Intern Med 43: 9C17, 2004. 0.1, respectively; 0.05; Physique 1B). Open in a separate window Physique 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 (* 0.05 control). The histologic distribution of SOCS1 and SOCS3 proteins was also evaluated in renal samples from diabetic nephropathy patients. In glomeruli, SOCS were observed mainly in podocytes and, to a lesser extent, in MCs. In the interstitial area, SOCS was expressed by proximal and distal tubular cells and inflammatory cells (Physique 2A). Quantification of positive staining in glomerular and tubulointerstitial compartments showed significant differences in diabetic patients compared with control subjects (Physique 2B). Additional micrographs of diabetic patients and animals are shown in Supplemental Physique I. Open in a separate window Physique 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, = 5 per group, * 0.05 control). SOCS Induction by Hyperglycemia Incubation of human glomerular MCs and human renal proximal tubular cells (HK2) under hyperglycemic conditions (medium made up of 30 mM d-glucose) time-dependently induced the gene and protein expression of SOCS compared with control conditions (medium with 10 mM d-glucose) (Physique 3, A and B). SOCS3 expression was maximal at 4 to 8 hours and then decreased to baseline, whereas SOCS1 was expressed later and remained elevated after longer incubation occasions (48 to 56 hours). In agreement with previous findings,26,27 hyperglycemia induced tyrosine phosphorylation of JAK2 and STAT1, peaking at 24 hours (Physique 3B). In all of the experiments presented in this study, no significant effects of hyperosmolarity were seen in cells incubated in medium with 20 mM d-mannitol (Figures 3A, ?A,5A,5A, ?A,5C,5C, and Supplemental Physique IIB). Open in a separate window Physique 3. HG induces SOCS expression in cultured renal cells. (A) HK2 cells were incubated for different time intervals in culture medium made up of 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 basal conditions (10 mM d-glucose) are mean SEM of NAV-2729 three to five experiments in duplicate (* 0.05 basal). Open in a separate window Physique 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 made up of HG or mannitol (M). The mRNA expression of the indicated genes was measured by real-time PCR, and values were normalized by NAV-2729 18S endogenous control. (C) NAV-2729 Cell proliferation assay in cells transfected with SOCS expression vectors (S1wt and S3wt) or vacant plasmid (p513) after 48 hours of incubation in RIEG basal conditions (B), HG or mannitol (M). Fold increases basal are mean SEM of three to six experiments. * 0.05 basal, # 0.05 control (Ad-null or empty plasmid). NAV-2729 SOCS Proteins Prevent HG-Induced JAK/STAT Activation To investigate the modulation of the.