and angiogenesis in diabetic mice. The immature structure and function of neo-vascular increase glomerular filtration barrier and elevate permeability, resulting in 24-hour proteinuria, glomerular sclerosis, interstitial fibrosis by promoting extracellular matrix deposition. 34,39 In summary, PTPN2, as a critical regulator for metabolic disorder and inflammation, participated in DN. The kidney protective roles with PTPN2 overexpression indicate a potential role for PTPN2 analogue in treating DN.