Decreased glomerular expression of agrin in diabetic nephropathy and podocytes, cultured in high glucose medium.

AIM: A decrease in glomerular heparan sulfate (HS) proteoglycan (PG), without apparent decrease in HSPG core protein expression, has been reported to occur in diabetic nephropathy (DN). In most studies however, agrin, the major HSPG core protein in the glomerular basement membrane, has not been studied. This prompted us to study the glomerular expression of agrin in parallel to the expression of HS-glycosaminoglycans (GAG) in biopsies of patients with DN. Furthermore, the influence of glucose on agrin production in cultured podocytes and the expression of agrin in fetal kidneys was investigated. METHODS: Cryostat sections of renal biopsies from patients with DN (n = 8) and healthy controls (HC, n = 8), were stained for agrin and HS-GAG. Sections of fetal kidneys were double stained for agrin and CD35 or CD31. Stainings were performed by indirect immunofluorescence (IIF). The production of agrin by cultured human podocytes was tested by ELISA and IIF. RESULTS: The expression of agrin, detected by AS46, was significantly reduced in biopsies from patients with DN compared to HC (p < 0.01). Similar findings were observed when monoclonal antibody JM72 was used (p < 0.05). In addition, a significant reduction in the glomerular expression of HS-GAG was detected with JM403 in these patients (p < 0.01). Agrin is expressed in cultured podocytes, the expression hereof was reduced when the cells were cultured in the presence of 25 mM D-glucose (p < 0.01). In biopsies of human fetal kidneys, glomerular expression of agrin coincided with the expression of CD31. In early stages of glomerular differentiation there was a strong staining for agrin and CD31 while CD35 was only slightly positive. CONCLUSIONS: Our data argue against a selective dysregulation in HSPG sulfation in DN, but suggest a pivotal role for hyperglycemia in the downregulation of agrin core protein production.

Insulin resistance and hyperinsulinemia are already present in patients with incipient renal disease.

In uremic patients resistance to the action of insulin has been documented, but it is not known at what stage of renal disease it appears. We therefore examined 29 patients with IgA glomerulonephritis (IgAGN) and 21 patients with adult polycystic kidney disease (ADPKD) in different stages of renal failure, and in addition, healthy age-matched subjects. Insulin sensitivity and other variables of glucose metabolism were assessed using a frequent sampling intravenous glucose tolerance test (minimal-model technique). Glomerular filtration rate (GFR) was assessed in renal patients using the inulin-clearance technique. Mean insulin sensitivity index (SI), that is, insulin sensitivity, was significantly lower (P < 0.001) in all patients combined than in matched healthy subjects (N = 16; 14 males, mean age 42 +/- 3 years; mean SI 8.6 +/- 0.8 min-1 uU/ml). The mean SI was not significantly different in patients with renal disease of immune (IgAGN) or non-immune (ADPKD) origin, and it was not correlated with GFR (r = 0.01, P < 0.52), intact PTH (r = -0.23, P < 0.11) or calcitriol concentration (r = -0.03, P < 0.82). Consequently, the mean SI was similar in renal patients with GFR within the normal range (N = 19; 17 males, mean age 41 +/- 2 years; mean GFR 119 +/- 5 ml/min/1.73 m2; 5.1 +/- 0.7 min-1 uU/ml), in patients with mild to moderate renal failure (N = 16; 15 males, 46 +/- 3 years; 67 +/- 4 ml/min/1.73 m2; 5.1 +/- 0.7 min-1 microU/ml) and in patients with advanced renal failure (N = 15; 13 males, 46 +/- 3 years; 25 +/- 2 ml/min/1.73 m2; 4.7 +/- 0.6 min-1 uU/ml). Mean fasted plasma insulin concentration, the area under the curve for plasma insulin concentration (AUC) and total insulin delivery (TID) during the glucose tolerance test were significantly higher in patients than in healthy subjects, reflecting hyperinsulinemia in renal patients. Further, fasted plasma insulin concentration (r = -0.32, P < 0.009), AUC (r = -0.62, P < 0.0001) and TID (r = -0.34, P < 0.004) in patients were significantly correlated with insulin sensitivity (SI). The present data document that insulin resistance and concomitant hyperinsulinemia are present early in the course of renal disease, that is, even in patients with GFR within the normal range, irrespective of the type of renal disease. This observation may have potential implications with respect to the high cardiovascular morbidity and mortality in patients with renal disease.