The overall goal of our research is to contribute to understanding the role of the kidneys in the maintenance of body fluid volume and arterial blood pressure. Specifically, we study the renal mechanisms responsible for the match between glomerular filtration of plasma and tubular salt and fluid reabsorption that is ultimately responsible for the precise balance between salt intake and excretion. One of the key mechanisms that mediates between renal absorption and filtration function is the so-called tubuloglomerular feedback, a regulatory pathway in which a measure of tubular reabsorptive capacity serves as the signal to alter glomerular filtration.? 1. Substantial effort has gone into the development of new mouse models designed to enhance the understanding of the function of the juxtaglomerular apparatus and the renin-angiotensin system. To generate mice with an overexpression of the adenosine1 receptor (A1AR) in blood vessels we have placed the mouse A1AR gene under the control of the alpha-actin promoter that has been shown to direct expression to vascular smooth muscles cells. Transgenic mice will be generated in the laboratory of Dr. Jeffrey Kopp. Two approaches are being used to affect the expression of ClCkb, the chloride channel expressed in the cortical ascending loop of Henle. Transgenic mice are being generated that express a panel of 6 different siRNAs directed against the ClCkb mRNA. SiRNAs will also be injected in the Kopp laboratory. In addition, we have made a construct to inactivate the ClCkb gene by homologous recombination. A first attempt to obtain stem cells with the recombined gene has been unsuccessful, but a second transfection has been performed and the cells are currently being genotyped. Work on NKCC2 has continued with a new effort to obtain mice with deletion of the F isoform of NKCC2. In previous experiments we have been unable to obtain germline transmission of the gene mutation, and a new blastocyst injection has been done in the meantime in laboratory of Dr. Chuxia Deng. We also have made a construct designed to replace both the B and A variants of NKCC2 including intronic sequences with a DNA sequence of equal length in an attempt to simultaneously delete the A and B NKCC2 isoforms. The construct has been transfected into stem cells, and genotyping of stem cell DNA is under way. Finally, we have used a commercially available stem cell line with a knockout mutation of the newly discovered renin receptor gene to generate chimeric mice carrying the mutated gene.? 2. JGA-mediated effects are sensitive to loop diuretics suggesting that NaCl transport through the NKCC2 cotransporter in the apical membrane of macula densa (MD) cells may be critical for eliciting JGA dependent responses. NKCC2 is expressed along the thick ascending limb (TAL) of the loop of Henle in three isoforms representing splice variants of a single gene. As previously shown by RT-PCR and in situ hybridization, the B isoform of NKCC2 is expressed in MD cells, the A isoform in both cortical and medullary TAL, and the F isoform in medullary TAL exclusively. By inserting a stop codon into either the B, A, or F region of exon 4 of the NKCC2 gene by homologous recombination, we have so far been able to obtain both NKCC2B-/- and NKCC2A-/- mice, and have found that both strains are viable and show no morphological abnormalities. After removal of the neomycin selection cassette alternative splicing appears to be unaffected. We have summarized our results on NKCC2B-deficient mice in a recent publication, and are currently completing the phenotypic evaluation of the NKCC2A-/- animals. Ambient urine osmolarity (mosm/l) was reduced in NKCC2A-/- (1236 vs. 1673, p=.02), and basal plasma renin concentration (PRC; ng Ang I/ml/hr) tended to be lower in NKCC2A-/- compared to WT (621 vs. 1211, p=.061). The renin inhibitory response to an acute increase in luminal NaCl concentration was eliminated in the knockout animals suggesting that the A isoform is required for the detection of elevated NaCl. Cl reabsorption along the loop of Henle was normal at low flow rates and significantly diminished at high flows. Maximum tubuloglomerular feedback (TGF) responses were reduced in NKCC2A mice, and TGF curves were left-shifted. Thus, the presence of two NKCC2 isoforms with different Cl affinities in the same tubule widens the NaCl concentration range over which the transporter can operate.? 3. To study the role of the COX-2/PGE2 pathway in the control of renin secretion we determined the response of plasma renin concentration (PRC) to acute i.p. administration of furosemide (40 mg/kg), hydralazine (2 mg/kg), isoproterenol (10 mg/kg), candesartan (50 microg), or quinaprilate (50 microg) in conscious wild type (WT) and COX-2-/- mice on 3 different genetic backgrounds (mixed, C57BL/6, 129J). Basal PRC was significantly lower in COX-2-/- than WT mice independent of genetic background (51%, 10%, and 17% of WT in mixed, 129J, and C57BL/6). All five acute interventions caused significant increases of PRC in both COX-2+/+ and -/- mice, but the response was consistently less in COX-2-deficient mice (e.g. deltaPRC in ng Ang I/ml hr caused by furosemide, isoproterenol, hydralazine, quinaprilate, or candesartan 4699, 3534, 2522, 9453, 66455 in 129J WT, and 201, 869,140, 902, 2660 in 129J COX-2-/-). A low NaCl diet and enalapril for one week caused a 14fold elevation of PRC in COX-2-/- mice, and was associated with a greatly increased PRC response to acute furosemide (delta PRC 201 before and 15984 after low Na/enalapril). As measured by radiotelemetry, blood pressure and heart rate responses to furosemide, hydralazine, isoproterenol, candesartan, or quinaprilate were not different between COX-2 genotypes. Thus, chronic absence of COX-2 reduces renin expression, release, and PRC, and is associated with a reduced ability to alter PRC during acute stimulation regardless of the nature of the stimulus. These studies do not support the notion of a specific role for COX-2 in macula densa control of renin secretion. They rather suggest that renin gene expression is the primary determinant of the amount of renin that is available for acute release, and that COX-2 is merely one of the factors that affect renin expression. ? 4. Glomerular hyperfiltration early in the disease is thought to be a critical contributory factor in the pathogenesis of diabetic nephropathy in humans. Tubuloglomerular feedback is believed to be responsible for hyperfiltration since glucose-driven proximal fluid reabsorption increases in diabetes and may cause dilatation of afferent arterioles subsequent to a lowering of distal NaCl delivery. We have used the Akita mouse model of type 1 diabetes to determine whether hyperfiltration occurs early in the disease. Furthermore, we have bred Akita diabetic mice into an A1AR-/- background and thereby generated diabetic mice without feedback regulation of GFR. To be able to perform these studies we developed a technique that permits repeat measurements of GFR in conscious mice using excretion kinetics after a single dose of FITC-inulin. We find that Akita mice have a largely normal GFR at 14 weeks of age whereas Akita/A1AR-/- mice show a doubling of GFR at this age. Thus, early hyperfiltration is not a characteristic of this diabetic mouse model. As the observations in the diabetic A1AR-/- mice suggest, the operation of TGF actually prevents GFR from increasing rather than causing hyperfiltration.
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