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Collagen cross-linking induced by lysyl oxidase has been implicated in liver and lung fibrosis. To define the role of this process in kidney fibrosis, we investigated the renal expression of lysyl oxidase and the content in collagen cross-links at various stages of chronic Adriamycin nephropathy in Sprague-Dawley rats. Lysyl oxidase expression was determined by RT-PCR; collagen pyridinium residues, indicating lysyl oxidase induced cross-links, were evaluated by HPLC. These parameters followed a synergic albeit asynchronous outcome: (a) lysyl oxidase mRNA levels in total kidney, glomeruli and medulla from Adriamycin-treated rats increased up to 3 times compared to controls between week 8 and 12, then returning within the normal range; (b) the pyridinium residue content did not show any significant difference between Adriamycin-treated and control rats, until diffuse interstitial fibrosis developed (16 weeks), showing at this time a 2- to 3-fold increment. Lysyl oxidase was expressed by several renal cell lines and in tubular-epithelial cells it was up-regulated in vitro by TGF beta-1, a recognized fibrogenetic factor in Adriamycin nephropathy. Our observations demonstrated that an increased expression of lysyl oxidase in the kidney precedes the development of diffuse fibrotic lesions and that, at this stage, collagenic structures contain highly cross-linked components, the final product of lysyl oxidase activity. The evidence of lysyl oxidase up-regulation in tubular epithelial cells by the same factor implicated in Adriamycin toxicity in the kidney suggests a common pathogenetic mechanism. Collagen cross-link formation by lysyl oxidase may be implicated in the pathogenesis of irreversible, fibrotic renal lesions.

Ultrasonography is currently the method of choice to examine the pediatric patient because it is widely available, noninvasive and inexpensive. Particularly, Doppler US is the method of choice to study renal artery stenoses, which are among the most frequent causes of arterial hypertension in children, in both renal allografts and native kidneys. This study was aimed at investigating the value of the indirect signs of stenosis observed during the assessment of intrarenal vessels--i.e., changes in the waveform, resistive index and acceleration index. We examined 63 renal allograft recipients and a 12-year-old boy with a native kidney and arterial hypertension. In all cases the Doppler curves of the intrarenal vessels were studied both qualitatively and quantitatively. In this paper we report on 5 cases (4 allografts and 1 native kidney) with major waveform changes in the downstream renal circulation. Our findings confirmed the resistive index and the acceleration index measured with Doppler US in the intrarenal vessels to be major diagnostic tools in renal artery stenoses in the pediatric patient when the renal arteries are difficult to assess. However, the technique exhibits some limitations and allows an unquestionable diagnosis only in severe stenoses.

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.

Protein restriction ameliorates proteinuria in acute adriamycin (ADR) nephrosis and decreases the renal levels of xanthine oxidase (XO), a putative mediator of ADR nephrotoxicity. Hypothetically, the effect of protein restriction on renal XO levels may be due to variations in plasma and tissue proteic amino acids (AA). To elucidate this point, the levels of AA in plasma and in renal homogenates were determined in rats with ADR nephrosis and fed diets with different protein contents: (a) high (35%) casein; (b) standard (21%) casein; (c) low (9%) casein; (d) low casein plus a synthetic mixture of Val, Leu and Ile. The protein content of the diet determined certain marked variations in plasma AA: high levels of Val, Leu and Ile were found in rats fed on a high protein diet, while the same AA were low, in rats on low protein regimen. Supplementation of the low protein diet with a synthetic mixture of branched-chain AA (Val, Leu and Ile) normalized the plasma levels of these AA. In spite of these changes, tissue AA were similar in all groups, regardless of the protein contents of the diets. Furthermore, the levels of renal XO and proteinuria were unrelated to variations in plasma AA, since both parameters were low in protein-restricted and protein-restricted AA-supplemented rats while high in rats fed a high or normoproteic diet. These data demonstrate that low protein diets induce marked alterations in plasma AA composition which are similar in may respects to those found in protein malnutrition.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

It is well accepted that the molecular charge and conformation of serum proteins are major determinants of their glomerular filtration, but few studies characterizing the molecular features of circulating proteins in renal diseases are currently available. In 11 children affected by minimal change nephropathy (MCN) we determined the electrical charge and the fluorescence quantum yield of Tyrosine (Tyr) and Tryptophan (Trp) (taken as index of conformation) of serum and urinary albumin before and after steroid-induced remission of proteinuria. In all proteinuric children at the onset of the disease, urinary albumin was formed by one band with an isoelectric point (pI) of 4.7 (pI of the native protein), and by numerous other, less anionic bands with pIs between 4.8 and 5.5 accounting for about 50% of the total amount of this protein. The normalization of proteinuria which followed steroid therapy was characterized by the disappearance in urines of the less anionic fraction and by the appearance of numerous isoforms with a pI still more anionic (pI less than 4.7) than normal. At the same time, in the proteinuric phase, the fluorescence quantum yield of Trp of urinary albumin was markedly quenched, returning to near normal levels after steroid-induced remission of proteinuria. These data indicate that in MCN the charge-dependent renal selectivity properties are partially maintained and that the less anionic isoforms of albumin are a main component of urinary albumin. Together with the electrical charge, the conformation of albumin as a major determinant of its urinary excretion in MCN must also be considered.

The major features of the Branchio-Oto-Renal syndrome (BOR syndrome), an autosomal dominant disorder, are branchial remnants, ear anomalies, deafness and renal dysplasia. We report two family groups affected by the BOR syndrome: in two-thirds of the affected children renal abnormalities led to severe renal insufficiency in early life. The necessity for a meticulous search for renal anomalies in individuals with aural and/or branchial abnormalities is emphasized. In affected families, genetic counselling is suggested.

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.