Kidney injury rates after unilateral nephrectomy in childhood-a systematic review and meta-analysis.

BACKGROUND: Unilateral nephrectomy is a relatively common procedure in children which results in a solitary functioning kidney (SFK). Living with an SFK predisposes to kidney injury, but it remains unknown which children are most at risk. We aimed to investigate kidney injury rates in patients who underwent unilateral nephrectomy in childhood and to investigate differences among nephrectomies performed for a congenital anomaly, malignancy or other condition. METHODS: MEDLINE and EMBASE were searched for studies reporting kidney injury rates [i.e. proteinuria, hypertension and/or a decreased glomerular filtration rate (GFR)] of patients who underwent unilateral nephrectomy during childhood. Studies including five or more patients with at least 12 months of follow-up were eligible. Analyses were performed using random effects models and stratified by indication for nephrectomy. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines were used for reporting. RESULTS: Over 5000 unique articles were screened, of which 53 studies reporting on >4000 patients were included in the analyses. Proteinuria, hypertension and a decreased GFR were present in 15.3, 14.5 and 11.9% of patients, respectively. Heterogeneity among the studies was large in several subgroups, impairing quantitative meta-analyses. However, none of our analyses indicated differences in injury rates between a congenital anomaly or malignancy as an indication for nephrectomy. CONCLUSIONS: Unilateral nephrectomy during childhood results in signs of kidney injury in >10% of patients, with no clear difference between the indications for nephrectomy. Therefore, structured follow-up is necessary in all children who underwent nephrectomy, regardless of the indication.

Complement Factor H Serum Levels Determine Resistance to Pneumococcal Invasive Disease.

Streptococcus pneumoniae is a major cause of life-threatening infections. Complement activation plays a vital role in opsonophagocytic killing of pneumococci in blood. Initial complement activation via the classical and lectin pathways is amplified through the alternative pathway amplification loop. Alternative pathway activity is inhibited by complement factor H (FH). Our study demonstrates the functional consequences of the variability in human serum FH levels on host defense. Using an in vivo mouse model combined with human in vitro assays, we show that the level of serum FH correlates with the efficacy of opsonophagocytic killing of pneumococci. In summary, we found that FH levels determine a delicate balance of alternative pathway activity, thus affecting the resistance to invasive pneumococcal disease. Our results suggest that variation in FH expression levels, naturally occurring in the human population, plays a thus far unrecognized role in the resistance to invasive pneumococcal disease.

Differential Expression of Specific Dermatan Sulfate Domains in Renal Pathology.

Dermatan sulfate (DS), also known as chondroitin sulfate (CS)-B, is a member of the linear polysaccharides called glycosaminoglycans (GAGs). The expression of CS/DS and DS proteoglycans is increased in several fibrotic renal diseases, including interstitial fibrosis, diabetic nephropathy, mesangial sclerosis and nephrosclerosis. Little, however, is known about structural alterations in DS in renal diseases. The aim of this study was to evaluate the renal expression of two different DS domains in renal transplant rejection and glomerular pathologies. DS expression was evaluated in normal renal tissue and in kidney biopsies obtained from patients with acute interstitial or vascular renal allograft rejection, patients with interstitial fibrosis and tubular atrophy (IF/TA), and from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy (MGP) or systemic lupus erythematosus (SLE), using our unique specific anti-DS antibodies LKN1 and GD3A12. Expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 was decreased in the interstitium of transplant kidneys with IF/TA, which was accompanied by an increased expression of type I collagen, decorin and transforming growth factor beta (TGF-ß), while its expression was increased in the interstitium in FSGS, MGP and SLE. Importantly, all patients showed glomerular LKN1 staining in contrast to the controls. Expression of the IdoA-Gal-NAc4SDS domain recognized by GD3A12 was similar in controls and patients. Our data suggest a role for the DS domain recognized by antibody LKN1 in renal diseases with early fibrosis. Further research is required to delineate the exact role of different DS domains in renal fibrosis.

The challenge of managing hemophilia A and STEC-induced hemolytic uremic syndrome.

BACKGROUND: The hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy leading to acute kidney injury in children. In most cases it is triggered by an infection caused by Shiga-like toxin-producing Escherichia coli (STEC). Endothelial damage plays a central role in the pathogenesis of disease. Hemophilia A is a genetic disorder leading to factor VIII (FVIII) deficiency, an important factor in the coagulation system. CASE: Here we describe a hemophilia A patient who developed HUS due to a STEC O26 infection. The patient developed not only acute kidney injury, but also severe gastro-intestinal and neurological complications. Increased amounts of recombinant FVIII (rFVIII) had to be administered during the acute phase of the disease to reach acceptable blood levels of FVIII, in order to control the hemorrhagic colitis and to prevent severe neurological complications. CONCLUSION: The patient's treatment schedule of rFVIII during the HUS period was a serious challenge, and we cannot exclude that it contributed to the severity of the HUS by enhancing the thrombotic microangiopathic process. The role of factor VIII administration in the severe outcome of this disease is discussed.

Clinical features and heteroplasmy in blood, urine and saliva in 34 Dutch families carrying the m.3243A > G mutation.

The m.3243A>G mutation has become known as the MELAS mutation. However, many other clinical phenotypes associated with this mutation have been described,most frequently being Maternally Inherited Diabetes and Deafness (MIDD). The m.3243A>G mutation, can be detected in virtually all tissues, however heteroplasmy differs between samples. Recent reports indicate, a preference to perform mutation analysis in Urinary Epithelial Cells(UEC). To test this, and to study a correlation between the mutational load in different tissues with two mitochondrial scoring systems (NMDAS and NPMDS) we investigated 34 families carrying the m.3243A>G mutation. Heteroplasmy was determined in three non-invasively collected samples,namely leucocytes, UEC and buccal mucosa. We included 127 patients, of which 82 carried the m.3243A>G mutation.None of the children (n011) had specific complaints. In adults(n071), a median NMDAS score of 15 (IQR 10-24) was found. The most prevalent symptoms were hearing loss(68 %), gastro-intestinal problems (59 %), exercise intolerance(54 %) and glucose intolerance (52 %). Ten patients had neurologic involvement. Buccal mucosa had the best correlation with the NMDAS in all adults (r00.437, p<0.001),whereas UEC had the strongest correlation with the NMDAS in severely affected patients (r00.593, p00.002). Heteroplasmy declined significantly with increasing age in all three samples (leucocytes r0-0.705 (p<0.001), UEC r0-0.374 (p00.001), buccal mucosa r0-0.460 (p<0.001). In our cohort of 82 patients, the m.3243A>Gmutation causes a wide variety of signs and symptoms, MIDD being far more prevalent than MELAS. Looking at the characteristics of the three noninvasively available tissues for testing heteroplasmy we confirm that UEC are the preferred sample to test [corrected].

Contiguous gene deletion of ELOVL7, ERCC8 and NDUFAF2 in a patient with a fatal multisystem disorder.

Contiguous gene syndromes affecting the mitochondrial oxidative phosphorylation system have been rarely reported. Here, we describe a patient with apparent mitochondrial encephalomyopathy accompanied by several unusual features, including dysmorphism and hepatopathy, caused by a homozygous triple gene deletion on chromosome 5. The deletion encompassed the NDUFAF2, ERCC8 and ELOVL7 genes, encoding complex I assembly factor 2 (also known as human B17.2L), a protein of the transcription-coupled nucleotide excision repair (TC-NER) machinery, and a putative elongase of very long-chain fatty acid synthesis, respectively. Detailed evaluation of cultured skin fibroblasts revealed disturbed complex I assembly, depolarization of the mitochondrial membrane, elevated cellular NAD(P)H level, increased superoxide production and defective TC-NER. ELOVL7 mRNA was not detectable in these cells and no alterations in fatty acid synthesis were found. By means of baculoviral complementation we were able to restore the aberrations, thereby establishing causative links between genotype and cell-physiological phenotype. This first chromosomal microdeletion illustrates that beside primary defects in mitochondrial genes also additional genes possibly contribute to the disease phenotype, providing an additional explanation for the broad clinical symptoms associated with these disorders.

Proteomic profiling and identification in peritoneal fluid of children treated by peritoneal dialysis.

BACKGROUND: Proteomic technologies offer a high-throughput analysis of the expression of proteins in biological samples. The global analysis of the proteins in peritoneal dialysis (PD) fluid will provide a better understanding of the biological processes of the peritoneal membrane. METHODS: The dialysate of nine paediatric PD patients was collected from peritoneal equilibrium tests with 3.86% glucose. Proteins were separated on a 10% SDS-PAGE gel and in-gel digested with trypsin. Peptide mixtures were analysed using nanoLC-MS/MS and results were searched against the NCBI database. RESULTS: A total number of 189 proteins were identified in the PD fluid of nine patients, with 88 proteins shared by all patients. These 88 proteins accounted for 47% of the identified proteins and >90% of the total protein content in the analysed samples. Proteins were subdivided into eight different classes according to function. CONCLUSIONS: This study gives a representative overview of the proteins present in PD fluid. The proteins in PD fluid reflect plasma proteins as well as local peritoneal processes. Potentially interesting proteins are revealed.

Removal of heparan sulfate from the glomerular basement membrane blocks protein passage.

Heparan sulfate (HS) within the glomerular basement membrane (GBM) is thought to play a major role in the charge-selective properties of the glomerular capillary wall. Recent data, however, raise questions regarding the direct role of HS in glomerular filtration. For example, in situ studies suggest that HS may prevent plasma macromolecules from clogging the GBM, keeping it in an "open" state. We evaluated this potential role of HS in vivo by studying the passage of protein through the glomerular capillary wall in the presence and absence of HS. Intravenous administration of neuraminidase removed neuraminic acid--but not HS--from the GBM, and this led to albuminuria. Concomitant removal of HS with heparinase III, confirmed by ultrastructural imaging, prevented the development of albuminuria in response to neuraminidase treatment. Taken together, these results suggest that HS keeps the GBM in an open state, facilitating passage of proteins through the glomerular capillary wall.

Anti-proteinuric effects of glycosaminoglycan-based drugs.

Heparan sulfate (HS) is a member of the family of glycosaminoglycans (GAGs) that is generally bound to a core protein to form a proteoglycan (PG). HSPGs may be cell-membrane associated (glypicans and syndecans) or located within the extracellular matrix (agrin, perlecan and type XVIII collagen). The sulfate and carboxylic groups in HS are responsible for the negative charge of the sugar chain. HS is abundantly present in the filter unit of the kidney, especially in the glomerular basement membrane (GBM), and is assumed to repel negatively charged proteins, including albumin, thereby preventing their filtration. Alterations in HS expression in the GBM have been reported in a number of renal pathologies, including diabetic nephropathy, minimal change nephropathy and membranous glomerulopathy.A decreased HS expression in the GBM generally correlates with an increase in the level of proteinuria. Progressive proteinuria may result in end-stage renal failure when untreated. Based on these findings, GAG-based drugs have been used to treat proteinuria and some, notably sulodexide, have shown beneficial effects. The biosynthesis of HS and its possible role in renal filtration are discussed, an overview of GAG-based drugs and their effect on proteinuria is provided, and possible mechanisms by which GAG-based drugs ameliorate proteinuria are discussed.

Adult and paediatric patients with minimal change nephrotic syndrome show no major alterations in glomerular expression of sulphated heparan sulphate domains.

BACKGROUND: Minimal change nephrotic syndrome (MCNS) is the most frequent form of nephrotic syndrome in childhood. In the glomerular basement membrane (GBM) of adult patients with MCNS, a reduced expression of a specific heparan sulphate (HS) domain has been reported. In children with MCNS, urinary activity of the HS-degrading enzyme heparanase was increased. It is, therefore, possible that a decreased GBM HS expression is associated with the pathogenesis of proteinuria in patients with MCNS. METHODS: In this study, HS in glomeruli of five adult and six paediatric patients with MCNS were analysed by immunofluorescence staining using four different antibodies, each defining a specific sulphated HS domain. The pediatric patients were subdivided into three groups depending on the presence or absence of podocyte foot process effacement, the level of proteinuria and prednisone administration at the time of the biopsy. In addition, kidneys of rats with adriamycin nephropathy (ADRN), a model for MCNS, were included in the study. RESULTS: Expression of sulphated HS domains was not aberrant in adult or paediatric patients compared with control subjects. Children with and without proteinuria had the same HS content. In contrast, rats with ADRN showed a decreased glomerular expression of sulphated HS domains. CONCLUSIONS: These results suggest that in patients with MCNS proteinuria is not associated with major changes in glomerular expression of sulphated HS domains.

Cytosolic signaling protein Ecsit also localizes to mitochondria where it interacts with chaperone NDUFAF1 and functions in complex I assembly.

Ecsit is a cytosolic adaptor protein essential for inflammatory response and embryonic development via the Toll-like and BMP (bone morphogenetic protein) signal transduction pathways, respectively. Here, we demonstrate a mitochondrial function for Ecsit (an evolutionary conserved signaling intermediate in Toll pathways) in the assembly of mitochondrial complex I (NADH:ubiquinone oxidoreductase). An N-terminal targeting signal directs Ecsit to mitochondria, where it interacts with assembly chaperone NDUFAF1 in 500- to 850-kDa complexes as demonstrated by affinity purification and vice versa RNA interference (RNAi) knockdowns. In addition, Ecsit knockdown results in severely impaired complex I assembly and disturbed mitochondrial function. These findings support a function for Ecsit in the assembly or stability of mitochondrial complex I, possibly linking assembly of oxidative phosphorylation complexes to inflammatory response and embryonic development.

Investigation of the complex I assembly chaperones B17.2L and NDUFAF1 in a cohort of CI deficient patients.

Dysfunction of complex I (NADH:ubiquinone oxidoreductase; CI), the largest enzyme of the oxidative phosphorylation (OXPHOS) system, often results in severe neuromuscular disorders and early childhood death. Mutations in its seven mitochondrial and 38 nuclear DNA-encoded structural components can only partly explain these deficiencies. Recently, CI assembly chaperones NDUFAF1 and B17.2L were linked to CI deficiency, but it is still unclear by which mechanism. To better understand their requirement during assembly we have studied their presence in CI subcomplexes in a cohort of CI deficient patients using one- and two-dimensional blue-native PAGE. This analysis revealed distinct differences between their associations to subcomplexes in different patients. B17.2L occurred in a 830 kDa subcomplex specifically in patients with mutations in subunits NDUFV1 and NDUFS4. Contrasting with this seemingly specific requirement, the previously described NDUFAF1 association to 500-850 kDa intermediates did not appear to be related to the nature and severity of the CI assembly defect. Surprisingly, even in the absence of assembly intermediates in a patient harboring a mutation in translation elongation factor G1 (EFG1), NDUFAF1 remained associated to the 500-850 kDa subcomplexes. These findings illustrate the difference in mechanism between B17.2L and NDUFAF1 and suggest that the involvement of NDUFAF1 in the assembly process could be indirect rather than direct via the binding to assembly intermediates.

In vivo degradation of heparan sulfates in the glomerular basement membrane does not result in proteinuria.

Heparan sulfates (HS) are long, unbranched, negatively charged polysaccharides that are bound to core proteins. HS in the glomerular basement membrane (GBM) is reported to be important for charge-selective permeability. Aberrant GBM HS expression has been observed in several glomerular diseases, such as diabetic nephropathy and membranous glomerulopathy, and a decrease in HS generally is associated with proteinuria. This study, with the use of a controlled in vivo approach, evaluated whether degradation of HS in rat GBM resulted in acute proteinuria. Rats received two intravenous injections of either heparinase III to digest HS or neuraminidase to remove neuraminic acids (positive control). Urine samples were taken at various time points, and at the end of the experiment, kidneys were removed and analyzed. Injection with heparinase III resulted in a complete loss of glomerular HS as demonstrated by immunofluorescence staining using anti-HS antibodies and by electron microscopy using cupromeronic blue in a critical electrolyte concentration mode. In the urine, a strong increase in HS was found within 2 h after the first injection. Staining for agrin, the major HS proteoglycan core protein in the GBM, was unaltered. No urinary albumin or other proteins were detected at any time point, and no changes in glomerular morphology were noticed. Injection of rats with neuraminidase, however, resulted in a major increase of urinary albumin and was associated with an increase in urinary free neuraminic acid. An increased glomerular staining with Peanut agglutinin lectin, indicative of removal of neuraminic acid, was noted. In conclusion, removal of HS from the GBM does not result in acute albuminuria, whereas removal of neuraminic acid does.

Identification of mitochondrial complex I assembly intermediates by tracing tagged NDUFS3 demonstrates the entry point of mitochondrial subunits.

Biogenesis of human mitochondrial complex I (CI) requires the coordinated assembly of 45 subunits derived from both the mitochondrial and nuclear genome. The presence of CI subcomplexes in CI-deficient cells suggests that assembly occurs in distinct steps. However, discriminating between products of assembly or instability is problematic. Using an inducible NDUFS3-green fluorescent protein (GFP) expression system in HEK293 cells, we here provide direct evidence for the stepwise assembly of CI. Upon induction, six distinct NDUFS3-GFP-containing subcomplexes gradually appeared on a blue native Western blot also observed in wild type HEK293 mitochondria. Their stability was demonstrated by differential solubilization and heat incubation, which additionally allowed their distinction from specific products of CI instability and breakdown. Inhibition of mitochondrial translation under conditions of steady state labeling resulted in an accumulation of two of the NDUFS3-GFP-containing subcomplexes (100 and 150 kDa) and concomitant disappearance of the fully assembled complex. Lifting inhibition reversed this effect, demonstrating that these two subcomplexes are true assembly intermediates. Composition analysis showed that this event was accompanied by the incorporation of at least one mitochondrial DNA-encoded subunit, thereby revealing the first entry point of these subunits.

Aberrant heparan sulfate profile in the human diabetic kidney offers new clues for therapeutic glycomimetics.

BACKGROUND: Diabetic nephropathy poses an increasing health problem in the Western world, and research to new leads for diagnosis and therapy therefore is warranted. In this respect, heparan sulfates (HSs) offer new possibilities because crude mixtures of these polysaccharides are capable of ameliorating proteinuria. The aim of this study is to immuno(histo)chemically profile HSs from microalbuminuric kidneys from patients with type 1 diabetes and identify specific structural HS alterations associated with early diabetic nephropathy. METHODS: Renal cryosections of control subjects and patients with type 1 diabetes were analyzed immunohistochemically by using a set of 10 unique phage display-derived anti-HS antibodies. HS structures defined by relevant antibodies were characterized chemically by means of enzyme-linked immunosorbent assay and probed for growth factor binding and presence in HS/heparin-containing drugs. RESULTS: In all patients, HS structure defined by the antibody LKIV69 consistently increased in basement membranes of proximal tubules. This structure contained N- and 2-O-sulfates and was involved in fibroblast growth factor 2 binding. It was present in HS/heparin-containing drugs shown to decrease albuminuria in patients with diabetes. The HS structure defined by the antibody HS4C3 increased in the renal mesangium of some patients, especially those who developed macroalbuminuria within 8 to 10 years. This structure contained N- and 6-O-sulfates. For 8 other antibodies, no major differences were observed. CONCLUSION: Specific structural alterations in HSs are associated with early diabetic nephropathy and may offer new leads for early diagnosis and the rational design of therapeutic glycomimetics.

Teaching molecular genetics: Chapter 3--Proteomics in nephrology.

The novel discipline of proteomics has experienced a rapid growth in the recent past and has great potentials for the future. The study of proteins on a genomic scale enables a large number of proteins to be analysed simultaneously. Moreover, proteomic analysis reveals the presence of protein isoforms and post-translational modifications, both of which have the potential to regulate protein complex formation, activity and function. As such, the assessment of the proteome, unlike genomic analysis, provides a view of biological processes at their level of occurrence. The knowledge thus gained is important not only for a better understanding of renal physiology and pathophysiology, but also for the identification of disease markers and the development of new therapies. This review applies the science of proteomics to nephrology: our aim is to give an overview of the discipline, providing background information and outlining the scope, advantages and limitations of proteomics.

Localization and functional characterization of glycosaminoglycan domains in the normal human kidney as revealed by phage display-derived single chain antibodies.

Glycosaminoglycans (GAG) play an important role in renal homeostasis. They are strongly negatively charged polysaccharides that bind and modulate a myriad of proteins, including growth factors, cytokines, and enzymes. With the aid of specific phage display-derived antibodies, the distribution of heparan sulfate (HS) and chondroitin sulfate (CS) domains in the normal human kidney was studied. HS domains were specifically located in basement membranes and/or surfaces of renal cells and displayed a characteristic distribution over the nephron. A characteristic location in specific parts of the tubular system was also observed. CS showed mainly an interstitial location. Immunoelectron microscopy indicated specific ultrastructural location of domains. Only partial overlap with any of seven different proteoglycan core proteins was observed. Two HS domains, one highly sulfated (defined by antibody HS4C3) and one low sulfated (defined by antibody RB4Ea12), were studied for their cell biologic relevance with respect to the proliferative effect of FGF-2 on human mesangial cells in vitro. Fibroblast growth factor 2 (FGF-2) binding was HS dependent. Addition of purified HS4C3 antibody but not of the RB4Ea12 antibody counteracted the binding and the proliferative effect of FGF-2, indicating that the HS4C3 domain is involved in FGF-2 handling by mesangial cells. In conclusion, specific GAG domains are differentially distributed in the normal human kidney and are likely involved in binding of effector molecules such as FGF-2. The availability of tools to identify and study relevant GAG structures allows the development of glycomimetica to halt, for instance, mesangial proliferation and matrix production as seen in diabetic nephropathy.

Heparan sulfate proteoglycans in glomerular inflammation.

Heparan sulfate proteoglycans (HSPGs) are glycoproteins consisting of a core protein to which linear heparan sulfate side chains are covalently attached. These heparan sulfate side chains can be modified at different positions by several enzymes, which include N-deacetylases, N- and O-sulfotransferases, and an epimerase. These heparan sulfate modifications give rise to an enormous structural diversity, which corresponds to the variety of biologic functions mediated by heparan sulfate, including its role in inflammation. The HSPGs in the glomerular basement membrane (GBM), perlecan, agrin, and collagen XVIII, play an important role in the charge-selective permeability of the glomerular filter. In addition to these HSPGs, various cell types express HSPGs at their cell surface, which include syndecans, glypicans, CD44, and betaglycan. During inflammation, HSPGs, especially heparan sulfate, in the extracellular matrix (ECM) and at the surface of endothelial cells bind chemokines, which establishes a local concentration gradient recruiting leukocytes. Endothelial and leukocyte cell surface HSPGs also play a role in their direct adhesive interactions via other cell surface adhesion molecules, such as selectins and beta2 integrin. Activated leukocytes and endothelial cells exert heparanase activity, resulting in degradation of heparan sulfate moieties in the ECM, which facilitates leukocyte passage into tissues and the release of heparan sulfate-bound factors. In various renal inflammatory diseases the expression of agrin and GBM-associated heparan sulfate is decreased, while the expression of CD44 is increased. Heparan sulfate or heparin preparations affect inflammatory cell behavior and have promising therapeutic, anti-inflammatory properties by preventing leukocyte adhesion/influx and tissue damage.

Heparan sulphate proteoglycans in Alzheimer's disease and amyloid-related disorders.

Proteoglycans are associated with all kinds of amyloid deposits in the human body. These complex macromolecules, in particular heparan sulphate proteoglycans, have also been implicated in several features of the pathogenesis of Alzheimer's disease (AD), including the genesis of senile plaques, cerebrovascular amyloid, and neurofibrillary tangles. In this review we focus on the role of proteoglycans and glycosaminoglycans in amyloidogenesis in general and in AD in particular. Heparan sulphate proteoglycans may promote amyloid-beta peptide (Abeta) or tau fibrillisation on the one hand, and provide resistance against proteolytic breakdown on the other. Knowledge about the role of proteoglycans in AD pathology may eventually be of therapeutic use, because small polysulphated compounds, which can interfere with the interaction between proteoglycan and Abeta, have been shown to stop or even prevent amyloidogenesis.

Dominant isolated renal magnesium loss is caused by misrouting of the Na+,K+-ATPase gamma-subunit.

Hereditary primary hypomagnesemia comprises a clinically and genetically heterogeneous group of disorders in which hypomagnesemia is due to either renal or intestinal Mg(2+) wasting. These disorders share the general symptoms of hypomagnesemia, tetany and epileptiformic convulsions, and often include secondary or associated disturbances in calcium excretion. In a large Dutch family with autosomal dominant renal hypomagnesemia, associated with hypocalciuria, we mapped the disease locus to a 5.6-cM region on chromosome 11q23. After candidate screening, we identified a heterozygous mutation in the FXYD2 gene, encoding the Na(+),K(+)-ATPase gamma-subunit, cosegregating with the patients of this family, which was not found in 132 control chromosomes. The mutation leads to a G41R substitution, introducing a charged amino acid residue in the predicted transmembrane region of the gamma-subunit protein. Expression studies in insect Sf9 and COS-1 cells showed that the mutant gamma-subunit protein was incorrectly routed and accumulated in perinuclear structures. In addition to disturbed routing of the G41R mutant, Western blot analysis of Xenopus oocytes expressing wild-type or mutant gamma-subunit showed mutant gamma-subunit lacking a posttranslational modification. Finally, we investigated two individuals lacking one copy of the FXYD2 gene and found their serum Mg(2+) levels to be within the normal range. We conclude that the arrest of mutant gamma-subunit in distinct intracellular structures is associated with aberrant posttranslational processing and that the G41R mutation causes dominant renal hypomagnesemia associated with hypocalciuria through a dominant negative mechanism.