James M. Bates

Validation of Uromodulin as a Candidate Gene for Human Essential Hypertension

A recent genome-wide association study identified a locus on chromosome 16 in the promoter region of the uromodulin (UMOD) gene that is associated with hypertension. Here, we examined the hypertension signal with functional studies in Umod knockout (KO) mice. Systolic blood pressure was significantly lower in KO versus wild-type (WT) mice under basal conditions (KO: 116.6±0.3 mm Hg versus WT: 136.2±0.4 mm Hg; P<0.0001). Administration of 2% NaCl did not alter systolic blood pressure in KO mice, whereas it increased in WT mice by ≈33%, P<0.001. The average 24-hour urinary sodium excretion in the KO was greater than that of WT mice (P<0.001). Chronic renal function curves demonstrate a leftward shift in KO mice, suggesting that the relationship between UMOD and blood pressure is affected by sodium. Creatinine clearance was increased during salt loading with 2% NaCl in the KO mice, leading to augmented filtered Na+ excretion and further Na+ loss. The difference in sodium uptake that exists between WT and KO strains was explored at the molecular level. Urinary tumor necrosis factor-&agr; levels were significantly higher in KO mice compared with WT mice (P<0.0001). Stimulation of primary thick ascending limb of the loop of Henle cells with exogenous tumor necrosis factor-&agr; caused a reduction in NKCC2A expression (P<0.001) with a concurrent rise in the levels of UMOD mRNA (P<0.001). Collectively, we demonstrate that UMOD regulates sodium uptake in the thick ascending limb of the loop of Henle by modulating the effect of tumor necrosis factor-&agr; on NKCC2A expression, making UMOD an important determinant of blood pressure control.

Tamm-Horsfall Protein Acts as a General Host-Defense Factor against Bacterial Cystitis

Aims: Tamm-Horsfall protein (THP) is urine’s most abundant protein, but its biological function has remained elusive. Recently, THP-deficient (THP–/–) mice were shown to have difficulty clearing Escherichia coli from the urinary bladder. It has remained unclear if interaction between THP and E. coli is specific for E. coli or if THP has a versatile ability to clear a variety of bacteria from the bladder, and act as a broad host-defense mechanism against urinary tract infection (UTI). In this study, we examined the role of THP as a protective factor against UTI caused by bacteria other than E. coli, namely Klebsiella pneumoniae and Staphylococcus saprophyticus by determining if the THP–/– mouse has difficulty clearing these bacteria from its bladder. Methods: THP gene knockout mice were generated by the technique of homologous recombination. K. pneumoniae and S. saprophyticus were introduced transurethrally, in separate experiments, into the bladders of the THP–/– and genetically similar wild-type (THP+/+) mice. Urine was collected at periodic intervals and cultured to quantitate the degree of bacteriuria. Bladders were surgically removed and examined histomorphometrically to determine the intensity of inflammation. Results: Results showed that both with K. pneumoniae and with S. saprophyticus, the THP–/– mice had more severe bacteriuria in comparison with THP+/+ mice. The inflammatory changes in the bladder were also markedly more intense in THP–/– mice with each of the bacterial species. Conclusions: These findings support the hypothesis that THP helps eliminate K. pneumoniae and S. saprophyticus from the urinary tract and acts as a general host-defense factor against UTI.

Tamm-Horsfall Protein Protects Against Urinary Tract Infection by Proteus mirabilis

PURPOSE Proteus mirabilis is a common cause of urinary tract infection. We determined the role of Tamm-Horsfall protein as a host defense factor against the cystitis and pyelonephritis caused by P. mirabilis. MATERIALS AND METHODS We generated Tamm-Horsfall protein gene knockout mice using homologous recombination. We introduced P. mirabilis transurethrally into the bladder of Tamm-Horsfall protein deficient (THP(-/-)) and genetically similar WT (THP(+/+)) mice. We cultured urine to quantitate the degree of bacteriuria. We examined bladders and kidneys grossly and histomorphometrically to determine the intensity of inflammation. RESULTS THP(-/-) mice had more severe bacteriuria and cystitis than THP(+/+) mice. THP(-/-) mice had more pyelonephritic abscesses than THP(+/+) mice. The severity of histological pyelonephritis on semiquantitative histomorphometric analysis appeared to be greater in THP(-/-) mice. The difference between the 2 groups approached but did not attain statistical significance (p = 0.053). CONCLUSION Tamm-Horsfall protein acts as a host defense factor against P. mirabilis induced urinary tract infection.

Nucleotide sequence and peptide motifs of mouse uromodulin (Tamm-Horsfall protein) - the most abundant protein in mammalian urine☆

The mouse uromodulin cDNA sequence was sequenced. The predicted peptide sequence is 642 amino acids long and contains several modular components including four epidermal growth factor like repeats, one betaglycan-like domain (ZP domain), and a consensus sequence for attachment of a glycosyl-phosphatidyl-inositol anchor. An arginine-glycine-aspartate tripeptide reported for rat and human sequence is absent in the mouse. There are several potential sites for post-translational modification.

Tamm-Horsfall protein facilitates catheter associated urinary tract infection

BackgroundUrinary catheters are associated, commonly with bacteriuria and frequently with urinary tract infection. Tamm-Horsfall Protein (THP) is urines most abundant protein and is known to bind to uropathogenic bacteria. The role of THP in the pathogenesis of catheter associated urinary tract infection (CAUTI) is not clear. We examined the role of THP in facilitating bacterial binding to urinary catheters in vivo and in vitro.FindingsTwenty one urinary catheters were obtained from 20 hospitalized patients. THP was eluted from the catheter surface and catheter segments were cultured. Additional studies were performed in vitro on unused silicone and latex catheters to determine the binding of THP, and the effect of THP on the binding of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), to the catheter surface.On catheters obtained from patients, the THP deposition was significantly more on culture positive catheters than on culture negative catheters. In the in vitro studies, THP bound to both silicone and latex catheters, and THP enhanced the adherence of E. coli and P. aeruginosa to both types of catheters.ConclusionTHP binds to urinary catheters and facilitates the binding of uropathogenic bacteria to catheters.

Abnormal zinc metabolism in unilateral maldescended testes of a mutant rat strain.

Abstract The status of zinc in a mutant rat strain with heritable maldescended testes was examined. In rats with unilateral maldescended testis, the ectopic testis consistently had decreased zinc content (121.0 ± 23.0 μg zinc/g dry wt), while the eutopic testis had zinc content similar to that of normal rats (182.0 ± 5.0 μg zinc/g dry wt). Uptake of zinc by the ectopic testis was comparable to normal. Sephadex gel chromatography showed greatly reduced zinc content of one of the endogenous zinc binding fractions with a mol wt of 30,000 of cytosol of the ectopic testis in spite of a near normal protein content. Incorporation of zinc-65 into this fraction was also shown to be greatly reduced in ectopic testis. Sodium dodecylsulphate-polyacrylamide gel electrophoresis demonstrated that a protein of 23,000 Da was greatly reduced in quantity. This 23-kDa protein in ectopic testis may play a role in reduced testicular function of the ectopic testis.

Consultants for the American Journal of Nephrology 2005

Sandra Garber Cybele Ghossein Richard Glassock Alan Go Laurence Greenbaum Karen Griffi n S. Grigoryev Krishnamurthy Gudehithlu Peter Hart Koichi Hayashi Peter Heering Susan Hou John Hoyer Randall Hudson Todd Ing Eunice John Richard Johnson Michelle Josephson Pradeep Kadambi Ramesh Khanna Orly Kohn Jeff Kopp Mark Kraus Jerome Lane Craig Langman James Lash David Leehey Oliver Lenz Edgar Lerma Jerrold Levine Wilfred Lieberthal Stuart Linas Jill Lindberg Natalia Litbarg Gerard London Rodger Loutzenhiser Friedrich Luft K. Matsumoto Peter McCullough Patrick Murray Naoyuki Nakao Kevin Abbott Rajiv Agarwal Sharon Anderson Gema Ariceta John Asplin Simon Atkinson Asad Bakir George Bakris Vinod Bansal Amelia Bartholomew Amy Barton Pai Daniel Batlle Vecihi Batuman Enrico Benedetti Angelito Bernardo Anil Bidani Peter Blake Anthony Bleyer W. Kline Bolton Michael Braun Carolyn Brecklin Harold Bregman Ellen Brooks Vito Campese Sule Cataltepe Nina Clark Jay Cohn Richard Cohn Judith Cook Andrey Cybulsky Mohamed Daha Farhard Danesh Janice Douglas George Dunea Lance Dworkin Beatrice Edwards Leon Ferder Steven Fishbane Kenneth Fisher Mary Foster Barry Freedman

PLACENTAL COPPER TRANSPORT IN THE BRINDLED MOUSE

The X-linked mouse mutant brindled is a model for Menkes syndrome. In young hemizygotes, reduced liver and brain copper concentrations are associated with neurologic dysfunction. In fetuses copper concentrations in placenta and kidney are higher in brindled than controls while those in liver and carcass are lower. To treat the copper deficiency in brindled young, heterozygotes were injected at 16 or 18 days gestation with copper, 6 mcg/g/dose, as cupric chloride, 18 and 6 hours before sacrifice. Placental, carcass, and hepatic copper concentrations in brindled fetuses increased (p>0.006). Injection of methylprednisolone, 5 mcg/g, 20 hours before the copper, to increase fetal hepatic copper storage through metallothionein induction, resulted only in further increase in the carcass copper concentrations. These data suggest: placental copper transport in brindled fetuses is impaired; hepatic copper binding capacity of control and brindled fetuses is limited and cannot be augmented by pretreatment with methylprednisolone; extra-hepatic copper binding may be increased with methylprednisolone, which implicates induction of extra-hepatic metallothionein in both groups of animals.

234 ABERRANT ZINC BINDING IN TESTES OF TESTICULAR FEMINIZATION RATS

Testicular feminization (Tfm) is an inherited form of male pseudohermaphroditism. In the rat, this syndrome is manifested by an absence of androgen dependent differentiation, small cryptorchid testes and infertility. Previous studies in our laboratory had established that cryptorchid testes of these rats showed drastically reduced zinc content and failure of zinc retention. The present studies attempt to further characterize this aberrant metabolism of zinc in the Tfm rats. Testes from normal littermates were used as control in all experiments. Distribution of endogenous zinc in the testicular cytosol was examined by chromatography through a Sephadex G-75 column (1.5×90 cm) equilibrated with 10 mM Tris-HCl, pH 8.0. Both zinc content and 280 nm absorbance of fractions collected were determined. Normal control testes showed typically four zinc peaks: peak 1 at void volume, peak 2 corresponded to Mr 30k, peak 3 corresponded to Mr 10k and peak 4 at the wash volume. Peak 2, however, was missing when cytosol of cryptorchid testes of the Tfm rats were analyzed. Column fractions corresponding to zinc peak 2 of the cryptorchid and normal control testes were pooled and concentrated separately and examined by electrophoresis through 12% polyacrylamide gel containing 0.1% SDS. A protein band with Mr 23k was missing in the cryptorchid testes. Whether the missing protein of the cryptorchid testis was related to the absence of zinc peak 2 and the decreased testicular zinc content in the Tfm rat is under further investigation. (Supported in part by NIH grant HD 16730).