Activation of AMP kinase plays a role in the increased apoptosis in the renal proximal tubule in cystinosis.

In cystinosis, renal proximal tubule (RPT) function is compromised, due to mutations in ctns, which encodes for the transporter cystinosin, which removes cystine from lysosomes. Altered RPT function in cystinosis has been attributed to decreased ATP, as well as increased apoptosis. In this report, the role of AMPK was examined. AMPK was activated in primary rabbit RPT cells with a cystinosin knockdown, using cystinosin siRNA. The activation of AMPK was associated with a 50% decrease in ATP and a 1.7-fold increase in the ADP/ATP level. Cisplatin-induced apoptosis also increased in primary RPT cells with a cystinosin knockdown. The role of AMPK in the increased sensitivity to cisplatin was examined. The increased sensitivity to cisplatin was prevented in primary RPT cells with a cystinosin knockdown by the AMPK inhibitor Compound C. The effect of siRNAs against AMPKα1 and AMPKα2 was also studied. The siRNAs knocked down AMPKα, and prevented AMPKα activation by 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR). The siRNAs against AMPKα1 and AMPKα2 also prevented the increased sensitivity to cisplatin in the primary RPT cells with a cystinosin knockdown. These results suggest that signaling through AMPK plays a role in the enhanced apoptosis in the RPT in cystinosis.

Chitotriosidase plasma activity in nephropathic cystinosis.

Chitotriosidase is a fully active chitinase produced and secreted by activated phagocytes. Plasma chitotriosidase activity is a well-established marker of total disease burden in Gaucher disease that has proved useful in monitoring the response to both enzyme replacement and substrate reduction therapies in patients with Gaucher disease. Increased chitotriosidase plasma activity has also been observed in several other lysosomal and non lysosomal disorders. Cystinosis, a rare multisystemic lysosomal storage disease, is characterized by the intralysosomal accumulation of free cystine in many cell types including phagocytes. We here report on plasma chitotriosidase activity in a child with nephropathic cystinosis. Increased plasma chitotriosidase activity (481 nmol/h per ml; normal range 0-150 units) was found on diagnosis and prior to the initiation of oral cysteamine (Cystagon) treatment. Serial estimations of plasma chitotriosidase activity showed that it correlated with leukocyte cystine content and decreased to 100 nmol/h per ml following 14 months' treatment. This novel observation suggests that cystinosis should be included in the differential diagnosis of disorders associated with increased plasma chitotriosidase activity. Furthermore, it suggests that serial estimations of plasma chitotriosidase activity could be of value in monitoring the response to oral cysteamine treatment in cystinosis.

Kearns-Sayre syndrome associated with de Toni-Debré-Fanconi syndrome due to cytochrome-c-oxidase (COX) deficiency.

We report a 43-year-old female with complete Kearns-Sayre syndrome, focal deficiency of cytochrome-c-oxidase (COX) and extensive deletion of the mtDNA in muscle fibers, which showed progressive insufficiency of the renal tubule: first hyperphosphaturia and hyperaminoaciduria and, later, also glucosuria (de Toni-Debré-Fanconi syndrome), a syndrome to date rarely diagnosed in association with complete Kearns-Sayre syndrome. In our opinion, this case, in view of the relationships between retinal and kidney disorders, suggests a search for de Toni-Debré-Fanconi syndrome in all patients with Kearns-Sayre syndrome also by quantitative and chromatographic methods for the assessment of aminoacids, phosphates and sugars in the urine.

N-acetyl-beta-glucosaminidase and beta-galactosidase activity in children receiving antiepileptic drugs.

To evaluate renal tubular function in children receiving antiepileptic drugs the urinary activity of two lysosomal enzymes, N-acetyl-beta-glucosaminidase and beta-galactosidase, were measured. The enzyme levels were determined before the administration of antiepileptic drugs and 8 months after. Fourteen epileptic children received valproate, and 17 received carbamazepine. The urinary activity of these enzymes in 25 healthy control patients also was examined. Increased N-acetyl-beta-glucosaminidase activity was found in 50% of patients taking valproate and in 17.6% of patients taking carbamazepine. Increased beta-galactosidase activity was found in 28.5% of patients taking valproate and 11.7% of patients taking carbamazepine compared with the results before treatment. On the basis of these results, it is suggested that patients taking antiepileptic drugs, especially valproate, may demonstrate minor signs of tubular dysfunction. In those patients who use these drugs at increased dosage levels or for long periods, the possibility of tubular dysfunction may be increased, and these dysfunctions may manifest in clinical symptoms.

Clinical heterogeneity in respiratory chain complex III deficiency in childhood.

Six children are presented with an isolated complex III deficiency in muscle tissue. More specifically, oxidation rates and ATP+CrP production rates from both pyruvate and succinate as substrates and/or the activity of decylubiquinol:cytochrome c oxidoreductase were all markedly reduced. Complex III deficiency was also present in liver of two patients tested, but could not be demonstrated in cultured fibroblasts of four patients tested. Mitochondrial DNA, extracted from muscle, was analyzed; no deletions or common point mutations were found. Four patients presented with a multi-organ disorder. Among these patients three presented at neonatal age with neurological signs and lactate elevation in blood and CSF, of whom two had severe neonatal Fanconi syndrome. One child, aged seven years, had encephalomyopathy, ophthalmoplegia, retinopathy and Wolff-Parkinson-White syndrome. The remaining two patients exhibited myopathy only, within the first year of life. Thus, like in other respiratory chain disorders, patients with complex III deficiency may present at any age and show variable symptoms and outcome, ranging from neonatal death to failure to thrive only. Apparently there are no clinical findings which are specific for complex III deficiency.

Time course of renal glutathione levels in experimental Fanconi syndrome: an enzyme-based approach.

Time-dependent alterations in glutathione (GSH) concentration and the activities of several key enzymes of GSH metabolism were studied in a rat model of experimental Fanconi syndrome induced by i.p. injection of sodium maleate (400 mg/kg BW). The changes in the parameters tested were monitored 0, 2, 4, and 12 h after sodium maleate administration. A significant decrease in renal GSH level was observed 2 and 4 h after sodium maleate treatment (27% and 38% of control values, respectively). The renal GSH depletion did not appear to be due to the decreased production rate or to an increased degradation of the tripeptide. This suggestion is based on the findings that the activities of the GSH synthesis (gamma-glutamyl cysteine synthetase and glutathione reductase) and those of the catabolic pathways (gamma-glutamyl transpeptidase) were unaltered at the same time points. The unchanged activity of gamma-glutamyl transpeptidase also suggests preserved luminal membrane integrity in experimental Fanconi syndrome. The decreased activity of glutathione peroxidase, which utilizes GSH as a cosubstrate in the course of inactivation of free radicals, in the first hours after treatment could facilitate lipid peroxidation reactions in this model of acute renal failure. The observed changes in all parameters tested were transient, with recovery to baseline levels in a period of 12 h after sodium maleate administration. At the same time a pronounced functional impairment still existed. The beneficial effect of fast recovery of renal GSH level on the functional and morphological restitution in experimental Fanconi syndrome is suggested.

Na+,K(+)-ATPase expression in maleic-acid-induced Fanconi syndrome in rats.

Na+,K(+)-ATPase activity and its alpha 1 subunit protein and mRNA in kidney cortex were monitored in rats developing Fanconi syndrome after the administration of maleate. Na+,K(+)-ATPase activity was significantly lower than in saline-injected controls, although this was partially mediated by a general, non-specific decrease in the cortex protein content. 2. The low activity of the sodium pump correlated with low abundance of alpha 1 subunit mRNA and protein levels. Hsp60 protein levels were also decreased in kidney cortex from maleate-treated rats. 3. Kidney cortex brush-border membrane vesicles from maleate-treated rats showed a marked decrease in Na(+)-dependent alanine and glucose transport, which was not dependent on the Na(+)-transmembrane gradient itself, a finding which is consistent with a more stable effect at the plasma membrane level. 4. The effect of maleate may be partially non-specific and involve a great variety of proteins, but seems to be restricted to selected tissues because alpha 1 subunit Na+,K(+)-ATPase and hsp60 protein amounts were not significantly modified in livers from rats developing Fanconi syndrome. 5. These results show that maleate administration induces a low activity of selected concentrative transport systems and a decrease in Na+,K(+)-ATPase activity and expression. The combination of both effects may explain the increased excretion of most organic solutes present in rats developing Fanconi syndrome.

[Renal proximal disfunction based on activity of n-acetyl-beta-d-glucosaminidase in urine of patients with kidney failure]. / Uszkodzenie kanalika proksymalnego na podstawie aktywnosci N-acetylo-beta-D-glukozaminidazy w moczu u chorych z niewydolnoscia nerek.

N-acetyl-beta-D-glucosaminidase (NAG) urine activities of 63 patients with stable and unstable chronic renal failure have been investigated. The values of NAG activity obtained from these patients were compared with NAG activity of 33 normal controls. Abnormal NAG values (> 70 nmol/mg of creatinine) were found in 60 (95.2%) patients with chronic renal failure and the median of all values was 327.8 nmol/mg of creatinine. It was 14-fold greater than the median of values for normal controls. There were any significant differences of NAG values between the patients with massive proteinuria (> 1.5 g/24 h), moderate proteinuria and those without 24 hour proteinuria or non-significant proteinuria (respectively 423.5 +/- 286.3 vs 414.4 +/- 334.8 vs 453.0 +/- 451.3 nmol/mg of creatinine). There was no significant difference between the two subgroups of patients with NAG values above and below 280 nmol/mg of creatinine in age, gender, serum urea and uric acid levels. However, the incidence of patients with NAG values higher than 280 nmol/mg of creatinine was statistically significant in unstable course of renal insufficiency and raised serum creatinine levels. It is suggested that the measurement of NAG excretion may be helpful to monitor unstable process in renal failure.

The renal adenosine triphosphatases: functional integration and clinical significance.

The ion-transporting ATPases determine the chemical composition of cells both directly and through their secondary effects. The Na,K-ATPase generates the transmembrane sodium gradient which provides the primary energy for uptake and extrusion of a wide variety of solutes by renal tubular epithelia. The H-ATPase and the H,K-ATPase acidify the urine, and also generates bicarbonate for excretion by the cortical collecting duct. Calcium ATPase regulates the intracellular calcium, which in turn impacts on the myriad of cellular functions for which calcium serves as an intracellular messenger. If one considers the impact of potential pump dysfunction in a purely speculative mode, the list of disorders which might be potentially ascribed to 'pump disease' would be enormous. This article reviews those disorders of renal transport already considered to be 'pump diseases'.

Human trehalase: characterization, localization, and its increase in urine by renal proximal tubular damage.

By using polymerase chain reaction, cDNA encoding human renal trehalase has been isolated. The partial amino acid sequence deduced by the cDNA showed homologies in rabbit, Tenebrio molitor and silkworm trehalase. Northern blots showed renal trehalase mRNA to be about 2.0 kb. To examine the properties of renal and urinary human trehalase, the trehalase cDNA was inserted in the pMAL-cRI vector downstream from the malE gene, which encodes maltose-binding protein. Transfection of the recombinant pMAL-cRI in Escherichia coli provided high levels of expression of the maltose binding protein-trehalase fusion protein. A rabbit was immunized with purified fusion protein, and antihuman trehalase antibodies were obtained. Immunoblot analysis disclosed that renal and urinary trehalase exhibited a molecular mass of about 75 kDa. Analysis by indirect fluorescent microscopy demonstrated that the enzyme located in only proximal tubular cells. Urinary trehalase activity was low in the healthy infants and elevated in patients with asphyxia. Markedly high activity was observed in a patient with Lowe syndrome. The immunoreactive urinary trehalase with 75 kDa was increased dependent on the elevation of the activity. On the basis of these findings, we conclude that the increase of urinary trehalase reflects the extent of renal tubular damage, and we propose that urinary trehalase can be a specific marker of renal tubular damage.

Diseases of renal adenosine triphosphatase.

Most renal transport is a primary or secondary result of the action of one of three membrane bound ion translocating ATPase pumps. The proximal tubule mechanisms for the reabsorption of salt, volume, organic compounds, phosphate, and most bicarbonate reabsorption depend upon the generation and maintenance of a low intracellular sodium concentration by the basolateral membrane Na-K-ATPase pump. The reabsorption of fluid and salt in the loop of Henle is similarly dependent on the energy provided by Na-K-ATPase activity. Some proximal tubule bicarbonate reabsorption and all distal nephron proton excretion is a product of one of two proton translocating ATPase pumps, either an electrogenic H-ATPase or an electroneutral H-K-ATPase. In this article, the authors review the biochemistry and physiology of pump activity and consider the pathophysiology of proximal and distal renal tubular acidosis, the Fanconi syndrome, and Bartter's syndrome as disorders of ATPase pump function.

Fanconi's syndrome with hepatorenal glycogenosis associated with phosphorylase b kinase deficiency.

OBJECTIVE: To describe two patients with Fanconi's nephropathy secondary to glycogen storage disease and speculate on the possible etiology. DESIGN: Convenience sample. SETTING: Tertiary care, referral center. PATIENTS: Two related children referred for failure to thrive, rickets, and hepatomegaly. INTERVENTION: Dietary and therapeutic measures for rickets and renal tubular acidosis. MEASUREMENTS AND RESULTS: The main laboratory findings were fasting hypoglycemia and massive glucosuria, with evidence of multiple renal tubular dysfunction characteristic of the Fanconi syndrome. Liver and kidney biopsy specimens were consistent with glycogen storage disease. Enzymatic assay of liver homogenates revealed marked deficiency of phosphorylase b kinase in one patient and absent activity in the other. CONCLUSION: Phosphorylase b kinase deficiency may be causally related to hepatorenal glycogenosis with the Fanconi syndrome. More patients with this syndrome need to be studied before a definitive causal role is implicated.

Maleic acid-induced proximal tubulopathy: Na:K pump inhibition.

Maleic acid (MA) administration to experimental animals induces a rapid, reversible, complex dysfunction of the renal tubule resembling Fanconi's syndrome. The intent of this work was to characterize the changes in the Na:K pump along the nephron during the development and recovery from MA injury to better define the site of damage and to correlate the observed changes in Na:K pump function with alterations in metabolic function. Male Sprague Dawley rats were studied before and 2 and 24 h after the injection of MA (100 mg/kg iv). MA induced an early and reversible decline in Na:K pump activity in the proximal convoluted tubule (PCT) from 2,324 +/- 61 to 1,446 +/- 55 pmol/mm.h (P < 0.001). This decrement was transient because enzyme activity returned to near baseline by 24 h after MA administration. The changes in Na:K pump activity were restricted to the PCT because no change in pars rectae, in medullary thick ascending limb, or in medullary collecting tubules was observed. PCT obtained from MA-treated rats 2 h after drug injection showed a decline in 14CO2 formation from radiolabeled glutamine, implying impaired oxidation of the carbon skeleton of the amino acid. This decline was transient with recovery of oxidative rates to normal 24 h after MA administration. It was concluded that a reversible, segment-specific impairment in PCT Na:K pump occurs early after the administration of MA. The decline in PCT Na:K pump activity is paralleled by a decrement in oxidative metabolism and may underlie the many consequences of this model of proximal tubulopathy that are reflections of impairment in sodium-dependent transport processes.

delta-Aminolevulinic acid dehydratase: effects of succinylacetone in rat liver and kidney in an in vivo model of the renal Fanconi syndrome.

Succinylacetone (SA) is a known inhibitor of the heme biosynthetic pathway in liver. We have demonstrated previously the SA enhancement of delta-aminolevulinic acid dehydratase (ALAD) in renal tubules, while this enzyme is known to be impaired by SA in the liver. The present studies, based on in vivo treatment of animals with SA, show equivalent degree of inhibition of specific ALAD activity in liver and kidney. Both tissues evidenced an ability to restore enzyme activity with time, the recovery occurring much more slowly in kidney than in liver. The discrepant in vitro and in vivo effect of SA on renal ALAD may be due to differences between a direct inhibitor-enzyme interaction and inhibitor actions in the living cell, respectively. Persistent tissue levels of SA, consistent with demonstrated SA in plasma and urine, might account for continuing inhibition, with the greatest tissue accumulation in kidney where the substance must be cleared for excretion.

Mitochondrial myopathy with lactic acidaemia, Fanconi-De Toni-Debré syndrome and a disturbed succinate: cytochrome c oxidoreductase activity.

A patient with severe muscular hypotonia, failure to thrive, a metabolic acidosis and a renal tubular dysfunction is presented. The disease followed a fatal course. Blood lactate and pyruvate levels as well as lactate/pyruvate ratios were strongly elevated. There were a massive excretion of lactate in urine, a generalized hyperaminoaciduria, a proteinuria and a mellituria. The carnitine concentration was diminished in blood and muscle tissue. Biochemical investigations of skeletal muscle and liver tissue revealed a defect in the respiratory chain at the level of succinate: cytochrome c oxidoreductase. The defect could not be demonstrated in cultured fibroblasts.

Coordinately increased lysozymuria and lysosomal enzymuria induced by maleic acid.

During the acute renal tubular dysfunction of Fanconi syndrome and type 2 renal tubular acidosis (FS/RTA2) induced by maleic acid in the unanesthetized dog, we observed: 30 minutes after the onset of FS/RTA2, the urinary excretion of lysosomal enzymes, N-acetyl-beta-glucosaminidase (NAG), beta-glucuronidase (beta-gluc) and beta-galactosidase (beta-galac), increased simultaneously with the anticipated increase in renal clearance of lysozyme; the severities of all these hyperenzymurias increased rapidly, progressively, and in parallel, all reaching a peak some 60 to 80 minutes after their onset; thereafter, while the FS/RTA2 continued undiminished in severity, the severity of the hyperenzymurias decreased rapidly, greatly, progressively, and in parallel; and sodium phosphate loading strikingly attenuated the FS/RTA2 and the hyperenzymurias. Thus, the maleic acid-induced FS/RTA2 is attended by an acute reversible-complex derangement in the renal tubular processing of proteins that: affects not only lysozyme which is normally filtered, but also NAG and other lysosomal enzymes, which are not; and is to some extent functionally separable from that of FS/RTA2. The findings suggest that the derangements in renal processing of lysozyme and lysosomal enzymes are linked, and that a phosphate-dependent metabolic abnormality in the proximal tubule can participate in the pathogenesis of both these derangements and the FS/RTA2.

Renal filtration and catabolism of complement protein D.

Complement protein D, a serine protease participating in the formation of the C3 convertase of the alternative complement pathway, has the lowest molecular weight (23,750) and serum concentration of all complement proteins. In normal serum, D is the rate-limiting protease of the alternative pathway of complement activation. We report that the serum concentrations of D in 20 patients with chronic renal failure (mean +/- S.D., 0.42 +/- 0.28 mg per deciliter) and in 16 patients on long-term dialysis (1.53 +/- 0.39 mg per deciliter) were significantly higher (P less than 0.001) than in 22 healthy adults (0.18 +/- 0.04 mg per deciliter). In chronic renal failure the serum concentration of D correlated with that of creatinine (r = 0.75, P less than 0.001). The serum concentrations of D found in patients with renal failure reached and in some cases exceeded those at which the protease is no longer rate-limiting. Thus, enhanced activity of the alternative pathway of complement should be expected in patients with advanced renal failure. Urinary D was undetectable (less than 0.2 micrograms per deciliter) in 17 normal adults and either undetectable or below the concentration expected from the degree of proteinuria in 10 patients with nephrotic syndrome. However, in a patient with Fanconi's syndrome the urinary concentration of D (1.3 mg per deciliter) was an order of magnitude higher than the serum concentration, representing 0.5 per cent of the total protein. The urinary D in this patient had normal hemolytic activity, antigenicity, and size. These results indicate that D is filtered through the glomerular membrane and is probably catabolized in the proximal renal tubules.

Renal refractoriness to PTH in experimental Fanconi syndrome: evidence for intact adenylate cyclase activation.

Previous studies demonstrated renal refractoriness to parathyroid hormone (PTH) in maleate-induced Fanconi syndrome (FS) in rats. In membrane fraction of renal cortex of parathyroidectomized (PTX) rats with FS, PTH increased adenylate cyclase (AC) activity from a basal value of 7.4 +/- 0.6 (mean +/- SE) to 16.8 +/- 3.5 pmoles cAMP/mg prot/min (P less than 0.01), similar stimulation by PTH was observed in control incubation with normal kidneys. In membrane fraction of renal cortex of PTX rats incubated with maleate, PTH increased AC from 5.7 +/- 0.42 to 10.7 +/- 1.08 (P less than 0.01) similar to control incubation without maleate. In cortical slices from PTX rates with FS incubated in vitro, PTH increased cAMP content only from 4.0 +/- 0.21 to 5.27 +/- 0.27 pmole cAMP/mg prot (P less than 0.005), while in slices from the control group the increment by PTH was from 3.9 +/- 0.43 to 10.7 +/- 0.93 pmoles cAMP/mg prot (P less than 0.001). For the difference in the increment between the control and FS group, P less than 0.001. In cortical tissue of PTX rats with FS, PTH injection failed to increase cAMP content: basal value 7.4 +/- 0.8 and with PTH 9.2 +/- 0.8 pmole cAMP/mg prot (P NS), as compared with controls: basal value 9.5 +/- 0.5 and with PTH 23.8 +/- 1.6 (P less than 0.001). ATP content of cortical slices fell from a control value of 2.3 +/- 0.18 in PTX rats to 1.0 +/- 0.14 nmol/mg prot in PTX rats with FS.(ABSTRACT TRUNCATED AT 250 WORDS)

Familial Fanconi syndrome with malabsorption and galactose intolerance, normal kinase and transferase activity. A report on two siblings.

Two siblings of Turkish-Assyrian extraction, whose parents were first cousins, had poor appetite, slow weight gain and retarded psychomotor development. When given milk the galactose concentration in blood increased. An oral galactose load showed a markedly reduced capacity to metabolize galactose. Fanconi syndrome was present as in classical galactosemia. A galactose-free diet reduced the aminoaciduria but did not normalize the renal tubular function nor the children's general condition. Galactokinase and galactose-1-phosphate uridyltransferase activities in red blood cells were normal. The physical appearance of the children (sparse subcutaneous fat, thin extremities and distended abdomen) and the results of vitamin A and xylose absorption tests, were in accordance with a malabsorption condition. Glucose, however, seemed to be absorbed normally from the gut. There was no evidence of primary liver disease. Since the condition did not normalize with a galactose-free diet, an enzyme defect of galactose metabolism is unlikely. Instead, a more general transport defect with autosomal recessive inheritance is proposed.