Immunohistochemical Quantification of Heparan Sulfate Proteoglycan and Collagen IV in Skeletal Muscle Capillary Basement Membranes of Patients With Diabetic Nephropathy

Abstract

In IDDM patients, an increased permeability of the glomerular capillaries has been associated with a general loss of negatively charged heparan sulfate proteo-glycans (HSPGs) within basement membranes (BMs). In the present study, we used immunohistochemical staining to quantify heparan sulfate (HS), HSPG core protein, and collagen IV in capillary basement membranes of skeletal muscle biopsies taken from 9 healthy control subjects (C) and 20 IDDM patients: 7 with normal albumin excretion rate (<30 mg/24 h) (D₀), 5 with incipient nephropathy (albumin excretion rate 30–300 mg/24 h) (D_x), and 8 with clinical nephropathy (albumin excretion rate >300 mg/24 h) (D₂). In the capillaries, staining was measured by a scanning and integrating microspectrophotometer. A significant difference in the absorbance of HS was found among the four subgroups (means ± SD): 0.477 ± 0.082 (C), 0.627 ± 0.031 (D₀), 0.542 ± 0.098 (D₁, and 0.371 ± 0.118 (D₂) (P = 0.006). Similarly, an overall significant difference in the absorbance of collagen IV was demonstrated (means ± SD): 0.836 ± 0.111 (C), 0.838 ± 0.300 (D₀), 0.970 ± 0.173 (D₁), and 0.512 ± 0.248 (D₂) (P = 0.02). No statistical difference in the absorbance of core protein was demonstrated among the groups. Within the diabetic groups, HS was inversely correlated to albumin-uria (r = −0.76, P = 0.003) and albuminuria corrected for creatinine clearance (r = −0.69, P = 0.008). Because, in IDDM patients with albuminuria, alterations of the content of HS and collagen IV within the capillary BM have been demonstrated immunohistochemically, not only in the glomerular filtration barrier, but also in the skeletal muscle capillary BM, we suggest that these changes reflect universal quantitative or qualitative alterations within the capillary filtration barrier.