Glycation of Albumin, Not Glomerular Basement Membrane, Alters Permeability in an In Vitro Model BARBARA S. DANIELS AND ELIZABETH B. HAUSER
The effects of glycation of either albumin, a plasma protein, or GBM were examined in an in vitro model of GBM permeability. Albumin was incubated with glucose in vitro, and nonglycated and glycated albumin were separated by affinity chromatography. Rat GBM was glycated either in vivo after the induction of diabetes or in vitro after incubation with 25 mM glucose. 150 \ig of GBM was consolidated in an ultrafiltration cell, and albumin permeability across the GBM filter was assessed at an applied pressure (50 mmHg) selected to approximate glomerular capillary pressure in vivo. The sieving coefficient of glycated albumin was greater than the sieving coefficient of nonglycated albumin (0.25 ± 0.03 vs. 0.10 ± 0.02; P < 0.05). GBM glycated in vivo in diabetic rats exhibited native albumin and water permeability that was indistinguishable from that for GBM from control rats. Similarly, GBM glycated in vitro by incubation with 25 mM glucose exhibited water and albumin permeability identical to that for GBM incubated in buffer. Thus, the glycation of albumin, but not of GBM, leads to enhanced permeability in an in vitro GBM filtration system. Increased permeability of glycated albumin may contribute to albuminuria and/or renal injury in states of increased circulating glycated
From the Department of Medicine, University of Minnesota School of Medicine, Minneapolis, Minnesota. Address correspondence and reprint requests to Barbara S. Daniels, MD, Box 736 UMHC, University of Minnesota, Minneapolis, Minnesota 55455. Received for publication 8 October 1991 and accepted in revised form 7 May 1992. GBM, glomerular basement membrane; BSA, bovine serum albumin; J v , flux of water; Con A, concanavalin A; SDS, sodium dodecyl sulfate; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PMSF, phenuylmethylsulfonyl fluoride; cpm, counts per min; FITC, fluorescein isothiocyanate; CF, concentration of albumin in the filtrate; C M , concentration of albumin of the retained sample immediately adjacent to the membrane surface; CR, concentration of albumin assayed in the bulk retained sample; n GBM- number of points on glomerular basement membrane; n MM , number of points on mesangial matrix; B, polarization factor expressing the efficiency of stirring relative to membrane water permeability and macromolecule diffusivity; 6, sieving coefficient; HPLC, high-performance liquid chromatography; pi, pH of a substance at its isoelectric point; AGE, advanced glycosylation end product.
DIABETES, VOL. 41, NOVEMBER 1992
albumin such as diabetes and experimental galactosemia. Diabetes 41:1415-21,1992
T
he development of albuminuria and nephropathy in diabetes is related partly to the level of glycemic control. Patients with the best levels of glycemic control have only one-third the risk of developing nephropathy of those with the worse glycemic control (1). Protein glycation, the nonenzymatic reaction between glucose and an amino group in a protein, is one mechanism through which the structure and function of proteins may be altered in a manner dependent on the level of glycemic control and has been hypothesized to contribute to the genesis of neural and vascular complications (2). Experimental galactosemia, a model of protein glycation, results in albuminuria and histological abnormalities of the glomerular filtration surface, providing evidence that glycation may contribute to glomerular abnormalities in diabetes (3). Altered renal clearance of glycated albumin (4-7) has been reported in both normal, nondiabetic and diabetic subjects and could contribute to albuminuria and the initiation of glomerular injury in diabetes. Increased glycation of GBM has been described in both rats (8) and humans (9), and the glycation of important constituents of the basement membrane, such as laminin and collagen, alters their function (10,11) and conceivably could result in alterations in glomerular permeability. Glycation coexists with hemodynamic, metabolic, and hormonal abnormalities in diabetes, so determining the role of glycation in the genesis of abnormal glomerular permeability has been difficult. In addition, measurements of the renal clearance of a protein may not reflect changes in its glomerular permeance because the tubular reabsorption of glycated proteins may be altered independently of glomerular permeability. This study used an in vitro model of GBM filtration to determine the effects of the
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GLYCATION AND GBM PERMEABILITY
glycation of circulating and basement membrane proteins on GBM permeability. RESEARCH DESIGN AND METHODS Sprague Dawley rats were maintained on Purina rat chow with free access to water. Diabetes was induced with a single injection of streptozocin (65 mg/kg by tail vein). Blood glucose was measured 2 days later, and rats with levels >16.7 mM were assumed to be diabetic. Ultralente insulin (Lilly, Indianapolis, IN), 1.4 U subcutaneously, was administered daily to maintain the blood glucose at - 2 2 mM, and the rats were maintained for 1 mo. For the study with diabetic rats, control nondiabetic rats were followed concurrently. In a separate set of diabetic and control rats, urinary albumin excretion was determined on 24-h urine specimens 1 mo after the initiation of diabetes. Isolation of GBM. Rats were anesthetized with methohexital (100 mg/kg i.p.), and the kidneys were perfused in situ at 115 mmHg with 0.1 M Tris-buffered saline (pH 7.4) to remove blood. The renal capsule, extrarenal vessels, and papilla were dissected away and discarded. The cortex was diced into 1-mm3 bits and sequentially passed through 250-, 150-, and 75-|xm nylon filters to isolate glomeruli. The resulting glomerular preparation contained 95% of the glomeruli were devoid of Bowman's capsule and arterioles. GBM was isolated according to the method of Ligler and Robinson (12). Briefly, cellular elements were lysed with 0.5% A/-lauroylsarcosine, and residual gel-like nucleoprotein was removed with 0.015% DNase. All solutions were filtered (0.22-|x polysulfone filter, Millipore, Bedford, MA) immediately before use to remove contaminants. GBM isolated by this method in our laboratory retains immunofluorescence for heparan sulfate proteoglycan, type IV collagen, laminin, and fibronectin (13). Filtration system. We extensively modified a method for the in vitro determination of kidney basement membrane permeability (13) initially reported by Cotter and Robinson (14) such that microgram quantities of
The effects of glycation of either albumin, a plasma protein, or GBM were examined in an in vitro model of GBM permeability. Albumin was incubated with glucose in vitro, and nonglycated and glycated albumin were separated by affinity chromatography. Rat GBM was glycated either in vivo after the induction of diabetes or in vitro after incubation with 25 mM glucose. 150 \ig of GBM was consolidated in an ultrafiltration cell, and albumin permeability across the GBM filter was assessed at an applied pressure (50 mmHg) selected to approximate glomerular capillary pressure in vivo. The sieving coefficient of glycated albumin was greater than the sieving coefficient of nonglycated albumin (0.25 ± 0.03 vs. 0.10 ± 0.02; P < 0.05). GBM glycated in vivo in diabetic rats exhibited native albumin and water permeability that was indistinguishable from that for GBM from control rats. Similarly, GBM glycated in vitro by incubation with 25 mM glucose exhibited water and albumin permeability identical to that for GBM incubated in buffer. Thus, the glycation of albumin, but not of GBM, leads to enhanced permeability in an in vitro GBM filtration system. Increased permeability of glycated albumin may contribute to albuminuria and/or renal injury in states of increased circulating glycated
From the Department of Medicine, University of Minnesota School of Medicine, Minneapolis, Minnesota. Address correspondence and reprint requests to Barbara S. Daniels, MD, Box 736 UMHC, University of Minnesota, Minneapolis, Minnesota 55455. Received for publication 8 October 1991 and accepted in revised form 7 May 1992. GBM, glomerular basement membrane; BSA, bovine serum albumin; J v , flux of water; Con A, concanavalin A; SDS, sodium dodecyl sulfate; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PMSF, phenuylmethylsulfonyl fluoride; cpm, counts per min; FITC, fluorescein isothiocyanate; CF, concentration of albumin in the filtrate; C M , concentration of albumin of the retained sample immediately adjacent to the membrane surface; CR, concentration of albumin assayed in the bulk retained sample; n GBM- number of points on glomerular basement membrane; n MM , number of points on mesangial matrix; B, polarization factor expressing the efficiency of stirring relative to membrane water permeability and macromolecule diffusivity; 6, sieving coefficient; HPLC, high-performance liquid chromatography; pi, pH of a substance at its isoelectric point; AGE, advanced glycosylation end product.
DIABETES, VOL. 41, NOVEMBER 1992
albumin such as diabetes and experimental galactosemia. Diabetes 41:1415-21,1992
T
he development of albuminuria and nephropathy in diabetes is related partly to the level of glycemic control. Patients with the best levels of glycemic control have only one-third the risk of developing nephropathy of those with the worse glycemic control (1). Protein glycation, the nonenzymatic reaction between glucose and an amino group in a protein, is one mechanism through which the structure and function of proteins may be altered in a manner dependent on the level of glycemic control and has been hypothesized to contribute to the genesis of neural and vascular complications (2). Experimental galactosemia, a model of protein glycation, results in albuminuria and histological abnormalities of the glomerular filtration surface, providing evidence that glycation may contribute to glomerular abnormalities in diabetes (3). Altered renal clearance of glycated albumin (4-7) has been reported in both normal, nondiabetic and diabetic subjects and could contribute to albuminuria and the initiation of glomerular injury in diabetes. Increased glycation of GBM has been described in both rats (8) and humans (9), and the glycation of important constituents of the basement membrane, such as laminin and collagen, alters their function (10,11) and conceivably could result in alterations in glomerular permeability. Glycation coexists with hemodynamic, metabolic, and hormonal abnormalities in diabetes, so determining the role of glycation in the genesis of abnormal glomerular permeability has been difficult. In addition, measurements of the renal clearance of a protein may not reflect changes in its glomerular permeance because the tubular reabsorption of glycated proteins may be altered independently of glomerular permeability. This study used an in vitro model of GBM filtration to determine the effects of the
1415
GLYCATION AND GBM PERMEABILITY
glycation of circulating and basement membrane proteins on GBM permeability. RESEARCH DESIGN AND METHODS Sprague Dawley rats were maintained on Purina rat chow with free access to water. Diabetes was induced with a single injection of streptozocin (65 mg/kg by tail vein). Blood glucose was measured 2 days later, and rats with levels >16.7 mM were assumed to be diabetic. Ultralente insulin (Lilly, Indianapolis, IN), 1.4 U subcutaneously, was administered daily to maintain the blood glucose at - 2 2 mM, and the rats were maintained for 1 mo. For the study with diabetic rats, control nondiabetic rats were followed concurrently. In a separate set of diabetic and control rats, urinary albumin excretion was determined on 24-h urine specimens 1 mo after the initiation of diabetes. Isolation of GBM. Rats were anesthetized with methohexital (100 mg/kg i.p.), and the kidneys were perfused in situ at 115 mmHg with 0.1 M Tris-buffered saline (pH 7.4) to remove blood. The renal capsule, extrarenal vessels, and papilla were dissected away and discarded. The cortex was diced into 1-mm3 bits and sequentially passed through 250-, 150-, and 75-|xm nylon filters to isolate glomeruli. The resulting glomerular preparation contained 95% of the glomeruli were devoid of Bowman's capsule and arterioles. GBM was isolated according to the method of Ligler and Robinson (12). Briefly, cellular elements were lysed with 0.5% A/-lauroylsarcosine, and residual gel-like nucleoprotein was removed with 0.015% DNase. All solutions were filtered (0.22-|x polysulfone filter, Millipore, Bedford, MA) immediately before use to remove contaminants. GBM isolated by this method in our laboratory retains immunofluorescence for heparan sulfate proteoglycan, type IV collagen, laminin, and fibronectin (13). Filtration system. We extensively modified a method for the in vitro determination of kidney basement membrane permeability (13) initially reported by Cotter and Robinson (14) such that microgram quantities of
