Concentration of urine in mammals is regulated by the antidiuretic hormone vasopressin. Binding of vasopressin to its V2 receptor leads to the insertion of water channels in apical membranes of principal cells in collecting ducts. In nephrogenic diabetes insipidus (NDI), the kidney fails to concentrate urine in response to vasopressin. A male patient with an autosomal recessive form of NDI was found to be a compound heterozygote for two mutations in the gene encoding aquaporin-2, a water channel. Functional expression studies in Xenopus oocytes revealed that each mutation resulted in nonfunctional water channel proteins. Thus, aquaporin-2 is essential for vasopressin-dependent concentration of urine.

Reabsorption of filtered solutes from the glomerular filtrate and excretion of waste products and xenobiotics are the main functions of the renal proximal tubular (PT) epithelium. A human PT cell line expressing a range of functional transporters would help to augment current knowledge in renal physiology and pharmacology. We have established and characterized a conditionally immortalized PT epithelial cell line (ciPTEC) obtained by transfecting and subcloning cells exfoliated in the urine of a healthy volunteer. The PT origin of this line has been confirmed morphologically and by the expression of aminopeptidase N, zona occludens 1, aquaporin 1, dipeptidyl peptidase IV and multidrug resistance protein 4 together with alkaline phosphatase activity. ciPTEC assembles in a tight monolayer with limited diffusion of inulin-fluorescein-isothiocyanate. Concentration and time-dependent reabsorption of albumin via endocytosis has been demonstrated, together with sodium-dependent phosphate uptake. The expression and activity of apical efflux transporter p-glycoprotein and of baso-lateral influx transporter organic cation transporter 2 have been shown in ciPTEC. This established human ciPTEC expressing multiple endogenous organic ion transporters mimicking renal reabsorption and excretion represents a powerful tool for future in vitro transport studies in pharmacology and physiology.

In the pathogenesis of the hemolytic uremic syndrome (HUS), endothelial damage of glomeruli and arterioles of the kidney appears to play a central role. Previous studies have shown that verocytotoxin-1 (VT-1) cytotoxicity on human vein endothelial cells require additional stimuli, in particular the inflammatory mediator tumor necrosis factor alpha (TNF alpha). In this study the effects of VT on human glomerular microvascular endothelial cells (GMVEC) were examined. A reproducible method was developed for the isolation and purification of large numbers of highly purified GMVEC. The obtained GMVEC were over 99% pure; their endothelial origin was demonstrated by the expression of the endothelial antigens von Willebrand factor, EN-4, PECAM-1 and V,E-cadherin. Upon stimulation with TNF alpha the cells expressed the endothelial-specific adhesion molecule E-selectin. A limited number of fenestral structures was observed by scanning electron microscopy (SEM), suggesting glomerular origin of the endothelial cells. Cytotoxicity of VT-1 to GMVEC was evaluated by determination of the number of viable adherent cells and by assay of overall protein synthesis after exposure to varying concentrations of VT-1. In non-stimulated GMVEC, cytotoxicity of VT-1 was inversely related to the degree and duration of confluence, subconfluent cells being the most sensitive. In highly confluent GMVEC, VT cytotoxicity required pre-exposure of the cells to the inflammatory mediator TNF alpha, which induced an increase in the number of VT receptors on GMVEC. Thin layer chromatography of extracted glycolipids from the GMVEC showed binding of VT-1 to globotriaosylceramide (Gb3), known to be the functional receptor for VT. There were no major differences in protein synthesis inhibition with equal concentrations VT-1 and VT-2. In conclusion, in this study we provide a reproducible method to isolate, purify and culture well characterized human GMVEC on a routine basis. In vitro studies with these GMVEC demonstrate that VT cytotoxicity depends on the degree of confluence and the additional preexposure to the inflammatory mediator TNF alpha. These observations provide further insight into the complex events that may occur in glomeruli in the pathogenesis of HUS.

Gitelman syndrome (familial hypokalemia-hypomagnesemia syndrome) is an autosomal recessive inherited renal disorder characterized by defective tubular reabsorption of magnesium and potassium. In this study a group of 18 unrelated and 2 related Gitelman patients, collected from six different countries have been screened for mutations in the human thiazide-sensitive sodium-chloride cotransporter (SLC12A3) gene. Fourteen novel SLC12A3 mutations are presented along with six mutations described earlier, and three neutral polymorphisms. Among the tested patients are two who carry a total of three heterozygous SLC12A3 mutations. Two-thirds of the total number of mutant SLC12A3 alleles are amino acid substitutions. Most SLC12A3 gene mutations, 14 out of a total of 20, are localized at the intracellular carboxy-terminal domain of the NCCT protein. The pathogenicity of individual SLC12A3 mutations is based upon their predicted effect on SLC12A3 protein, and segregation in family members. Evolutionary conservation of substituted amino acid residues and their frequency in control chromosomes is presented. Identical mutations have been found in Gitelman families from different geographical origin, suggesting ancient mutations originating from a common ancestor. As yet, we have not found any evidence for a possible genotype-phenotype correlation.

Background and objectives: Mutations in podocyte genes are associated with steroid-resistant nephrotic syndrome (SRNS), mostly affecting younger age groups. To date, it is unclear whether these patients benefit from intensified immunosuppression with cyclosporine A (CsA). The aim of this study was to evaluate the influence of podocyte gene defects in congenital nephrotic syndrome (CNS) and pediatric SRNS on the efficacy of CsA therapy and preservation of renal function.Design, settings, participants, & measurements: Genotyping was performed in 91 CNS/SRNS patients, irrespective of age at manifestation or response to CsA.Results: Mutations were identified in 52% of families (11 NPHS1, 17 NPHS2, 11 WT1, 1 LAMB2, 3 TRPC6). Sixty-eight percent of patients with nongenetic SRNS responded to CsA, most of them achieved complete remission. In contrast, none of the patients with genetic CNS/SRNS experienced a complete remission and only two (17%) achieved a partial response, both affected by a WT1 mutation. Preservation of renal function was significantly better in children with nongenetic disease after a mean follow-up time of 8.6 years (ESRD in 29% versus 71%).Conclusions: The mutation detection rate in our population was high (52%). Most patients with genetic CNS/SRNS did not benefit from CsA with significantly lower response rates compared with nongenetic patients and showed rapid progression to end-stage renal failure. These data strongly support the idea not to expose CNS/SRNS patients with inherited defects related to podocyte function to intensified immunosuppression with CsA.

Primary hypomagnesaemia is composed of a heterogeneous group of disorders characterized by renal or intestinal Mg(2+) wasting, often associated with disturbances in Ca(2+) excretion. We identified a putative dominant-negative mutation in the gene encoding the Na(+), K(+)-ATPase gamma-subunit (FXYD2), leading to defective routing of the protein in a family with dominant renal hypomagnesaemia.

Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.

A group of 22 unrelated patients with sporadic or non-X-linked Alport syndrome were screened for mutations in the non-collagenous domain of the type IV collagen alpha 3 (COL4A3) chain gene. The five 3'-exons of this gene, located on chromosome 2qter, were tested by single strand conformation polymorphism analysis and direct sequencing. One patient was heterozygous and another homozygous (Mochizuki et al., Nature Genetics, in press) for a deletion of five nucleotides. A third patient appeared to be a compound heterozygote for two different nonsense mutations. In two patients and the father of a deceased patient we found a heterozygous substitution of an evolutionary conserved leucine by proline. However, segregation data of the mutation and a COL4A3/COL4A4 CA-repeat marker in their families argued against a causative role of the missense mutation. Even drastic changes of strongly conserved amino acids, as in the Leu36Pro case, may not be significant. Autosomal recessive inheritance due to pathogenic COL4A3 mutations accounts for at least 13% of Alport syndrome cases in this sample. It is concluded that COL4A3 is a major gene in the genetically and clinically heterogeneous Alport syndrome.