Number Of Anionic Sites Research Articles

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.

Tranilast Prevents the Progression of Experimental Diabetic Nephropathy through Suppression of Enhanced Extracellular Matrix Gene Expression

The present study was performed to investigate the effects of the antiallergic drug tranilast on the development of diabetic nephropathy in streptozotocin (50 mg/kg)-induced diabetic spontaneously hypertensive rats (SHR). Diabetic SHR were given standard chow or chow containing tranilast at a dose of 1400 mg/kg for 24 weeks. The effects of tranilast on urinary albumin excretion, mesangial expansion, expression of transforming growth factor-beta (TGF-beta) and type I collagen mRNAs, number of anionic sites on the glomerular basement membrane (GBM), and urinary TGF-beta and 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion were assessed. Tranilast did not affect the blood glucose concentration or blood pressure in diabetic SHR. Urinary albumin excretion rate and creatinine clearance were markedly increased in diabetic SHR. Tranilast treatment decreased albuminuria and hyperfiltration. Tranilast inhibited the diabetes-induced expansion of mesangial and tuft areas, as well as the increase in urinary TGF-beta and 8-OHdG excretion, loss of anionic sites of GBM, and overexpression of TGF-beta as determined immunohistochemically. The levels of TGF-beta and type I collagen mRNA expression were increased in the renal cortex in untreated diabetic SHR at 24 weeks, as determined by real-time quantitative polymerase chain reaction. Tranilast treatment inhibited the up-regulation of TGF-beta and type I collagen mRNA expression by 65 and 36%, respectively, in diabetic SHR. In conclusion, tranilast decreased albuminuria by suppressing glomerular hyperfiltration, mesangial expansion, and loss of the charge barrier via regulation of extracellular matrix gene expression and oxidative stress. Tranilast may be clinically useful in the treatment of diabetic nephropathy.

Preventive effect of cerivastatin on diabetic nephropathy through suppression of glomerular macrophage recruitment in a rat model.

We investigated the effect of cerivastatin, a statin, on the development of diabetic nephropathy in spontaneously hypertensive rats (SHR) with streptozotocin-induced diabetes. Diabetic SHR were given standard chow or chow containing cerivastatin at a dose of 0.1 mg/kg or 1.0 mg/kg for 12 weeks. Effects of cerivastatin on urinary albumin excretion, mesangial expansion, glomerular macrophage infiltration, and the number of anionic sites on the glomerular basement membrane (GBM) were assessed. Cerivastatin did not affect the blood glucose concentration, blood pressure or serum cholesterol concentration in diabetic SHR. However, cerivastatin treatment caused a dose-dependent decrease of albuminuria and hyperfiltration. At 1.0 mg/kg, cerivastatin inhibited the diabetes-induced expansion of mesangial and tuft areas on histological examination of the kidneys, as well as the loss of anionic sites from the GBM evaluated with polyetyleneimine and the intraglomerular infiltration of ED1-positive macrophages evaluated by immunohistochemistry. Whole-kidney expression of mRNA for MCP-1 and TGF-beta, estimated by the real-time quantitative RT-PCR, was increased (both 2.6-fold) in untreated diabetic SHR at 12 weeks. Cerivastatin treatment (1.0 mg/kg) inhibited the up-regulated expression of MCP-1 and TGF-beta mRNA (decreased to 48% and 34%, respectively) in diabetic SHR. In this hypertensive model of diabetic nephropathy, cerivastatin decreased albuminuria through suppression of glomerular hyperfiltration, mesangial expansion, and the loss of charge barrier independently of a cholesterol-lowering effect. These preventive effects could be at least partly due to inhibition of macrophage recruitment and activation, and inhibition of TGF-beta overexpression.

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.

Abnormal Distribution of Anionic Sites in the Glomerular Basement Membrane in Glomerulonephritis of Dogs Infected with Dirofilaria immitis.

Ultrastructural alteration of anionic sites (ASs) in the glomerular basement membrane (GBM) was studied in glomerulonephritis characterized by linear capillary IgG deposition in four dogs infected with Dirofilaria immitis and two normal control dogs using polyethyleneimine. ASs were identified as small dense particles distributed regularly in the lamina rara externa (LRE), but there were no ASs in the lamina densa (LD) of the GBM of the control dogs. In the glomeruli of the infected dogs, ASs were distributed regularly or irregularly in the thickened LD. ASs were in addition localized over the characteristic continuous bands of subendothelial dense deposits. The number of ASs of the LRE increased in all four infected cases as compared to the controls (p<0.01).

Initial Ultrastructural Changes in Pore Size and Anionic Sites of the Glomerular Basement Membrane in Streptozotocin-Induced Diabetic Rats and Their Prevention by Insulin Treatment

Background/Aim: The present study was conducted to elucidate the mechanism(s) of the development of early diabetic nephropathy, examining ultrastructural changes employing electron microscopy, especially changes in pore size of the glomerular basement membrane (GBM) of streptozotocin (STZ)-induced diabetics rats. Methods: Urinary albumin excretion rate (UAE), pore size of the lamina densa of the GBM visualized directly by the tissue negative staining method, and number of anionic sites (AS) in the corresponding portion of the lamina rara externa were determined for 6 weeks in diabetic rats without and with insulin treatment. Results: The UAE of the diabetic rats increased with time and was significantly greater than that of the nondiabetic control rats after 4 weeks (p < 0.01), while insulin treatment suppressed the increased UAE of diabetic rats. The median values in both short diameter and long dimension of the pores in the diabetic group were markedly increased at the 2nd week as compared with those in the nondiabetic control rats, whereas no significant change was found in the pore size of the diabetic rats with insulin treatment. Moreover, the number of AS in the GBM of the diabetic rats was significantly (p < 0.001) decreased from the 2nd week onward. Insulin treatment also prevented a decrease in AS number in diabetic rats. Conclusions: It is suggested from these results that an impairment of barrier size selectivity occurs at a very early stage of STZ-induced diabetes in rats, which may enhance the abnormality of the charge-selective properties of the GBM. In addition, insulin treatment may protect this barrier system through normalizing blood glucose control in STZ-diabetic rats.

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.

Correlation between Histological Changes and Loss of Anionic Charge of the Glomerular Basement Membrane in Early-Onset Pre-Eclampsia

This study morphometrically evaluates the glomerular anionic charge and examines the renal pathology in African women with early-onset pre-eclampsia. Polyethyleneimine-labelled anionic sites were decreased within the glomerular basement membrane in the early-onset pre-eclampsia group as compared with the control group (p < or = 0.02). A strong correlation (r = -0.76) was obtained between the number of anionic sites and the severity of proteinuria. Renal biopsy specimens revealed the coexistence of decreased glomerular charge with pathology of glomerular basement membrane, endothelial cells, foot processes, and mesangial cell proliferation. The loss of glomerular charge induces structural alterations of the glomerular filtration barrier and may be the mechanism responsible for proteinuria in early-onset pre-eclampsia.

Impairment of endothelial and subendothelial sites by a circulating plasma factor associated with minimal change disease

The pathogenesis of albuminuria in minimal change disease (MCD) is unknown. A human plasma factor (denoted as 100KF) is able to induce minimal change-like glomerular alterations, i.e. loss of glomerular sialoglycoproteins and decreased expression of glomerular ecto—ATPase, following in vitro incubation with kidney tissue. In addition, increased (selective) permeability for plasma proteins occurs after perfusion of 100KF into the rat kidney ex vivo . As in kidney tissue from subjects with minimal change disease, subendothelial injury has also been observed, i.e. reduced anionic sites in the lamina rara interna, the question was raised whether injury induced by the plasma factor 100KF involves vascular endothelium or subendothelial matrix of the glomerular capillary wall. Permeability studies were carried out by using confluent endothelial monolayers (HUVEC) cultured on a standard two—compartment system. The permeability for a macromolecular marker (horse radish peroxidase) was tested following incubation of the monolayers with either the native plasma factor 100KF or the control factor (heat—inactivated 100KF), in combination with histochemical evaluation of the cells for ecto—ATPase expression. Also quantification of glomerular anionic sites at the ultrastructural level was carried out, after ex vivo perfusion of 100KF or control factor into rat kidneys. The plasma factor 100KF is able to increase the permeability of human endothelial monolayers for macromolecules in a dose—dependent manner (relative increase 122.4±24, 178.4±34 and 236.1 ±58% after preincubation with 0.05; 0.5 and 1.5 mg/ml 100KF respectively), concomitant with induction of minimal change-like histochemical alterations such as reduced expression of ecto—ATPase. The number of anionic sites in the lamina rara interna of the glomerular capillary wall is significantly diminished following perfusion with the plasma factor 100KF versus control factor (7.58±1.60 versus 12.57±2.05 per 1000nm; P <0.02); in contrast to the lamina rara externa (22.71±3.15 versus 22.27±2.92 per 1000 nm; statistically not significant). Endothelial cells and subendothelial matrix along the glomerular filtration barrier may be considered as target structures for the plasma factor 100KF, leading to initial minimal change-like alterations associated with glomerular albumin leakage.

Impairment of endothelial and subendothelial sites by a circulating plasma factor associated with minimal change disease.

The pathogenesis of albuminuria in minimal change disease (MCD) is unknown. A human plasma factor (denoted as 100KF) is able to induce minimal change-like glomerular alterations, i.e. loss of glomerular sialoglycoproteins and decreased expression of glomerular ecto-ATPase, following in vitro incubation with kidney tissue. In addition, increased (selective) permeability for plasma proteins occurs after perfusion of 100KF into the rat kidney ex vivo. As in kidney tissue from subjects with minimal change disease, subendothelial injury has also been observed, i.e. reduced anionic sites in the lamina rara interna, the question was raised whether injury induced by the plasma factor 100KF involves vascular endothelium or subendothelial matrix of the glomerular capillary wall. Permeability studies were carried out by using confluent endothelial monolayers (HUVEC) cultured on a standard two-compartment system. The permeability for a macromolecular marker (horse radish peroxidase) was tested following incubation of the monolayers with either the native plasma factor 100KF or the control factor (heat-inactivated 100KF), in combination with histochemical evaluation of the cells for ecto-ATPase expression. Also quantification of glomerular anionic sites at the ultrastructural level was carried out, after ex vivo perfusion of 100KF or control factor into rat kidneys. The plasma factor 100KF is able to increase the permeability of human endothelial monolayers for macromolecules in a dose-dependent manner (relative increase 122.4 +/- 24, 178.4 +/- 34 and 236.1 +/- 58% after preincubation with 0.05; 0.5 and 1.5 mg/ml 100KF respectively), concomitant with induction of minimal change-like histochemical alterations such as reduced expression of ecto-ATPase. The number of anionic sites in the lamina rara interna of the glomerular capillary wall is significantly diminished following perfusion with the plasma factor 100KF versus control factor (7.58 +/- 1.60 versus 12.57 +/- 2.05 per 1000 nm; P < or = 0.02); in contrast to the lamina rara externa (22.71 +/- 3.15 versus 22.27 +/- 2.92 per 1000 nm; statistically not significant). Endothelial cells and subendothelial matrix along the glomerular filtration barrier may be considered as target structures for the plasma factor 100KF, leading to initial minimal change-like alterations associated with glomerular albumin leakage.

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.

Reduction of heparan sulphate-associated anionic sites in the glomerular basement membrane of rats with streptozotocin-induced diabetic nephropathy.

Heparan sulphate-associated anionic sites in the glomerular basement membrane were studied in rats 8 months after induction of diabetes by streptozotocin and in age- adn sex-matched control rats, employing the cationic dye cuprolinic blue. Morphometric analysis at the ultrastructural level was performed using a computerized image processor. The heparan sulphate specificity of the cuprolinic blue staining was demonstrated by glycosaminoglycan-degrading enzymes, showing that pretreatment of the sections with heparitinase abolished all staining, whereas chondroitinase ABC had no effect. The majority of anionic sites (74% in diabetic and 81% in control rats) were found within the lamina rara externa of the glomerular basement membrane. A minority of anionic sites were scattered throughout the lamina densa and lamina rara interna, and were significantly smaller than those in the lamina rara externa of the glomerular basement membrane (p<0.001 and p<0.01 for diabetic and control rats, respectively). Diabetic rats progressively developed albuminuria reaching 40.3 (32.2-62.0) mg/24 h after 8 months in contrast to the control animals (0.8 (0.2-0.9) mg/24 h, p<0.002). At the same time, the number of heparan sulphate anionic sites and the total anionic site surface (number of anionic sites x mean anionic site surface) in the lamina rara externa of the glomerular basement membrane was reduced by 19% (p<0.021) and by 26% (p<0.02), respectively. Number and total anionic site surface in the remaining part of the glomerular basement membrane (lamina densa and lamina rara interna) were not significantly changed. We conclude that in streptozotocin-diabetic rats with an increased urinary albumin excretion, a reduced heparan sulphate charge barrier/density is found at the lamina rara externa of the glomerular basement membrane.

Reduction of heparan sulphate-associated anionic sites in the glomerular basement membrane of rats with streptozotocin-induced diabetic nephropathy

Heparan sulphate-associated anionic sites in the glomerular basement membrane were studied in rats 8 months after induction of diabetes by streptozotocin and in age- and sex-matched control rats, employing the cationic dye cuprolinic blue. Morphometric analysis at the ultrastructural level was performed using a computerized image processor. The heparan sulphate specificity of the cuprolinic blue staining was demonstrated by glycosaminoglycan-degrading enzymes, showing that pretreatment of the sections with heparitinase abolished all staining, whereas chondroitinase ABC had no effect. The majority of anionic sites (74% in diabetic and 81% in control rats) were found within the lamina rara externa of the glomerular basement membrane. A minority of anionic sites were scattered throughout the lamina densa and lamina rara interna, and were significantly smaller than those in the lamina rara externa of the glomerular basement membrane (p<0.001 and p<0.01 for diabetic and control rats, respectively). Diabetic rats progressively developed albuminuria reaching 40.3 (32.2–62.0) mg/24 h after 8 months in contrast to the control animals (0.8 (0.2–0.9) mg/24 h, p<0.002). At the same time, the number of heparan sulphate anionic sites and the total anionic site surface (number of anionic sites × mean anionic site surface) in the lamina rara externa of the glomerular basement membrane was reduced by 19% (p<0.021) and by 26% (p<0.02), respectively. Number and total anionic site surface in the remaining part of the glomerular basement membrane (lamina densa and lamina rara interna) were not significantly changed. We conclude that in streptozotocin-diabetic rats with an increased urinary albumin excretion, a reduced heparan sulphate charge barrier/density is found at the lamina rara externa of the glomerular basement membrane.

Effects of antiplatelet drug dilazep dihydrochloride on anionic sites and extracellular matrix (ECM) components in glomerular basement membrane of STZ-induced diabetic rats.

A study of anionic sites in the glomerular basement membrane (GBM) of streptozotocin (STZ)-induced diabetic rats with or without treatment by an antiplatelet drug, dilazep dihydrochloride, is described. Expression of glomerular extracellular matrix (ECM) components was examined by immunofluorescence. Renal specimens were immersed in polyethyleneimine (PEI) as a cationic probe and then examined by electron microscopy. Renal specimens were also incubated with rabbit antirat type IV collagen, laminin, and fibronectin antisera and then stained with fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG antiserum. Mean values of proteinuria in the dilazep-treated diabetic rats were significantly decreased compared with those in nontreated diabetic rats. There was no significant correlation between the levels of proteinuria and those of creatinine clearance (CCr). Number of anionic sites on the GBM in the dilazep-treated diabetic rats were greater than those in diabetic rats. There was no significant difference in the staining of such ECM components between both rat groups. The authors concluded that the dilazep dihydrochloride might prevent anionic charges on the GBM and decrease the urinary excretion of proteins in STZ-induced diabetic rats.

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.

Protective effect of heparin on renal glomerular anionic sites of streptozotocin-injected rats

Albuminuria at the rate of 500 μg/24 h was observed in rats treated with 50 mg/kg body wt. i. v. streptozotocin (STZ). When STZ was combined with heparin, administered twice daily (250 IU/kg subcutaneous injection) after 24 h following STZ injection, the daily urinary albumin excretion (U-AE) was less than half the amount found in non-heparinized rats (280.3 μg/24 h). The observation period in both instances was 8 weeks. When animals were permitted to develop albuminuria over the first 4 weeks, subsequent heparin administration for another 4 weeks lowered U-AE from 500 μg/24 h to less than half the amount (227.8 μg/24 h). A significantly negative correlation ( P < 0.001) existed between U-AE and the number of anionic sites (AS) in the lamina rara externa of glomerular basement membranes, as visualized by electron microscopy. The number of AS per 1000 nm in STZ-injected rats (15.5 ± 0.2) was lower than that in control rats (23.1 ± 0.6); however, in heparinized animals, regardless of STZ, the values were not significantly different from normal. Heparin by itself had no effect on the number of AS in normal rats. All STZ-injected animals became hyperglycemic (400–550 mg/dl), but received no insulin. Heparin had no effect on plasma glucose levels. From these results, it is concluded that heparin suppressed both of an increase in U-AE and a decrease in AS count of glomerular basement membranes in STZ-injected rats.

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.

Interaction of two fragments of Cibacron Blue F3GA having anthraquinone chromophores with basic polypeptides

The interaction of two anthraquinone dyes, bromaminic acid 1-amino-4-bromo anthraquinone-2-sulphonic acid (BA) and 1-amino-4-(4'-aminophenylamino)-anthraquinone-2,3'-disulphonic acid (ASSO), with poly-L-lysine (PLL), poly-L-ornithine (PLO) and poly-L-arginine (PLA) has been studied. The flexibility of the side chain (chain length) and the geometry of the charge centre (tetrahedral/planar) of the polypeptide were found to affect the interaction. Further, both dyes induce ordered conformation (alpha or beta) for the three polypeptides, but the final conformation was found to depend on the polypeptide as well as the number of anionic sites on the ligand. While the bidentate ASSO-polypeptide complexes acquire beta-conformation, the monodentate BA distinguished between the three substrates and induces beta-conformation only for PLO.

Heparan sulfate proteoglycan in the glomerular basement membrane in type 1 diabetes mellitus

Heparan sulfate proteoglycans (HSPG) are negatively charged constituents of the renal extracellular matrix including the glomerular basement membrane (GBM) and mesangial matrix. Biochemical and functional studies of patients with type-1 insulin dependent diabetes mellitus (IDDM) suggest that alterations of HSPG may occur in diabetic nephropathy. We have utilized a specific cytochemical method and electron microscopy to quantitate the distribution of HSPG in the GBM of 10 normal people and in 16 IDDM patients with a spectrum of clinical and structural changes. Enzyme incubation studies of normal infant kidney demonstrated that heparitinase removed 94% of the stainable anionic sites in the lamina rara externa (LRE) and 77% of the sites in the lamina rara interna (LRI) of the GBM. In contrast, incubation in the enzyme chondroitinase ABC did not reduce the number of sites in the LRE but reduced the number of sites in the LRI by 26%. The HSPG anionic sites in normal subjects were distributed in the LRE as 20.9 +/- 1.3, and in the LRI as 13.1 +/- 2.2 per micron GBM length. Anionic sites were slightly reduced (19.6 +/- 1.3, P less than 0.04) in the LRE of IDDM patients with normal urinary albumin excretion rates (UAE), or microalbuminuria, and were reduced in both the LRE and LRI of IDDM patients with clinical proteinuria (13.1 +/- 2.3, P less than 0.001 and 8.9 +/- 2.1, P less than 0.001, respectively). The number of anionic sites in the LRE and LRI, respectively, correlated with UAE (r = +0.78, P less than 0.001, r = +0.58, P less than 0.02), with GBM thickness (LRE, r = +0.81, P less than 0.001; LRI, r = +0.67, P less than 0.01) and with the volume fraction of mesangium (LRE, r = +0.59, P less than 0.02; LRI, r = +0.58, P less than 0.03). These data confirm earlier biochemical findings of a reduction of HSPG in the GBM in advanced diabetic nephropathy but do not provide evidence for the loss of HSPG in the GBM as a mechanism for early microalbuminuria.