Tissue-specific regulation of the mouse Pkhd1 (ARPKD) gene promoter.

Autosomal recessive polycystic kidney disease, an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene. Expression of PKHD1 is tissue specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of mouse Pkhd1 directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor (HNF)-1ß, which is required for activity in transfected cells. Mutation of the HNF-1ß-binding site abolishes the expression of the lacZ reporter gene in renal collecting ducts. Transgenes containing the 2.0-kb promoter and 2.7 kb of additional genomic sequence extending downstream to the second exon are expressed in the kidney, intrahepatic bile ducts, and male reproductive tract. This pattern overlaps with the endogenous expression of Pkhd1 and coincides with sites of expression of HNF-1ß. We conclude that the proximal 2.0-kb promoter is sufficient for tissue-specific expression of Pkhd1 in renal collecting ducts in vivo and that HNF-1ß is required for Pkhd1 promoter activity in collecting ducts. Additional genomic sequences located from exons 1-2 or elsewhere in the gene locus are required for expression in extrarenal tissues.

Kidney cysts, pancreatic cysts, and biliary disease in a mouse model of autosomal recessive polycystic kidney disease.

Mutations in PKHD1 cause autosomal recessive polycystic kidney disease (ARPKD). We produced a mouse model of ARPKD by replacing exons 1-3 of Pkhd1 with a lacZ reporter gene utilizing homologous recombination. This approach yielded heterozygous Pkhd1 (lacZ/+) mice, that expressed beta-galactosidase in tissues where Pkhd1 is normally expressed, and homozygous Pkhd1 (lacZ/lacZ) knockout mice. Heterozygous Pkhd1 (lacZ/+) mice expressed beta-galactosidase in the kidney, liver, and pancreas. Homozygous Pkhd1 (lacZ/lacZ) mice lacked Pkhd1 expression and developed progressive renal cystic disease involving the proximal tubules, collecting ducts, and glomeruli. In the liver, inactivation of Pkhd1 resulted in dilatation of the bile ducts and periportal fibrosis. Dilatation of pancreatic exocrine ducts was uniformly seen in Pkhd1 (lacZ/lacZ ) mice, with pancreatic cysts arising less frequently. The expression of beta-galactosidase, Pkd1, and Pkd2 was reduced in the kidneys of Pkhd1 (lacZ/lacZ ) mice compared with wild-type littermates, but no changes in blood urea nitrogen (BUN) or liver function tests were observed. Collectively, these results indicate that deletion of exons 1-3 leads to loss of Pkhd1 expression and results in kidney cysts, pancreatic cysts, and biliary ductal plate malformations. The Pkhd1 (lacZ/lacZ ) mouse represents a new orthologous animal model for studying the pathogenesis of kidney cysts and biliary dysgenesis that characterize human ARPKD.

Advances in genetic hypertension.

PURPOSE OF REVIEW: Mendelian forms of hypertension are rare genetic disorders that cause severe hypertension. This review will explore the recently identified molecular mechanisms and pathogenesis of genetic disorders that cause hypertension in children. RECENT FINDINGS: Hypertension is now believed to be a polygenic disorder resulting from the interaction of multiple genes and the environment. A few forms of severe hypertension have been linked to single genes. The genes responsible for these disorders have all been cloned and all participate in pathways involved in heightened renal sodium reabsorption. The increased sodium reabsorption arises in the distal nephron and leads to volume expansion and hypertension. SUMMARY: Investigating forms of monogenic hypertension has advanced the understanding of sodium transport and volume control by the kidney. Future studies will identify novel genes, pathways and treatment targets important in the fight against primary hypertension.

Claudins 6, 9, and 13 are developmentally expressed renal tight junction proteins.

The adult proximal tubule is a low-resistance epithelium where there are high rates of both active transcellular and passive paracellular NaCl transport. We have previously demonstrated that the neonatal rabbit and rat proximal tubule have substantively different passive paracellular transport properties than the adult proximal tubule, which results in a maturational change in the paracellular passive flux of ions. Neonatal proximal tubules have a higher P(Na)/P(Cl) ratio and lower chloride and bicarbonate permeabilities than adult proximal tubules. Claudins are a large family of proteins which are the gate keepers of the paracellular pathway, and claudin isoform expression determines the permeability characteristics of the paracellular pathway. Previous studies have shown that claudins 1, 2, 3, 4, 5, 7, 8, 10, 11, 12, 15, and 16 are expressed in the adult mouse kidney. To determine whether there are developmental claudin isoforms, we compared the claudin isoforms present in the neonatal and adult kidney using RT-PCR to detect mRNA of claudin isoforms. Claudin 6, claudin 9, and claudin 13 were either not expressed or barely detectable in the adult mouse kidney using traditional PCR, but were expressed in the neonatal mouse kidney. Using real-time RT-PCR, we were able to detect a low level of claudin 6 mRNA expression in the adult kidney compared with the neonate, but claudin 9 and claudin 13 were only detected in the neonatal kidney. There was the same maturational decrease in these claudin proteins with Western blot analysis. Immunohistochemistry showed high levels of expression of claudin 6 in neonatal proximal tubules, thick ascending limb, distal convoluted tubules, and collecting ducts in a paracellular distribution but there was no expression of claudin 6 in the adult kidney. Using real-time RT-PCR claudin 6 and 9 mRNA were present in 1-day-old proximal convoluted tubules and were virtually undetectable in proximal convoluted tubules from adults. Claudin 13 was not detectable in neonatal or adult proximal convoluted tubules. In summary, we have identified developmentally expressed claudin isoforms, claudin 6, claudin 9, and claudin 13. These paracellular proteins may play a role in the maturational changes in paracellular permeability.