Glomerular macrophages and the mesangial proliferative response in the experimental nephrotic syndrome.

Mesangial cell proliferation, which is a harbinger of glomerulosclerosis, occurs in both immune and nonimmune glomerulopathies. The proximity of infiltrating glomerular macrophages to the contractile mesangial cells during acute puromycin aminonucleoside (PA) nephrosis suggests the possibility of a paracrine effect on mesangial cell growth. To test this, three maneuvers to either raise or lower the glomerular macrophage number during acute PA nephrosis (2 weeks after PA) were employed: 1) an essential fatty acid-deficient (EFAD) diet; 2) a cholesterol-supplemented diet (CSD); and 3) a single dose (600 rad) whole-body X-irradiation (XI) given to CSD-fed PA rats. Both the glomerular macrophage number and proliferation within the mesangium were evaluated immunohistochemically with ED-1, a mouse monoclonal anti-rat macrophage label, and 19A2, a mouse monoclonal anti-proliferating cell nuclear antigen (PCNA)/cyclin antibody, respectively. Immunohistochemical detection of 5'-bromo-2'-deoxyuridine (BrdU) incorporation confirmed that proliferation was occurring within the mesangial zones. The EFAD diet significantly reduced both the glomerular macrophage and PCNA/cyclin-positive cell number at 2 weeks after PA with a positive correlation (r = 0.89, P < 0.05). The CSD maneuver significantly increased both the glomerular macrophage and PCNA/cyclin cell number with a strong degree of correlation (r = 0.95, P < 0.01). X-irradiation administered to CSD-fed PA rats significantly lowered both the glomerular macrophage and PCNA/cyclin-positive cell number at 2 weeks. In all groups, the glomerular tufts did not express muscle actin using HHF 35, a specific immunolabel, suggesting that the proliferation in this model is not related to direct mesangial cell injury. This study shows that maneuvers that modulate the glomerular macrophage number are also associated with corresponding changes in the number of proliferating cells within the mesangium, suggesting a paracrine growth stimulation by the infiltrating macrophage during acute PA nephrosis. The infiltrating glomerular macrophage may be an effector mechanism for the propagation of initial glomerular injury to glomerulosclerosis by augmenting mesangial cell proliferation early in the course of this nonimmune progressive glomerulopathy.     

Cyclosporin reduces proteinuria in rats with aminonucleoside nephrosis.

The effect of cyclosporin (CS) was assessed in Sprague-Dawley rats with puromycin aminonucleoside (PA) nephrosis induced by a single intraperitoneal injection of PA. Three groups of rats were injected intraperitoneally with CS (10 mg/kg body weight) daily, beginning 1 day before PA administration, or 5 or 10 days after PA administration, for 10 days. CS significantly reduced proteinuria in rats with PA nephrosis in comparison with untreated nephrotic controls. After discontinuation of the CS treatment, proteinuria gradually increased, reaching values similar to those in control nephrotic rats. CS pretreatment did not prevent the induction of PA-induced nephrotic syndrome. Light microscopy and assessment of anionic sites in the glomerular basement membrane revealed no differences between normal rats, nephrotic controls, and CS-treated rats. These results show that CS can reduce proteinuria in PA nephrosis, but cannot ameliorate the glomerular changes.     

Changes in the molecular sieve of glomerular basement membrane in rats with aminonucleoside nephrosis

Isolated and purified glomerular basement membranes (GBM) of normal and aminonucleoside (PAN) nephrosis rats were observed by electron microscopy after negative staining. Although GBM of normal rats appeared as a molecular sieve with uniform pores, GBM of nephrotic rats showed enlargement and elongation of the pores. For an average of fifty pores, the long dimension was 40.4+/-10.7 A and the short dimension 13.8+/-3.6 A in nephrosis whereas the long dimension was 12.3+/-2.5 A and the short dimension 8.4+/-1.0 A in normal rats. Changes in the pores in GBM were thought to result in increased permeability of serum protein and hence proteinuria.     

Relationship among altered glomerular barrier permselectivity, angiotensin II, and mesangial uptake of macromolecules

Clinical and experimental studies suggest that accumulation of phlogogenic macromolecules in the glomerular mesangium may lead to mesangial expansion and eventual glomerulosclerosis. In focal glomerulosclerosis and nephrotic syndrome entrapment of macromolecules is observed in areas of glomerulosclerosis. To determine whether mesangial uptake of radiolabeled, heat-aggregated IgG (AG125I), a biologically active macromolecular protein, is influenced by increased glomerular filtration barrier permeability, we evaluated the glomerular uptake of AG125I in three models of proteinuria: aminonucleoside of puromycin nephropathy (PAN), adriamycin nephropathy, and Heyman's nephropathy. Rats were studied approximately 1 week after onset of proteinuria. AG125I was measured in preparations of isolated glomeruli and compared to simultaneous blood, liver, and spleen levels. Only rats with PAN had a marked increase in glomerular AG125I compared to control rats, 7.8 versus 2.6 micrograms/mg of glomeruli, respectively. We then evaluated whether a continuous infusion of a competitive inhibitor of angiotensin II, saralasin (300 micrograms/kg of body weight/minute), influenced mesangial uptake of AG125I in PAN rats. Strikingly, glomerular AG125I in rats with PAN was reduced to levels comparable to that observed in control rats infused with only saralasin, 2.8 versus 3.0 micrograms/mg of glomeruli, respectively. This effect on glomerular AG125I content was independent of any significant effect of saralasin on blood, hepatic, or splenic levels of AG125I. Moreover, these changes in glomerular AG125I in saralasin-infused rats with PAN did not appear to directly correlate with changes in whole kidney function. These studies also demonstrated that proteinuria per se did not influence mesangial uptake of macromolecules. Thus, these data indicated that angiotensin II had an important effect on intraglomerular factors that modulate mesangial localization of phlogogenic macromolecules.     

Irreversible tubulointerstitial damage associated with chronic aminonucleoside nephrosis. Amelioration by angiotensin I converting enzyme inhibition.

Chronic aminonucleoside nephrosis is variably associated with tubulointerstitial damage, depending on the route and frequency of drug administration. Recently, different groups have shown this injurious tubulointerstitial process to be reversible, coinciding with the resolution of heavy proteinuria to normal values. The authors have previously shown that a single jugular intravenous administration of puromycin aminonucleoside (PA) to male Munich-Wistar rats produces a triphasic pattern of glomerular injury and proteinuria, which culminates in focal glomerulosclerosis 70 weeks after drug administration. The authors now report the later progression of the tubulointerstitial morphologic abnormalities associated with acute nephrosis (phase I), despite spontaneous resolution of glomerular injury during the intermediate period (phase II) in this model. Although treatment of rats with the angiotensin I converting enzyme inhibitor enalapril (50 mg/l drinking water) over the 70-week period did not affect the magnitude of proteinuria during the acute nephrotic phase, enalapril prevented the recurrence of proteinuria (phase III), as well as significantly reducing the severity of interstitial fibrosis, extent of tubular dilatation, and number of intratubular casts on semiquantitative scoring at the conclusion of the study. In addition, enalapril-treated rats had less low-molecular-weight protein excretion during the recurrent phase of proteinuria, suggesting a preservation of tubular functional capacity to reabsorb these proteins. In vitro cytotoxicity studies showed only the glomerular visceral epithelial cell to be sensitive to PA, in contrast with rat tubular epithelium and other cellular controls. Although the exact pathogenetic mechanism responsible for the development of the tubulointerstitial damage remains unknown, PA in vitro does not adversely affect rat tubular epithelium; there is however a clear correlation between the magnitude of recurrent proteinuria and the severity of tubulointerstitial morphologic abnormalities, as suggested by the beneficial effect of converting enzyme inhibition on both of these untoward processes.     

Age-related nephropathy and proteinuria in rats with intact kidneys exposed to diets with different protein content

Tubulointerstitial damage and glomerular sclerosis are findings commonly observed in the experimental models of adriamycin and puromycin aminonucleoside nephrosis. It has been suggested that in such models proteinuria might be an important mediator of tubulo-interstitial damage which in turn may determine the progression of the disease favoring the development of glomerulosclerosis. The objective of the present investigation was to establish the temporal relationship between proteinuria, tubulo-interstitial damage and glomerulosclerosis in aging rats with intact kidneys exposed to diets with different protein content. There were six groups of rats studied. Animals of groups 1, 5, and 6 (N = 10) were fed diets containing 20, 35, and 6% protein, respectively, for 20 months and sacrificed at the end of the experimental period. Rats in groups 3 and 4 (N = 6) exhibited marked and mild proteinuria, respectively, after 14 months of maintenance on standard diet, and followed for two additional months after the onset of proteinuria with the aim of evaluating the pattern of renal damage after a relatively short period of proteinuria. Rats in group 2 (N = 10) were fed standard diet and sacrificed before (5 months) and at the onset of proteinuria (10 months). Protein excretion and plasma creatinine were measured for each animal every month. Pathologic examination was performed by light and electron microscopy. At the onset of proteinuria neither renal structural nor functional abnormalities were detected. After 20 months, rats fed standard diet developed tubulo-interstitial damage (score: 1.29 +/- 1.05) and focal glomerular sclerosis (percentage of glomeruli with focal segmental glomerular sclerosis: 16.70 +/- 16.40). A significant correlation was found between the degree of tubulo-interstitial damage and the percentage of glomeruli with focal glomerular sclerosis (r = 0.99, p less than 0.01). Development of tubulo-interstitial damage and focal glomerular sclerosis were correlated with heavy and sustained proteinuria. The high protein diet significantly worsened proteinuria (at month 20: 247.08 +/- 101.73 mg/day), tubulo-interstitial changes (score: 1.99 +/- 0.70), focal glomerular sclerosis (percentage of glomeruli with focal segmental glomerular sclerosis: 21.50 +/- 9.44) and was associated with deteriorating renal function (at month 20, plasma creatinine: 1.20 +/- 0.50 mg/dl).(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in glomerular heparan sulfate in puromycin aminonucleoside nephrosis.

Changes in glomerular anionic sites in puromycin aminonucleoside nephrosis (PAN) in the rat are controversial. The authors examined glomerular anionic sites in PAN by in vivo staining with polyethyleneimine (PEI). They also quantitated and characterized glomerular heparan sulfate (HS), which is known to be a major glomerular polyanion in PAN, using in vivo incorporation of 35S-sulfate. PAN rats had a mean protein excretion of 96 +/- 23 mg per 24 hours. Staining of anionic sites with PEI showed 15.3 +/- 2.8 sites per 1000-nm length of glomerular basement membrane in controls, 13.7 +/- 1.9 sites in PAN rats (P greater than 0.05), and 50% of rats with early PAN had absent staining. Total 35S-sulfate incorporation was similar in both the controls and established PAN rats (2900 +/- 150 dpm/mg dry wt of glomeruli versus 3005 +/- 260, P greater than 0.05) but decreased in early PAN rats (2025 +/- 148). The percentage of 35S-sulfate incorporated into chondroitin sulfate was similar in all three groups of animals. HS uronic acid was also similar (1.8 +/- 0.2 g/mg dry wt of glomeruli versus 1.7 +/- 0.3, P greater than 0.05) but decreased in early PAN (1.1 +/- 0.2). The distribution of 35S-sulfate activity within the HS subfractions was examined by ion-exchange chromatography and showed a shift in percent present from 1.0 M to 1.25 M fraction in established and early PAN animals (control 1.0 M 37% +/- 3.2% versus PAN 19% +/- 3.4%, P less than 0.01, and 1.25 M 36% +/- 2.9% versus 53% +/- 2.9%, P less than 0.01). These results demonstrate that glomerular heparan sulfate is unchanged in established PAN but decreased in early PAN. SO4 incorporation is unchanged in established PAN and diminished in early PAN. Thus, early in PAN HS synthesis is impaired, but in established PAN the HS is normal, and changes in glomerular HS cannot explain the increased permeability.     

Impact of cyclosporin on podocyte ZO-1 expression in puromycin aminonucleoside nephrosis rats

Puromycin aminonucleoside (PAN)-induced nephrosis is a well-described model of human idiopathic nephrotic syndrome, but the mechanism of PAN's effect is not completely understood. To investigate whether proteinuria in the PAN model is associated with an alteration of zonula occludens-1 (ZO-1) expression within the glomeruli, and whether cyclosporin A (CsA) has an effect on proteinuria and ZO-1 expression in this model, eighteen Sprague Dawley (SD) rats were assigned into three groups. Twelve rats received a single intraperitoneal injection of PAN (15 mg/100 g). The other six rats received an equal volume of saline (normal control group; control). CsA solution was administered intraperitoneally once a day for 20 days after the PAN injection (n=6, PAN+CsA). The remaining six rats received PAN, but they didn't receive CsA (n=6, PAN). Compared to control rats (35.1+/-5.4 mg/day), the 24-hour urinary protein excretion on day 18 was significantly higher in the PAN rats (1021.9+/-128.9 mg/day, p<0.01), and the CsA treatment partly reversed the increase in proteinuria in the PAN rats (556.4+/-102.3 mg/day, p<0.05). Glomerular ZO-1 protein expressions were significantly increased in the PAN rats as compared to the control group on day 20 (176%, p<0.01). CsA treatment for 20 days in the PAN rats inhibited the increase in ZO-1 protein expression by 71.1% (p<0.05). CsA treatment significantly diminished the glomerular ZO-1 expression in the PAN rats as assessed by immunohistochemistry. CsA treatment significantly reduced proteinuria and the diminished glomerular ZO-1 expression in a PAN nephrosis rat model. These findings suggest the potential role of the slit diaphragm associated proteins in the development of the nephrotic syndrome, and CsA decreased the proteinuria probably by a direct action on the expression of these proteins in podocytes. Further investigations are needed to clarify the role of slit diaphragm associated proteins in the development of PAN nephrosis.     

Renal apolipoproteins in nephrotic rats.

Recent experimental data suggest a role for lipids in the pathogenesis of glomerulosclerosis. In this study, we examined the main apolipoproteins (apo) of high density lipoproteins (A-I, A-IV, E), low density lipoproteins (B), and very low density lipoproteins (B,E) in plasma and kidney tissue of rats with puromycin aminonucleoside or adriamycin nephrosis. In full-blown nephrosis, plasma concentrations of apo A-I and apo B were significantly elevated, apo A-IV and apo E levels did not change. Immunohistological studies in plastic sections revealed increased apo A-I, apo A-IV, and apo E immunoreactivity in glomerular visceral epithelial cells both in puromycin aminonucleoside and adriamycin nephrosis. This was confirmed by immunoelectronmicroscopy. In addition, apo B and apo E were encountered in increased amounts in the mesangium and colocalized with Oil Red O-positive lipid deposits, particularly in puromycin aminonucleoside nephrosis rats. Double-staining showed a preferential localization of apo B and apo E at sites of increased mesangial matrix in close proximity to ED1-positive foam cells, i.e., the mesangial macrophages. The close topographic association between apo B and apo E, lipid deposits, and macrophages in the mesangium lend further support to the concept of lipid-mediated glomerular injury in nephrosis.     

Iimitations of podocyte adaptation for glomerular injury in puromycin aminonucleoside nephrosis

Glomerular synechiae that occurred in nephrotic rats with a single intraperitoneal injection of puromycin aminonucleoside were analyzed by immunohistochemistry, radiolabeled thymidine (PHI-thymidine) autoradiography, as well as light, electron and immunoelectron microscopy. To discriminate podocytes from parietal epithelial cells (PEC) and monocytes, monoclonal antibodies (mAb) against podocalyxin and ED1 were used. The cell kinetics of glomerular epithelial cells were autoradiographically assessed with isotope labeling procedures before and during nephrosis (co-labeled), and a mAb against proliferating cell nuclear antigen (PCNA). All the cell types except the podocyte of normal kidneys were labelled with rHI-thymidine at different rates. Detachment of degenerated podocytes from the outside of the glomerular basement membrane (GBM) is the first step of synechia, and detached sites are confronted by PEC that were hypertro-phied and frequently radiolabeled. Evidence that podocytes in glomeruli of nephrotic rats can proliferate was shown by the presence of mitoses, ^rHt]-thymidine uptake in the co-labeled experiment, and by PCNA staining, but reepithelialization over bare segments of the GBM with proliferated podocytes is doubtful. It was concluded that glomerular synechia resulted from the limits of podocyte adaptation to glomerular injuries.     

Glomerular macrophage modulation affects mesangial expansion in the rat after renal ablation.

Recent studies have provided evidence for the involvement of macrophages (m phi) in various types of human and experimental glomerular disease. The aim of the present study was to examine the effect of m phi depletion on glomerular injury after 3/4 renal ablation in the rat. This "remnant kidney" model is a widely used experimental model of focal and segmental glomerulosclerosis. Sustained glomerular m phi depletion was induced in remnant kidney rats by a regimen of sublethal triple systemic X-irradiation with shielding of the kidney remnants. Groups of 8 X-irradiated and 8 non-irradiated rats were studied at 5, 9, and 13 weeks after renal ablation. X-irradiated rats showed severe peripheral blood leukopenia at 5 and 9 weeks which had normalized at 13 weeks. The number of remnant glomerular m phi (immunohistochemistry with the monoclonal antibody ED1) in X-irradiated rats at 5 weeks was significantly lower when compared to non-irradiated remnant kidney rats. A rebound effect occurred at 9 and 13 weeks with increased m phi in remnant glomeruli of X-irradiated rats. Light microscopic examination disclosed significantly lower semiquantitative scores for mesangial cellularity and mesangial matrix expansion in remnant glomeruli of X-irradiated rats at 5 weeks when compared to non-irradiated remnant kidney rats. Mesangial matrix expansion had increased in X-irradiated rats at 9 and 13 weeks after ablation coincident with elevated glomerular m phi at these intervals. Multiple linear regression analysis indicated a highly significant contribution of m phi to the best fitting regression model predicting mesangial matrix expansion (multiple r2 = 0.81). In conclusion, these data provide evidence for a contributory role of m phi in the evolution of glomerular injury in the rat after renal ablation.     

Focal and segmental glomerulosclerosis following a single intravenous dose of puromycin aminonucleoside.

Focal and segmental glomerulosclerosis (FSGS) represents a final pathologic pattern of a number of human renal disorders. Among laboratory models, repeated intraperitoneal injections of the aminonucleoside of puromycin (PA) produces a histologic pattern not unlike the human process. A single intravenous dose of this drug usually results in glomerular morphologic changes in rats resembling those in human nephrotic syndrome with minimal changes. This report describes acute and chronic glomerular injury that begins as early as 8 days after a single central administration of PA and progresses to FSGS within an 18-week period. It seems likely that minimal change disease and FSGS are two pathologic processes in the same continuum of disease. In this model, the severity and persistence of the glomerular lesion may represent irreversible glomerular epithelial cell (GEC) injury secondary to the toxic effects of PA.     

Experimental nephrosis: interassociation of proteinuria with impaired lymphocyte blastogenesis.

The drug puromycin aminonucleoside (PA), used to induce an experimental nephrosis in rats, inhibited the blastogenic response of normal rat spleen cells when cultured in vitro with autologous or heterologous serum. Only at final PA concentrations of less than 5 microgram/ml did PHA induce normal blastogenesis. When PA was unilaterally perfused through the rat kidney a nephrosis developed, characterized by massive proteinuria. Microscopically, the foot process fusion and mesangial cell increase were similar to that seen in human steroid-responsive nephrotic syndrome. Once proteinuria had developed, there was marked suppression of the lymphocyte blastogenic response of the nephrotic rat spleen cells when cultured in autologous sera. Neither proteinuria nor inhibited blastogenesis was found in animals perfused with a buffered salt solution. Animals which were perfused with PA, nephrectomized 2 days after perfusion, and did not show proteinuria, had suppressed lymphocyte blastogenesis after stimulation with PHA. However, the degree of stimulation by the spleen cells of these animals was similar to that from control perfused and nephrectomized animals. Therefore, the aberration in lymphocyte response was consistent with the development of the nephrosis and proteinuria.     

Glomerular cells and macrophages in the progression of experimental focal and segmental glomerulosclerosis.

To determine the characteristics of glomerular cell involvement in experimental focal and segmental glomerulosclerosis (FGS) in rats induced by repeated injections of aminonucleoside of puromycin (PAN) and protamine sulfate (PS), glomeruli were isolated and grown in tissue culture. When compared with saline controls the FGS rats had decreased rates of glomerular attachment and significantly reduced outgrowths of type 1 (epithelial) and type II (mesangial) cells. However, the FGS rats had greater numbers of type III cells (macrophages) present in the outgrowths than controls. The production of interleukin-1 (IL-1) in the glomerular culture supernatant, measured by thymocyte stimulating activity, was determined at various stages throughout the evolution of FGS and in saline controls. The FGS glomeruli early in the disease course (day 10) had higher levels of IL-1 activity than glomerular outgrowths from control rats but significantly lower levels of IL-1 were produced late in the disease (day 80). Glomerular macrophages were present throughout the evolution of the disease but in greater numbers early (day 10). In this experimental model of FGS, IL-1 production, has been demonstrated early in the disease process, could originate from macrophages and/or mesangial cells, and could be involved in the progressive glomerulosclerosis.     

Stable analogs of prostaglandins E1 and F2 alpha ameliorate the proteinuria of aminonucleoside-of-puromycin nephrosis in Lewis rats.

Prostaglandins have been implicated by previous investigators in the pathogenesis of the nephrotic syndrome. A single subcutaneous injection of 1 mg/kg of stable analogs of prostaglandins E1 or F2 alpha (15[S]-15-methyl -PGE1 [M-PGE1] and -PGF2 alpha [M-PGF2 alpha]) was found in the present study to dramatically decrease proteinuria on Day 10 of puromycin aminonucleoside (PAN) nephrosis in Lewis rats. The decrease in proteinuria was mediated at least in part by a decrease in glomerular filtration rate (GFR), as quantitated by inulin clearances in nephrotic control and prostaglandin-treated rats. M-PGE1, moderately, and M-PGF2 alpha, to a lesser degree, also decreased the GFR in normal rats. Interestingly, the GFR was dramatically decreased in nephrotic as compared with nonnephrotic control rats, which suggests that PAN nephrosis may not be an ideal experimental model for human minimal change nephrosis in which the GFR is usually not severely compromised. The prostaglandin-induced decrease in GFR in both nephrotic and normal rats was coincident with a drop in systemic blood pressure. Nephrotic rats, however, had a slightly higher baseline blood pressure than normals, and the hypotensive effects of both prostaglandins were much less in nephrotic than in normal rats. The decrease in proteinuria was not related to a cytoprotective effect, as indicated by the failure of daily doses of 5 micrograms/kg M-PGE1 to reduce proteinuria 6, 8, or 10 days after injection of puromycin aminonucleoside. The similar antiproteinuric effects of prostaglandin synthesis inhibitors and of pharmacologic doses of prostaglandins are somewhat paradoxical but are reminiscent of the similarly paradoxical mutual antiinflammatory effects of these agents. The high doses of prostaglandins required to reduce proteinuria as well as their reduction of blood pressure and GFR will limit their clinical usefulness in the nephrotic syndrome.     

Alterations in proteoglycan metabolism in the nephrotic syndrome induced by the aminonucleoside of puromycin

The synthesis of intact proteoglycans and their glycosaminoglycan (GAG) side chains by isolated rat glomeruli in vitro were studied both at the onset (5 days) and at the point of maximal proteinuria (7 days) of the nephrotic syndrome induced with the aminonucleoside of puromycin. Glomeruli from nephrotic animals incorporated 1.5-fold and 3.0-fold more 35SO4 label into GAG than glomeruli from control animals at 5 and 7 days, respectively, with heparan-35SO4 GAG being responsible for the majority of the increment. Both nephrotic and control incubations contained 60% of the label in the incubation medium, 40% in the glomerular fractions, and less than 1% in the glomerular basement membrane. Glomerular basement membrane from nephrotic rats had no change in their total heparan-35SO4 GAG content. The majority of intact proteoglycan(s) from the glomerular matrix and from the incubation medium of nephrotic and control animals was found in the most buoyantly dense fraction of CsCl gradients (fraction 1). 35S-labeled material isolated from glomeruli of nephrotic animals showed a consistent shift toward lower density gradient fractions, indicating a decrease in their overall carbohydrate to protein ratio. Diethylaminoethyl chromatography of fraction 1 proteoglycan showed a single biphasic peak with the nephrotic rat having an increase in the proportion contributed by the earlier component of the peak. Fraction 1 proteoglycan(s) from the nephrotic experiment was found to have a smaller average hydrodynamic size by Sepharose CL-2B chromatography without a significant change in the corresponding 35S-GAG chain sizes (molecular weight 14,000) by Sepharose CL-6B chromatography. 35S-macromolecules from glomeruli of nephrotic and control rats that appeared in the middle of the CsCl gradients (fraction 3) had similar Sepharose CL-2B elution volumes, whereas the corresponding 35S-GAG chains from incubations of glomeruli from nephrotic animals were smaller. Increased synthesis of heparan sulfate proteoglycan by glomeruli from puromycin aminonucleoside-induced nephrotic rats may be compensatory to loss of another component of the glomerular filtration barrier or may result from abnormal interaction of proteoglycan(s) from nephrotic animals with other glomerular matrix constituents.     

An ultrastructural study of the effect of the steroid in puromycin aminonucleoside nephrosis rats

Summary In order to investigate the significance of the histological change in glomerular epithelial cells in minimal change nephrotic syndrome in man (MCNS) and to help in clarifing the mechanism of action of a steroid in this disease, methylprednisolone was administered to rats with puromycin aminonucleoside nephrosis (PAN). This is an experimental nephrosis having a close resemblance morphologically and physiologically, to human MCNS. Morphological changes in the glomerulus were observed ultrastructurally. The administration of the steroid to PAN rats showed remarkable changes including, rapid disappearance of proteinuria in PAN rats in a manner similer to that seen in human MCNS, and significantly faster recovery of changes in glomerular epithelial cells when compared with spontaneous recovery. From the present study, it is clear that the steroid is effective in rapidly restoring the normal shape of glomerular epithelial cells in PAN rats. The filtration barrier in the glomerular capillary wall (GCW) is also thought to have recovered and proteinuria is cured. Based on these considerations, it may be suggested that proteinuria in human MCNS is caused by changes in glomerular epithelial cells, and that the clinical treatment of proteinuria in MCNS is effective when glomerular epithelial cells have functionally recovered.     

Glomerular epithelial detachment, not reduced charge density, correlates with proteinuria in adriamycin and puromycin nephrosis

To identify the structural change coincident with increased glomerular permeability in both adriamycin (ADR) and puromycin-aminonucleoside (PAN) nephrosis, we explored the temporal correlation between developing proteinuria, the reduction in glomerular polyanions, and the detachment of epithelium from glomerular basement membrane (GBM). Sprague-Dawley rats received a single tail-vein injection of PAN (150 mg/kg), ADR (7.5 mg/kg) or saline. Nephrotic-range proteinuria appeared between days 2 to 5 in the PAN-treated and days 5 to 10 in the ADR-treated rats. The GBM heparan sulfate charge density and epithelial membrane sialic acid (SA) content were determined before, during and after the rise in proteinuria. Polyethyleneimine was used to detect heparan sulfate and the number staining of renal cortical slices was used to detect heparin sulfate and the number of sites/microns GBM in the lamina rara externa were counted on electron micrographs (magnification x60,000). Controls had a regular distribution of polyethyleneimine 20.19 +/- 1.72 sites/microns (X + SD). In ADR rats, the polyethyleneimine density decreased by day 5, 18.61 +/- 1.79 sites/microns (p less than 0.05) which persisted to day 15, 17.38 +/- 1.27 sites/microns (p less than 0.02). PAN rats, by day 1, had significant reduction, 14.94 +/- 1.47 sites/microns (p less than 0.05), which persisted to day 20. The total membrane-bound SA content of isolated glomeruli was analyzed with a modified Warren's method. The SA content in control glomeruli was 46.8 +/- 8.0 nmol/mg glomerular protein (X +/- SD). In ADR rats, there was significant decrease in SA content to 84 +/- 3% of control (p less than 0.01) at day 15. In PAN rats, by day 2, the SA content was decreased to 73 +/- 18% of control (p less than 0.05). In both models, scanning and transmission electron microscopy revealed epithelial foot process fusion, loss of slit diaphragms, and vacuolization before increased proteinuria. Epithelial detachment from the GBM and rupture of vacuoles occurred coincidently with rapid development of nephrotic proteinuria in both models. In summary, reduced GBM heparan sulfate and epithelial SA content do not correlate with the onset of altered glomerular permeability, whereas epithelial detachment is coincident with the development of massive proteinuria in both ADR and PAN nephrosis.