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. 2017 Feb;28(2):575-585.
doi: 10.1681/ASN.2016020237. Epub 2016 Sep 8.

Suppressor of Cytokine Signaling-1 Peptidomimetic Limits Progression of Diabetic Nephropathy

Carlota Recio  1   2   3 , Iolanda Lazaro  1   2 , Ainhoa Oguiza  1   2   3 , Laura Lopez-Sanz  1   2 , Susana Bernal  1   2 , Julia Blanco  4 , Jesus Egido  2   3 , Carmen Gomez-Guerrero  5   2   3
Affiliations

Suppressor of Cytokine Signaling-1 Peptidomimetic Limits Progression of Diabetic Nephropathy

Carlota Recio et al. J Am Soc Nephrol. 2017 Feb.

Abstract

Diabetes is the main cause of CKD and ESRD worldwide. Chronic activation of Janus kinase and signal transducer and activator of transcription (STAT) signaling contributes to diabetic nephropathy by inducing genes involved in leukocyte infiltration, cell proliferation, and extracellular matrix accumulation. This study examined whether a cell-permeable peptide mimicking the kinase-inhibitory region of suppressor of cytokine signaling-1 (SOCS1) regulatory protein protects against nephropathy by suppressing STAT-mediated cell responses to diabetic conditions. In a mouse model combining hyperglycemia and hypercholesterolemia (streptozotocin diabetic, apoE-deficient mice), renal STAT activation status correlated with the severity of nephropathy. Notably, compared with administration of vehicle or mutant inactive peptide, administration of the SOCS1 peptidomimetic at either early or advanced stages of diabetes ameliorated STAT activity and resulted in reduced serum creatinine level, albuminuria, and renal histologic changes (mesangial expansion, tubular injury, and fibrosis) over time. Mice treated with the SOCS1 peptidomimetic also exhibited reduced kidney leukocyte recruitment (T lymphocytes and classic M1 proinflammatory macrophages) and decreased expression levels of proinflammatory and profibrotic markers that were independent of glycemic and lipid changes. In vitro, internalized peptide suppressed STAT activation and _target gene expression induced by inflammatory and hyperglycemic conditions, reduced migration and proliferation in mesangial and tubuloepithelial cells, and altered the expression of cytokine-induced macrophage polarization markers. In conclusion, our study identifies SOCS1 mimicking as a feasible therapeutic strategy to halt the onset and progression of renal inflammation and fibrosis in diabetic kidney disease.

Keywords: Chronic inflammation; apolipoprotein E; diabetic nephropathy; fibrosis; macrophages; transcription factors.

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Figures

Figure 1.
Figure 1.
SOCS1 peptide inhibits STAT activation in diabetic kidneys. (A) Stat1, Stat3, and Socs1 gene expression in renal cortex from nondiabetic and diabetic mice in early (aged 16 weeks) and late (aged 34 weeks) disease checkpoints was analyzed using real-time PCR, normalized by 18S endogenous control and expressed in arbitrary units (a.u.). (B–D) Immunostaining of P-STAT1 and P-STAT3 in kidney sections from nondiabetic and diabetic mice (early model). Representative micrographs (B) and quantification of positive cells in glomeruli (C) and tubulointerstitium (D) are shown. Horizontal dotted lines represent the mean values for nondiabetic mice. (E) Western blot of P-STAT1 and P-STAT3 in renal cortical lysates in the early and late models of diabetes. Shown are representative images and the summary of normalized quantification, expressed in a.u. Diab, diabetic; ND, nondiabetic; Veh, vehicle. *P<0.05, **P<0.01, and ***P<0.001 versus Veh (16 weeks); †P<0.05 and ††P<0.01 versus Veh (34 weeks); #P<0.05 and ##P<0.01 versus Mu. Original magnification, ×200 in B.
Figure 2.
Figure 2.
SOCS1 peptide protects from diabetes-associated renal injury in apoE-deficient mice. (A) Albuminuria levels in apoE-deficient mice at early (age 16 weeks) and late (age 34 weeks) diabetes. (B) Gene expression of kidney injury molecule (Kim-1) in renal cortex was analyzed by real-time PCR, normalized by 18S endogenous control, and expressed in arbitrary units (a.u.). (C) Representative images of PAS-stained kidney sections from mice in the early (age 16 weeks; a–c) and late (age 34 weeks; d–f) diabetes models: nondiabetes (a), diabetes+vehicle (b and d), diabetes+S1 (c and e), and diabetes+Mu (f). Diabetic mice exhibited glomerular hypertrophy/PAS+ area expansion (arrows) and tubular atrophy/glycogen deposition (arrowheads). Milder damage was observed in S1 groups. (D) Glomerular area quantification in the experimental groups. (E) PAS+ mesangial area analysis. Veh, vehicle. *P<0.05, **P<0.01, and ***P<0.001 versus Veh (16 weeks); †P<0.05 and †††P<0.001 versus Veh (34 weeks); #P<0.05 and ##P<0.01 versus Mu. Horizontal dotted lines represent the mean values for nondiabetic mice in A, B, D, and E. Original magnification, ×200 in C.
Figure 3.
Figure 3.
Effect of SOCS1 peptide on diabetes-induced renal fibrosis. (A) Representative images of picrosirius red–sensitive collagen staining in renal sections from mice in the early (age 16 weeks; a–c) and late (age 34 weeks; d–f) diabetes models: nondiabetes (a), diabetes+vehicle (b and d), diabetes+S1 (c and e) and diabetes+Mu (f). Quantification of fibrosis (% picrosirius red area) in glomerular (Glom.) (B) and tubulointerstitial (TI) (C) compartments. Horizontal dotted lines represent the mean values for nondiabetic mice. (D) Real-time PCR analysis of type I collagen (Col I), fibronectin (Fn), and Tgfβ in renal cortex. Normalized values are expressed in arbitrary units (a.u.). (E) Western blot analyses of fibronectin (FN) and TGFβ expression in renal cortical lysates from diabetic mice. Shown are representative blots and the summary of normalized densitometric quantification. Bars represent the mean±SEM of 5–10 animals per group. Veh/V, vehicle. *P<0.05 and **P<0.01 versus Veh (16 weeks); †P<0.05, ††P<0.01, and †††P<0.001 versus Veh (34 weeks); #P<0.05 and ##P<0.01 versus Mu. Original magnification, ×100 in A.
Figure 4.
Figure 4.
SOCS1 peptide decreases inflammation in diabetic mice. (A–C) Histologic analysis of T lymphocytes and macrophages in kidney sections from diabetic mice (early model). (A) Representative micrographs. Quantification of CD3+ and F4/80+ cells in glomeruli (B) and interstitium (C). Horizontal dotted lines represent the mean values for nondiabetic mice. (D) Real-time PCR analysis of inflammatory genes in renal cortex. Normalized values are expressed in arbitrary units (a.u.). (E) Kidney chemokine levels measured by ELISA. (F) Gene expression levels of arginase isoforms (ArgII and ArgI) in diabetic kidneys. Real-time PCR data normalized by 18S are expressed in a.u. (G) Representative immunoblots and summary of the relative levels of ArgII and ArgI protein expression in renal lysates from diabetic mice. (H) Flow cytometry analysis of relative CD115+ monocyte population (Ly6Chigh and Ly6Clow) in peripheral blood. Bars represent the mean±SEM of 5–10 animals per group. Veh/V, vehicle. *P<0.05, **P<0.01, and ***P<0.001 versus Veh (16 weeks); †P<0.05 versus Veh (34 weeks); #P<0.05 versus Mu. Original magnification, ×200 in A.
Figure 5.
Figure 5.
SOCS1 peptide inhibits STAT activation and _target gene expression in vitro. Western blot analysis for P-STAT1 and P-STAT3 proteins in total cell extracts from MC and MCT stimulated with cytokines (60 minutes) (A) and HG (6 hours) (B) in the presence or absence of peptides (100 μg/ml). Representative immunoblots are shown and densitometry data expressed as fold increases over basal conditions (arbitrarily set to 1). Real-time PCR analysis of indicated genes in MCT at 24 hours of stimulation with cytokines (C) and HG (D). (E) CCL2 and CCL5 concentrations in MC supernatants measured by ELISA. (F) Western blot of fibronectin (FN) levels in culture supernatants. Bars represent the mean±SEM of 4–7 independent experiments. *P<0.05 versus basal; †P<0.05 versus stimulus; #P<0.05 versus Mu.
Figure 6.
Figure 6.
In vitro effects of SOCS1 peptide on cell migration, proliferation, and differentiation. (A) Analysis of MC migration by scratch-wound-healing assay. Representative phase-contrast images of cells migrating into the wounded area (dotted lines) at 0 hours and 16 hours of cytokine incubation in the absence or presence of peptides (S1 and Mu sequences, 100 μg/ml). The graph shows the results from quantification of covered healing areas over time. (B) Dose-dependent curves of peptides on cell viability (basal conditions) and proliferation (cytokine stimulation) in MCT (MTT assay, 48 hours). (C) Effect of peptides (100 µg/ml) on MC growth. (D) Antiproliferative effect of S1 peptide on HG-stimulated MCT. (E) Real-time PCR analysis of arginase isoforms (ArgII and ArgI) in bone marrow–derived macrophages. Data expressed as percentage or fold increases over basal conditions are mean±SEM (n=4–6 experiments). *P<0.05 versus basal; †P<0.05 versus stimulus; #P<0.05 versus Mu.
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