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1.  Uromodulin Upregulates TRPV5 by Impairing Caveolin-Mediated Endocytosis 
Kidney international  2013;84(1):130-137.
Uromodulin (UMOD) is synthesized in the thick ascending limb and secreted into urine as the most abundant protein. Association studies in humans suggest protective effects of UMOD against calcium-containing kidney stones. Mice carrying mutations of Umod found in human uromodulin-associated kidney disease (UAKD) and Umod deficient mice exhibit hypercalciuria. The mechanism for UMOD regulation of urinary Ca2+ excretion is incompletely understood. We examined if UMOD regulates TRPV5 and TRPV6, channels critical for renal transcellular Ca2+ reabsorption. Coexpression with UMOD increased whole-cell TRPV5 current density in HEK293 cells. In biotinylation studies UMOD increased TRPV5 cell-surface abundance. Extracellular application of purified UMOD upregulated TRPV5 current density within physiological relevant concentration ranges. UMOD exerted a similar effect on TRPV6. TRPV5 undergoes constitutive caveolin-mediated endocytosis. UMOD had no effect on TRPV5 in a caveolin-1 deficient cell line. Expression of recombinant caveolin-1 in these cells restored the ability of UMOD to upregulate TRPV5. Secretion of UAKD-mutant UMOD was markedly reduced and coexpression of mutant UMOD with TRPV5 failed to increase its current. Immunofluorescent studies demonstrated lower TRPV5 expression in Umod−/− mice compared to wild-type. UMOD upregulates TRPV5 by acting from extracellular and by decreasing endocytosis of TRPV5. The stimulation of Ca2+ reabsorption via TRPV5 by UMOD may contribute to protection against kidney stone formation.
doi:10.1038/ki.2013.63
PMCID: PMC3700562  PMID: 23466996
2.  Autosomal Dominant Mutation in the Signal Peptide of Renin in a Kindred with Anemia, Hyperuricemia, and CKD 
Homozygous or compound heterozygous Renin (REN) mutations cause renal tubular dysgenesis (RTD), which is characterized by death in utero due to renal failure and pulmonary hypoplasia. The phenotype resembles the fetopathy caused by angiotensin-converting enzyme inhibitor or angiotensin receptor blocker intake during pregnancy. Recently, heterozygous REN mutations were shown to result in early-onset hyperuricemia, anemia and chronic renal failure. So far, only three different heterozygous REN mutations were reported.
We performed mutation analysis of the REN gene in 39 kindreds with hyperuricemia and chronic kidney disease (CKD) previously tested negative for mutations in the UMOD and HNF1β genes. We identified one kindred with a novel c.28T>C (p.W10R) REN mutation in the signal sequence, concluding that REN mutations are rare events in CKD patients. Affected individuals over four generations were identified carrying the novel REN mutation and were characterized by significant anemia, hyperuricemia and CKD. Anemia was severe and disproportional to the degree of renal impairment. Moreover all heterozygous REN mutations are localized in the signal sequence. Therefore, screening of the REN gene for CKD patients with hyperuricemia and anemia may be focusing on exon 1 sequencing, which encodes the signal peptide.
doi:10.1053/j.ajkd.2011.06.029
PMCID: PMC3366501  PMID: 21903317
4.  Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy  
The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1–NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are “ciliopathies”. Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.
doi:10.1172/JCI40076
PMCID: PMC2827951  PMID: 20179356
5.  Nephronophthisis 
Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease, which represents the most frequent genetic cause for end-stage renal disease up to the third decade of life. Nephronophthisis is caused by mutations in eleven different genes called nephrocystins (NPHP1-11, NPHP1L). With an increasing number of identified genes our knowledge of nephronophthisis is changing and improving our understanding of the pathomechanisms in nephronophthisis. Recent publications described ciliary expression of nephrocystins together with other cystoproteins like polycystins 1 and 2, and fibrocystin. These findings have shifted our focus to a pathomechanism involving defects in ciliary function (ciliopathy) and planar cell polarity (PCP). In addition, discoveries of new nephrocystin genes have shown that the disease spectrum of nephronophthisis is much broader than previously anticipated. Different forms of mutations within the same NPHP gene can cause different disease severity. In this review we will highlighten the different hypotheses concerning the pathomechanisms for nephronophthisis and we will underline the clinical variability of nephronophthisis. The clinical spectrum has become even more complex with the possibility of oligogenicity in NPHP.
doi:10.1007/s00467-010-1585-z
PMCID: PMC4160028  PMID: 20652329
nephronophthisis; cystic kidney disease; ciliopathy; Senior-Loken syndrome; Joubert syndrome; Meckel-Gruber syndrome; molecular genetics
6.  Mutation analysis of the Uromodulin gene in 96 individuals with urinary tract anomalies (CAKUT) 
Uromodulin (UMOD) mutations were described in patients with medullary cystic kidney disease (MCKD2), familial juvenile hyperuricemic nephropathy (FJHN), and glomerulocystic kidney disease (GCKD). UMOD transcription is activated by the transcription factor HNF1B. Mutations in HNF1B cause a phenotype similar to FJHN/GCKD but also congenital anomalies of the kidney and the urinary tract (CAKUT). Moreover, we recently detected UMOD mutations in 2 patients with CAKUT. As HNF1B and UMOD act in the same pathway and cause similar phenotypes we here examined, whether UMOD mutations would be found in patients with CAKUT.
Mutation analysis of UMOD was performed in 96 individuals with CAKUT by direct sequencing of exons 4 and 5 and by heteroduplex analysis following CEL I digestion assay of the exons 3 and 6–12.
The mean age of patients was 11.4 years and in 36.4% of patients the family history was positive for CAKUT. In the CEL I assay 12 aberrant bands were detected in 103 of 960 PCR products and were sequenced. Two previously known and eight new SNPs were detected. As no UMOD mutations were identified in these 96 patients with CAKUT, UMOD mutations do not seem to be a significant cause of CAKUT in this cohort.
doi:10.1007/s00467-008-1016-6
PMCID: PMC3155267  PMID: 18846391
Uromodulin; Tamm-Horsfall protein; urinary tract malformation; CAKUT; mutation analysis
7.  Uromodulin is expressed in renal primary cilia and UMOD mutations result in decreased ciliary uromodulin expression 
Human Molecular Genetics  2010;19(10):1985-1997.
Uromodulin (UMOD) mutations are responsible for three autosomal dominant tubulo-interstitial nephropathies including medullary cystic kidney disease type 2 (MCKD2), familial juvenile hyperuricemic nephropathy and glomerulocystic kidney disease. Symptoms include renal salt wasting, hyperuricemia, gout, hypertension and end-stage renal disease. MCKD is part of the ‘nephronophthisis–MCKD complex’, a group of cystic kidney diseases. Both disorders have an indistinguishable histology and renal cysts are observed in either. For most genes mutated in cystic kidney disease, their proteins are expressed in the primary cilia/basal body complex. We identified seven novel UMOD mutations and were interested if UMOD protein was expressed in the primary renal cilia of human renal biopsies and if mutant UMOD would show a different expression pattern compared with that seen in control individuals. We demonstrate that UMOD is expressed in the primary cilia of renal tubules, using immunofluorescent studies in human kidney biopsy samples. The number of UMOD-positive primary cilia in UMOD patients is significantly decreased when compared with control samples. Additional immunofluorescence studies confirm ciliary expression of UMOD in cell culture. Ciliary expression of UMOD is also confirmed by electron microscopy. UMOD localization at the mitotic spindle poles and colocalization with other ciliary proteins such as nephrocystin-1 and kinesin family member 3A is demonstrated. Our data add UMOD to the group of proteins expressed in primary cilia, where mutations of the gene lead to cystic kidney disease.
doi:10.1093/hmg/ddq077
PMCID: PMC2860893  PMID: 20172860
8.  A Systematic Approach to Mapping Recessive Disease Genes in Individuals from Outbred Populations 
PLoS Genetics  2009;5(1):e1000353.
The identification of recessive disease-causing genes by homozygosity mapping is often restricted by lack of suitable consanguineous families. To overcome these limitations, we apply homozygosity mapping to single affected individuals from outbred populations. In 72 individuals of 54 kindred ascertained worldwide with known homozygous mutations in 13 different recessive disease genes, we performed total genome homozygosity mapping using 250,000 SNP arrays. Likelihood ratio Z-scores (ZLR) were plotted across the genome to detect ZLR peaks that reflect segments of homozygosity by descent, which may harbor the mutated gene. In 93% of cases, the causative gene was positioned within a consistent ZLR peak of homozygosity. The number of peaks reflected the degree of inbreeding. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations within a single ZLR peak of homozygosity as short as 2 Mb, containing an average of only 16 candidate genes. As many specialty clinics have access to cohorts of individuals from outbred populations, and as our approach will result in smaller genetic candidate regions, the new strategy of homozygosity mapping in single outbred individuals will strongly accelerate the discovery of novel recessive disease genes.
Author Summary
Many childhood diseases are caused by single-gene mutations of recessive genes, in which a child has inherited one mutated gene copy from each parent causing disease in the child, but not in the parents who are healthy heterozygous carriers. As the two mutations represent the disease cause, gene mapping helped understand disease mechanisms. “Homozygosity mapping” has been particularly useful. It assumes that the parents are related and that a disease-causing mutation together with a chromosomal segment of identical markers (i.e., homozygous markers) is transmitted to the affected child through the paternal and the maternal line from an ancestor common to both parents. Homozygosity mapping seeks out those homozygous regions to map the disease-causing gene. Homozygosity mapping requires families, in which the parents are knowingly related, and have multiple affected children. To overcome these limitations, we applied homozygosity mapping to single affected individuals from outbred populations. In 72 individuals with known homozygous mutations in 13 different recessive disease genes, we performed homozygosity mapping. In 93% we detected the causative gene in a segment of homozygosity. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations. This will strongly accelerate the discovery of novel recessive disease genes.
doi:10.1371/journal.pgen.1000353
PMCID: PMC2621355  PMID: 19165332

Results 1-8 (8)