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Chronic peritoneal dialysis (CPD), widely used in uremic children, may have contrasting effects on the nutritional status of patients. Metabolic and nutritional abnormalities due to the combined effects of uremia per se, glucose absorption from the dialysate and catabolic factors, such as protein and amino acid losses into dialysate, poor appetite, and recurrent episodes of peritonitis are the most important. Although CPD allows for fewer dietary restrictions and supplies an extra amount of calories by glucose absorbed with the peritoneal fluid, when protein and energy intakes are assessed the protein intake was almost sufficient or more than that prescribed, whereas the energy intake was low. In CPD children the standard deviation score for weight, height, triceps skinfold thickness, and midarm circumference has been reported as more severely impaired in children less than ten years old. Anthropometric parameters did not worsen during CPD treatment. Plasma proteins and albumin are reported as being low in CPD children. The dietary intake and protein losses have been considered to be the most important determinants of the albumin level in CPD patients. The reported average dialysate losses of free amino acids (AA) during CPD in children vary from 0.02 to 0.03 g/kg/day in different studies. The patterns of plasma AA in CPD is represented by reduced levels of branched chain AA and of other essential amino acids and increased concentrations of some nonessential AA. Several factors may influence plasma AA profile: uremia per se, hormonal alterations, protein and AA losses, and dietary intake. A more specific uremic AA pattern is found in muscle, the largest pool of free AA in the body. Studies on muscle AA in adults on CPD are conflicting: some authors have reported several muscle AA alterations, but others have shown an almost normal pattern. Low valine and leucine muscle levels have been reported in children on CPD.

This multicenter study investigated the characteristics of circulating IgA molecules in 77 children: 42 had primary IgA nephropathy (IgAN), 20 were non-IgA glomerulonephritides (CGN) and 15 had urological problems (U). Fifteen assays were employed including the detection of macromolecular IgA [IgA immune complexes (IgAIC) by the conglutinin (K) assay, heavy molecular weight IgA in 2.5% polyethylene glycol (PEG), mixed IgA/IgGIC (Jacalin assay), IgA-Fibronectin (IgA-F) aggregates]IgA antibodies to alimentary antigens (gliadin, glycgli, glutein, ovalbumin, bovine serum albumin) and IgA binding to mesangial antigens (fibronectin, laminin, type IV collagen) or polycations (poly-L-lysine). Total IgA and IgA reacting with jacalin, supposed to bear an altered galactosylation, were measured as well. Mean levels of each kind of macromolecular IgA were significantly increased in children with IgAN in comparison to U disease (K-IgAIC p < 0.05, PEG-IgAIC p < 0.01, IgA/IgGIC p < 0.004, IgA-F aggregates p < 0.0003). However, IgA-F were the only macromolecular IgA significantly higher in IgAN than in CGN (p < 0.0005). IgA-F aggregates did not correlate with any urinary sign of activity, while K-IgAIC data were significantly related with microscopic hematuria (p < 0.05) and past history of gross hematuria (p < 0.02). Children with IgAN had mean levels of IgA reacting with the lectinic fractions of gliadin significantly higher than CGN (p < 0.01) and U groups (p < 0.003). IgAN displayed an enhanced production of IgA reacting with mesangial matrix components vs CGN (p < 0.03) and U (p < 0.0003) groups and showed altered interactions with positively charged molecules (poly-L-Lysine, p < 0.01) and carbohydrate residues (jacalin p < 0.05). In IgAN there is an increased circulation of altered IgA favouring the formation of macromolecular IgA, including true IgAIC or IgA aggregated by carbohydrate interactions. The affinity for the mesangial matrix glycoproteins and for the mesangial area electrical charge might further enhance the trapping and deposition of the immune material containing IgA. IgA-F aggregates seem to be a marker of this event, while complement binding molecules in IgAIC correspond to the hematuric manifestation of the nephritogenic process.

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.

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.

It is generally thought that in primary IgA nephropathy (IgAN) an altered immune response favours high levels of serum IgA directed against environmental or mesangial antigens (Ag) leading to circulating IgA containing immune complexes (IgAIC). The aim of this multicenter collaborative study was to evaluate some of the IgA immunologic abnormalities in children with IgAN. We investigated 42 children with biopsy-proved IgAN, 21 children with non-IgA glomerulonephritides (CD) and 15 with previous urologic disorders without any evidence of immunologic disease (U). The following methods were used: detection of macromolecular IgA (IgAIC by the conglutinin solid-phase assay, heavy MW IgA measured in 2.5% polyethylene glycol precipitate, IgA-fibronectin aggregates, mixed IgA-IgGIC); serum levels of IgA to alimentary Ags (gliadin, glyc-gli, ovalbumin, bovine serum albumin) and mesangial Ags (fibronectin, laminin, type IV collagen) and reactivity of IgA with the lectin jacalin. In children affected with IgAN serum levels of macromolecular IgA were significantly higher in comparison to U group (p < 0.05-p < 0.0005). IgA-fibronectin aggregates only were significantly higher also than CD group (p < 0.0005). Mean levels of antigliadin IgA were significantly higher in IgAN than in controls (CD p < 0.01 and U p < 0.03) and similar data were found for IgA to mesangial matrix (laminin and fibronectin), which were significantly greater in IgAN than in CD and U (p < 0.01-p < 0.0002). Serum IgA in children with IgAN showed a greater affinity for both polycations and glycosylated molecules. Children affected by IgAN present abnormalities in serum IgA similar to those observed in adults with the same disease.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

To compare the efficacy (induction of remission) and safety of cyclosporine (CsA) with those of supportive therapy in patients with steroid-resistant idiopathic nephrotic syndrome (INS), we organized an open, prospective, randomized, multicentric, controlled study for parallel groups, stratified for adults and children. Forty-five patients with steroid-resistant INS were randomly assigned to supportive therapy or CsA (5 mg/kg/day for adults, 6 mg/kg/day for children) for six months, then tapered off by 25% every two months until complete discontinuation. Four patients were lost to follow-up. During the first year 13/22 CsA-treated patients versus three of 19 controls attained remission of the nephrotic syndrome (P < 0.001). A symptom score was assessed at time 0 and at six months. The mean score significantly decreased in the CsA group (P < 0.001), but remained unchanged in the controls. At month 6 the mean urinary protein excretion, the mean serum proteins and plasma cholesterol had significantly improved in the CsA group but were not changed in the controls. There were no significant differences in serum creatinine and creatinine clearance between treatments (interaction time* treatments, P = 0.089 and P = 0.935, respectively) at month 6 versus basal. The CsA-related side-effects were mild; no significant difference in blood pressure between the two groups was seen at any time. This study shows that CsA can bring about remission in some 60% of patients with steroid-resistant INS. In patients with normal renal function and without severe hypertension, CsA at the therapeutic scheme adopted did not produce severe renal or extrarenal toxicity.

Incidence of chronic renal failure in children is not yet clearly known. In recent years it has been evaluated on the basis of the number of patients accepted into dialysis-transplantation programs and is thus underestimated, as registries do not list children who are not treated for technical reasons, lack of facilities or health policy. The number of new patients per year per million child population varies widely. Differences among countries are mainly related to economic development. In developed countries the incidence of CRF remains stable or decreases slowly owing to early diagnosis, improved conservative treatment, prevention of genetically-transmitted diseases, whereas the prevalence increases steadily as a consequence of improved replacement therapy. Causes of primary renal diseases have been analyzed in several series totaling over 9400 children. The most frequent cause is chronic primary glomerulonephritis followed by pyelonephritis, including obstructive uropathies and vesico-ureteral reflux. Differences in geographical distribution of etiologies are also analyzed. The relative contribution of chronic peritoneal dialysis and hemodialysis in the treatment of children with ESRF varies from country to country. Several problems regarding CRF in children are briefly discussed: prevention of renal failure, extension of treatment opportunities to more children, quality of replacement therapy, and clinical rehabilitation of children.

Early tubular alterations were studied in 53 children with insulin-dependent diabetes mellitus (IDDM), 32 of whom were followed at regular 6-monthly intervals for 3 years. The urinary levels of retinol-binding protein (RBP), beta 2-microglobulin and brush border antigens (BBA) (determined by monoclonal enzyme immunoassay) were taken as indices of functional and cellular tubular alterations; urinary albumin was considered an early marker of glomerular alterations. All indices of tubular alterations were higher in IDDM children than in 368 normal children, while albuminuria was unchanged. Urinary levels of BBA, however, varied widely during follow-up, with 25 of the 32 IDDM patients who were followed at regular intervals having pathological values for BBA on at least one occasion, followed by normalization. Metabolic alteration was found to be the main cause of this variability, since a high statistical correlation was found between urinary BBA and fructosamine (P < 0.001) and between RBP and the stable fraction of glycosylated haemoglobin (P < 0.001). The data confirm that transient tubular proteinuria occurs in diabetic children before any other marker of renal involvement such as microalbuminuria. The maintenance of good metabolic control is essential to normalize this early abnormality that can be considered a reversible sign of functional renal involvement.

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.

Nutritional status, assessed by anthropometric and biochemical methods, and muscle water, protein and amino acid composition, were evaluated in a control group of 10 children with normal renal function who were undergoing elective surgery, and in 15 children with end-stage chronic renal failure. Samples of the rectus abdominis muscle were taken when surgery was performed in the control children and when a peritoneal catheter was implanted in the uremic children. Height and body weight were reduced in the uremic children compared to the controls but skinfold thickness, arm muscle circumference and serum proteins (total protein, albumin, transferrin, pseudocholinesterase) were essentially normal. The muscle contents of total, extracellular and intracellular water, and of alkali-soluble protein (ASP), DNA and the ASP-DNA ratio were not significantly different in uremic children from those in the controls. Plasma leucine, isoleucine, tyrosine, valine, and serine levels were significantly decreased, whereas plasma citrulline, 1-methylhistidine and 3-methylhistidine levels were increased. Muscle isoleucine and valine levels and the valine/glycine ratio were low in the uremic children. Our results demonstrate that children with chronic renal failure and growth retardation may maintain a satisfactory nutritional status but exhibit amino acid abnormalities typical of uremia.

Cells from the cysts of patients with autosomal dominant polycystic kidney disease (PKD) were grown in vitro under standard conditions without the aid of collagen-pretreated surfaces, and both the synthesis and composition of the extracellular matrix were investigated. At confluence, PKD cells presented the typical features of epithelial cells, but showed a different collagen composition from fibroblasts. Compared with normal tubular epithelia (NTE), PKD monolayers produced an excess of extracellular matrix, which accounted for 30% of the total incorporation of [3H] proline, although this value was considerably lower (by a factor of 10) in the case of NTE. Immunohistochemical and electrophoretic techniques revealed a complex collagen composition in the extracellular matrix which included [alpha (III)]3 and collagen IV. However, part of the collagen components remained unidentified in spite of the fact that they exhibited a typical M(r) of alpha 1(I) and alpha 2(I) in the presence of urea. Immunoprecipitation with monospecific antibodies and Northern blotting with specific probes failed to recognize alpha 1(I) and alpha 2(I), but demonstrated their presence in fibroblasts. Purification and cyanogen bromide digestion demonstrated a strong interhomology in fingerprint peptide composition among the uncharacterized collagens synthesized by PKD cells, thus suggesting a common identity. These observations document a markedly augmented production of extracellular matrix by PKD cultured cells in vitro, and show the presence of collagens which do not share homologies with the major collagen molecules. A better characterization of extracellular matrix composition is central to any comprehension of the cytogenetic mechanisms in vivo.

This study was designed to evaluate the occurrence and the type of proteinuria in 82 children with vesico-ureteric reflux (VUR) with or without renal scars. The urinary excretion of the high molecular weight protein albumin was taken as an index of glomerular alterations and the excretion of retinol-binding protein (RBP), beta 2-microglobulin and brush border antigens (BBA) (measured by monoclonal antibody-based enzyme-linked immunosorbent assay) was taken as an index of tubular alterations. All such markers were increased in children with VUR and were related to the degree of renal function. Patients showing reduced creatinine clearance had very high levels of albuminuria, microproteinuria and BBA, with all these variables reciprocally correlated. In children with normal renal function however, only microproteins (not albumin or BBA) were slightly increased, thus indicating an isolated tubular defect without involvement of the proximal segment of the tubule. However, microprotein excretion did not correlate with the grade of scarring (99mtechnetium-dimercaptosuccinic acid scan), both RBP and beta 2-microglobulin excretion being normal in 75% of children with radioisotopic signs of renal lesions but increased in 17% of children without scars. Therefore, tubular proteinuria identifies different groups of children with VUR but is not related to renal scarring. Prospective studies will define the usefulness of proteinuria as a reliable indicator of renal outcome.

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)

It has been suggested that the generation of toxic radicals plays an important role in toxicity by Adriamycin (ADR) on cancer cell lines and in vivo. We have examined the role of free radicals in determining toxicity and resistance to ADR of rat glomerular epithelial cells in culture; this method provides a good model for analyzing the mechanisms responsible for ADR experimental nephrosis in rats. Three points were established: a) the intra- or extracellular site of ADR toxicity; b) the role of the superoxide anion and of the hydroxyl radical in determining intra- and -extracellular cytotoxicity; and c) the implication of oxido-reduction cycling as a potential route for ADR semiquinone transformation. Free ADR was found to induce the same inhibition of [3H]thymidine incorporation into DNA as ADR bound to an agarose macroporous bed which prevents the intracellular incorporation of the drug. Specific scavenging of free radical activity by the enzymes catalase and superoxide dismutase, the hydroxyl radical inhibitors dimethyl sulfoxide and dimethylthiourea (DMTU) and by chelation of intracellular free iron with deferoxamine produced only a partial restoration of [3H]thymidine incorporation into DNA, which was maximal for DMTU (30% of normal incorporation). DMTU treatment was unsuccessful in preventing the extracellular cytostatic effect of ADR. Finally, glomerular epithelial cell killing (51Cr-release method) by 5-iminodaunorubicin, an ADR analogue with a modified quinone function that prohibits oxido-reduction cycling, was higher than unmodified ADR. These results indicate that ADR may exert its cytotoxic effects on glomerular epithelial cells by interaction at the cell surface, whereas the intracellular compartment, principally DNA, does not seen to be the target of ADR effects. They also suggest that the free radicals are in part responsible for ADR intracellular cytotoxicity, but other mechanisms should also be hypothesized. Finally, the participation of the ADR semiquinone radical in oxido-reduction cycling seems not important for the induction of the cellular damage.

Normal pubertal progression is associated with quantitative and qualitative changes in gonadotrophin release. Uraemic children show a delayed or disturbed puberty. We have therefore examined nocturnal gonadotrophin and sex steroid secretion in seven males and three females [age 11-15 years, pubertal stage (PS) 1-3] with chronic renal failure on conservative treatment. In addition to immunoreactive luteinizing hormone (i-LH) we have measured the biological activity of LH (b-LH). Nine children aged 12-17 years with PS 1-3 and normal renal function served as a control group. In two uraemic children, i-LH, b-LH, follicle stimulating hormone and sex steroids were evaluated before and 7 days after pulsatile LH-releasing hormone (LHRH) administration (150 ng/kg body weight subcutaneously every 120 min). Mean i-LH levels were higher in uraemic children than in controls. An increase in i-LH during sleep was found in all controls and in eight of ten uraemic subjects. Mean b-LH levels were lower during sleep and the b/i LH ratio was reduced in uraemic children with PS 2-3 whether asleep or awake compared with controls. Pulsatile administration of LHRH provoked a rise of i-LH and b-LH levels with an increased b/i LH ratio, suggesting an intact pituitary responsiveness. These preliminary data indicate that the gonadotrophin control of LH is abnormal in uraemic children, and that biopotency of LH secretion might be improved after short-term pulsatile LHRH administration.

The contribution of different families of lymphocytotoxic antibodies in the serologic reactivity of 45 highly sensitized dialysis patients (HSDP) (panel reactivity antibody value-PRA greater than 80%) was assessed by analyzing patients' sera for the presence of auto- and alloreactive IgM and alloreactive IgG antibodies. A total of 220 sera was screened at different incubation temperatures, before and after treatment with the reducing agent dithiothreitol, against a large variety of cell targets by means of complement dependent cytotoxicity (CDC) and antiglobulin augmented (AHG) CDC assays. The results allowed to subdivide the HSDP under study into four groups: Group 1 consisted of 13 untransplanted patients and 14 patients with a prior failed graft whose PRA values did not change following DTT treatment. Alloreactive IgG antibodies alone, with anti-HLA specificity, were present in the sera of this patient group. Group 2 consisted of 3 untransplanted patients whose sera did not contain any autolymphocytotoxic antibody but appeared completely unreactive to panel lymphocytes following DTT treatment, thus confirming the presence of alloreactive IgM only endowed with antiHLA reactivity. Group 3 consisted of 4 untransplanted and 4 patients with a prior failed graft whose sera were found to contain in addition to autoreactive IgM also alloreactive IgG antibodies. Their PRA values declined after DTT treatment on average from 96.2% to 45% and from 95% to 52.5%, respectively. Group 4 consisted of 6 untransplanted patients whose PRA reactivity to both autologous and panel lymphocytes completely disappeared following DTT treatment, thus indicating that their sera contained exclusively autolymphocytotoxic IgM antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Two puromycin aminonucleoside (PAN) excretion products were purified by HPLC from urine of PAN-treated rats and characterized by nuclear magnetic resonance as N6-dimethyl-3'amino-3'deoxyadenosine (DA-Ado) and N6-methyl-3'amino-3'deoxyadenosine (MA-Ado), respectively, the former corresponding to unmodified PAN. DA-Ado was not a substrate for adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) or xanthine oxidase (XO), while MA-Ado was consecutively converted into hypoxanthine by a mixture of ADA and PNP. A different rate of transformation of DA-Ado and MA-Ado into hypoxanthine by isolated glomeruli was observed and was higher for the monomethylated analogue by a factor of 3 (79% vs. 21%); this was ascribed to the rate-limiting level of a demethylase activity acting on DA-Ado. Furthermore, DA-Ado was not transformed by glomerular epithelial cells in culture, while a little amount of MA-Ado was converted into hypoxanthine after six hours of incubation. In spite of this different metabolic behavior, the same order of cytotoxicity on glomerular epithelial cells in culture was observed for MA-Ado, DA-Ado and commercial PAN. All these molecules induced a dose response inhibition of [3H]thymidine incorporation into DNA after exposure for two hours and a marked alteration of cell viability which was not inhibited by free radical scavengers and deferoxamine. This study provides the first evidence for a glomerular metabolism of PAN and its urinary metabolite MA-Ado involving their transformation via the purine cycle enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

We review 30 cases of pediatric systemic lupus erythematosus followed over an 8-year period at our institution. The female to male ratio was 3.3:1; the age at diagnosis ranged between 3.5 and 16 years. On first admission, renal involvement was detected in the majority of the patients, as assessed by laboratory findings and/or clinical manifestations. Other frequently observed symptoms were fever, skin rashes, arthralgias and/or arthritis and serositis. All of the patients were treated with corticosteroids and most of them also received immunosuppressive drugs in order to control disease activity. Two patients were lost to the follow-up, five died and only one of the 23 evaluable patients is off therapy after a median follow-up of 5 years. This study confirms that pediatric systemic lupus erythematosus is a very aggressive disease.

An 85 KDa protein was purified by a multistep procedure (ultracentrifugation, HPLC, SDS-PAGE) from sera and amyloid deposits of patients on chronic hemodialysis and was characterized as a novel protein on the basis of its NH2 terminus (KVQLVE-V). This protein was formed by two subunits with Mr of 55 and 30 KDa and had affinity for Thyoflavin T, a fluorescent dye which was employed for labelling the protein prior HPLC. The 85 KDa was the only fluorescent component of ultracentrifugates from the serum of hemodialyzed patients while in amyloid fibrils it coexisted in roughly equimolar amounts with beta 2-microglobulin. This new high molecular weight protein which accumulates in uremia, could be co-responsible with beta 2-microglobulin for hemodialysis-related osteoarticular amyloidosis.

The study involved eight metabolically stable children, with chronic renal failure on continuous ambulatory peritoneal dialysis (CAPD) whom we followed for 12-18 months. For the first 6 months CAPD was performed with dextrose; for the subsequent 6-12 months the morning exchange was substituted with a 1% amino-acid (AA) solution. The following parameters did not change during the study: serum creatinine, uric acid, inorganic phosphate, serum bicarbonate, potassium, cholesterol, triglycerides, total protein, albumin and transferrin. The only parameter that changed was blood urea nitrogen, which increased moderately. The anthropometric parameters did not show significant variation before and after AA dialysis. The plasma AA profile, which under basal conditions showed lower levels of several essential AAs, improved during the treatment period, with a partial correction of the imbalance. It is possible that this correction of plasma AAs may positively influence the metabolism of some organs such as the brain, muscle and those of the hepatosplanchnic region. The intracellular pool of free AAs, measured in polymorphonuclear leucocytes, was severely altered before the treatment and after 6 and 12 months showed only minor variations. It is possible that some modifications in the proportion of the different AAs in the dialysis solution or an improvement in the concentration or in the number of exchanges per day are necessary in order to change the nutritional status and to modify the intracellular AA pool.

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)

In 109 patients with insulin-dependent diabetes mellitus (IDDM), we measured the urinary excretion of albumin, the low molecular weight proteins (LMWP) retinol-binding protein (RBP) and beta 2-microglobulin (beta 2m), and brush-border antigens (BBA) revealed by monoclonal antibodies. All such markers of kidney damage and/or dysfunction were higher in diabetic patients than in 44 controls. Increased urinary levels of BBA (p = 0.0001) were associated with higher values of albumin (p = 0.0002), RBP (p = 0.0005) and, to a lesser extent, of beta 2m (p = 0.1), different combinations of values above the reference limits being observed. Some 30 and 40% of patients with and without microalbuminuria, respectively, also exhibited signs of tubulopathy. Although under certain circumstances tubular defects may give rise to small increases in albuminuria, the most likely explanation for our findings is the coexistence of glomerular and tubular damage in some patients with IDDM. Neither the prognostic value nor the pathophysiological meaning of tubular damage and/or dysfunction can be assessed by the present study, owing to its cross-sectional design. Tubular markers thus deserve further studies to clarify whether in diabetic patients they indicate a more severe or diffuse kidney impairment.

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)

Abnormalities of tubular membrane structure and composition have been proposed as the primary defect in nephronophthisis (NEF). In order to characterize the protein composition of tubular cells in NEF, in vitro methods were developed to culture and propagate tubular cells obtained from biopsy fragments. Accordingly, microdissected cortical slices (1 x 3 mm) were first digested with collagenase and DNAse and then grown in RPMI medium supplemented with 10% NU serum and conditioned serum deriving from 3T3 cultures. At confluence, cultured cells from NEF showed characteristics which were typical of normal tubules, i.e. presence of cytokeratin and positivity for succinic dehydrogenase and alkaline phosphatase stainings, and presented no morphological alterations compared to cultured cells from normal tubular epithelium. Moreover, no difference was observed for fibronectin, collagen IV and laminin stains. Analysis by two-dimensional electrophoresis of cellular extracts revealed several changes in protein composition of NEF, the main one being the decrease in NEF cells of a polypeptide with a molecular weight of 120 kD and a pI of 4.8; this polypeptide was a constant finding in normal kidneys. These observations demonstrated that human tubular epithelial cells can be successfully cultured from very small biopsy fragments, which represents a new approach to the study of molecular disorders involving tubular cells in inherited disease. Cultured cells from NEF maintain the same morphological, immunological and cytochemical characteristics as normal tubular cells, but present a few alterations in polypeptide composition which may have pathogenetic relevance. A more careful analysis of these alterations is needed to define the molecular disorder(s) involving the tubule in NEF.

The changes in plasma and dialysate amino acids (AA) in 7 continuous ambulatory peritoneal dialysis (CAPD) children after dialysis with a 1% AA solution were compared with a glucose-containing solution. During the AA-exchange, the plasma levels of individual AA reached their peaks after 1 h, with their percentage increments significantly correlated (p less than 0.001) with the ratio of the amount of AA in the bag to the basal plasma concentration. The plasma concentration of methionine, valine, phenylalanine, and isoleucine remained higher than the basal value at 4 h. The amount of AA absorbed was 66% after 1 h, and 86% after 4 h and 6 h, corresponding to 2574 +/- 253 mumol/kg body wt. During glucose-dialysis (1.36%), levels of histidine, methionine, valine, phenylalanine, and isoleucine were significantly decreased in plasma after 1 h, and stayed low throughout the dialysis period. The loss of AA with the peritoneal effluent was 116 +/- 69 mumol/kg/body wt. From this study, it seems that using an AA dialysis solution, with 1 exchange per day, might limit the daily glucose load and compensate for AA losses by supplying an extra amount of AA and by reducing the loss of other AA not contained in dialysis solutions. The AA pattern in plasma following AA-dialysis resembles that observed after a protein meal, with no signs of persistently high, nonphysiological levels.

The renal selectivity properties towards albumin were evaluated in ten diabetic patients with arterial hypertension before and after the pharmacological normalisation of blood pressure, and were compared to 12 subjects with essential hypertension. While all patients of the control group were normoalbuminuric during hypertension, six of the diabetic group were microalbuminuric when hypertensive and became almost normoalbuminuric after blood pressure pharmacological control. All microalbuminuric diabetic patients presented altered properties of renal selectivity as epitomised by a non-preferential urinary excretion of glycosyl albumin (GA) (urinary GA/serum GA less than or equal to 1). At variance the selectivity properties were normal in normoalbuminuric diabetic patients and in essential hypertension. It was concluded that in diabetes mellitus arterial hypertension is associated with microalbuminuria when the renal properties of selectivity are altered, but does not implicate any proteinuric effect in those cases where the GBM function is preserved.

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.

The reaction of 2-amino-2-deoxy-D-glucose with lysine in water under simulated physiological conditions gave several browning products, with characteristic optical (lambda max 340 nm) and fluorescent properties (emission at 430 nm for excitation at 362 nm). The major product was isolated and characterized by mass spectrometry and n.m.r. spectroscopy as 2,5-bis(tetrahydroxybutyl)pyrazine derived by the condensation of two molecules of 2-amino-2-deoxy-D-glucose.

The uraemic syndrome is characterized by several endocrinological disturbances. This study was undertaken in order to evaluate the GH response to growth hormone-releasing hormone (GRH) in children with chronic renal failure (CRF) and to compare the results with those observed after insulin hypoglycaemia. Twenty-two children with CRF, 10 undergoing continuous ambulatory peritoneal dialysis (CAPD) and 12 on conservative treatment (CT), age ranges 2-15 years, were studied and the data were compared with those from 14 children with normal renal function and normal hormonal behaviour, affected by short stature (NC), and those form 13 healthy adult volunteers (NA). The GRH test (l micrograms/kg body weight, iv) was carried out in 8 CAPD, 8 CT, 9 NC and 10 NA subjects. The blood samples were taken every 30 min for 3 h in CAPD and CT and for 2 h in NC and NA starting at 09.00 h. The following hormones were measured: GH, LH, FSH, Prl, TSH and cortisol (F). The insulin test (0.1 U/kg body weight, iv) was carried out in 5 CAPD, 5 CT, 10 NC and 9 NA on blood samples taken every 30 min for 2 h, measuring GH and glycaemia. No adverse effects were observed after the infusion of GRH. GRH administration induced a prompt response in all subjects, but GH plasma levels were significantly higher in uraemic children than in adults (peak value of 43.5 +/- 8.2, 45.0 +/- 8.4, 27.8 +/- 6.0; 13.5 +/- 2.6 micrograms/ml in CAPD, CT, NC and NA, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

A pregnant woman affected with autosomal dominant polycystic kidney disease (ADPKD) had a history of an affected fetus, diagnosed by sonography at 29 weeks of pregnancy. The proband's father was also affected. DNA analysis performed on chorionic villi at 11 weeks during a second pregnancy predicted an affected fetus, and sonographic examination at 14 weeks confirmed the diagnosis.

Plasma protein and amino acid concentrations have been reported to be abnormal in patients with chronic renal failure, whether on conservative or regular dialysis treatment. These abnormalities may be related to impaired protein and amino acid metabolism associated with uremia, to dietary deficiencies of calories and proteins or to amino acid and protein losses due to peritoneal dialysis or hemodialysis. Plasma free amino acid concentrations were evaluated in 17 children undergoing hemodialysis (HD) and 13 children treated by continuous ambulatory peritoneal dialysis (CAPD). Plasma levels of free amino acids showed a reduction of EAA and of the ratio EAA/NEAA. There were some abnormalities in plasma amino acid concentrations; these included decreased levels of valine, threonine, lysine, serine, tyrosine, arginine, alpha-ABA. Aspartate, glycine, citrulline, and, only in HD, cystine and methionine were increased. Plasma protein and amino acid concentrations in CAPD patients are similar to those found in HD patients; thus they result poorly affected by different dialysis techniques and the uremic state itself seems to play a more decisive role.