Suppressors of Cytokine Signaling Abrogate Diabetic Nephropathy

BASIC RESEARCH www.jasn.org

Guadalupe Ortiz-Mun˜oz,* Virginia Lopez-Parra,* Oscar Lopez-Franco,* Paula Fernandez-Vizarra,* Ben˜at Mallavia,* Claudio Flores,† Ana Sanz,* Julia Blanco,‡ Sergio Mezzano,† Alberto Ortiz,* Jesus Egido,* and Carmen Gomez-Guerrero*

*Renal and Vascular Research Laboratory, Fundacion Jimenez Diaz, Autonoma University, Madrid, Spain; †Division of Nephrology, School of Medicine, Austral University, Valdivia, Chile; and ‡Department of Pathology, Hospital Clinico San Carlos, Madrid, Spain

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 BASIC RESEARCH 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. Overexpres- sion 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.

J Am Soc Nephrol 21: 763–772, 2010. doi: 10.1681/ASN.2009060625

Diabetic nephropathy, the most common cause vent the renal complications of diabetes except of ESRD, is thought to result from interaction for therapies that may slow the progression between metabolic and hemodynamic factors. through intensive control of glycemia and blood The pathologic changes in diabetic nephropathy pressure.1 Therefore, it is of great clinical impor- include renal hypertrophy and extracellular ma- tance to identify new therapeutic targets that trix accumulation, which contribute to glomerular sclerosis, which leads to proteinuria and renal Received June 17, 2009. Accepted January 4, 2010. failure through the tubular interstitial fibrosis.1 Published online ahead of print. Publication date available at The basic underlying mechanisms of diabetic ne- www.jasn.org. phropathy involve high-glucose (HG)-induced Correspondence: Dr. Carmen Gomez-Guerrero, Renal and Vas- production of cytokines and growth factors, cular Research Laboratory, Fundacion Jimenez Diaz, Avda. Reyes which promote leukocyte infiltration, renal cell Catolicos, 2, 28040 Madrid (Spain). Phone: 34-91-5504800, ext. proliferation, and matrix production.2–4 No 3168; Fax: 34-91-5442636; E-mail: [email protected] treatment options are currently available to pre- Copyright ᮊ 2010 by the American Society of Nephrology

J Am Soc Nephrol 21: 763–772, 2010 ISSN : 1046-6673/2105-763 763 BASIC RESEARCH www.jasn.org might lead to the prevention of diabetes-induced nephrop- RESULTS athy. Hyperglycemia triggers a series of intracellular events in Renal Expression of SOCS in Diabetic Nephropathy glomerular and tubular cells, including reactive oxygen spe- SOCS1 and SOCS3 were increased in the renal cortex of strep- cies generation, protein kinase C and mitogen-activated tozotocin (STZ)-induced diabetic rats at the mRNA (Figure protein kinase activation, and transcription factor induc- 1A) and protein level (n-fold versus controls: SOCS1, 4.5 Ϯ 0.7; tion.2,5–8 Acting through these different intracellular path- SOCS3, 2.1 Ϯ 0.2; P Ͻ 0.05; Figure 1B). Immunohistochem- ways, HG enhances proinflammatory and profibrotic fac- istry revealed a broad distribution of SOCS1 and SOCS3 in tors as well as cellular hypertrophy.1,2,9 Recent findings also glomerular mesangial cells (MCs), podocytes, and proximal suggest that exposure of renal cells to HG activates the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling cascade.6,10–13 The JAK/STAT pathway is an essential intracellular mechanism of cytokines and other stimuli that regulates gene expression and cellular activation, proliferation, and differentiation. Four JAK and seven STAT family members constitute the JAK/STAT system, and cell-specific JAK/ STAT combinations have been paired with each receptor type.14,15 Receptor engagement activates the associated JAK, which phosphorylates the receptor cytoplasmic domain to allow recruitment and tyrosine phosphorylation of STAT. Activated STATs dimerize and translocate into the nucleus to activate specific gene expression. The JAK/STAT pathway is controlled through different mechanisms16: receptor in- ternalization, protein tyrosine phosphatases, protein inhibitors of activated STAT, and suppressors of cytokine signaling (SOCS). SOCS comprise a family of eight intracellular, cytokine-inducible proteins (CIS; SOCS1 to SOCS7), each of which has a variable N-terminal domain, a central SH2 domain, and a conserved C-terminal SOCS box.14,15 SOCS family members, particularly SOCS1 and SOCS3, control the magnitude and duration of JAK/STAT signaling through several mechanisms, including direct JAK inhibition, STAT binding, and targeting receptor complex and other signaling proteins for proteasomal degradation.14,15,17 In addition to cytokines, SOCS1 and SOCS3 are induced by many pathologic stimuli (e.g., angiotensin II, chemokines, insulin, immunoglobulins, and lipoproteins), thus indicating their involvement in many biologic processes.15,18–21 Members of the JAK/STAT pathway have been claimed as new molecular targets of anti-inflammatory treatment in acute and chronic inflammatory diseases,22 and their activation is involved in the development of atherosclerosis, hypertension, and the renal and vascular complications of diabetes.12,23,24 In this study, we explored the negative regulation of the JAK/STAT pathway during diabetes-induced renal damage, focusing on the two main members of SOCS Figure 1. Renal SOCS expression increases after diabetes induc- family (SOCS1 and SOCS3). We first evaluated the renal tion in rats. (A) SOCS mRNA expression in renal samples from expression of SOCS1 and SOCS3 in human and experimen- control and diabetic rats was determined by real-time PCR. (B) Representative Western blots for SOCS1, SOCS3, P-JAK2, P- tal diabetic nephropathy. Next, we analyzed whether SOCS STAT1, and P-STAT3 in control and diabetic rats. (C) Represen- proteins modulate JAK/STAT-mediated gene expression tative micrographs showing positive SOCS immunostaining in and cell responses to hyperglycemia in cultured renal cells. glomeruli (magnification: ϫ400) and tubulointerstitium (ϫ200) of Finally, we investigated the effects of SOCS renal delivery by diabetic rats. Lower panel: Quantitative analysis data in control recombinant adenovirus on the progression of diabetic ne- and diabetic groups. Data are mean Ϯ SEM of three to five phropathy in rats. animals per group (*P Ͻ 0.05 versus control).

764 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 763–772, 2010 www.jasn.org BASIC RESEARCH tubular cells of diabetic rats (Figure 1C). Moreover, and con- imal at 4 to 8 hours and then decreased to baseline, whereas sistent with previous reports,13,25 diabetes induction resulted SOCS1 was expressed later and remained elevated after in the activation of the JAK/STAT pathway, as assessed by longer incubation times (48 to 56 hours). In agreement with JAK2, STAT1, and STAT3 tyrosine phosphorylation (n-fold previous findings,26,27 hyperglycemia induced tyrosine versus controls: 5.2 Ϯ 1.5, 8.5 Ϯ 2.5, and 1.8 Ϯ 0.1, respectively; phosphorylation of JAK2 and STAT1, peaking at 24 hours P Ͻ 0.05; Figure 1B). (Figure 3B). In all of the experiments presented in this The histologic distribution of SOCS1 and SOCS3 proteins study, no significant effects of hyperosmolarity were seen in was also evaluated in renal samples from diabetic nephropathy cells incubated in medium with 20 mM D-mannitol (Figures patients. In glomeruli, SOCS were observed mainly in podo- 3A, 5A, 5C, and Supplemental Figure IIB). cytes and, to a lesser extent, in MCs. In the interstitial area, SOCS was expressed by proximal and distal tubular cells and SOCS Proteins Prevent HG-Induced JAK/STAT inflammatory cells (Figure 2A). Quantification of positive Activation staining in glomerular and tubulointerstitial compartments To investigate the modulation of the JAK/STAT pathway by showed significant differences in diabetic patients compared SOCS in renal cells, overexpression of SOCS1 and SOCS3 with control subjects (Figure 2B). Additional micrographs of proteins was induced by plasmid transfection (S1wt, S3wt, diabetic patients and animals are shown in Supplemental Fig- and control p513) or adenovirus infection (Ad-S1, Ad-S3, ure I. and control Ad-null). In human MCs, SOCS overexpression significantly inhibited HG-induced JAK2 and STAT1 ty- In Vitro SOCS Induction by Hyperglycemia rosine phosphorylation (Figure 4A). Similarly, SOCS-ex- Incubation of human glomerular MCs and human renal pressing adenovirus prevented the STAT1 phosphorylation proximal tubular cells (HK2) under hyperglycemic condi- and STAT-transcriptional activity by HG in HK2 cells (Fig- tions (medium containing 30 mM D-glucose) time-depen- ure 4, B and C). dently induced the gene and protein expression of SOCS compared with control conditions (medium with 10 mM Inhibition of STAT-Dependent Genes and Cell D-glucose) (Figure 3, A and B). SOCS3 expression was max- Proliferation by SOCS Among the genes upregulated by HG in renal cells, we examined the effect of SOCS on the expression of classical STAT-responsive inflammatory genes, including chemokines, adhesion molecules, and cytokines.12,22 Adenovirus- mediated SOCS overexpression efficiently inhibited the induction of monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 and IL-6 by hyperglycemia in human MCs (Figure 5A) and HK2 cells (Supplemen- tal Figure IIA) as compared with control adenovirus. More- over, treatment with Ad-S1 and Ad-S3 prevented the expression of transforming growth factor-␤ (TGF␤), collagen IV, and fibronectin, which are profibrotic genes known to be activated by HG via JAK/STAT during diabetic ne- phropathy6,8–10,13,27,28 (Figure 5B and Supplemental Figure IIB). SOCS also significantly reduced the accumulation of fibronectin protein in HK2 cells (% inhibition at 48 hours in HK2: Ad-S1, 71 Ϯ 5; Ad-S3, 67 Ϯ 7, P Ͻ 0.05, n ϭ 3; not shown). Furthermore, the proliferative effect of HG on human MCs and HK2 cells was prevented by SOCS overexpression (Figure 5C).

In Vivo Gene Transfer of SOCS Figure 2. SOCS proteins are expressed in renal biopsies of dia- To test the efficiency of the in vivo gene transfer, adenoviruses betic patients. (A) Representative micrographs of SOCS1 and expressing green fluorescence protein (Ad-GFP) or SOCS pro- SOCS3 immunostaining in serial sections of kidney biopsies from teins (Ad-S1 and Ad-S3) were injected into normal rats. As a patient with progressive diabetic nephropathy and a control subject. SOCS expression was increased in glomerular (magnifi- shown in Figure 6, A and B, intense green fluorescence was cation: ϫ400) and tubulointerstitial (ϫ200) areas of a diabetic seen in kidney (glomerular and tubular cells) and liver of Ad- patient compared with the control (ϫ100). (B) Quantitative anal- GFP transfected rats as compared with control adenovirus. ysis data in control and diabetic groups (mean Ϯ SEM, n ϭ 5 per Moreover, real-time PCR and Western blot analyses con- group, *P Ͻ 0.05 versus control). firmed that the expression of SOCS1 and SOCS3 in renal tissue

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remained increased even at 7 days of gene transfer (Figure 6, C and D).

Effects of SOCS Gene Delivery in Diabetic Rats Adenovirus expressing SOCS (Ad-S1 and Ad-S3) or the control virus (Ad-null) was injected into STZ-induced diabetic rats 3 weeks after the onset of diabetes, and animals were studied after 4 weeks of gene delivery. At this time point, SOCS transgene expression did not vary considerably among the diabetic groups (not shown). Interestingly, treatment with Ad-S1 and Ad-S3 tended to normalize creatinine clearance, urinary albumin excretion, and weight loss in diabetic rats (Table 1). Histologic assessment in renal samples revealed that SOCS gene delivery ameliorated the Figure 3. HG induces SOCS expression in cultured renal cells. (A) HK2 cells pathologic changes associated with diabetes, in- were incubated for different time intervals in culture medium containing HG ϩ cluding glomerular hypertrophy, mesangial ex- (30 mM D-glucose, black bars) or mannitol (10 mM D-glucose 20 mM pansion, and tubular atrophy/dilation (Figure 7, D-mannitol; gray bars). Gene expression of SOCS1 (upper panel) and SOCS3 A and B). Accordingly, fractional kidney weight (lower panel) was analyzed by real-time PCR and values were normalized by 18S expression. (B) Representative immunoblots and densitometric analysis and glomerular volume (parameters of kidney showing time course of SOCS1, SOCS3, P-JAK2, and P-STAT1 induction by hypertrophy) were significantly lower in diabetic HG in human MCs. Fold increases versus basal conditions (10 mM D-glucose) rats treated with Ad-S1 and Ad-S3 versus Ad-null are mean Ϯ SEM of three to five experiments in duplicate (*P Ͻ 0.05 versus or sham-operated rats (Table 1). Furthermore, basal). analysis of collagen distribution by picrosirius red staining indicated that SOCS administration normalized the intraglomerular and tubulointerstitial fibrosis in diabetic rats (Figure 7, A and C). SOCS gene delivery also decreased the number of glomerular and interstitial macrophages in the diabetic kidneys (CD68 immunostaining; Figure 7, A and D).

SOCS Reduces STAT-Mediated Gene Expression in Diabetes Diabetic rats treated with Ad-S1 and Ad-S3 showed a decrease in the tyrosine phosphorylation of STAT1 and STAT3 (Figure 8, A and B). Furthermore, the renal expression of STAT-dependent genes associated with inflammation (MCP-1; regulated upon activation, normal T cell expressed and secreted; and intercellular adhesion molecule-1) and fibrosis (TGF␤ and col- Figure 4. SOCS inhibit JAK/STAT activation by HG in renal cells. (A) Human lagen IV) during diabetic nephropathy (Figure MCs were transfected with SOCS expression vectors (S1wt and S3wt) or empty 8C) were significantly reduced by SOCS treat- plasmid (p513). (B) Tubular HK2 cells were infected with SOCS-expressing ade- ment. SOCS gene transfer also diminished the novirus (Ad-S1, Ad-S3) or control adenovirus (Ad-null). After 24 hours, cells were protein levels of MCP-1 and fibronectin (Figure stimulated for an additional 24 hours with HG. Representative immunoblots and 8B) and the positive staining for TGF␤ in diabetic densitometric analysis of the indicated proteins are shown. Fold increases versus kidneys (% of total area: sham, 16.0 Ϯ 1.8; Ad- basal conditions (10 mM D-glucose) are mean Ϯ SEM of three independent null, 14.8 Ϯ 2.7; Ad-S1, 2.8 Ϯ 1.8; Ad-S3, 1.8 Ϯ experiments. (C) Adenovirus-treated HK2 cells were transfected with the STAT- Ͻ responsive luciferase vectors and then stimulated for 24 hours with HG. Data of 0.9; P 0.05 versus sham/null; Figure 8A). In relative luciferase units (Firefly/Renilla/mg protein) are mean Ϯ SEM of three to these studies, no significant differences were ob- five experiments. *P Ͻ 0.05 versus basal, #P Ͻ 0.05 versus control (empty served between sham-operated and Ad-null plasmid or Ad-null). groups.

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In agreement with previous reports in renal diseases,13,20,25,26,28 we observed JAK2, STAT1 and STAT3 activation in diabetic rats. However, this is the first time that the expression of negative regulators SOCS has been demonstrated in an in vivo setting of diabetic nephropathy. Thus, SOCS1 and SOCS3 expression was found increased in renal samples from diabetic patients and STZ-induced diabetic rats when compared with control subjects. Histologic examination revealed that SOCS proteins are produced by glomerular and tubular cells, thus indicating that both renal compartments are involved in the JAK/STAT-mediated responses of diabetic kidney. In this study, we also demonstrate activation of JAK/STAT/ SOCS axis by hyperglycemia in vitro. Consistent with previous data,6,10,13,26,27 HG increased the tyrosine phosphorylation of JAK/STAT members in human MCs and HK2 cells. Further- more, HG induced transcriptional activation of STAT1, STAT2, and STAT3. Along with STAT activation, HG tran- siently induced SOCS expression (gene and protein) in renal cells; SOCS1 expression was always preceded by SOCS3 induction. This suggests a dual negative regulatory system of SOCS in HG-stimulated renal cells, similar to the control of cytokine signaling17: SOCS3 first modulating the STAT-mediated response and SOCS1 as a regulator of activated pathways after longer exposure to hyperglycemia. Previous findings suggest that the JAK/STAT pathway, es- pecially the JAK2/STAT1/STAT3-dependent axis, contributes to HG-mediated renal cell responses, including enhanced expression of genes involved in leukocyte infiltration, cell growth, and fibrosis.6,9,11,22,28 Moreover, diverse approaches Figure 5. SOCS overexpression prevents HG-induced gene ex- including JAK2 inhibition, STAT1 antisense oligonucleotides, pression and cell proliferation. (A) Human MC and (B) tubular HK2 STAT3 gene knockdown, and some drugs have been proven to cells treated with SOCS-expressing adenovirus (Ad-S1, Ad-S3) or counteract the harmful effects of JAK/STAT activation in cul- control adenovirus (Ad-null) were then incubated for 24 to 48 tured renal cells6,10,13,18,22,29 and in the development of diabetes hours in medium containing HG or mannitol (M). The mRNA 13,25,26,28 27 expression of the indicated genes was measured by real-time in vivo. Recently, Shi et al. reported that SOCS1 in- PCR, and values were normalized by 18S endogenous control. (C) hibits JAK/STAT activation and gene expression by hypergly- Cell proliferation assay in cells transfected with SOCS expression cemia in human MCs. In agreement with this, we demonstrate vectors (S1wt and S3wt) or empty plasmid (p513) after 48 hours of that SOCS1 and SOCS3 overexpression (plasmid and adeno- incubation in basal conditions (B), HG or mannitol (M). Fold in- virus vector systems) effectively prevented the JAK2/STAT1/ creases versus basal are mean Ϯ SEM of three to six experiments. STAT3 phosphorylation and the STAT transcriptional activity *P Ͻ 0.05 versus basal, #P Ͻ 0.05 versus control (Ad-null or empty induced by HG in human MCs and tubular cells. However, we plasmid). cannot discard that in addition to SOCS, other intracellular mechanisms (e.g., the constitutive inhibitors protein tyrosine DISCUSSION phosphatases and protein inhibitors of activated STAT12,13,16) may act in conjunction or independently in controlling the The JAK/STAT pathway regulates a wide range of genes in- JAK/STAT pathway in renal cells. volved in cell proliferation, inflammation, and fibrosis and Excessive production of cytokines and growth factors is in- is an important mechanism by which hyperglycemia con- volved in the pathogenesis of diabetic nephropathy, with cell tributes to nephropathy associated with diabetes.9,12,28 In growth and fibrosis being the major contributors to the patho- this study, we show that SOCS proteins, negative regulators logic changes associated with this disease.2–4 JAK/STAT path- of the JAK/STAT pathway, are induced by hyperglycemia in way activation mediates the mitogenic and fibrotic actions of renal cells. Furthermore, SOCS proteins reduce harmful cytokines, angiotensin II, and hyperglycemia in the kidney.12 JAK/STAT-mediated cell responses in the diabetic kidney, Moreover, several groups, including ours, have previously thus suggesting the potential benefit of SOCS to halt the demonstrated that the SOCS family regulates signaling by cy- progression of diabetic nephropathy. tokines, angiotensin II, immune complexes, and HG in human

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kines, thus unveiling the importance of the JAK/ STAT/SOCS axis in inflammatory responses to hyperglycemia. Evidence is emerging for the involvement of SOCS in inflammatory diseases (e.g., rheumatoid arthritis, cerebral ischemia, heart failure, hypertension, and vascular and renal injury).15,20,34–36 A recent report correlates the increased expression of SOCS1 and SOCS3 in mononuclear cells with decreased renal function in chronic kidney disease patients, thus proposing SOCS as a new cardiovascu- lar risk marker in these patients.37 Moreover, SOCS3 expression increased in insulin-sensitive tissues from type 2 diabetic patients, although it re- mains to be established whether this is a cause or a consequence of insulin resistance.38 Different studies demonstrated that inhibition of SOCS expression leading to sustained STAT activation contrib- Figure 6. Adenovirus-mediated gene delivery increases SOCS expression in utes to the progression of chronic inflammatory control rats. Representative fluorescence photomicrographs of (A) kidney and (B) diseases.18,20,36,39–41 By contrast, transgenic expres- liver of rats 5 days after Ad-GFP injection showing intense fluorescence com- sion of SOCS1 in ␤ cells protects from T cell- pared with mild autofluorescence in the control group (Ad-null). Arrows and mediated damage in diabetic mice,42 whereas arrowheads in panel A indicate positive cells in glomerular and tubulointerstitial areas, respectively. (C) Real-time PCR analysis of SOCS gene expression in renal adenovirus-mediated SOCS expression re- 15,34,43 tissues 3 and 7 days after adenovirus injection (Ad-S1 and Ad-S3). Fold increases duces arthritis and innate immune re- 44 versus control conditions are mean Ϯ SEM of six experiments (*P Ͻ 0.05 versus sponses. Recently, a mimetic peptide containing control). (D) Representative immunoblots of SOCS1 and SOCS3 protein expres- the kinase inhibitory region of SOCS1 was proven sion in kidney of rats treated with Ad-S1 and Ad-S3. to effectively protect against virus infection.45 On the basis of these observations and our in vitro data, we speculate that SOCS are induced in the diabetic MCs.14,18,20,27 In this work, we confirm that the SOCS endog- kidney as a compensatory but not sufficient mechanism to sup- enous pathway controls JAK/STAT-dependent events in HG- press renal damage. Therefore, strategies to upregulate SOCS ex- stimulated MCs; however, this is interestingly the first descrip- pression in the kidney may have therapeutic value for experimen- tion of the involvement of SOCS1 and SOCS3 in the control of tal diabetic nephropathy. Because transient expression and tubular cell responses to hyperglycemia, including JAK/STAT immune response are the major limitations of adenovirus-medi- activation, cytokine and matrix protein expression, and cell ated gene therapy in chronic diseases,43,44,46 we studied the effects growth. This reinforces the concept that, in addition to glo- of a single-treatment strategy on early diabetic nephropathy. merular cells, tubular cells are a primary target of hyperglyce- Transgene overexpression was detected up to 7 days after adeno- mia and chronic exposure to elevated glucose levels contrib- virus injection, thereafter gradually diminishing within the next 4 utes to the tubulointerstitial changes seen in overt diabetic weeks, a normal phenomenon related to the one-time gene trans- nephropathy.2,6,8 fection in vivo.46 Meanwhile, the transient expression of SOCS in Although diabetic nephropathy is traditionally considered a diabetic rats exerts a beneficial effect on renal damage that tran- nonimmune disease, accumulating evidence indicates that im- scends the duration of adenovirus-mediated SOCS expression. In munoinflammatory mechanisms play a crucial role in its de- fact, renal delivery of SOCS1 and SOCS3 attenuated STAT1 and velopment and progression. In this sense, macrophages have STAT3 activity, ameliorated albuminuria and renal lesions, and been implicated in the pathogenesis of diabetic nephropathy reduced the expression of STAT-dependent genes involved in leu- by direct interaction with mesangial and tubular cells or by kocyte infiltration, cell proliferation, and fibrosis during diabetic releasing factors involved in cell proliferation and matrix pro- nephropathy. SOCS gene therapy also reduced the glomerular duction.3 Moreover, expression of genes involved in the re- hyperfiltration associated with early diabetic nephropathy, prob- cruitment, infiltration, and activation of leukocytes has been ably by decreasing hyperplasia and hypertrophy, components of observed in experimental diabetic nephropathy in parallel with the increased glomerular volume that are altered by hyperglyce- disease progression and in cultured renal cells under hypergly- mia via the JAK/STAT pathway.6,12,21,23 cemic conditions4,30,31; their inhibition abolished diabetes-in- In summary, our data provide direct evidence for linkages duced renal damage.32,33 Our study demonstrates that SOCS between the JAK/STAT/SOCS axis and hyperglycemia-in- are able to inhibit the renal expression of STAT-dependent duced cell responses in the diabetic kidney. Moreover, the genes, including adhesion molecules, chemokines, and cyto- SOCS family of intracellular inducible proteins plays a critical

768 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 763–772, 2010 www.jasn.org BASIC RESEARCH

Table 1. Functional and structural data from control and diabetic rats Diabetes Control Sham Ad-Null Ad-S1 Ad-S3 Blood glucose (mg/dl) 95 Ϯ 8 581 Ϯ 8a 567 Ϯ 20a 576 Ϯ 18a 587 Ϯ 9a Body weight (g) 283 Ϯ 13 209 Ϯ 14a 196 Ϯ 7a 235 Ϯ 15ab 230 Ϯ 14ab Creatinine clearance (ml/min/kg) 1.42 Ϯ 0.28 3.34 Ϯ 0.26a 3.23 Ϯ 0.17a 2.01 Ϯ 0.14b 2.12 Ϯ 0.22b Urinary albumin (mg albumin/mg creatinin) 0.19 Ϯ 0.04 9.72 Ϯ 3.16a 9.47 Ϯ 1.48a 2.76 Ϯ 0.64ab 4.75 Ϯ 1.07ab Kidney-to-body-weight ratio (g/kg) 8.87 Ϯ 0.43 12.90 Ϯ 0.57a 13.41 Ϯ 0.77a 9.71 Ϯ 1.07b 9.42 Ϯ 0.85b Glomerular volume (␮m3 ϫ 106) 0.45 Ϯ 0.03 1.04 Ϯ 0.06a 1.00 Ϯ 0.03a 0.69 Ϯ 0.04ab 0.61 Ϯ 0.05ab aP Ͻ 0.05 versus control nondiabetic rats. bP Ͻ 0.05 versus sham-operated diabetic rats.

CONCISE METHODS

Reagents D-glucose, D-mannitol, and STZ were obtained from Sigma Chemical (St. Louis, MO). Primary antibodies for immunohistochemistry included SOCS1, SOCS3, and TGF␤1 (Santa Cruz Bio- technology, Santa Cruz, CA); phosphotyrosine (P)-STAT1 (Biosource International, Cama- rillo, CA); and P-STAT3 (Cell Signaling, Bev- erly, MA). Primary antibodies for Western blot included SOCS1 and P-STAT1 (Zymed Labora- tories, South San Francisco, CA); SOCS3, P-JAK2, and P-STAT3 (Santa Cruz); fibronectin (Chemicon, Temecula, CA); MCP-1 (Pepro- tech, Rocky Hill, NJ); and ␣-tubulin (Sigma).

Construction of Adenoviral Vectors The first generation of adenoviruses regulated by the cytomegalovirus promoter containing mouse SOCS1 and SOCS3 genes (Ad-S1 and Ad-S3) were prepared using the AdEasyTM vector system (Qbiogene, Inc., Carlsbad, CA). In brief, mouse SOCS1 and SOCS3 cDNAs were excised from the p513HA vector20 and inserted into pShuttle-cytomegalovirus transfer vector. The resulting plasmids were cotransformed into Figure 7. SOCS gene delivery ameliorates diabetes-induced renal damage. Renal bacteria together with pAdEasy-1 (E1 and E3 histopathology in control nondiabetic rats, diabetic rats without treatment (sham- deleted). The recombinant adenoviral con- operation), treated with control adenovirus (Ad-null), and with SOCS-expressing adenovirus (Ad-S1 and Ad-S3). (A) Representative micrographs of renal sections stained structs were cleaved with PacI and transfected with periodic acid–Schiff (glomerular capillary dilation, mesangial expansion, and tu- into HEK293 cells to produce viral particles. Pu- bular glycogen deposits are observed; magnification: ϫ400), picrosirius red (collagen rification and titration of each virus were made deposition; ϫ200), and CD68 antibody (macrophage marker; ϫ400). (B) Semiquanti- using Adeno-X Virus Kits (BD Biosciences tative assessment of renal lesions (periodic acid–Schiff score: 0 to 3). (C) Quantification Clontech, Palo Alto, CA). Ad-GFP and Ad-null ϩ of collagen-positive areas. (D) Number of CD68 macrophages in all of the study were used as control vectors. groups. Upper and lower panels represent measurements in glomerular and tubulo- Ϯ ϭ interstitial areas, respectively. Data are mean SEM of n 12 rats analyzed per group Cell Cultures Ͻ Ͻ (*P 0.05 versus control nondiabetic rats, #P 0.05 versus sham-operated diabetic Human MCs were cultured in RPMI 1640 with rats). 25 mM HEPES (pH 7.4) supplemented with role in the regulation of inflammation, abnormal cell prolifer- 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM ation, and matrix synthesis at early stages of diabetic nephrop- glutamine (Life Technologies, Paisley, Scotland, United Kingdom) athy. Compensatory suppression of the JAK/STAT pathway by and 5 ␮g/ml transferrin, 5 ␮g/ml bovine insulin, and 5 ng/ml sodium increasing SOCS may have a therapeutic potential in this dis- selenite (Sigma), as described.18,20 The HK2 cell line (American Type ease. Culture Collection, Rockville, MD) was cultured in RPMI 1640 with

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activity was assayed using a luminometer, and data were normalized for protein content.18,20

Cell Proliferation Assay Quiescent cells on 96-well plates were incubated under hyperglycemic conditions for 48 hours. Cell proliferation was measured by colori- metric assay.21

Human Renal Biopsies Kidney samples were obtained from patients with type 2 diabetes mellitus (n ϭ 5) that were submitted to renal biopsy because of ne- phrotic proteinuria.30 Clinical data at the biopsy are as follows: mean age ϭ 59 years (range 50 to 65 years); mean serum creatinine ϭ 2.31 mg/dl (range 1.2 to 3.4 mg/dl); mean proteinuria ϭ 7.6 g/d (range 2.3 to 10.5 g/d). Biopsies showed a clear picture of diffuse or nodular glomerulosclerosis with a marked tubulointerstitial involvement. Samples from minimal change disease patients were used as a control group (n ϭ 5).

Experimental Diabetic Nephropathy Diabetes was induced in male Wistar rats (220 to 250 g) by intraperi- toneal injection of STZ (50 mg/kg in 10 mM citrate buffer, pH 4.5) as described.31 Blood glucose levels were measured 48 hours after STZ injection. Only animals with glucose levels Ͼ350 mg/dl were included in the studies. To prevent animal death,2Uofinsulin were adminis- tered weekly 7 days after STZ injection (blood glucose levels Ͼ 400 mg/dl). After 6 months, diabetic rats (n ϭ 5) were killed and renal samples were processed. Age-matched nondiabetic rats were used as a control group (n ϭ 5).

Figure 8. SOCS gene delivery reduces STAT activation and Renal Delivery of SOCS-Expressing Adenovirus The adenovirus-mediated transduction in vivo was performed by in- expression of inflammatory and fibrotic genes in the diabetic kidney. (A) Representative micrographs of P-STAT1, P-STAT3, jection into the left renal vein. In brief, rats were anesthetized and the ϫ 9 and TGF␤ immunodetection in diabetic rat groups as specified. left kidney was exposed by midline incision. Adenoviruses (1 10 (B) Western blot analyses of P-STAT1, P-STAT3, MCP-1, and viral particles per rat) were infused into the renal vein over 7 minutes fibronectin (FN) levels in the renal cortex of control nondiabetic and then blood flow to the kidney was restored. For assessing the rats and STZ-induced diabetic rats (sham-operated, Ad-null, Ad- efficiency and localization of adenovirus, initial experiments were S1, and Ad-S3). Representative immunoblots and quantitative performed in control rats infused with Ad-GFP, Ad-null, Ad-S1, and analyses are shown. (C) Real-time PCR analysis of the indicated Ad-S3 (n ϭ 6 per group) and kidneys were removed after 3, 5, and 7 genes in renal samples was assessed in duplicate and normalized days of delivery. For studying the effects of SOCS on diabetic ne- by 18S endogenous control. Fold increases versus control rats are phropathy, 3 weeks after the STZ injection diabetic rats were ran- mean Ϯ SEM (*P Ͻ 0.05 versus controls, #P Ͻ 0.05 versus sham- domly distributed into four groups (n ϭ 12 per group): sham opera- operated diabetic rats). tion, control adenovirus (Ad-null), SOCS1 adenovirus (Ad-S1), and 10% FCS. Quiescent, subconfluent cells were stimulated in culture SOCS3 adenovirus (Ad-S3). Nondiabetic rats were used as a control ϭ medium supplemented with D-glucose (final concentration 30 mM) group (n 12). Four weeks after injection, blood and 24-hour urine using D-mannitol as an osmolarity control. Transient expression of samples were collected and the animals were killed. After saline per- SOCS1 and SOCS3 proteins in cells was performed by plasmid trans- fusion, kidneys were dissected, weighed, and processed for histology fection (S1wt, S3wt, and control vector p513)18,21 and adenovirus and RNA expression. Creatinine clearance was calculated from the infection (Ad-S1, Ad-S3, and Ad-null; multiplicity of infection ϭ 40) formula of UV/P ϫ 1/1440 minutes, where U represents the urine for 24 hours before stimulation. concentration, V is the urine volume, and P is the plasma concentration. The results were then corrected for body weight. The concentra- Luciferase Assay tion of urine albumin was measured by ELISA (Cell Trend, Lucken- Cells were cotransfected with luciferase plasmids containing the walde, Germany) and values were normalized by creatinine STAT1, STAT1/2, and STAT3 binding sites and the pRenilla-TK vec- concentration. All studies were performed in accordance to the Euro- tor and then stimulated for an additional 24 hours. Dual luciferase pean Union normative.

770 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 763–772, 2010 www.jasn.org BASIC RESEARCH

Histology and Immunohistochemistry DISCLOSURES Cryostat sections of unfixed, snap-frozen specimens from Ad-GFP- None. injected rats were analyzed under the fluorescence microscope. Par- affin-embedded kidney sections (5-␮m thick) were used for histologic analysis. Renal lesions in periodic acid–Schiff and Masson’s trichrome stained sections were scored on a scale of 0 to 3 according to REFERENCES the extent of glomerular changes (hypertrophy, hypercellularity, and mesangial expansion) and tubular dilation as follows: 0 ϭ absent, 1 ϭ 1. Forbes JM, Fukami K, Cooper ME: Diabetic nephropathy: Where hemodynamics meets metabolism. Exp Clin Endocrinol Diabetes 115: mild, 2 ϭ moderate, and 3 ϭ severe. Renal fibrosis was examined by 69–84, 2007 polarized light microscopy after picrosirius red staining. Immunode- 2. 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772 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 763–772, 2010 Online Figure I (Ortiz-Muñoz et al.) A SOCS1 SOCS3

B SOCS1 SOCS3

Renal expression of SOCS proteins in human and experimental diabetic nephropathy. Representative micrographs of SOCS1 and SOCS3 immunostaining in sections of kidney biopsies of progressive diabetic nephropathy patients (A) and STZ- induced diabetic rats (B). SOCS expression was increased in glomerular

(magnification x400) and tubulointerstitial (x200) areas. Online Figure II (Ortiz-Muñoz et al.)

A C 3.5 Ad-null 2.0 SOCS1 S1(B) 3.0 * * Ad-S1 S1(HG) null(HG) * Ad-S3 2.5 Rn 1.2 * null(B) 2.0 # * # # 0.4 1.5 # 2.8 # * # SOCS3 # # # # S3(B) 1.0 2.0 S3(HG) null(HG) mRNA (n-fold vs basal) 0.5 Rn 1.2 null(B) 24 48 24 48 24 48 hours 0.4 MCP-1 ICAM-1 IL-6 25 30 35 40 Cycle B 2.5 Ad-null Ad-S1 2.0 * Ad-S3 * 1.5

# # # # 1.0 mRNA (n-fold basal) vs mRNA 0.5 MHGMHG TGFβ Fibronectin

Effects of SOCS1 and SOCS3 on hyperglycemia-induced gene expression. Human tubular HK2 cells (A, C) and mesangial cells (B) treated with SOCS expressing adenovirus (Ad-S1, Ad-S3) or control adenovirus (Ad-null) were then incubated for 24-48 h in basal conditions or 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. Fold increases vs basal are mean±SEM of 5 experiments.

Symbols: *, P<0.05 vs basal; #, P <0.05 vs control (Ad-null or empty plasmid). (C)

Representative curves of SOCS1/SOCS3 fluorescence intensity vs cycle number in adenovirus-infected HK2 cells after 24 h stimulation with HG.