Friedhelm Hildebrandt

Mutations in INVS encoding inversin cause nephronophthisis type 2, linking renal cystic disease to the function of primary cilia and left-right axis determination

Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, leads to chronic renal failure in children. The genes mutated in NPHP1 and NPHP4 have been identified, and a gene locus associated with infantile nephronophthisis (NPHP2) was mapped. The kidney phenotype of NPHP2 combines clinical features of NPHP and polycystic kidney disease (PKD). Here, we identify inversin (INVS) as the gene mutated in NPHP2 with and without situs inversus. We show molecular interaction of inversin with nephrocystin, the product of the gene mutated in NPHP1 and interaction of nephrocystin with β-tubulin, a main component of primary cilia. We show that nephrocystin, inversin and β-tubulin colocalize to primary cilia of renal tubular cells. Furthermore, we produce a PKD-like renal cystic phenotype and randomization of heart looping by knockdown of invs expression in zebrafish. The interaction and colocalization in cilia of inversin, nephrocystin and β-tubulin connect pathogenetic aspects of NPHP to PKD, to primary cilia function and to left-right axis determination.

Barttin is a Cl- channel beta-subunit crucial for renal Cl- reabsorption and inner ear K+ secretion.

Renal salt loss in Bartters syndrome is caused by impaired transepithelial transport in the loop of Henle. Sodium chloride is taken up apically by the combined activity of NKCC2 (Na+-K--2Cl- cotransporters) and ROMK potassium channels. Chloride ions exit from the cell through basolateral ClC-Kb chloride channels. Mutations in the three corresponding genes have been identified that correspond to Bartters syndrome types 1–3. The gene encoding the integral membrane protein barttin is mutated in a form of Bartters syndrome that is associated with congenital deafness and renal failure. Here we show that barttin acts as an essential β-subunit for ClC-Ka and ClC-Kb chloride channels, with which it colocalizes in basolateral membranes of renal tubules and of potassium-secreting epithelia of the inner ear. Disease-causing mutations in either ClC-Kb or barttin compromise currents through heteromeric channels. Currents can be stimulated further by mutating a proline-tyrosine (PY) motif on barttin. This work describes the first known β-subunit for CLC chloride channels and reveals that heteromers formed by ClC-K and barttin are crucial for renal salt reabsorption and potassium recycling in the inner ear.

The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4

The molecular basis of nephronophthisis, the most frequent genetic cause of renal failure in children and young adults, and its association with retinal degeneration and cerebellar vermis aplasia in Joubert syndrome are poorly understood. Using positional cloning, we here identify mutations in the gene CEP290 as causing nephronophthisis. It encodes a protein with several domains also present in CENPF, a protein involved in chromosome segregation. CEP290 (also known as NPHP6) interacts with and modulates the activity of ATF4, a transcription factor implicated in cAMP-dependent renal cyst formation. NPHP6 is found at centrosomes and in the nucleus of renal epithelial cells in a cell cycle–dependent manner and in connecting cilia of photoreceptors. Abrogation of its function in zebrafish recapitulates the renal, retinal and cerebellar phenotypes of Joubert syndrome. Our findings help establish the link between centrosome function, tissue architecture and transcriptional control in the pathogenesis of cystic kidney disease, retinal degeneration, and central nervous system development.

Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible

Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene (PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCε1 expression in developing and mature glomerular podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif–containing GTPase-activating protein 1 as a new interaction partner of PLCε1. Two siblings with a missense mutation in an exon encoding the PLCε1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.

Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left–right asymmetry

Primary ciliary dyskinesia (PCD, MIM 242650) is characterized by recurrent infections of the respiratory tract due to reduced mucociliary clearance and by sperm immobility. Half of the affected offspring have situs inversus (reversed organs), which results from randomization of left-right (LR) asymmetry. We previously localized to chromosome 5p a PCD locus containing DNAH5, which encodes a protein highly similar to the Chlamydomonas γ-dynein heavy chain. Here we characterize the full-length 14-kb transcript of DNAH5. Sequence analysis in individuals with PCD with randomization of LR asymmetry identified mutations resulting in non-functional DNAH5 proteins.

Mutation of BSND causes Bartter syndrome with sensorineural deafness and kidney failure

Antenatal Bartter syndrome (aBS) comprises a heterogeneous group of autosomal recessive salt-losing nephropathies. Identification of three genes that code for renal transporters and channels as responsible for aBS has resulted in new insights into renal salt handling, diuretic action and blood-pressure regulation. A gene locus of a fourth variant of aBS called BSND, which in contrast to the other forms is associated with sensorineural deafness (SND) and renal failure, has been mapped to chromosome 1p. We report here the identification by positional cloning, in a region not covered by the human genome sequencing projects, of a new gene, BSND, as the cause of BSND. We examined ten families with BSND and detected seven different mutations in BSND that probably result in loss of function. In accordance with the phenotype, BSND is expressed in the thin limb and the thick ascending limb of the loop of Henle in the kidney and in the dark cells of the inner ear. The gene encodes a hitherto unknown protein with two putative transmembrane α-helices and thus might function as a regulator for ion-transport proteins involved in aBS, or else as a new transporter or channel itself.

Patients with Mutations in NPHS2 (Podocin) Do Not Respond to Standard Steroid Treatment of Nephrotic Syndrome

Nephrotic syndrome (NS) represents the association of proteinuria, hypoalbuminemia, edema, and hyperlipidemia. Steroid-resistant NS (SRNS) is defined by primary resistance to standard steroid therapy. It remains one of the most intractable causes of ESRD in the first two decades of life. Mutations in the NPHS2 gene represent a frequent cause of SRNS, occurring in approximately 20 to 30% of sporadic cases of SRNS. On the basis of a very small number of patients, it was suspected that children with homozygous or compound heterozygous mutations in NPHS2 might exhibit primary steroid resistance and a decreased risk of FSGS recurrence after kidney transplantation. To test this hypothesis, NPHS2 mutational analysis was performed with direct sequencing for 190 patients with SRNS from 165 different families and, as a control sample, 124 patients with steroid-sensitive NS from 120 families. Homozygous or compound heterozygous mutations in NPHS2 were detected for 43 of 165 SRNS families (26%). Conversely, no homozygous or compound heterozygous mutations in NPHS2 were observed for the 120 steroid-sensitive NS families. Recurrence of FSGS in a renal transplant was noted for seven of 20 patients with SRNS (35%) without NPHS2 mutations, whereas it occurred for only two of 24 patients with SRNS (8%) with homozygous or compound heterozygous mutations in NPHS2. None of 29 patients with homozygous or compound heterozygous mutations in NPHS2 who were treated with cyclosporine A or cyclophosphamide demonstrated complete remission of NS. It was concluded that patients with SRNS with homozygous or compound heterozygous mutations in NPHS2 do not respond to standard steroid treatment and have a reduced risk for recurrence of FSGS in a renal transplant. Because these findings might affect the treatment plan for childhood SRNS, it might be advisable to perform mutational analysis of NPHS2, if the patient consents, in parallel with the start of the first course of standard steroid therapy.

The ciliary gene RPGRIP1L is mutated in cerebello-oculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome

Cerebello-oculo-renal syndrome (CORS), also called Joubert syndrome type B, and Meckel (MKS) syndrome belong to the group of developmental autosomal recessive disorders that are associated with primary cilium dysfunction. Using SNP mapping, we identified missense and truncating mutations in RPGRIP1L (KIAA1005) in both CORS and MKS, and we show that inactivation of the mouse ortholog Rpgrip1l (Ftm) recapitulates the cerebral, renal and hepatic defects of CORS and MKS. In addition, we show that RPGRIP1L colocalizes at the basal body and centrosomes with the protein products of both NPHP6 and NPHP4, known genes associated with MKS, CORS and nephronophthisis (a related renal disorder and ciliopathy). In addition, the RPGRIP1L missense mutations found in CORS individuals diminishes the interaction between RPGRIP1L and nephrocystin-4. Our findings show that mutations in RPGRIP1L can cause the multiorgan phenotypic abnormalities found in CORS or MKS, which therefore represent a continuum of the same underlying disorder.

Nephrocystin-5, a ciliary IQ domain protein, is mutated in Senior-Loken syndrome and interacts with RPGR and calmodulin

Nephronophthisis (NPHP) is the most frequent genetic cause of chronic renal failure in children. Identification of four genes mutated in NPHP subtypes 1–4 (refs. 4–9) has linked the pathogenesis of NPHP to ciliary functions. Ten percent of affected individuals have retinitis pigmentosa, constituting the renal-retinal Senior-Loken syndrome (SLSN). Here we identify, by positional cloning, mutations in an evolutionarily conserved gene, IQCB1 (also called NPHP5), as the most frequent cause of SLSN. IQCB1 encodes an IQ-domain protein, nephrocystin-5. All individuals with IQCB1 mutations have retinitis pigmentosa. Hence, we examined the interaction of nephrocystin-5 with RPGR (retinitis pigmentosa GTPase regulator), which is expressed in photoreceptor cilia and associated with 10–20% of retinitis pigmentosa. We show that nephrocystin-5, RPGR and calmodulin can be coimmunoprecipitated from retinal extracts, and that these proteins localize to connecting cilia of photoreceptors and to primary cilia of renal epithelial cells. Our studies emphasize the central role of ciliary dysfunction in the pathogenesis of SLSN.

Mutations in a novel gene, NPHP3, cause adolescent nephronophthisis, tapeto-retinal degeneration and hepatic fibrosis.

Nephronophthisis (NPHP), a group of autosomal recessive cystic kidney disorders, is the most common genetic cause of progressive renal failure in children and young adults. NPHP may be associated with Leber congenital amaurosis, tapeto-retinal degeneration, cerebellar ataxia, cone-shaped epiphyses, congenital oculomotor apraxia and hepatic fibrosis. Loci associated with an infantile type of NPHP on 9q22–q31 (NPHP2), juvenile types of NPHP on chromosomes 2q12–q13 (NPHP1) and 1p36 (NPHP4) and an adolescent type of NPHP on 3q21–q22 (NPHP3) have been mapped. NPHP1 and NPHP4 have been identified, and interaction of the respective encoded proteins nephrocystin and nephrocystin-4 has been shown. Here we report the identification of NPHP3, encoding a novel 1,330-amino acid protein that interacts with nephrocystin. We describe mutations in NPHP3 in families with isolated NPHP and in families with NPHP with associated hepatic fibrosis or tapeto-retinal degeneration. We show that the mouse ortholog Nphp3 is expressed in the node, kidney tubules, retina, respiratory epithelium, liver, biliary tract and neural tissues. In addition, we show that a homozygous missense mutation in Nphp3 is probably responsible for the polycystic kidney disease (pcy) mouse phenotype. Interventional studies in the pcy mouse have shown beneficial effects by modification of protein intake and administration of methylprednisolone, suggesting therapeutic strategies for treating individuals with NPHP3.