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Examines directly the effects of chronic chloride depletion metabolic alkalosis (CDAlk) on renal ammonium production, urinary ammonium excretion, and proximal tubule ammonium transport in the rat. Effects of alkalosis on ammonium transport and excretion under conditions of potassium and chloride depletion in a setting of constant glomerular filtration rate.

Focuses on the regulation of H+-K+-adenosinetriphosphatase (H+-K+-ATPase) expression in the kidney. Development of highly specific probes for the catalytic subunit using reverse transcriptase-PCR with gastric- or colonic-specific primers; Suggestion that H+-K+-ATPase activity and expression might be modulated in response to chronic modulation of potassium and/or acid-base balance.

Studies the direct effects of endothelin-1 on proximal tubular sodium-inorganic phosphate (Na+-Pi) cotransport and sodium-hydrogen exchange in vitro in brush-border membrane vesicles. Specific activities of alkaline phosphatase and Na+-K+-ATPase; Effects of incubation of renal cortical slices with parathyroid hormone or vehicle.

The article reflects on Renal tubular acidosis (RTA), a medical condition that involves an accumulation of acid in the body due to a failure of the kidneys. It informs that RTA involving the proximal tubule (PRTA) consists of two major categories including disorders of proximal tubule reabsorption and isolated abnormalities in renal acidification. It mentions that missense mutation T485S alters the sodium/bicarbonate symporter 1NBCe1-A electrogenicity that causes RTA.

The authors comments on the interactive effects of kidney disease and cardiovascular disease (CVD). They discuss the results of two reports on the issue. The first report analyzed two large community-based cohorts at relatively low risk for kidney disease to determine whether prevalent CVD was a risk factor for the development and progression of chronic kidney disease. The second report evaluated the impact of renal risk factors on the prevalence of CVD.

We previously reported that the deletion of the pH sensor GPR4 causes a non-gap metabolic acidosis and defective net acid excretion (NAE) in the GPR4 knockout mouse (GPR4-/-) (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, and Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). Since the major regulatory site of NAE in the kidney is the collecting duct (CD), we examined acid-base transport proteins in intercalated cells (ICs) of the CD and found comparable mRNA expression of kidney anion exchanger 1 (kAE1), pendrin, and the a4 subunit of H(+)-ATPase in GPR4-/- vs. +/+. However, NH4Cl loading elicited adaptive doubling of AE1 mRNA in GPR4+/+, but a 50% less pronounced response in GPR4-/-. In GPR4+/+, NH4Cl loading evoked a cellular response characterized by an increase in AE1-labeled and a decrease in pendrin-labeled ICs similar to what was reported in rabbits and rats. This response did not occur in GPR4-/-. Microperfusion experiments demonstrated that the activity of the basolateral Cl(-)/HCO3- exchanger, kAE1, in CDs isolated from GPR4-/- failed to increase with NH4Cl loading, in contrast to the increase observed in GPR4+/+. Therefore, the deficiency of GPR4 blunted, but did not eliminate the adaptive response to an acid load, suggesting a compensatory response from other pH/CO2/bicarbonate sensors. Indeed, the expression of the calcium-sensing receptor (CaSR) was nearly doubled in GPR4-/- kidneys, in the absence of apparent disturbances of Ca2+ homeostasis. In summary, the expression and activity of the key transport proteins in GPR4-/- mice are consistent with spontaneous metabolic acidosis, but the adaptive response to a superimposed exogenous acid load is blunted and might be partially compensated for by CaSR. [ABSTRACT FROM AUTHOR]

The carboxy terminus (CT) of the colonic H+-K+-ATPase is required for stable assembly with the β-subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein inter- actions involved in the localization and function of HKα2, we selected 84 amino acids in the CT of the α-subunit of mouse colonic H+-K+- ATPase (CT-HKα2) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HKα2 with CD63, recombinant CT-HKα2 and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HKα2 protein (but not CT-HKα1) coprecipitated with CD63, confirming stable assembly of HKα2 with CD63. In HEK-293 transfected with HKα2 plus β1-Na+-K+-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKα2/NKα1 and 86Rb+ uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKα2-NKβ1 complex in the cell membrane. [ABSTRACT FROM AUTHOR]

Background: The present experiments were designed to study the importance of the carboxy-terminus of HKalpha2, for both function and integrity of assembly with beta1-Na+,K+-ATPase.Methods: For this purpose, stop codons were created, by polymerase chain reaction (PCR), at different positions in the carboxy-terminus of HKalpha2. Subsequently, chimeras between HKalpha2 and the carboxy-terminus of alpha1-Na+,K+-ATPase or with the carboxy-terminus of the gastric H+,K+-ATPase were created. Human embryonic kidney HEK-293 cells were used as expression systems for functional studies using 86Rb+ uptake and alpha/beta assembly using specific antibodies.Results: The results demonstrate that the entire carboxy-terminus of HKalpha2 is required for optimal protection of the alpha/beta complex from degradation and for functionality as evidenced by 86Rb+ uptake. The results also demonstrate that there was flexibility in the sequence of the carboxy-terminus. The last two tyrosines (Y1035Y1036) of HKalpha2 could be mutated to alanines and the carboxy-terminus of HKalpha2 could be replaced by the carboxy-terminus of alpha1-Na+,K+-ATPase while preserving transport activity.Conclusion: The entire carboxy-terminus of HKalpha2 is required for stable assembly with beta1-Na+,K+-ATPase and functionality. [ABSTRACT FROM AUTHOR]

Studies the use of antibodies to investigate changes in expression of HK alpha 2 in renal cortex, renal medulla and distal colon in two pathophysiological conditions. Methodology of the study; Results of the specificity of anti-HK alpha 2; Discussion of the study.

Although the kidney plays the major role in the regulation of systemic K+ homeostasis, the colon also participates substantively in K+ balance. The colon is capable of both K+ absorption and secretion, the magnitude of which can be modulated in response to dietary K+ intake. The H+-K+-adenosinetriphosphatase (H+-K+-ATPase) has been proposed as a possible mediator of K+ absorption in distal colon, but inhibitor profiles obtained in recent studies suggest that two, and perhaps more, distinct H+-K+-ATPase activities may be present in mammalian distal colon. We have developed highly specific probes for the catalytic a-subunits of colonic and gastric H+-K+-ATPase, &#39 45;¹-Na+-K+-ATPase, and β-actin, which were used in Northern analysis of total RNA from whole distal colon and stomach obtained from one of three experimental groups of rats: 1) controls, 2) chronic dietary K+ depletion, and 3) chronic metabolic acidosis. The probe for the colonic but not the gastric H+-K+-ATPase a-isoform hybridized to distal colon total RNA in all groups. A significant increase in colonic H+-K+-ATPase mRNA abundance was observed in response to chronic dietary K+ depletion but not to chronic metabolic acidosis. The α¹-isoform of Na+-K+-ATPase, which is also expressed in distal colon, did not respond consistently to either chronic dietary K+ depletion or chronic metabolic acidosis. The gastric probe did not hybridize to total RNA from distal colon but, as expected, hybridized to total stomach RNA. However, the abundance of gastric H+-K+-ATPase or Na+-K+-ATPase in stomach was not altered consistently by either chronic dietary K+ depletion or metabolic acidosis. Under the conditions of this study, it appears that the mRNA encoding the colonic α-isoform is upregulated by chronic dietary K+ restriction, a condition shown previously to increase K+ absorption in the distal colon. [ABSTRACT FROM AUTHOR]

Gestational potassium retention, most of which occurs during late pregnancy, is essential for fetal development. The purpose of this study was to examine mechanisms underlying changes in potassium handling by the kidney and colon in pregnancy. We found that potassium intake and renal excretion increased in late pregnancy while fecal potassium excretion remained unchanged and that pregnant rats exhibited net potassium retention. By quantitative PCR we found markedly increased H+-K+- ATPase type 2 (HKA2) mRNA expression in the cortex and outer medullary of late pregnant vs. virgin. Renal outer medullary potassium channel (ROMK) mRNA was unchanged in the cortex, but apical ROMK abundance (by immunofluorescence) was decreased in pregnant vs. virgin in the distal convoluted tubule (DCT) and connecting tubule (CNT). Big potassium-α (BKα) channel-α protein abundance in intercalated cells in the cortex and outer medullary collecting ducts (by immunohistochemistry) fell in late pregnancy. In the distal colon we found increased HKA2 mRNA and protein abundance (Western blot) and decreased BKα protein with no observed changes in mRNA. Therefore, the potassium retention of pregnancy is likely to be due to increased collecting duct potassium reabsorption (via increased HKA2), decreased potassium secretion (via decreased ROMK and BK), as well as increased colonic reabsorption via HKA2. [ABSTRACT FROM AUTHOR]

Acid-secreting intercalated cells respond to changes in systemic pH through regulation of apical H+ transporters. Little is known about the mechanism by which these cells sense changes in extracellular pH (pHo). Pyk2 is a nonreceptor tyrosine kinase activated by autophosphorylation at Tyr402 by cellspecific stimuli, including decreased pH, and is involved in the regulation of MAPK signaling pathways and transporter activity. We examined whether the Pyk2 and MAPK signaling pathway mediates the response of transport proteins to decreased pH in outer medullary collecting duct cells. Immunoblot analysis of phosphorylated Pyk2 (Tyr402), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) was used to assay protein activation. To examine specificity of kinase activation and its effects, we used Pyk2 small interfering RNA to knockdown Pyk2 expression levels, the Src kinase inhibitor 4-amino-5- (4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP 1) to inhibit Pyk2 phosphorylation, and the MEK inhibitor U0126 to inhibit ERK1/2 phosphorylation. The pH-sensitive fluorescent probe 2=-7=-bis(carboxyethyl)- 5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) was used to assay H+ transporter activity. The activity of H+ transporters was measured as the rate of intracellular pH (pHi) recovery after an NH4Cl prepulse. We show that Pyk2 is endogenously expressed and activated by acid pH in mouse-derived outer medullary collecting duct (mOMCD1) cells. Incubation of mOMCD1 cells in acid media [extracellular pH (pHo) 6.7] increased the phosphorylation of Pyk2, ERK1/2, and p38. Reduction in pHi induced by an NH4Cl prepulse also increased the phosphorylation of Pyk2, ERK1/2, and p38. Consistent with our previous studies, we found that mOMCD1 cells exhibit H+-ATPase and H+,K+-ATPase activity. Pyk2 inhibition by Pyk2 siRNA and PP 1 prevented Pyk2 phosphorylation as well as H+-ATPase-mediated recovery in mOMCD1 cells. In addition, ERK1/2 inhibition by U0126 prevented acid-induced ERK1/2 phosphorylation and H+-ATPase-mediated pHi recovery but not phosphorylation of p38. We conclude that Pyk2 and ERK1/2 are required for increasing H+-ATPase, but not H+,K+-ATPase, activity at decreased pHi in mOMCD1 cells. [ABSTRACT FROM AUTHOR]

A-kinase anchoring proteins (AKAPs) have emerged as important regulatory molecules that can compartmentalize cAMP signaling transduced by β2-adrenergic receptors (β2ARs); such compartmentalization ensures speed and fidelity of cAMP signaling and effects on cell function. This study ainaed to assess the role of AKAPs in regulating global and compartmentalized β2AR signaling in human airway smooth muscle (ASM). Transcriptome and proteomic analyses were used to characterize AKAP expression in ASM. Stable expression or injection of peptides AKAP-IS or Ht31 was used to disrupt AKAP-PKA interactions, and global and compartmentalized cAMP accumulation stimulated by β-agonist was assessed by radioimmunoassay and membrane-delineated flow through cyclic nucleotide-gated channels, respectively. ASM expresses multiple AKAP family members, with gravin and ezrin among the most readily detected. AKAP-PKA disruption had minimal effects on whole-cell cAMP accumulation stimulated by β-agonist (EC50 and Bmax) concentrations, but significantly increased the duration of plasma membrane-delineated cAMP (τ=251±51 s for scrambled peptide control vs. 399±79 s for Ht31). Direct PKA inhibition eliminated decay of membrane-delineated cAMP levels. AKAPs coordinate compartmentalized cAMP signaling in ASM cells by regulating multiple elements of β2AR-mediated cAMP accumulation, thereby representing a novel target for manipulating β2AR signaling and function in ASM. [ABSTRACT FROM AUTHOR]

The H+-K+-ATPase α-subunit (HKα2) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα2 plasma membrane expression is regulated by PKA (Codina J, Liu J, Bleyer AJ, Penn RB, DuBose TD Jr. J Am Soc Nephrol 17: 1833-1840, 2006) and in a separate study demonstrated that genetic ablation of the proton-sensing Gs-coupled receptor GPR4 results in spontaneous metabolic acidosis (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). In the present study, we investigated the ability of chronic acidosis and GPR4 to regulate HKα2 expression in HEK-293 cells. Chronic acidosis was modeled in vitro by using multiple methods: reducing media pH by adjusting bicarbonate concentration, adding HCl, or by increasing the ambient concentration of CO2. PKA activity and HKα2 protein were monitored by immunoblot analysis, and HKα2 mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα2 mRNA; however, PKA activity and HKα2 protein abundance increased when media pH decreased from 7.4 to 6.8. Furthermore, this increase was independent of the method used to create chronic acidosis. Heterologous expression of GPR4 was sufficient to increase both basal and acid-stimulated PKA activity and similarly increase basal and acid-stimulated HKα2 expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα2 protein by increasing PKA activity without altering HKα2 mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα2 and acid-base balance. [ABSTRACT FROM AUTHOR]

The effect of β-subunit assembly on function and localization of the colonic H+,K+-ATPase α-subunit. Background. Previous experiments from our laboratory have demonstrated that HKα2 coimmunoprecipitated with β1-Na+,K+-ATPase. Although HKα2 is expressed abundantly in the apical membrane of distal colon, the demonstration that β1 localizes to this same membrane in distal colon has not been demonstrated previously. Methods. Immunolocalization was performed in distal colon using a polyclonal antibody against HKα2 and a monoclonal antibody against β1. Results. The results demonstrate that HKα2 localizes to the apical membrane. Two pools of β1-Na+,K+-ATPase were detected. The first localized to the apical membrane. The second pool was detected in the basolateral membrane when distal colon sections were deglycosylated with glycosidase F. Therefore, our results demonstrate that β1 localizes to the apical membrane with HKα2, and supports the view that β1 is the physiologic β-subunit for HKα2. We tested, therefore, the efficiency of the two β-subunits expressed in distal colon (β1 and β3) to support the activity of HKα2. Human embryonic kidney HEK-293 cells were transiently cotransfected with HKα2 plus β1 or HKα2 plus β3. Subsequently, 86Rb+-uptake and plasma membrane localization were evaluated. The results demonstrate that both HKα2/β1 and HKα2/β3 support 86Rb+-uptake. However, 86Rb+-uptake measured in the cells cotransfected with HKα2 plus β1 exceeded that measured in cells expressing HKα2/β3. Fluorescence microscopy using enhanced green fluorescent protein cloned at the amino-terminus of HKα2 demonstrated protein migration to the plasma membrane in cells cotransfected with EGFP-HKα2 plus β1. In contrast, in cells cotransfected with EGFP-HKα2 plus β3, the vast majority of the protein remained confined to intracellular compartments. The significantly higher 86Rb+-uptake corresponded to additional localization of HKα2 to the plasma membrane when coexpressed with β1 compared to β3. Conclusion. Taken together, these and previous results from our laboratory indicate that β1-Na+,K+-ATPase is likely to represent the most physiologic and efficient subunit for HKα2 assembly in distal colon. [ABSTRACT FROM AUTHOR]