Lysyl oxidase regulates MMTV promoter: indirect evidence of histone H1 involvement.

Lysyl oxidase (LOX) is the enzyme that facilitates the cross-linking of collagen and elastin, although other functions for this enzyme have been indicated. Of these other functions, we describe herein the ability of LOX to regulate several gene promoters, like collagen III, elastin, and cyclin D1. We have previously demonstrated a specific binding between LOX and histone H1, in vitro.

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)).

Rare functional variants of podocin (NPHS2) promoter in patients with nephrotic syndrome.

Podocin (NPHS2) is a component of the glomerular slit-diaphragm, with major regulatory functions in renal permeability of proteins. Loss of podocin and decrease in resynthesis may influence the outcome of proteinuric renal disease such as segmental glomerulosclerosis (FSGS), and promoter functionality plays a key role in this process. NPHS2 promoter variants with functional activity may be a part of the problem of podocin resynthesis.

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.

beta-catenin signaling and regulation of cyclin D1 promoter in NRK-49F cells transformed by down-regulation of the tumor suppressor lysyl oxidase.

Lysyl oxidase is the enzyme that is essential for collagen and elastin cross-linking. Previous investigations showed that lysyl oxidase is down-regulated in many human tumors and ras-transformed cells. Recently, we proved that antisense down-regulation of lysyl oxidase in NRK-49F cells induced phenotypic changes and oncogenic transformation, characterized by p21(ras) activation and beta-catenin/cyclin D1 up-regulation.

Demonstration of in vitro interaction between tumor suppressor lysyl oxidase and histones H1 and H2: definition of the regions involved.

Lysyl oxidase (LOX) is the enzyme that cross-links extracellular collagen and tropoelastin and is involved in tumor suppressor activity. Based on the existent homologies between lysine-rich regions of tropoelastin and the "lysine-rich" histone H1, we tested the possibility that H1 could be a new nuclear target. Our study shows that LOX could actually interact specifically not only with histone H1, but also with histone H2.

Altered adhesion features and signal transduction in NRK-49F cells transformed by down-regulation of lysyl oxidase.

Lysyl oxidase (LOX) down-regulation induced an oncogenic phenotype in NRK-49F. This event was accompanied by a constitutive activation of ras oncogene and down-regulation of PDGF beta receptor, among other important phenotypic and molecular modifications. In the present paper we show that ras activation is not accompanied by a constitutive activation of the MAP kinases as expected.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.