Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in glomerular basement membrane heparan sulphate

Diabetologia, Feb 1995

In the present study we investigated whether glomerular hyperfiltration and albuminuria in streptozotocin-induced diabetic nephropathy in male Wistar-Münich rats are associated with changes in the heparan sulphate content of the glomerular basement membrane. Rats with a diabetes mellitus duration of 8 months, treated with low doses of insulin, showed a significant increase in glomerular filtration rate (p<0.01) and effective renal plasma flow (p<0.05), without alterations in filtration fraction or mean arterial blood pressure. Diabetic rats developed progressive albuminuria (at 7 months, diabetic rats (D): 42±13 vs control rats (C): 0.5±0.2 mg/ 24 h, p<0.002) and a decrease of the selectivity index (clearance IgG/clearance albumin) of the proteinuria (at 7 months, D: 0.20±0.04 vs C: 0.39±0.17, p<0.05), suggesting loss of glomerular basement membrane charge. Light- and electron microscopy demonstrated a moderate increase of mesangial matrix and thickening of the glomerular basement membrane in the diabetic rats. Immunohistochemically an increase of laminin, collagen III and IV staining was observed in the mesangium and in the glomerular basement membrane, without alterations in glomerular basement membrane staining of heparan sulphate proteoglycan core protein or heparan sulphate. Giomerular basement membrane heparan sulphate content, quantitated in individual glomerular extracts by a new inhibition ELISA using a specific anti-glomerular basement membrane heparan sulphate monoclonal antibody (JM403), was not altered (median (range) D: 314 (152–941) vs C: 262 (244–467) ng heparan sulphate/mg glomerulus). However, the amount of glomerular 4-hydroxyproline, as a measure for collagen content, was significantly increased (D: 1665 (712–2014) vs C: 672 (515–1208) ng/mg glomerulus, p<0.01). Consequently, a significant decrease of the heparan sulphate/4-hydroxyproline ratio (D: 0.21 (0.14–1.16) vs C: 0.39 (0.30–0.47), p<0.05) was found. In summary, we demonstrate that in streptozotocin-diabetic rats glomerular hyperfiltration and a progressive, selective proteinuria are associated with a relative decrease of glomerular basement membrane heparan sulphate. Functionally, a diminished heparan sulphate-associated charge density within the glomerular basement membrane might explain the selective proteinuria in the diabetic rats.

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Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in glomerular basement membrane heparan sulphate

Diabetologia Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in glomerular basement membrane heparan sulphate J. van den B o r n t 0 A . A . van Kraats 0 M. A . H. B a k k e r 0 K. J. M. A s s m a n n 0 L. P. W. J. van d e n H e u v e l 0 L H. V e e r k a m p 0 J. H. M. B e r d e n 0 0 1Department of Nephrology,University Hospital St. Radboud , Nijmegen , The Netherlands 2Department of Pathology,University Hospital St. Radboud , Nijmegen , The Netherlands 3Department of Pediatrics, University Hospital St. Radboud , Nijmegen , The Netherlands 4Department of Biochemistry,University of Nymegen , Nijmegen , The Netherlands Summary In the present study we investigated whether glomerular hyperfiltration and albuminuria in streptozotocin-induced diabetic nephropathy in male Wistar-Mtinich rats are associated with changes in the heparan sulphate content of the glomerular basement membrane. Rats with a diabetes mellitus duration of 8 months, treated with low doses of insulin, showed a significant increase in glomerular filtration rate (p < 0.01) and effective renal plasma flow (p < 0.05), without alterations in filtration fraction or mean arterial blood pressure. Diabetic rats developed progressive albuminuria (at 7 months, diabetic rats (D): 42+ 13 vs control rats (C): 0.5 + 0.2 mg/ 24 h, p < 0.002) and a decrease of the selectivity index (clearance IgG/clearance albumin) of the proteinuria (at 7 months, D: 0.20 + 0.04 vs C: 0.39 +_0.17, p < 0.05), suggesting loss of glomerular basement membrane charge. Light- and electron microscopy demonstrated a moderate increase of mesangial matrix and thickening of the glomerular basement membrane in the diabetic rats. Immunohistochemically an increase of laminin, collagen III and IV staining was observed in the mesangium and in the glomerular basement membrane, without alterations in glomerular basement membrane staining of heparan sulphate proteoglycan core protein or heparan sulphate. Glomerular basement membrane heparan sulphate content, quantitated in individual glomerular extracts by a new inhibition E L I S A using a specific anti-glomerular basement membrane heparan sulphate monoclonal antibody (JM403), was not altered (median (range) D: 314 (152-941) vs C: 262 (244467) ng heparan sulphate/mg glomerulus). However, the amount of glomerular 4-hydroxyproline, as a measure for collagen content, was significantly increased (D: 1665 (712-2014) vs C: 672 (5151208) ng/mg glomerulus, p < 0.01). Consequently, a significant decrease of the heparan sulphate/4-hydroxyproline ratio (D: 0.21 (0.14-1.16) vs C: 0.39 (0.30-0.47), p < 0.05) was found. In summary, we demonstrate that in streptozotocin-diabetic rats glomerular hyperfiltration and a progressive, selective proteinuria are associated with a relative decrease of glomerular basement membrane heparan sulphate. Functionally, a diminished heparan sulphate-associated charge density within the glomerular basement membrane might explain the selective proteinuria in the diabetic rats. [Diabetologia (1995) 38: 161-172] Glomerular basement membrane; heparan sulphate; collagen; glomerular filtration rate; albuminuria - 9 Springer-Verlag1995 Abbreviations: BW, Body weight; ERPF, effective renal plasma flow; GAG, glycosaminoglycan;GBM, glomerular basement membrane; GFR, glomerular filtration rate; HS, heparan sulphate; HSPG, heparan sulphate proteoglycan; IDDM, insulin-dependent diabetes mellitus; STZ, streptozotocin. Heparan sulphate (HS) is the anionic glycosaminoglycan side chain of heparan sulphate proteoglycan (HSPG), the major proteoglycan present in the glomerular basement m e m b r a n e (GBM) [ 1, 2 ]. Strong evidence indicates that this proteoglycan plays an important role in the maintenance of the charge-selective permeability properties of the glomerular capillary wall by electrostatic repulsion of anionic plasma proteins, especially albumin. This is clearly demonstrated by studies eliminating the presence or charge of G B M HS by heparitinase perfusion of the kidneys [3], intrarenal or intravenous injection of cationic probes [ 4-7 ] or antibodies against HS [8], all leading to an increased permeability of the GBM for albumin. Diabetic nephropathy ultimately develops in 3040 % of patients suffering from insulin-dependent diabetes mellitus (IDDM). Since diabetic nephropathy is initially characterized by microalbuminuria [ 9, 10 ], the significance of GBM H S P G for its development has been analysed in several studies. Moreover, the altered selectivity index of the proteinuria in patients with "incipient" diabetic nephropathy [ 11-14 ] suggested a decreased charge-dependent permeability of the GBM. Studies in humans revealed a relatively decreased content of HS and H S P G within the GBM of kidneys from I D D M patients [ 15-17 ]. A recent study using the cationic probe cuprolinic blue demonstrated a reduction of HS-associated anionic sites within the GBM, which wa (...truncated)


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Dr. J. van den Born, A. A. van Kraats, M. A. H. Bakker, K. J. M. Assmann, L. P. W. J. van den Heuvel, J. H. Veerkamp, J. H. M. Berden. Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in glomerular basement membrane heparan sulphate, Diabetologia, 1995, pp. 161-172, Volume 38, Issue 2, DOI: 10.1007/BF00400090