Cis and trans regulatory elements in NPHS2 promoter: implications in proteinuria and progression of renal diseases.

Podocin (NPHS2) expression in podocytes is associated with variable degrees of proteinuria and progression to renal failure in different glomerular diseases that suggests different expression profiles in NPHS2 promoter. Three functional polymorphisms in NPHS2 promoter (-51T, -116T, and -535 insCTTTTTT(3)) were found determining strong downregulation (-73, -59, and -82%, respectively) of the reporter gene expression when transfected in podocytes. Electrophoretic mobility shift assay experiments showed that all wild-type variants (-51G, -116C, and -535 insCTTTTTT(2)) formed specific DNA-protein complexes with podocyte nuclear extracts that were abolished by the presence of the rare forms (-51T, -116T, and -535 insCTTTTTT(3)). In the case of -51G, upstream stimulatory factor-1 (USF1) was identified as the specific trans element in accord to binding inhibition experiments and USF1 RNAi silencing. Haplotype analysis of 204 normal controls and 545 patients with renal diseases (308 immunoglobulin (Ig)A nephropathy and 237 focal segmental glomerulosclerosis) evidenced that -116/-51 and -535/P2OL formed two blocks in strong linkage disequilibrium in both normal and pathological cohorts. The high NPHS2 promoter profile -116C/-51G haplotype was more frequent in patients with IgA nephropathy (P-value=0.005) and was associated with a better clinical outcome in terms of proteinuria and creatinine levels. Overall our study describes functional variants of NPHS2 promoter and characterizes trans-acting elements that modulate podocin expression in the kidney. High producer NPHS2 promoter haplotypes seem protective in patients with chronic glomerular diseases.

Circulating anti-actin and anti-ATP synthase antibodies identify a sub-set of patients with idiopathic nephrotic syndrome.

Idiopathic nephrotic syndrome (iNS) with resistance or dependence to steroids is a common disease in children but in spite of an increasing clinical impact its pathogenesis is unknown. We screened for the presence of circulating antibodies against glomerular (podocytes, mesangium) and tubular cells (tubular epithelia) a cohort of 60 children with iNS including 8 patients with a familial trait of iNS or with proven mutation of NPHS1-NPHS2 and 12 with good sensitivity to steroids. Positive sera were found in 8 cases, all belonging to the category without familial trait/molecular defects. The targets of antibodies were characterized with Western blot and MALDI-Mass utilizing beta-hexyl cell extracts separated with two-dimensional electrophoresis. In all cases antibodies of the IgM class were directed against ATP synthase beta chain alone (4 cases) or in combination with actin (3 cases); one child presented IgG against aldose reductase. The clinical picture was nephrotic syndrome with steroid resistance or dependence and variable cyclosporin sensitivity; 3 patients developed end stage renal failure. The basic pathology picture was focal segmental glomerulosclerosis (FSGS) in 4 cases and mesangial proliferative glomerulonephrites with deposition of IgM in 2. Overall, patients with circulating auto-antibodies could not be readely differentiated on clinical grounds with the exception of 3 children who developed positivity for antinuclear antibodies during the follow-up. Affinity-purified IgM from one patient who underwent plasmapheresis for therapeutical pourposes (but not from a normal pool) induced proteinuria in Sprague-Dawley rats and concomitant human IgM deposition within glomeruli. This is the first report of circulating anti-actin/ATP synthase beta chain antibodies in a subset of patients with iNS. Both pathological significance and clinical impact given by the presence of these antibodies and the relationship with other conditions such as lupus-erythematosus, characterized by their presence, must be defined.

A DNA element in the alpha1 type III collagen promoter mediates a stimulatory response by angiotensin II.

BACKGROUND: Angiotensin II (Ang II) plays an important role in extracellular matrix deposition and tissue scarring in the kidney and the heart. The mechanism for extracellular matrix stimulation by Ang II is currently hypothetical, with one possibility pointing to a direct effect on cell synthesis of specific collagens. METHODS: We studied the molecular mechanism for activation of type III collagen synthesis by Ang II in an in vitro cell model of myofibroblasts by evaluating (1) alpha1(III) collagen mRNA expression; (2) alpha1(III) collagen promoter activity; (3) DNA/protein binding with characterization of binding sites; (4) expression of transcription factors; and (5) the role of a short DNA segment as Ang II responsive element. RESULTS: We found a specific dose-dependent stimulation of alpha1(III) collagen mRNA expression and a parallel effect on alpha1(III) collagen promoter activity. Transfection of constructs containing alpha1(III) collagen promoter fragments of different lengths localized the site of activation within the shortest 178 bp construct. By gel-retardation experiments, we observed the formation of a DNA-protein complex with crude extracts from Ang II-stimulated cells and an oligonucleotide spanning the 3 to 20 sequence. This complex was due to a sequence-specific interaction and was abolished by a 3 bp substitution mutation. The introduction of this mutation into the 178 bp construct abolished the stimulatory effect of Ang II. CONCLUSIONS: These results demonstrate that Ang II stimulates the expression of alpha1(III) collagen mRNA in myofibroblasts in vitro by activating the alpha1(III) collagen promoter at the level of a factor recognition site localized immediately downstream of the transcription start site. This mechanism could be involved in Ang II-induced renal and heart fibrosis.

Characterization of a DNA binding site that mediates the stimulatory effect of cyclosporin-A on type III collagen expression in renal cells.

BACKGROUND: Previous work from our laboratory demonstrated upregulation of type III collagen by cyclosporin A (CsA) in a cellular model of renal fibroblasts 'in vitro', suggesting that a mechanism of gene transcriptional activation might be responsible for collagen accumulation in renal fibrosis resulting from chronic CsA treatment. METHODS: We analysed in the same cellular model: (i) COL3A1 mRNA expression by RT-PCR; (ii) COL3A1 promoter activity by transfection of renal fibroblasts with constructs containing promoter fragments of different length fused to a reporter gene; (iii) expression of transcription factors by western blot analysis; (iv) DNA-protein binding by gel retardation assays with nuclear extracts from CsA-treated and untreated cells; and (v) site-directed mutagenesis of COL3A1 promoter to verify the role of a short DNA segment as CsA responsive element. RESULTS: CsA induced a 3-5-fold increase in COL3A1 mRNA that was paralleled by a stimulation of the COL3A1 promoter. Degradation of COL3A1 mRNA was comparable in CsA-treated and -untreated cells. The target region was first limited to a 178 bp fragment from -117 to +61 (pFV1). By gel retardation, utilizing several oligonucleotides that covered the whole length of pFV1, we detected a factor able to bind the promoter DNA (oligo 31) in nuclear extracts after 3 h treatment with CsA. The binding was absent in untreated cells and it was not detected when a 10-base mutation was introduced in oligonucleotide 31. Finally, the same substitution mutation at the site of binding of this factor abolished the stimulatory effect of CsA on COL3A1 promoter. Some transcription factors, whose potential binding sites are included in the above promoter fragment, were induced by CsA treatment either soon (3 h) or late (24-72 h) after treatment and were detected by western blot analysis. CONCLUSIONS: CsA induces the synthesis of type III collagen by stimulating a pathway leading to activation of COL3A1 promoter and upregulation of COL3A1 mRNA. A short promoter fragment, proximal to the transcription start site, is the target of CsA stimulation.

Cell-specific regulation of alpha1(III) and alpha2(V) collagen by TGF-beta1 in tubulointerstitial cell models.

BACKGROUND: TGF-beta1 modulates the cellular expression of extracellular matrix (ECM) in several renal cell systems in vitro and is considered a determinant of ECM accumulation in tubulointerstitial fibrosis. METHODS: We evaluated the effects of TGF-beta1 on collagen transcription, expression, and removal of the relevant collagens by rat tubuloepithelial cells (NRK 52E) and both rat and monkey interstitial fibroblasts (NRK 49F, CV1) in vitro. RESULTS: TGF-beta1 upregulated the expression of alpha1(III) collagen by fibroblasts (+300%) without affecting its removal. In parallel, a threefold increment of COL3A1 mRNA was found. Experiments of cell transfection employing CV1 fibroblasts as the unique suitable model, and chimaeric constructs of COL3A1 and COL5A2 promoters fused to the luciferase reporter gene, demonstrated a twofold stimulation of a large 1436 COL3A1 promoter construct and negligible effects on shorter fragments, suggesting the presence of a positive responsive element in a region of COL3A1 promoter between -1375 and -579. TGF-beta1 did not influence COL5A2 mRNA and the relative promoter activity in renal fibroblasts. With NRK 52E cell line, TGF-beta1 induced comparable increment of both alpha1(III) collagen expression (+300%) and COL3A1 mRNA (+300%) without affecting the COL3A1 promoter activity of any constructs. TGF-beta1 also upregulated the expression of alpha2(V) collagen chain (+500%) and COL5A2 mRNA (+500%) with a stimulatory effect (+100%) on a 1177 bp fragment of COL5A2 promoter. In this case a relevant inhibitory effect of TGF-beta1, on removal of alpha2(V) by supernatants of NRK 52E was also observed, indicating a double regulatory role of the cytokine on both transcription and removal of this component of ECM. CONCLUSION: Taken together these data indicate that TGF-beta1 is a potent stimulator of alpha1(III) collagen expression by renal fibroblast cell lines in vitro, the basic mechanism being stimulation of COL3A1 transcription. With renal epithelial cell lines, TGF-beta1 mainly upregulated the expression of type V collagen with the most relevant effect on stimulation of collagen transcription and inhibition of its removal. Tubular epithelial cells and renal fibroblasts should play distinct roles in renal fibrosis induced by TGF-beta1 in vivo.

Cyclosporine enhances the synthesis of selected extracellular matrix proteins by renal cells "in culture". Different cell responses and phenotype characterization.

Glomerulosclerosis and interstitial fibrosis are 2 major side effects of protracted therapy with CsA in heart transplant patients and in nonrenal immunologic diseases. To investigate whether there is any cause-effect correlation between CsA and the synthesis of extracellular matrix in the kidney, we determined the amount and composition of collagens produced by various renal cells "in culture" upon exposure to increasing levels of CsA. The cellular models we used included primary cultures of both human and rat mesangial cells (hMC, rMC), human and rat renal fibroblasts (hFib, rFib), and human tubular epithelia as well as cell lines of rat renal fibroblasts (NRK49F) and of tubular epithelia (NRK52E). In the case of primary cell cultures, CsA induced a marked increment of total collagen synthesis. This was highest for renal fibroblasts (+330% hFib, +110% rFib), followed by rMC (+170%), hMC (+100%), and human tubular epithelia (+130%). At the highest dosage of CsA (5 ng/ml), this corresponded to a net increment in collagen III synthesis by both hMC and hFib (+150% and +300%), while collagen I and collagen IV were unaffected. On hMC, CsA also induced a maximal increase in a component with 70 kDa molecular mass, which was produced only in a negligible amount by these cells in standard conditions. This low molecular mass collagen was tentatively characterized by cyanogen-bromide digestion and fingerprint analysis as a novel molecule showing a peptide composition without comparable features for any reported collagen map. NRK49F and NRK52E cell lines were not affected by CsA. Taken together, these observations demonstrate that CsA is able to induce the synthesis of specific collagens, mainly of collagen III and of a 70-kDa component, by various renal cells in cultures. Since the same cells are the renal site of production of extracellular matrix in pathological conditions, we hypothesize that this effect is a relevant one in the pathogenesis of glomerulosclerosis/interstitial fibrosis during protracted therapies with CsA.

Selective enhancement by cyclosporin A of collagen expression by mesangial cells 'in culture'.

Extracellular matrix deposition in mesangial areas leading to glomerulosclerosis is the major side effect of protracted therapies with cyclosporin A. In order to define any direct correlation between a chronic therapy with the drug and glomerulosclerosis we studied the effects of cyclosporin A on extracellular matrix production by human mesangial cells in culture. By immunoprecipitation and sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) of [3H]proline-labeled mesangial cells it was found that cyclosporin A induced a dose-dependent increase in total collagen synthesis (+80%), corresponding to a net increment in collagen III (+120%) and in a component with 70 kDa molecular weight which was produced only in negligible amount by mesangial cells under standard conditions. This collagen was characterized by cyanogen bromide digestion and finger print analysis as a novel molecule, not sharing any peptide composition similarities with the already characterized collagens. These data indicate that cyclosporin A stimulates the synthesis by mesangial cells of selected collagens, mainly collagen III and a new low molecular weight component. This mechanism may be relevant in cyclosporin A induced glomerulosclerosis occurring during protracted therapies with the drug.

Intact renal albumin downregulates the extracellular matrix expression by mesangial cells and renal fibroblasts in vitro.

Chronic Adriamycin (ADR) nephropathy is invariably associated with glomerulosclerosis and tubulointerstitial fibrosis. To investigate the hypothesis that severe albuminuria plays a role in the pathogenesis of both processes, we purified the protein from conditioned media of rats with advanced ADR nephropathy and tested the fibrogenic effect on renal fibroblasts and mesangial cells in vitro. Albumin was purified by pseudoligand chromatography and was identified on the basis of the NH2 amino terminus. Furthermore, it was differentiated from the urinary homologue, being more anionic and more fatted while maintaining a conserved peptide composition. The exposure of renal cells to renal albumin induced a dose-dependent reduction in collagen synthesis with a half-maximal decrease with 0.2 microgram/ml of albumin. With renal albumin levels of 0.4 microgram/ml the collagen incorporation of 3H-proline by mesangial cells and renal fibroblasts (primary cultures and cell lines) was reduced by 76, 81 and 45% respectively. A qualitative analysis by SDS-polyacrylamide electrophoresis and immunoprecipitation of radiolabelled collagens demonstrated a drastic and unselective decrease in all major collagens synthesized by mesangial cells and fibroblasts, including type I, III and V. Previous immunoprecipitation of the protein with anti-rat albumin antibodies completely reversed this phenomenon. Finally, albumin purified from urines of rats with ADR nephropathy downregulated the synthesis by renal cells of the same collagens but this effect was less evident compared to renal albumin. These findings demonstrate that renal albumin drastically reduces the synthesis of collagens by mesangial cells and renal fibroblasts, this effect being most evident on those components which constitute the extracellular matrix in glomerulosclerosis and interstitial fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple mechanisms for doxorubicin cytotoxicity on glomerular epithelial cells 'in vitro'.

This study was planned to define the metabolic pathways for free radical production by isolated glomeruli and glomerular epithelial cells in vitro after exposure to cytotoxic doses of doxorubicin. A net increment in glomerular superoxide anion (O2.) synthesis was observed at doxorubicin doses between 10 and 30 micrograms/ml, a drug level which also induced a parallel increment in uric acid synthesis. Since the synthesis of O2. with production of uric acid implies an activity of xanthine oxidase, a few experiments were performed with glomeruli which had been deprived of xanthine oxidase activity. In this case doxorubicin-inducible O2. and uric acid synthesis by glomeruli was practically nil. A similar stimulatory effect of O2. synthesis was induced by doxorubicin on glomerular epithelial cells and also in this case O2. synthesis was suppressed by pre-treating cells with deoxyconformicin, a selective inhibitor of adenosine deaminase. Finally, equimolar amounts of the drug were equally cytotoxic even when kept constantly outside the cell by a stable linkage with an agarose macroporous bed. In summary, these data demonstrate that O2. is generated by isolated glomeruli and glomerular epithelial cells 'in vitro' when exposed to cytotoxic amounts of doxorubicin and that purine degradation to uric acid furnish the metabolic pathways for glomerular O2. generation. However, doxorubicin is comparably cytotoxic on glomerular epithelial cells from outside cells thus suggesting that also a membrane perturbation may activate the series of events leading to cell injury.

Interaction between cationic dyes and erythrocyte membranes in minimal change nephropathy: an electrophoretic approach.

A study was undertaken to clarify the usefulness of two cationic dyes, alcian blue (AB) and ruthenium red (RR) in demonstrating the defect in cellular membranes noted in minimal change nephropathy (MCN). The binding of both dyes to RBC membranes purified from normal and nephrotic children was evaluated by electrophoretic titration curves. When examined separately, AB was found to precipitate spontaneously, producing macro-aggregates with no electrophoretic mobility at pH 5. This was presumed to be the result of hydrophobic interaction of the dye with itself. The same phenomenon was observed when this dye was incubated at 37 degrees C with RBC ghost's from normal children, when AB presented a sigmoidal curve with a net positive charge for pHs higher than 5.5 and lower than 5 and no electrophoretic mobility at pH 5. However, incubation of AB with RBC ghosts from children with MCN resulted in an improvement of the solubility of the dye which then migrated with a net positive charge along the whole gradient of pH from 3.5 to 9. The presence of zwitterionic neutral detergents such as CHAPS, but not of a charged substance such as protamine sulphate, inhibited precipitation at pH 5 when incubated with membranes from normal children, supporting the hydrophobic nature of the phenomenon. When RR was used instead of AB, it was fully protonated (i.e. did not precipitate) when analysed alone, but when incubated with normal RBC ghosts, it also revealed no electrophoretic mobility at pH 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary protein restriction on renal purines and purine-metabolizing enzymes in adriamycin nephrosis in rats: a mechanism for protection against acute proteinuria involving xanthine oxidase inhibition.

1. A low protein diet prevents the development of proteinuria and glomerular damage in adriamycin experimental nephrosis without affecting renal haemodynamics. In this study the hypothesis was tested as to whether protein restriction is able to modulate the purine metabolic cycle and related enzymes such as xanthine oxidase, one of the putative effectors of adriamycin nephrotoxicity. 2. Renal activities of xanthine oxidase and purine nucleoside phosphorylase were markedly depressed in adriamycin-treated rats fed a 9% casein (low protein) diet compared with the group fed a 22% casein (normal protein) diet both 1 day after adriamycin administration and at the time of appearance of heavy proteinuria (day 15), whereas the activity of renal adenosine deaminase was unchanged. 3. The concentrations of the metabolic substrates of xanthine oxidase, i.e. hypoxanthine and xanthine, were constantly lower in renal homogenates of rats fed a low protein diet compared with those on a normal protein diet. In urine, uric acid, the product of hypoxanthine-xanthine transformation, was lower 1 day after adriamycin injection in protein-restricted rats compared with the group on a normal protein diet which showed a marked increase in its excretion. At the same time, the urinary efflux of adenosine 5'-monophosphate, which is the precursor nucleotide of the above-mentioned nucleosides and bases, was very high in rats fed a low protein diet, whereas it was absent in the group on a normal protein diet. 4. The progressive increment in proteinuria of glomerular origin (i.e. increased excretion of albumin and transferrin) typical of adriamycin-treated rats fed a normal protein diet was inhibited in the protein-restricted animals, which were normoproteinuric on day 10 and were only slightly proteinuric on day 15. 5. Like protein restriction, the pharmacological suppression of renal xanthine oxidase by dietary tungstate and the scavenging by dimethylthiourea of the putative free radical deriving from the action of xanthine oxidase, were associated with a similar (quantitative and qualitative) inhibition of glomerular proteinuria. 6. These data demonstrate that dietary protein restriction is associated with a block in purine metabolism within the kidney due to a marked reduction in the activities of two main enzymes of the cycle, i.e. purine nucleoside phosphorylase and xanthine oxidase, the latter being a putative effector of adriamycin nephrotoxicity. The partial reduction of proteinuria induced by a low protein diet is quantitatively and qualitatively comparable with the reduction induced by the specific block of renal xanthine oxidase or by the scavenging of OH.deriving from hypoxanthine and xanthine transformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Renal purine efflux and xanthine oxidase activity during experimental nephrosis in rats: difference between puromycin aminonucleoside and adriamycin nephrosis.

1. The hypothesis was tested that the renal xanthine oxidase system provides a source of oxygen free radicals in puromycin aminonucleoside and adriamycin experimental nephrosis by generating uric acid from hypoxanthine and xanthine. 2. The concentrations in renal tissue of the putative intermediary products of puromycin aminonucleoside metabolism, hypoxanthine and xanthine, and of their precursors, adenosine and inosine, were lower in rats treated with puromycin aminonucleoside than in normal controls, whereas concentrations of the metabolites were normal after adriamycin intoxication. Their daily urinary excretion was lower in the 24 h after puromycin aminonucleoside administration compared with the baseline values and returned to near normal levels within 5 days. After adriamycin the 24 h urinary excretion of xanthine and uric acid was double the baseline levels (P less than 0.001). 3. When equimolar amounts of hypoxanthine were injected instead of puromycin aminonucleoside, the concentration of all bases increased slightly in renal tissue and their urinary efflux was double the baseline level: allantoin, uric acid, the unmodified nucleotide and xanthine were the most represented compounds in urine. 4. The enzymatic activities relative to xanthine oxidase (EC 1.1.3.22) and xanthine dehydrogenase (EC 1.1.1.204) in renal tissues were unchanged 1 day after puromycin aminonucleoside or hypoxanthine intoxication and only moderately increased in both groups at 13 days (the time of appearance of heavy proteinuria in the puromycin aminonucleoside-treated group). In contrast, xanthine oxidase and xanthine dehydrogenase activities were higher in adriamycin-treated rats at 1 and 15 days after the treatment (P less than 0.001). 5. Feeding rats with normoprotein diets containing tungsten induced a marked and constant decrease of renal xanthine oxidase and xanthine dehydrogenase activities to 20% of the baseline values in both puromycin aminonucleoside- and adriamycin-treated rats. Inhibition of renal xanthine oxidase and xanthine dehydrogenase activities by tungsten was associated with a marked reduction (P less than 0.001) of proteinuria in adriamycin-treated rats and the same occurred with allopurinol, a specific inhibitor of xanthine oxidase activity. In contrast, tungsten treatment did not reduce the proteinuria associated with puromycin aminonucleoside, which reached a maximum 13 days after puromycin aminonucleoside intoxication. Hypoxanthine-treated rats were normoproteinuric after 2 months of observation. 6. These data demonstrate an activation of renal xanthine oxidase and xanthine dehydrogenase after adriamycin intoxication which is relevant to the induction of proteinuria. They also argue against the involvement of the renal xanthine oxidase system as a source of free radicals in puromycin aminonucleoside nephrosis and suggest that the nucleotide cycle is not a normal route for puromycin aminonucleoside degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Low-protein diet and xanthine-metabolising enzymes in adriamycin nephrosis.

Proteinuria and renal xanthine metabolising enzymes, xanthine oxidase and xanthine dehydrogenase, were evaluated in Adriamycin-treated rats fed standard (21% casein) and low-protein (6% casein) diets. In rats fed a standard diet Adriamycin was associated with increased activities in the kidney of xanthine oxidase and xanthine dehydrogenase and induced massive proteinuria. The pharmacological block of both enzymes by allopurinol and tungsten block of both enzymes by allopurinol and tungsten reduced proteinuria to one-third of the original levels. Rats fed a low-protein diet presented decreased levels of renal xanthine oxidase and xanthine dehydrogenase and were only slightly proteinuric. Finally, rats shifted from a low-protein diet to a normal one developed massive proteinuria in spite of normal or slightly decreased levels of renal xanthine oxidase and xanthine dehydrogenase. We conclude that a low-protein diet is effective in decreasing the levels of xanthine metabolising enzymes that are in part responsible for the renal damage due to Adriamycin. This is not however the unique mechanism by which the low-protein diet protects against the development of proteinuria in Adriamycin nephrosis; other factors must also be hypothesised.

Analysis by isoelectric focusing of xanthine oxidase and NADH dependent enzymes in rat kidney.

Ultrathin isoelectric focusing was employed for analyzing xanthine oxidase and enzymes with NADH-dependent dehydrogenase activity in homogenates of rat kidney. After isoelectric focusing the enzymes were stained with specific assays where NBT is reduced upon incubation of the gel with xanthine (oxidase stain) and NADH (dehydrogenase stain) as substrates. A good separation of renal enzymes with dehydrogenase activities was obtained by using gels containing 2 M urea and by applying the sample at the anode. In these conditions 4 main isoforms with pI 6.4, 6.35, 6.5 and 6.6 were observed with the dehydrogenase stain but we were unable to demonstrate renal xanthine oxidase (XO) which seemed to be due to precipitation at the application point.

Preparative high performance chromatography of a major browning compound derived from lysine and glucose.

A major browning compound derived from lysine and glucose was purified by high performance chromatography on a RP8 column after several extractions in methanol plus acetonitrile. This compound was separated by a main contaminant corresponding to unreacted lysine by extracting the aminoacid after its derivatization with ninhydrin.

Peroxidative damage of the erythrocyte membrane in children with nephrotic syndrome.

The structural composition of erythrocyte ghosts was analysed in children affected by steroid-responsive (SRNS) and unresponsive nephrotic syndrome (SUNS). No variation of either intrinsic or extrinsic ghost proteins was found by discontinuous SDS-electrophoresis associated with a very sensitive double staining technique. By contrast, the composition of inner-layer phospholipids--phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS)--was altered in SRNS with minor changes also involving phosphatidic acid, phosphatidyl inositol and lysophosphatidyl choline. Signs of peroxidative damage were present in both SRNS and SUNS ghosts and inside the cells; these included high levels of fluorescent amino-iminopropene derivates of PE and PS, increased intraerythrocytic amounts of malonyldialdehyde and decreased levels of reduced glutathione. Taken together these results support the concept that in SRNS and SUNS erythrocytes are target cells for peroxidative damage. In SRNS peroxidation of membrane lipids results in a marked alteration of the phospholipid composition of erythrocyte ghosts.

Endogenous albumin as a marker of renal selectivity in steroid-unresponsive nephrotic syndrome.

Albumin electrical charge, conformation and hydrophobicity taken as indexes of renal selectivity were evaluated in 8 children affected by steroid-unresponsive nephrotic syndrome associated with glomerulosclerosis or mesangial hypercellularity. These characteristics related to urinary albumin have already been reported to vary markedly in steroid-responsive nephrotic syndrome of minimal-change nephropathy giving rise to new pathogenetic possibilities in this disease. In the steroid-unresponsive nephrotic children albumin was found to be more microheterogenous and cationic in urine than in serum and at the same time it was conformationally altered. Regarding these characteristics, the selectivity properties of the renal filter are similar in steroid-unresponsive nephrotic syndrome, suggesting a pathogenetic connection between these two renal disorders.