Anionic Sites In Glomerular Basement Membrane Research Articles

Podocyte-specific deletion of NDST1, a key enzyme in the sulfation of heparan sulfate glycosaminoglycans, leads to abnormalities in podocyte organization in vivo

Heparan sulfate proteoglycans have been shown to modulate podocyte adhesion to- and pedicel organization on- the glomerular basement membrane. Recent studies showed that foot process effacement developed in a mutant mouse model whose podocytes were unable to assemble heparan sulfate glycosaminoglycan chains. This study, a further refinement, explored the role of heparan N-sulfation on podocyte behavior. A novel mutant mouse (Ndst1-/-) was developed, having podocyte-specific deletion of NDST1, the enzyme responsible for N-sulfation of heparan sulfate chains. Podocytes having this mutation had foot process effacement and abnormal adhesion to Bowman's capsule. Although glomerular hypertrophy did develop in the kidneys of mutant animals, mesangial expansion was not seen. The lack of heparan N-sulfation did not affect the expression of agrin or perlecan proteoglycan core proteins. Loss of N-sulfation did not result in significant proteinuria, but the increase in the albumin/creatinine ratio was coincident with the development of the enlarged lysosomes in the proximal tubules. Thus, although the renal phenotype of the Ndst1-/- mouse is mild, the data show that heparan chain N-sulfation plays a key role in podocyte organization.

Losartan preserves glomerular basement membrane anionic charge sites in a rat model of nephropathy

Disruption of the size and charge selectivity of the glomerular basement membrane (GBM) leads to proteinuria. Blood pressure medications suppress proteinuria by preserving GBM function. We investigated the mechanism of losartan, an angiotensin II receptor blocker (ARB), in a rat model of nephropathy. Male Wistar rats were given 25 mg/kg per day of losartan or the same volume of saline (control) from 5 days before, to 14 days after, induction of nephropathy by injection of puromycin aminonucleoside (PAN). Serum blood urea nitrogen (BUN) and creatinine, blood pressure, urinary protein, glomerular morphology and the number of GBM anionic sites were measured in the 2 groups on days 0, 7 and 14. The losartan group had significantly lower urinary protein on days 7 and 14, and higher BUN on day 14, but there were no significant differences between the losartan and control groups in serum creatinine or blood pressure. Light microscopy indicated reduced mesangial cell proliferation and expansion of the mesangial area in the losartan group relative to controls. There were more GBM anionic sites in the losartan group on days 7 and 14. In addition, all anionic sites on the surface of foot processes of epithelial cells disappeared in the control group but remained in the losartan group. A rat model of nephropathy indicates that losartan reduces urinary protein and preserves the number of anionic sites on the GBM, but has no apparent effect on hemodynamics.

Renoprotective Action of a Matrix Metalloproteinase Inhibitor in Progressive Mesangioproliferative Nephritis

Background/Aim: Matrix metalloproteinases (MMPs) play pivotal roles in extracellular matrix turnover and are involved in chronic kidney disease. The renoprotective action of a synthetic MMP inhibitor, compound A, was investigated in chronic nephritis. Methods: Nephritis was induced by a single injection of anti-Thy1.1 antibody to unilaterally nephrectomized rats. The effects of compound A on proteinuria, blood urea nitrogen, and matrix-related gene expressions were evaluated. Collagen accumulation, as assessed by periodic acid-Schiff staining and hydroxyproline content, was determined. The integrity of glomerular epithelial cells and glomerular basement membrane was evaluated with desmin immunohistochemistry and electron microscopic detection of anionic charge sites, respectively. Results: Treatment with compound A notably attenuated proteinuria, ameliorated blood urea nitrogen, and prevented glomerulosclerosis. Gene upregulation of collagen and transforming growth factor β1 in the cortex was prevented in the treated animals. Glomerular epithelial cell injury was milder, and glomerular basement membrane anionic sites were protected with the treatment. Conclusion: A novel MMP inhibitor, compound A, exerts protective effects in progressive glomerulonephritis. Compound A ameliorates various aspects of renal injuries and may have therapeutic potential toward kidney diseases.

Contribution of Proteoglycans Towards the Integrated Functions of Renal Glomerular Capillaries: A Historical Perspective

Since the original colloidal iron tracer study by Farquhar and Palade almost 50 years ago,1 the permeability characteristics of mammalian glomerular capillary wall have been the subject of extensive investigations.2,3 In the ensuing 2 to 3 decades, various studies were performed to delineate whether glomerular basement membrane or the slit diaphragm of the capillary wall is the primary filtration barrier.4,5 Amid this contentious issue, in the mid to late 1970s various clearance and additional tracer studies indicated that the glomerular capillary wall behaves as a size- as well as charge-selective barrier, and the latter biophysical properties were attributed to the anionic sites within the basement membrane that are enriched with sulfated proteoglycans (PGs).6,7,8,9 The article by Harvey et al10 in this issue of The American Journal of Pathology suggests that agrin, a basement sulfated proteoglycan, imparts an electronegative charge to the glomerular basement membrane (GBM), but it may not be responsible for its charge-selective permeability properties. In light of these findings, we deemed it necessary to revisit the tracer work performed during the past few decades and reconcile with recent observations made in various mutant mice keeping in perspective the complex ultrastructure and integrated functions of the glomerular capillary wall.11

Mechanisms of albuminuria in the chronic nitric oxide inhibition model.

Chronic nitric oxide (NO) inhibition causes hypertension and renal injury. Concomitant salt overload promotes massive albuminuria. We investigated the mechanisms whereby these treatments impair glomerular permselectivity. Adult male Munich-Wistar rats received either a standard-salt (SS; 0.5% Na) or high-salt (HS; 3.1% Na) diet and either no treatment or the NO inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). At 30 days, albuminuria was moderate, the density of fixed anionic sites at the glomerular basement membrane (GBM), estimated by cationic ferritin binding, declined by approximately 35%, and the fractional clearance of 70-kDa neutral dextran (phi) rose moderately in rats receiving L-NAME and SS. Rats given L-NAME and HS exhibited massive albuminuria, whereas phi was nearly tripled. Depletion of GBM anionic sites was also seen in these rats. The GBM was thickened in both L-NAME-treated groups. These abnormalities were largely reversed after cessation of treatments. These results indicate that chronic L-NAME treatment promotes reversible albuminuria by impairing both glomerular size and charge selectivity. These effects likely reflect functional rather than structural disruption of the glomerular wall.

An impairment of barrier size and charge selectivity of glomerular basement membrane in streptozotocin-induced diabetes and prevention by pharmacological therapy.

We examined barrier size (pore size) and charge selectivity (anionic sites) of the glomerular basement membrane (GBM) in experimental diabetes. For estimation of the pore size we employed a new method, tissue negative staining. In diabetic rats, enlarged pores and decreased numbers of anionic sites of GBM were observed. Both insulin treatment and angiotensin-converting enzyme inhibitors prevented these changes. The renoprotective effect of the two drugs is discussed in the article.

Glomerular basement membrane polyanion distribution and nitric oxide in spontaneous hypertensive rats: effects of salt loading and antihypertensive therapy with propranolol

Cationic colloidal gold (CCG), a polycationic histochemical probe, was used to analyze the distribution of glomerular basement membrane (GBM) polyanions, mainly heparan sulfate proteoglycan in spontaneous hypertensive rats (SHR) with or without salt loading and antihypertensive treatment with propranolol. The changes of mean GBM width and anionic sites distribution were assessed by electron microscopy. Plasma and urinary nitrates (NO x) were measured by nitrite (NO 2) + nitrate (NO 3), stable metabolites of NO. SHR had decreased NO production and increased GBM width (27%) compared with the control Wistar-Kyoto (WKY) rats. The chronic high dietary salt intake resulted in a significant increase in blood pressure, proteinuria, and renal function in the SHR rats. The chronic high salt dietary intake resulted in a decrease in NO in the WKY and a further reduction in NO production in the SHR. The GBM anionic sites count was similar in the SHR and WKY nonsalt-loaded groups, 13.5 ± 0.5 and 12.8 ± 0.4 CCG counts/μm GBM, respectively, but significantly lower in both salt-loaded SHR and WKY, 9.9 ± 0.55 ( P < .01) and 9.6 ± 0.55 ( P < .01) CCG counts/ μm GBM, respectively. Antihypertensive treatment with propranolol in the salt-loaded SHR group resulted in lower blood pressure, a further decrease in NO production, but no significant changes in GBM width and anionic sites count. It is concluded that chronic high salt intake may be deleterious to the permselectivity of the GBM. A low NO production state that results from chronic salt loading in already hypertensive rats will result in more severe organ (renal) damage, most probably by the addition of the loss of GBM permselectivity to the existing pathomorphologic changes.

Glomerular Basement Membrane Anionic Sites in Adriamycin Nephropathy: Effect of Saline Loading and Nitric Oxide Modulation

Background: In previous studies we found that experimental Adriamycin (ADR) nephropathy is associated with the loss of glomerular basement membrane (GBM) anionic sites provided by heparan sulfate proteoglycans. Chronic saline loading in normal rats resulted in a similar effect on the GBM anionic sites. The L-arginine-nitric oxide synthase-nitric oxide system is involved in the pathogenesis of experimental chronic renal failure. The present study was performed to determine the combined effect of nitric oxide (NO) modulation and chronic saline loading in ADR nephropathy. The modulation of NO was done by chronic administration of L-arginine (NO donor) or N^w-nitro-L-arginine, a known nitric oxide synthase inhibitor. Methods: Systolic blood pressure was measured in awake rats by a tail-cuff method. Renal function was assessed by creatinine clearance, FeNa%, and daily protein excretion. The change of mean GBM widths and anionic sites distribution were assessed by electron microscopy. The localization of anionic sites was carried out by cationic colloidal gold. Plasma and urinary nitrates (NO_x) were measured by nitrite (NO₂) + nitrate (NO₃), stable metabolites of NO. Results: Two weeks after the ADR administration (3.5 mg/kg BW iv) the rats had severe renal failure (creatinine clearance 134 ± 31 µl/min/100 g BW vs. initial values 670 ± 29 µl/min/ 100 g BW, p < 0.001), high FeNa%, severe proteinuria, increased GBM width, significant reduction of GBM anionic sites and low urinary NO_x excretion. The saline loading resulted in further reduction of GBM anionic sites count and blood pressure elevation. The inhibition of NO did not change the course of ADR nephropathy. The main finding of the present study is that chronic administration of L-arginine significantly alleviates the renal failure in the ADR (+/– saline loading) nephropathy. The L-arginine-treated rat had higher creatinine clearance, lower FeNa% and protein excretion and complete normalization of GBM anionic sites distribution. Conclusions: Sodium loading has a deleterious effect on GBM permselectivity. L-Arginine prevents the reduction of GBM anionic sites, decreases proteinuria and alleviates the renal insufficiency in ADR nephropathy.

Protective Effects of a Small Dose of Captopril on the Reduction of Glomerular Basement Membrane Anionic Sites in Spontaneously Hypertensive Rats with Streptozotocin-Induced Diabetes

Angiotensin-converting enzyme (ACE) inhibitors have been used in several clinical trials to slow a progressive decline in glomerular function in patients with diabetic nephropathy independent of their effects on blood pressure. The purpose of this study was to clarify the mechanisms(s) through which an ACE inhibitor, captopril, exerts its protective effect on renal function using spontaneously hypertensive rats (SHR) with streptozotocin (STZ)-induced diabetes. Male SHRs were made diabetic by intravenous injection of STZ (45 mg/kg). One hundred or 25 mg/kg of captopril was administered daily for 4 weeks to them. Urine albumin excretion (UAE) rate was markedly increased in diabetic SHRs, while captopril treatment resulted in a significant suppression of UAE in diabetic SHRs, independent of both its daily dose and effects on blood pressure as well as glycemic control. Examination by electron microscope revealed that the number of anionic sites (AS) in the lamina rara externa per 1000 nm of glomerular basement membrane (GBM) was significantly decreased (22.9 ± 0.2 to 16.1 ± 0.3, p < 0.001), after induction of diabetes, whereas, significant recovery (18.2 ± 0.1, p < 0.001) could be obtained even by the smaller dose (25 mg/kg) of captopril which did not exert either antihypertensive or antidiabetic effect on diabetic SHRs. Thus, we demonstrate here the direct evidence that captopril, an ACE inhibitor, can protect against damage on GBM of diabetic SHR without controlling blood pressure as well as blood glucose level.

Effect of an α-glucosidase inhibitor on glomerular basement membrane anionic sites in streptozotocin induced mildly diabetic rats

The present study was conducted in order to examine the effect of acarbose, a potent α-glucosidase inhibitor, on renal function in rats with mild streptozotocin-diabetes. Male Wistar rats were made mildly diabetic by intravenous injection of streptozotocin (40 mg/kg) and were supplied a standard solid chow containing 0.1% acarbose for 8 weeks. Diabetic rats showed mild hyperglycemia under non-fasting condition and their urine albumin excretion (UAE) rate was markedly increased compared to non-diabetic control rats, while acarbose treatment resulted in a significant suppression of blood glucose level and UAE in diabetic rats. Examination by electronmicroscope revealed that the number of anionic sites in the lamina rara externa per 1000 nm of glomerular basement membrane (GBM) was significantly decreased in diabetic rats compared to control value (15.7 ± 0.9 vs. 20.9 ± 0.3 P < 0.001), whereas, significant recovery (19.6 ± 0.6 P < 0.01) was observed after 8 weeks of acarbose treatment. In conclusion, acarbose treatment suppressed blood glucose level of mildly-insulin deficient animal model without insulin treatment and prevented from a reduction in the number of anionic sites in GBM which might ameliorate an increased permeability of GBM leading to albuminuria.

Effect of interleukin-8 on glomerular sulfated compounds and albuminuria.

To evaluate the effect of interleukin-8 (IL8) on glomerular basement membrane (GBM) sulfated compounds and albuminuria, we infused IL8 in 1% bovine serum albumin (BSA) for 5 days into the left renal artery of Holtzman male rats at the rate of 10 microliters/h using an osmotic pump. Control rats received 1% BSA. A significant increase in urinary albumin/creatinine ratio was seen on the last day of IL8 infusion (0.38 +/- 0.11, mean +/- SEM) when compared with albumin/creatinine ratio prior to infusion (0.19 +/- 0.04, P = 0.04). No significant differences in urinary albumin excretion prior to and after infusion of 1% BSA were observed. On the last day of infusion, rats were injected with 35sulfate (1.0 mCi/200 g body weight) intraperitoneally and killed after 8 h. Glomeruli were isolated and GBM obtained. After 5 days of IL8 administration, there was a significant increase in 35sulfate uptake by GBM of the infused kidney (76 +/- 10 cpm/dry glomerular weight, mean +/- SEM) compared with the uptake seen in the contralateral kidney (53 +/- 9, P = 0.05). The in vivo infusion of IL8 increased the 35sulfate uptake by GBM and augmented the urinary albumin/creatinine ratio, suggesting that IL8 may induce albuminuria by altering the metabolism of the GBM sulfated compounds. This hypothesis needs to be confirmed by studies on glomerular charge selectivity and GBM anionic sites during the course of the infusion. Moreover, the persistence of the effect needs to be evaluated by prolonging the infusion for more than 5 days.

Ultrastructural and functional analyses of nephropathy in calmodulin‐induced diabetic transgenic mice

Background Previous animal models of diabetic nephropathy have used diabetic animals for which the underlying defect was either uncertain or the diabetes was induced by potentially specific toxins. In this report, we describe the renal abnormalities in a transgenic mouse model that develops early-onset diabetes due to overexpression of calmodulin in pancreatic beta cells. Methods Renal tissues were collected from normal and transgenic mice at 112, 182, and 300 days. These were prepared for light microscopic observation, stained with polyethylenimine (for anionic sites), or rendered acellular by detergent extraction prior to observation by transmission and scanning electron microscopy. Morphometric analysis of glomerular basement membrane thickness was carried out by the “orthogonal intercept” method. Twelve-hour urine samples of fed and fasting mice were collected for urine volume and glucose and protein analyses. Blood glucose, blood urea nitrogen, serum insulin, and creatinine were determined in 60-90-day-old and 255-day-old mice by established methods. Results Morphometric analyses revealed age-related and transgene-related increases in glomerular basement membrane thickness. A 22% increase in transgenic diabetics over controls was seen at 112 days of age that developed to increases of 43% and 37% at 182 and 300 days of age, respectively. Mesangial matrix area was also increased markedly in transgenic mice. Surprisingly, even in the oldest diabetic mice, there was no reduction in anionic sites. Moreover, despite an eightfold increase in urine volume, these mice did not become significantly proteinuric. Conclusions These results indicate that proteinuria of diabetes may be delayed or prevented by maintenance of a normal complement of glomerular basement membrane anionic sites. They also demonstrate that transgenic mice can provide a valuble model for discriminating between different aspects of diabetic nephropathy. Anat. Rec. 247:9–19 © 1997 Wiley-Liss, Inc.

Ultrastructural and functional analyses of nephropathy in calmodulin-induced diabetic transgenic mice.

Previous animal models of diabetic nephropathy have used diabetic animals for which the underlying defect was either uncertain or the diabetes was induced by potentially specific toxins. In this report, we describe the renal abnormalities in a transgenic mouse model that develops early-onset diabetes due to overexpression of calmodulin in pancreatic beta cells. Renal tissues were collected from normal and transgenic mice at 112, 182, and 300 days. These were prepared for light microscopic observation, stained with polyethylenimine (for anionic sites), or rendered acellular by detergent extraction prior to observation by transmission and scanning electron microscopy. Morphometric analysis of glomerular basement membrane thickness was carried out by the "orthogonal intercept" method. Twelve-hour urine samples of fed and fasting mice were collected for urine volume and glucose and protein analyses. Blood glucose, blood urea nitrogen, serum insulin, and creatinine were determined in 60-90-day-old and 255-day-old mice by established methods. Morphometric analyses revealed age-related and transgene-related increases in glomerular basement membrane thickness. A 22% increase in transgenic diabetics over controls was seen at 112 days of age that developed to increases of 43% and 37% at 182 and 300 days of age, respectively. Mesangial matrix area was also increased markedly in transgenic mice. Surprisingly, even in the oldest diabetic mice, there was no reduction in anionic sites. Moreover, despite an eightfold increase in urine volume, these mice did not become significantly proteinuric. These results indicate that proteinuria of diabetes may be delayed or prevented by maintenance of a normal complement of glomerular basement membrane anionic sites. They also demonstrate that transgenic mice can provide a valuable model for discriminating between different aspects of diabetic nephropathy.

Glomerular basement membrane polyanionic sites and nitric oxide in genetically salt-sensitive and resistant hypertensive rats.

Cationic colloid gold, a polycationic histochemical probe, was used to analyze the distribution of glomerular basement membrane (GBM) polyanions, including heparan sulfate protoglycan in genetic salt-sensitive (SBH/Y) and resistant (SBN/Y) hypertensive rats, with or without high dietary salt intake. GBM morphology, renal function and nitric oxide, as measured by plasma and urine nitrite (NO2) and nitrate (NO3) were also determined. In the salt-sensitive rats the high-salt dietary intake resulted in severe hypertension, proteinuria and decreased glomerular filtration rate. After 1 month of high-salt intake, the average width of the GBM of salt-sensitive rats was higher by 27% than that of salt-resistant rats. The number of GBM anionic sites (lamina rata externa and interna) was much lower in both salt-sensitive and salt-resistant groups after 1 month of salt loading, 8.04+/-0.36 and 7.8+/-0.25 counts/cm, respectively, compared to the respective values of non-salt-loaded animals, 20.58+/-1.08 counts/cm in the SBH/Y (p < 0.001) and 21+/-1.86 counts/cm in the SBN/Y (p < 0.001). A decreased nitric oxide production was found in the salt-sensitive rats before and after salt loading compared with the salt-resistant group. No correlation was found between the nitric oxide changes and the GBM modifications. It is concluded that high-salt intake may be deleterious to the permselectivity of the GBM. It is suggested that salt restriction in hypertension may have a beneficial effect in preventing GBM permselectivity changes and proteinuria.

Alterations in Glomerular Anionic Sites in the Autologous Phase of Canine Anti-Glomerular Basement Membrane Nephritis

There have been a few studies on canine nephrotoxic glomerulonephritis produced by anti-glomerular basement membrane serum (AGBM), but these reports have not focused on an alteration in the charge properties of glomerular basement membrane (GBM). In this study, rabbit AGBM or normal rabbit serum (NRS) was given intravenously (2 ml/kg body weight) to 16 male beagle dogs. An alteration of anionic sites (ASs) of GBM was studied quantitatively using polyethyleneimine as a cationic probe by electron microscopy at weeks 1, 2, 4, and 8 postinjection. In AGBM-treated dogs, severe or mild proteinuria continued until week 2. At weeks 4 and 8, there was no significant difference in the intensity of proteinuria between AGBM- and NRS-treated groups. Until week 2 postinjection, there were significantly fewer ASs of GBM in AGBM-treated dogs than in NRS-treated dogs. At week 8, however, there was no difference in ASs of GBM between AGBM- and NRS-treated dogs. The fact that a reduction of glomerular AS occurred in AGBM-treated dogs with severe or mild proteinuria and the recovery of AS in the GBM coincided with an improvement of proteinuria suggested that alteration of the glomerular ASs might play an important role in the pathogenesis of proteinuria in canine anti-GBM nephritis.

Effect of an aldose reductase inhibitor on glomerular basement membrane anionic sites in streptozotocin-induced diabetic rats

The present study was conducted in order to determine whether an aldose reductase inhibitor (ARI), epalrestat, prevents the progression of diabetic nephropathy in rats. Rats were made diabetic by intravenous injection of streptozotocin (STZ 50 mg/kg) and epalrestat (100 mg/kg) was administered orally through a gastric tube once daily for 4 weeks. Examination by electron microscope revealed that the number of anionic sites (AS) in the lamina rara externa per 1000 nm of glomerular basement membrane (GBM) was significantly decreased in diabetic rats compared to control values (17.6 ± 0.4 vs. 21.9 ± 0.4, P < 0.01), whereas, significant recovery (20.3 ± 0.7, P < 0.05) was observed after 4 weeks of epalrestat treatment. Urinary albumin excretion (UAE) rate was markedly increased in diabetic rats and the treatment resulted in its significant suppression from diabetic rats. In conclusion, administration of epalrestat to diabetic rats is capable of preventing a reduction in the number of AS in GBM which would ameliorate an increased permeability of the basement membrane leading to albuminuria.

Detection of hyaluronic acid in the glomerular basement membrane of murine chronic graft-versus-host disease

An ultrastructural investigation was made of the distribution of immune complexes (IC) and anionic sites using cationic colloidal gold in the glomerular basement membrane (GBM) of kidneys with murine graft-versus-host disease. At 8 weeks, a decrease in the number of anionic sites around deposits of IC in the GBM was noted. These anionic sites could be removed by treatment with hyaluronidase or chondroitinase, but not with heparitinase, suggesting that they consisted mainly of hyaluronic acid. These results support the theory that IC deposits are associated with a decrease in the number of GBM anionic sites which mainly consist of hyaluronic acid.

The Effect of Dilazep on Urinary Protein Excretion in Spontaneous Diabetic Chinese Hamster

The effect of dilazep, an adenosine potentiator and platelet aggregation inhibitor, on experimental diabetic nephropathy was investigated in spontaneous diabetic Chinese hamster. Prediabetic animals, 8 weeks of age, were divided into two groups. In one group, 5 mg/kg dilazep was injected i.p. once a day. In the other group, saline of the same amount was injected. Age- and sex-matched animals from a nondiabetic subline were used as controls. No difference was observed in body weight, mean blood pressure, fasting plasma glucose and glycated hemoglobin level between diabetic animals with and without dilazep administration throughout the entire period of experiment. Urinary protein excretions in untreated diabetic animals increased significantly compared to those of nondiabetic controls at 8 weeks (17.5 +/- 3.5 vs 2.0 +/- 0.1 mg/day), and at 24 weeks (25.3 +/- 5.1 vs 2.7 +/- 0.1 mg/day) of experiment. In diabetic animals with dilazep treatment, urinary protein excretions (4.1 +/- 0.7 at 8 weeks and 13.1 +/- 2.9 mg/day at 24 weeks of experiment) were significantly suppressed compared to those in untreated diabetic animals. Significant thickening of glomerular basement membrane (GBM) was observed in diabetic animals both with and without dilazep administration at 24 weeks of experiment compared to that in nondiabetic controls. The number of anionic sites in GBM, stained by polyethyleneimine, was reduced in untreated diabetic animals, but was not different in dilazep treated animals compared to that in nondiabetic controls. It was concluded that dilazep administration suppressed urinary protein excretion in diabetic Chinese hamster possibly through the preservation of charge barrier of the glomerulus.

Detection of glomerular anionic sites in post-embedded ultra-thin sections using cationic colloidal gold.

We detected glomerular anionic sites in fixed, LR Gold-embedded ultra-thin tissue sections using cationic colloidal gold. Manual and computer-assisted quantitation were compared, and the influence of pH and glycosaminoglycan-degrading enzymes on site expression was examined. Both quantitation methods produced similar results. Alteration of pH within a narrow range (pH 2.5-3.0) markedly affected the staining pattern. At pH 2.5, epithelial and endothelial glycocalyx and regular sites restricted to the lamina rara externa were stained. At pH 3.0 and above, glycocalyx was unstained but intracellular and nuclear staining was present; glomerular basement membrane (GBM) and mesangial matrix sites were abundant. After chondroitinase ABC or hyaluronidase digestion, GBM staining was eliminated at pH 2.0 and reduced at pH 7.0 (p less than 0.001), suggesting that degraded sites are associated with chondroitin sulfate or hyaluronic acid. By contrast, prolonged heparitinase I digestion was ineffective at either pH. Digestion of purified substrates revealed crossreactivity of heparitinase towards chondroitin sulfate and of chondroitinase towards hyaluronic acid. Since tissue sites were reduced by chondroitinase but not heparitinase, we suggest that degradation is due to hyaluronidase activity of chondroitinase and the anionic sites are associated with hyaluronic acid. However, the influence of pH indicates that lamina rara externa sites are structurally distinct from other GBM anionic sites.

Macromolecular sieving by glomerular basement membrane in vitro: Effect of polycation or biochemical modifications

To evaluate the effect of biochemical modifications not possible in vivo, filters of dog glomerular basement membrane (GBM) were constructed in ultrafiltration cells in vitro. The sieving coefficients (SCs) of three protein markers of differing size and charge (native, anionic bovine albumin—BSA; cationized BSA—cBSA; and immunoglobulin G—IgG) were determined using filters of differing amounts of control GBM, and under varying transmembrane pressures ( ΔP). Flow rates did not increase proportionately with increasing ΔP, indicating filter compressibility. Protein SCs did not change with changing ΔP, but did decrease with increasing filter thickness. Control filters showed a small but definite charge selectivity (SC cBSA − SC BSA >0); a much greater degree of size selectivity (SC cBSA − SC IgG) was observed. Hexadimethrine (HDM), a polycation which causes proteinuria in vivo, led to marked increases in protein SCs. In contrast, removal of the major population of intrinsic GBM negative charges by carboxyl group methylation only produced a small increase in the filtration of BSA, with no change in filtration of cBSA or IgG. Other biochemical modifications (heparinase or neuraminidase treatment) had no effect on filter permselectivity. Carboxyl group methylation essentially abolished filter binding of cationized ferritin, which showed substantial binding to control filters. These in vitro studies provide confirmatory evidence for a direct effect of HDM on the permselective properties of GBM. In addition, biochemical modification studies suggest a fundamental difference between the binding of an exogenous polycation to GBM anionic sites and the removal of intrinsic charges.