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To characterize the nature of NaCl transport in the thin ascending limb (tAL), we examined the transport properties of [Na.sup.+] and [Cl.sup.-] using in vitro microperfusion of the tAL in CLC-K1 null mice. In the presence of a transmural NaCl concentration gradient (100 mM higher in the lumen), the transepithelial diffusion voltage ([V.sub.d]) was 15.5 [+ or -] 1.0 and -7.6 [+ or -] 1.4 mV in CLC-K[1.sup.+/+] and CLC-K[1.sup.-/-] mice, respectively. Neither [Cl.sup.-] transport inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) nor acidification of the bathing fluid changed the [V.sub.d] values in CLC-K[1.sup.-/-] mice. The addition of 300 [micro]g/ml protamine, a selective blocker of paracellular conductance, to the bath increased the [V.sub.d] values by 5.6 [+ or -] 0.7 and 12.6 [+ or -] 1.5 mV (P < 0.001) in CLC-K[1.sup.+/+] and CLC-K[1.sup.-/-] mice, respectively. Although efflux coefficients of [sup.36]Cl were significantly decreased in CLC-K[1.sup.-/-] mice (188.3 [+ or -] 25.6 in 4 tubules vs. 17.2 [+ or -] 7.0 x [10.sup.-5] cm/s in 6 tubules), those of [sup.22]Na were not different between CLC-K[1.sup.+/+] and CLC-K[1.sup.-/-] mice. These results clearly indicate that the major component of Cl-transport sensitive to NPPB or pH is mediated by CLC-K1 in the tAL. gene targeting; renal medulla; urine-concentrating mechanism; chloride channel; paracellular shunt pathway; sodium chloride

Helix 5 and loop E of aquaporin-2 contribute to the high water permeability of the protein. This protein is a water channel regulated by vasopressin that concentrates urine.

Experiments were performed on homozygous Brattleboro rats, which lack the arginine vasopressin (AVP), to examine the possibility of an AVP-independent regulation of aquaporin 2 (AQP2) mRNA gene expression in these rats. Antidiuresis and expressed AQP2 mRNA and AQP2 protein were found in the renal medullary of the rats after exogenous administration of 1-deamino-8-D-AVP. Findings also indicated the absence of an AVP-independent mechanism for upregulating AQP-2 gene expression.

A study was conducted to localize the rat's CLC-K2, a kidney-specific member of the CLC chloride channel family, and the CLC-K2 messenger RNA (mRNA). Results revealed that CLC-K2 is expressed in the kidney's superficial cortex and outer medula while CLC-K2 mRNA can be found in the kidney's distal convoluted tubules, connecting tubules and cortical collection ducts. The latter's signal is also expressed in the medullary thick ascending limb of Henle's loop.

The CO1G6.1 is one of the eight cDNAs belonging to the major intrinsic protein superfamily found in the nematode Caenorhabditis elegans. The water channel function of the CO1G6.1 gene product is similar to that of aquaporin3 (AQP3) and glycerol facilitator (GlpF), but their osmotic water permeability and urea permeability are lower. Chimeric studies suggest that two segments of amino acid residues found in CO1G6.1, AQP3 and GlpF do not contribute to channel selectivity.

A study was conducted to investigate the involvement of G1 cyclin-dependent kinase activity in hypertrophy in cultured rat neonatal cardiomyocytes. Cardiomyocytes from isolated and put through Percoll gradient centrifugation before they were cultured in vitro. Cells were then treated with buffered Formalin to determine surface area. Experimental results indicated that D-tyke cyclin-dependent kinase was promoted during hypertrophic processes of cardiomyocytes that were serum-stimulated in culture.

Research indicates that the rat kidney-specific CIC-K1 chloride channel of the ascending limb of Henle's loop in the kidney is genetically influenced by a 40 bp purine-rich protein gene sequence. Experiments using gel-retardation analysis and inner medullary cells of simian virus 40 transgenic mice are described. Specific protein binding patterns are illustrated.

Research designed to establish the mercury inhibitory site in AQP3, an aquaporin (AQP) that has low homology to other AQPs, indicates that Cys-11 is the mercury-sensitive residue in AQP3. This residue could be independent of water channel function. Substitution of Cys-11 with tryptophan did not abolish water channel function. Mercury-sensitive cysteine may not be directly linked with the aqueous pore formation in AQP3. It is also possible that the side chain of Cys-11 may not extend into the pore.

The ability of corticosteroid to regulate aquaporin 3 (AQP3) expression in human airway epithelial cell lines was investigated. A549 lung cancer cells were used in an experiment to analyze protein and messenger RNA (mRNA) expression and water permeability of the cell membrane and the induction of AQP3 mRNA by dexamethasone. Results showed that mRNA and protein expression of AQP3 was stimulated and that cell membrane water permeability was enhanced by dexamethasone. Thus, corticosteroids were proven to upregulate the promoter activity of AQP3.

Two new approaches were applied to analyze the aquaporin-2 (AQP-2) promoters. The rat and mouse AQP-2 promoters were cloned and a negative cis-regulatory element was identified. Results of the study indicated that the tissue-specific expression of AQP-2 gene may in part be regulated by a novel negative acting cis-element. Trans-acting nuclear proteins were also identified by ultraviolet cross-linking.

Reverse transcriptase-polymerase chain reaction (RT-PCR) was utilized to examine the microlocalization of messenger RNA coding for adrenomedullin (AM) and the effects of the protein of cyclic adenylic acid. RT-PCR analysis of the intraglomerular site expression of AM in cultured rat mesanglial cells (MC) and microdissected nephron segments indicated the role of AM in the modulation of renal hemodynamics. AM also increased glomerular filtration rate and resultant water and sodium excretion via cyclic adenylic acid in the mesanglial cells.

The effects of dehydration on the distribution pattern and messenger ribonucleic acid (mRNA) expression of AQP3 were analyzed in rate kidney collecting ducts. Analysis of AQP3 mRNA expression in rat kidney collecting ducts indicated the role of dehydrationin inducing AQP3 expression at the protein level. Furthermore, analysis of AQP3 structure indicated that the water channel was a glycosylated protein with a structure that was similar to AQP1 and AQP2.

The cortical collecting duct (CCD) cells of dehydrated rats show the effects of arginine vasopressin (AVP) inhibition on the expression of aquaporin-2 (AQP-2) messenger ribonucleic acid (mRNA). Analysis of AQP-2 mRNA expression in CCD cells of dehydrated rats indicated the role of water deprivation and endogenous AVP in increasing the levels of AQP-2 in the apical plasma membranes. However, forced water load in dehydrated rats abolished AQP-2 mRNA expression and diffused AQP-2 into the cytoplasm.

The presence of aquaporin (AQP) messenger ribonucleic acids (mRNAs) during kidney morphogenesis was analyzed by ribonuclease protection assay (RPA) and immunohistochemistry in fetal rats. Analyis of fetal rat kidneys by RPA and immunohistochemistry indicated the presence of AQP1, AQP3, AQP2 and AQP4 mRNA in the apical side of the cortical collecting duct cells. Furthermore, the imbalanced expression of AQP2 and AQP3/AQP4 in fetal rat kidneys indicated the presence of other water channel families in the basolateral membrane collecting ducts.

The expression of aquaporin-2 (AQP2) messenger ribonucleic acid (mRNA) and the promoter activity of its 5'-flanking region was analyzed in cultured epithelial kidney cells. AQP2 mRNA was detected by Northern blot analysis in the cultured epithelial cell lines of the kidney such as mouse outer medullary collecting duct (MCD) and inner (MCD). However, minute levels of AQP2 mRNA was detected by ribonuclease protection assay in mouse outer MCD. Furthermore, a decrease in AQP2 mRNA expression was detected in rat inner MCD cell cultures after the initiation of transcription.

People infected with hepatitis C virus genotype 1b (HCV-1b) with mutations of the nonstructural protein 5A (NS5A) may be more responsive to interferon therapy than people infected with the non-mutated virus. HCV-1b is the most common strain of HCV, causes severe liver damage, and is generally resistant to interferon treatment. Researchers analyzed the viral RNA in blood samples from 84 people with HCV-1b infection who were treated interferon. A complete response to therapy was defined as the absence of HCV RNA in blood six months after treatment. A complete response occurred in all 16 people with a significant changes in the NS5A viral gene but in none of the 30 people without such changes, and in 5 of the 38 people with slight changes. People with low blood levels of HCV RNA before treatment also responded better to therapy.

Hydrogen-potassium-adenosinetriphosphatase, present in the luminal membrane of intercalated cells (ICs) and outer medullary collecting ducts (OMCD), stimulates K+ absorption in K+ -deficient conditions. Luminal infusion of K+ enhances (Na+)- and HCO3(super -)-independent intracellular pH rate by 1.6 plus or minus 0.4 and 1.9 plus or minus 0.4 X 10 to the -3rd power pH units/s in ICs and inner stripes of OMCD, respectively. The luminal Sch-28080 inhibits this increase in pH(sub i).

Experimental rat models were used to determine if aquaporin of the kidney collecting duct (AQP-CD) is involved in abnormal water retention which occurs in syndrome of inappropriate secretion of antidiuretic hormone (SIADH) and liver cirrhosis. The results show that arginine vasopressin (AVP) regulates AQP-CD expression at the transcriptional level, indicating that AQP-CD is the AVP-regulated water channel that is a major factor in pathological water retention in SIADH and liver cirrhosis.

It may be possible to determine what type of diabetes insipidus a patient has based on the changes in aquaporin-2 levels in the urine after taking vasopressin. Vasopressin is a hormone that increases urine concentration. Aquaporin-2 is a protein that transports water across membranes in the kidney. In 5 normal men, urinary levels of aquaporin-2 decreased from 11.2 pmol per milligram of creatinine following dehydration to 3.9 pmol per milligram of creatinine after drinking 1 liter of water. In another 6 normal people, aquaporin-2 levels went from 0.8 to 11.2 pmol per milligram of creatinine after receiving desmopressin, which is composed of desamino and arginine vasopressin. In 5 patients with central diabetes insipidus, aquaporin-2 levels increased by 4 to 6 times from a base level of 0.4 pmol per milligram of creatinine after receiving vasopressin. However, in 4 patients with genetically determined diabetes insipidus, aquaporin-2 levels did not change in response to vasopressin. Thus, patients with this rarer form of the disease do not appear to respond to vasopressin.

Reverse transcription and polymerase chain reaction (RT-PCR) followed by Southern blot analysis on the localization of mRNA encoding IP3 receptors on microdissected nephron segments of rats reveals that type 1 IP3 receptor mRNA distribution, although widespread, is not uniform along the nephron. On the other hand type, 2 IP3 receptor mRNA is entirely confined to the collecting ducts, which suggests a specific expression of type 2IP3 receptor in collecting ducts. Thus different types of IP3 receptors have different functions in the rat kidney.