Suppressors of cytokine signaling abrogate diabetic nephropathy

Abstract

Activation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) is an important mechanism by which hyperglycemia contributes to renal damage, suggesting that modulation of this pathway may prevent renal and vascular complications of diabetes. Here, we investigated the involvement of suppressors of cytokine signaling (SOCS) as intracellular negative regulators of JAK/STAT activation in diabetic nephropathy. In a rat model, inducing diabetes resulted in JAK/STAT activation and increased expression of SOCS1 and SOCS3. In humans, we observed increased expression of glomerular and tubulointerstitial SOCS proteins in biopsies of patients with diabetic nephropathy. In vitro, high concentrations of glucose activated JAK/STAT/SOCS in human mesangial and tubular cells. Overexpression of SOCS reversed the glucose-induced activation of the JAK/STAT pathway, expression of STAT-dependent genes (chemokines, growth factors, and extracellular matrix proteins), and cell proliferation. In vivo, intrarenal delivery of adenovirus expressing SOCS1 and SOCS3 to diabetic rats significantly improved renal function and reduced renal lesions associated with diabetes, such as mesangial expansion, fibrosis, and influx of macrophages. SOCS gene delivery also decreased the activation of STAT1 and STAT3 and the expression of proinflammatory and profibrotic proteins in the diabetic kidney. In summary, these results provide direct evidence for a link between the JAK/STAT/SOCS axis and hyperglycemia-induced cell responses in the kidney. Suppression of the JAK/STAT pathway by increasing intracellular SOCS proteins may have therapeutic potential in diabetic nephropathy.

Figure 1.

Renal SOCS expression increases after diabetes induction in rats. (A) SOCS mRNA expression in renal samples from control and diabetic rats was determined by real-time PCR. (B) Representative Western blots for SOCS1, SOCS3, P-JAK2, P-STAT1, and P-STAT3 in control and diabetic rats. (C) Representative micrographs showing positive SOCS immunostaining in glomeruli (magnification: ×400) and tubulointerstitium (×200) of diabetic rats. Lower panel: Quantitative analysis data in control and diabetic groups. Data are mean ± SEM of three to five animals per group (*P < 0.05 versus control).

Figure 2.

SOCS proteins are expressed in renal biopsies of diabetic patients. (A) Representative micrographs of SOCS1 and SOCS3 immunostaining in serial sections of kidney biopsies from a patient with progressive diabetic nephropathy and a control subject. SOCS expression was increased in glomerular (magnification: ×400) and tubulointerstitial (×200) areas of a diabetic patient compared with the control (×100). (B) Quantitative analysis data in control and diabetic groups (mean ± SEM, n = 5 per group, *P < 0.05 versus control).

Figure 3.

HG induces SOCS expression in cultured renal cells. (A) HK2 cells were incubated for different time intervals in culture medium containing HG (30 mM d-glucose, black bars) or mannitol (10 mM d-glucose + 20 mM d-mannitol; gray bars). Gene expression of SOCS1 (upper panel) and SOCS3 (lower panel) was analyzed by real-time PCR and values were normalized by 18S expression. (B) Representative immunoblots and densitometric analysis showing time course of SOCS1, SOCS3, P-JAK2, and P-STAT1 induction by HG in human MCs. Fold increases versus basal conditions (10 mM d-glucose) are mean ± SEM of three to five experiments in duplicate (*P < 0.05 versus basal).

Figure 4.

SOCS inhibit JAK/STAT activation by HG in renal cells. (A) Human MCs were transfected with SOCS expression vectors (S1wt and S3wt) or empty plasmid (p513). (B) Tubular HK2 cells were infected with SOCS-expressing adenovirus (Ad-S1, Ad-S3) or control adenovirus (Ad-null). After 24 hours, cells were stimulated for an additional 24 hours with HG. Representative immunoblots and densitometric analysis of the indicated proteins are shown. Fold increases versus basal conditions (10 mM d-glucose) are mean ± SEM of three independent experiments. (C) Adenovirus-treated HK2 cells were transfected with the STAT-responsive luciferase vectors and then stimulated for 24 hours with HG. Data of relative luciferase units (Firefly/Renilla/mg protein) are mean ± SEM of three to five experiments. *P < 0.05 versus basal, #P < 0.05 versus control (empty plasmid or Ad-null).

Figure 5.

SOCS overexpression prevents HG-induced gene expression and cell proliferation. (A) Human MC and (B) tubular HK2 cells treated with SOCS-expressing adenovirus (Ad-S1, Ad-S3) or control adenovirus (Ad-null) were then incubated for 24 to 48 hours in medium containing HG or mannitol (M). The mRNA expression of the indicated genes was measured by real-time PCR, and values were normalized by 18S endogenous control. (C) Cell proliferation assay in cells transfected with SOCS expression vectors (S1wt and S3wt) or empty plasmid (p513) after 48 hours of incubation in basal conditions (B), HG or mannitol (M). Fold increases versus basal are mean ± SEM of three to six experiments. *P < 0.05 versus basal, #P < 0.05 versus control (Ad-null or empty plasmid).

Figure 6.

Adenovirus-mediated gene delivery increases SOCS expression in control rats. Representative fluorescence photomicrographs of (A) kidney and (B) liver of rats 5 days after Ad-GFP injection showing intense fluorescence compared with mild autofluorescence in the control group (Ad-null). Arrows and arrowheads in panel A indicate positive cells in glomerular and tubulointerstitial areas, respectively. (C) Real-time PCR analysis of SOCS gene expression in renal tissues 3 and 7 days after adenovirus injection (Ad-S1 and Ad-S3). Fold increases versus control conditions are mean ± SEM of six experiments (*P < 0.05 versus control). (D) Representative immunoblots of SOCS1 and SOCS3 protein expression in kidney of rats treated with Ad-S1 and Ad-S3.

Figure 7.

SOCS gene delivery ameliorates diabetes-induced renal damage. Renal histopathology in control nondiabetic rats, diabetic rats without treatment (sham-operation), treated with control adenovirus (Ad-null), and with SOCS-expressing adenovirus (Ad-S1 and Ad-S3). (A) Representative micrographs of renal sections stained with periodic acid–Schiff (glomerular capillary dilation, mesangial expansion, and tubular glycogen deposits are observed; magnification: ×400), picrosirius red (collagen deposition; ×200), and CD68 antibody (macrophage marker; ×400). (B) Semiquantitative assessment of renal lesions (periodic acid–Schiff score: 0 to 3). (C) Quantification of collagen-positive areas. (D) Number of CD68⁺ macrophages in all of the study groups. Upper and lower panels represent measurements in glomerular and tubulointerstitial areas, respectively. Data are mean ± SEM of n = 12 rats analyzed per group (*P < 0.05 versus control nondiabetic rats, #P < 0.05 versus sham-operated diabetic rats).

Figure 8.

SOCS gene delivery reduces STAT activation and expression of inflammatory and fibrotic genes in the diabetic kidney. (A) Representative micrographs of P-STAT1, P-STAT3, and TGFβ immunodetection in diabetic rat groups as specified. (B) Western blot analyses of P-STAT1, P-STAT3, MCP-1, and fibronectin (FN) levels in the renal cortex of control nondiabetic rats and STZ-induced diabetic rats (sham-operated, Ad-null, Ad-S1, and Ad-S3). Representative immunoblots and quantitative analyses are shown. (C) Real-time PCR analysis of the indicated genes in renal samples was assessed in duplicate and normalized by 18S endogenous control. Fold increases versus control rats are mean ± SEM (*P < 0.05 versus controls, #P < 0.05 versus sham-operated diabetic rats).