Renal and colonic potassium transporters in the pregnant rat.

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.

Adaptation by the collecting duct to an exogenous acid load is blunted by deletion of the proton-sensing receptor GPR4.

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 Ca(2+) 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.

A-kinase anchoring proteins regulate compartmentalized cAMP signaling in airway smooth muscle.

A-kinase anchoring proteins (AKAPs) have emerged as important regulatory molecules that can compartmentalize cAMP signaling transduced by ß2-adrenergic receptors (ß(2)ARs); such compartmentalization ensures speed and fidelity of cAMP signaling and effects on cell function. This study aimed to assess the role of AKAPs in regulating global and compartmentalized ß(2)AR 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 ß-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 ß-agonist (EC(50) and B(max)) 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 ß(2)AR-mediated cAMP accumulation, thereby representing a novel target for manipulating ß(2)AR signaling and function in ASM.

Pyk2 regulates H+-ATPase-mediated proton secretion in the outer medullary collecting duct via an ERK1/2 signaling pathway.

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 (pH(o)). Pyk2 is a nonreceptor tyrosine kinase activated by autophosphorylation at Tyr402 by cell-specific 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 (pH(i)) recovery after an NH(4)Cl 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 (pH(o)) 6.7] increased the phosphorylation of Pyk2, ERK1/2, and p38. Reduction in pH(i) induced by an NH(4)Cl 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 pH(i) 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 pH(i) in mOMCD1 cells.

pH-dependent regulation of the α-subunit of H+-K+-ATPase (HKα2).

The H(+)-K(+)-ATPase α-subunit (HKα(2)) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα(2) 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 G(s)-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 HKα(2) 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 CO(2). PKA activity and HKα(2) protein were monitored by immunoblot analysis, and HKα(2) mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα(2) mRNA; however, PKA activity and HKα(2) 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 HKα(2) expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα(2) protein by increasing PKA activity without altering HKα(2) mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα(2) and acid-base balance.

Regulation of Potassium Homeostasis in CKD.

Disturbances of potassium homeostasis can cause either hyperkalemia or hypokalemia and result in serious consequences. Although the consequences of acute and chronic hyperkalemia and treatment of these conditions in CKD have been widely appreciated by nephrologists, more recent information has focused attention on the consequences of chronic hypokalemia. Several recent studies have documented a "U-shaped" relationship between the serum [K+] and higher mortality in several clinical studies. The causes of dyskalemias are placed into the unique perspective of patients with CKD and its evolution with progression of CKD to later stages and focuses on the pathophysiology of these disorders. Emphasis is placed on the high mortality associated with both low and high levels of potassium that are unique to patients with CKD. Recent information regarding sensors of changes in the serum [K+] that evoke changes in NaCl transport in the DCT1 and subsequent efferent responses by aldosterone-responsive cells in the DCT2 and cortical collecting duct to adjust K+ secretion by the renal outer medullary potassium channel is reviewed in detail. These sensing mechanisms can be interrupted by drugs, such as the calcineurin inhibitors to cause both hypertension and hyperkalemia in kidney transplant patients, or can be inherited as familial hypertensive hyperkalemia. The role and pathogenesis of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in causing hyperkalemia is a common stop point for cessation of these important drugs, but, and newer agents to lower the serum [K+] that might allow continuation of angiotensin-converting enzyme or angiotensin receptor blocker therapy are examined. Finally, the importance of emphasis on potassium-containing foods, such as fresh produce and fruit in the diets of patients with early-stage CKD, is examined as an under-appreciated area requiring more emphasis by nephrologists caring for these patients and may be unique to food-challenged patients with CKD.

Molecular regulation and physiology of the H+,K+ -ATPases in kidney.

Two H(+), K(+)-adenosine triphosphatase (ATPase) proteins participate in K(+) absorption and H(+) secretion in the renal medulla. Both the gastric (HKalpha(1)) and colonic (HKalpha(2)) H(+),K(+)-ATPases have been localized and characterized by a number of techniques, and are known to be highly regulated in response to acid-base and electrolyte disturbances. Both ATPases are dimers of composition alpha/beta that localize to the apical membrane and both interact with the tetraspanin protein CD63. Although CD63 interacts with the carboxy-terminus of the alpha-subunit of the colonic H(+),K(+)-ATPase, it interacts with the beta-subunit of the gastric H(+),K(+)-ATPase. Pharmacologically, both ATPases are distinct; for example, the gastric H(+),K(+)-ATPase is inhibited by Sch-28080, but the colonic H(+),K(+)-ATPase is inhibited by ouabain (a classic inhibitor of the Na(+)-pump) and is completely insensitive to Sch-28080. The alpha-subunit of the colonic H(+),K(+)-ATPase is the only subunit of the X(+),K(+)-ATPase superfamily that has 3 different splice variants that emerge by deletion or elongation of the amino-terminus. The messenger RNA and protein of one of these splice variants (HKalpha(2C)) is specifically up-regulated in newborn rats and becomes undetectable in adult rats. Therefore, HKalpha(2), in addition to its role in potassium and acid-base homeostasis, appears to play a significant role in early growth and development. Finally, because chronic hypokalemia appears to be the most potent stimulus for upregulation of HKalpha(2), we propose that the HKalpha(2) participates importantly in the maintenance of chronic metabolic alkalosis.

The role that graduate medical education must play in ensuring health equity and eliminating health care disparities.

Despite the 2002 Institute of Medicine report that described the moral and financial impact of health care disparities and the need to address them, it is evident that health care disparities persist. Recommendations for addressing disparities include collecting and reporting data on patient race and ethnicity, supporting language interpretation services, increasing awareness of health care disparities through education, requiring cultural competency training for all health care professionals, and increasing diversity among those delivering health care. The Accreditation Council on Graduate Medical Education places strong emphasis on graduate medical education's role in eliminating health care disparities by asking medical educators to objectively evaluate and report on their trainees' ability to practice patient-centered, culturally competent care. Moreover, one of the objectives of the Accreditation Council on Graduate Medical Education Clinical Learning Environment Review visits as part of the Next Accreditation System is to identify how sponsoring institutions engage residents and fellows in the use of data to improve systems of care, reduce health care disparities, and improve patient outcomes. Residency and fellowship programs should ensure the delivery of meaningful curricula on cultural competency and health care disparities, for which there are numerous resources, and ensure resident assessment of culturally competent care. Moreover, training programs and institutional leadership need to collaborate on ensuring data collection on patient satisfaction, outcomes, and quality measures that are broken down by patient race, cultural identification, and language. A diverse physician workforce is another strategy for mitigating health care disparities, and using strategies to enhance faculty diversity should also be a priority of graduate medical education. Transparent data about institutional diversity efforts should be provided to interested medical students, residents, and faculty. Graduate medical education has a clear charge to ensure a generation of physicians who are firmly grounded in the principles of practicing culturally competent care and committed to the reduction of health care disparities.

Incessant ventricular tachycardia and cardiogenic shock: a common presentation of an uncommon diagnosis.

Giant cell myocarditis is a rare and highly lethal disease that is characterized by a rapidly progressive course of biventricular dysfunction. The authors present a case of giant cell myocarditis that presented with incessant ventricular tachycardia and cardiogenic shock in which clinical improvement was achieved with immunosuppressive therapy.

Cardiovascular disease in chronic kidney disease.

The presence of kidney disease, manifested by low glomerular filtration rates (GFR) and/or large amounts of protein in the urine, is independently associated with increased rates of cardiovascular disease (CVD). The severity of kidney disease is associated with graded increases in risk for CVD and death. Chronic kidney disease (CKD) should be recognized and treatment initiated early to maximize the chances for slowing nephropathy progression and reducing proteinuria. We recommend screening for CKD in all patients with CVD, including computing an estimated GFR and evaluating for proteinuria using a spot urine albumin:creatinine ratio. Aggressive management of traditional cardiovascular risk factors should be employed in this high-risk population, specifically rigorous hypertension control (including the use of angiotensin-converting enzyme inhibitors (ACEI)/angiotensin receptor blocking agents (ARB)), management of hyperglycemia, hyperlipidemia and smoking cessation. Further studies are needed to identify the unique renal failure-related (non-traditional) risk factors that contribute to accelerated atherosclerosis in this population and performance of randomized trials to assess the effects of cardiovascular interventions in individuals with CKD.

Phosphorylation of S955 at the protein kinase A consensus promotes maturation of the alpha subunit of the colonic H+,K+ -ATPase.

All the alpha subunits of the Na+,K+ -ATPases and H+,K+ -ATPases have a protein kinase A (PKA) consensus sequence near or in the ninth transmembrane domain. The role of this domain in influencing alpha subunit synthesis/degradation, plasma membrane localization, and 86Rb+ uptake has not been established for the alpha subunit of the colonic H+,K+ -ATPase. This study examined the effect of mutating S955 (within the PKA consensus site of the alpha subunit of the colonic H+,K+ -ATPase [HKalpha2]) to alanine (S955/A) or aspartic acid (S955/D) on alpha subunit expression and function. The results demonstrate that a negatively charged amino acid at position 955 of HKalpha2 promotes higher expression levels of both whole-cell and plasma membrane-localized HKalpha2. Moreover, inhibition of PKA reduced expression of wild-type HKalpha2 and associated 86Rb+ uptake. Last, the activity of the HKalpha2 S955/A was rescued by treatment with 4-phenylbutyric acid, a compound that was shown previously to restore function to the cystic fibrosis transmembrane conductance regulator.