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. While there is now good evidence that adenosine plays a critical role in JGA-mediated responses, the source of the extracellular adenosine has remained unclear. We have hypothesized that adenosine is derived from released ATP through the successive action of phosphohydrolases and nucleotidases. Since the last step in adenosine formation is the dephosphorylation of 5?AMP by ecto-5?nucleotidase (CD73) we have generated a mouse with a CD73 knockout mutation with the support of the NIDDK stem cell lab directed by Dr. Chuxia Deng. These mice are viable, appear morphologically normal, and have normal plasma and urine chemistries. The JGA-dependent responses of vascular tone and renin release have been found to be severely attenuated while MD-dependent renin secretion appears to be normal. These data suggest that extracellular hydrolysis of AMP provides most of the adenosine necessary for JGA signaling events. 2. Renin formation in granular cells of the juxtaglomerular apparatus plays a critical role in the maintenance of body fluid volume and arterial blood pressure. There is evidence that agonists signaling through Gsa ?coupled receptors are important mediators of some the pathways mediating renin secretion. To study the consequences of eliminating JGA regulation of renin secretion, we have used a Cre-Lox based strategy to delete the Gsa-cAMP signaling pathway. In preliminary experiments we have used a 4kb renin promoter to express Cre recombinase in JGC selectively (Ren-Cre). With this construct we found expression of Cre recombinase mRNA in JGC, but the expression levels were low and the Cre activity as estimated by crossing Ren-Cre mice into a Rosa reporter mouse strain (129-Gt(Rosa)26 Sor, GFP; Jackson Labs.) was negligible. We are currently repeating these studies using a 12 kb human renin promoter (supplied by Dr. Germain, Paris) that has previously been shown to direct tissue-specific LacZ expression. In addition we are collaborating with Dr. Gomez (Univ. Virginia) who has used recombination in stem cells to generate mice expressing Cre-recombinase in JGC. We have crossed these Ren-Cre mice with mice generated by Drs. Weinstein and Chen at NIDDK in which Gsa is flanked by LoxP sites. We are expecting to obtain offspring with a renin-cre/Gsalphaflox/flox background within the next month. 3. JGA-mediated effects are sensitive to loop diuretics suggesting that NaCl transport through the NKCC2 cotransporter in the apical membrane of macula densa cells may be critical for eliciting JGA dependent responses. However, loop diuretics exert a host of secondary effects so that the specificity of this action and its mechanism have remained unclear. 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. In an attempt to produce isoform-specific NKCC2 knockout mice we have generated 3 constructs that contain about 5 kb of 5' homologous sequence, a Flag peptide sequence, a stop codon, a NeoR sequence between LoxP sites-, and about 5 kb of 3' homologous sequence. We have inserted these constructs in either the B, A, or F region of exon 4 of the NKCC2 gene by homologous recombination, and obtained recombinant stem cell clones for all three constructs. After blastocyst injection we have obtained chimeric mice carrying the B and F isoform mutation. Germline transmission has been established for the B isoform, and homozygous NKCC2-B-/- mice have been obtained. These mice produce a truncated NKCC2-B transcript while splicing of the A and F isoform transcripts occurs normally although at a reduced level. Initial functional studies in these mice do not show tubular absorption defects that is associated with diuresis or a reduced concentrating ability.
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