Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways.

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2-/-) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2-/- mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

Randomized, Controlled Trial of TRC101 to Increase Serum Bicarbonate in Patients with CKD.

BACKGROUND AND OBJECTIVES: Metabolic acidosis is common in patients with CKD and has significant adverse effects on kidney, muscle, and bone. We tested the efficacy and safety of TRC101, a novel, sodium-free, nonabsorbed hydrochloric acid binder, to increase serum bicarbonate in patients with CKD and metabolic acidosis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: One hundred thirty-five patients were enrolled in this randomized, double-blind, placebo-controlled, multicenter, in-unit study (designated the TRCA-101 Study). Patients had a mean baseline eGFR of 35 ml/min per 1.73 m2, a mean baseline serum bicarbonate of 17.7 mEq/L, and comorbidities, including hypertension (93%), diabetes (70%), and heart failure (21%). Patients ate a controlled diet and were treated for 14 days with placebo or one of four TRC101 dosing regimens (1.5, 3, or 4.5 g twice daily or 6 g once daily). After treatment, patients were discharged and followed for 7-14 days. RESULTS: All TRC101 treatment groups had a mean within-group increase in serum bicarbonate of ≥1.3 mEq/L (P<0.001) within 72 hours of the first dose and a mean increase in serum bicarbonate of 3.2-3.9 mEq/L (P<0.001) at the end of treatment compared with placebo, in which serum bicarbonate did not change. In the combined TRC101 treatment group, serum bicarbonate was normalized (22-29 mEq/L) at the end of treatment in 35% of patients and increased by ≥4 mEq/L in 39% of patients. After discontinuation of TRC101, serum bicarbonate decreased nearly to baseline levels within 2 weeks. All adverse events were mild or moderate, with gastrointestinal events most common. All patients completed the study. CONCLUSIONS: TRC101 safely and significantly increased the level of serum bicarbonate in patients with metabolic acidosis and CKD.

Research in academic medical centers: two threats to sustainable support.

Reductions in federal support and clinical revenue jeopardize biomedical research and, in turn, clinical medicine.

Treatment of severe metabolic alkalosis in a patient with congestive heart failure.

Metabolic alkalosis, isolated or in combination with another abnormality, is the most common acid-base disorder in patients with congestive heart failure. In most cases, it is a result of diuretic therapy, which causes activation of the renin-angiotensin system, chloride depletion, increased distal sodium delivery, hypokalemia, and increased urine acidification, all of which contribute to bicarbonate retention. In addition, the disease state itself results in neurohormonal activation (renin-angiotensin system, sympathetic nervous system, and endothelin) that further amplifies the tendency toward alkalosis. Treatment of metabolic alkalosis is based on the elimination of generation and maintenance factors, chloride and potassium repletion, enhancement of renal bicarbonate excretion (such as acetazolamide), direct titration of the base excess (hydrochloric acid), or, if accompanied by kidney failure, low-bicarbonate dialysis. In congestive heart failure, appropriate management of circulatory failure and use of an aldosterone antagonist in the diuretic regimen are integral to treatment.

Defining hypercalciuria in nephrolithiasis.

The classic definition of hypercalciuria, an upper normal limit of 200 mg/day, is based on a constant diet restricted in calcium, sodium, and animal protein; however, random diet data challenge this. Here our retrospective study determined the validity of the classic definition of hypercalciuria by comparing data from 39 publications analyzing urinary calcium excretion on a constant restricted diet and testing whether hypercalciuria could be defined when extraneous dietary influences were controlled. These papers encompassed 300 non-stone-forming patients, 208 patients with absorptive hypercalciuria type I (presumed due to high intestinal calcium absorption), and 234 stone formers without absorptive hypercalciuria; all evaluated on a constant restricted diet. In non-stone formers, the mean urinary calcium was well below 200 mg/day, and the mean for all patients was 127±46 mg/day with an upper limit of 219 mg/day. In absorptive hypercalciuria type I, the mean urinary calcium significantly exceeded 200 mg/day in all studies with a combined mean of 259±55 mg/day. Receiver operating characteristic curve analysis showed the optimal cutoff point for urinary calcium excretion was 172 mg/day on a restricted diet, a value that approximates the traditional limit of 200 mg/day. Thus, on a restricted diet, a clear demarcation was seen between urinary calcium excretion of kidney stone formers with absorptive hypercalciuria type I and normal individuals. When dietary variables are controlled, the classic definition of hypercalciuria of nephrolithiasis appears valid.

Competencies in premedical and medical education: the AAMC-HHMI report.

One hundred years ago, Flexner emphasized the importance of science in medicine and medical education. Over the subsequent years, science education in the premedical and medical curricula has changed little, in spite of the vast changes in the biomedical sciences. The National Research Council, in their report Bio 2010, noted that the premedical curriculum caused many students to lose interest in medicine and in the biological sciences in general. Many medical students and physicians have come to view the premedical curriculum as of limited relevance to medicine and designed more as a screening mechanism for medical school admission. To address this, the Association of American Medical Colleges and the Howard Hughes Medical Institute formed a committee to evaluate the premedical and medical school science curricula. The committee made a number of recommendations that are summarized in this essay. Most important were that competencies replace course requirements and that the physical sciences and mathematics be better integrated with the biological sciences and medicine. The goal is that all physicians possess a strong scientific knowledge base and come to appreciate the importance of this to the practice of medicine. While science education needs to evolve, Flexner's vision is as relevant today as it was 100 years ago.

Acid regulation of NaDC-1 requires a functional endothelin B receptor.

Metabolically generated acid is the major physiological stimulus for increasing proximal tubule citrate reabsorption, which leads to a decrease in citrate excretion. The activity of the Na-citrate cotransporter, NaDC-1, is increased in vivo by acid ingestion and in vitro by an acidic pH medium. In opossum kidney cells the acid stimulatory effect and the ability of endothelin-1 (ET-1) to stimulate NaDC-1 activity are both blocked by the endothelin B (ET(B)) receptor antagonist, BQ788. Acid feeding had no effect on brush border membrane NaDC-1 activity in mice in which ET(B) receptor expression was knocked out, whereas a stimulatory effect was found in wild-type mice. Using ET(A)/ET(B) chimeric and ET(B) C-terminal tail truncated constructs, ET-1 stimulation of NaDC-1 required a receptor C-terminal tail from either ET(A) or ET(B). The ET-1 effect was greatest when either the ET(B) transmembrane domain and C-terminal tail were present or the ET(B) C-terminal tail was linked to the ET(A) transmembrane domain. This effect was smaller when the ET(B) transmembrane domain was linked to the ET(A) C-terminal tail. Thus, the acid-activated pathway mediating stimulation of NaDC-1 activity requires a functional ET(B) receptor in vivo and in vitro, as does acid stimulation of NHE3 activity. Since increased NaDC-1 and NHE3 activities constitute part of the proximal tubule adaptation to an acid load, these studies indicate that there are similarities in the signaling pathway mediating these responses.

RhoA required for acid-induced stress fiber formation and trafficking and activation of NHE3.

Exposure to an acid load increases apical membrane Na(+)/H(+) antiporter (NHE3) activity, a process that involves exocytic trafficking of the transporter to the apical membrane. We have previously shown that an intact microfilament structure is required for this exocytic process (Yang X, Amemiya M, Peng Y, Moe OW, Preisig PA, Alpern RJ. Am J Physiol Cell Physiol 279: C410-C419, 2000). The present studies demonstrate that acid-induced stress fiber formation is required for stimulation of NHE3 activity. Formation of stress fibers is associated with acid-induced tyrosine phosphorylation and increases in protein abundance of two focal adhesion proteins, p125(FAK) and paxillin. The Rho kinase inhibitor Y27632 completely blocks acid-induced stress fiber formation and the increases in apical membrane NHE3 abundance and activity, but it has no effect on acid-induced tyrosine phosphorylation of p125(FAK) or paxillin. Herbimycin A completely blocks acid-induced tyrosine phosphorylation of p125(FAK) and paxillin but only partially blocks stress fiber formation and NHE3 activation. These studies demonstrate that Rho kinase mediates acid-induced stress fiber formation, which is required for NHE3 exocytosis, and increases in NHE3 activity. Acid-induced tyrosine phosphorylation of the focal adhesion proteins p125(FAK) and paxillin is not Rho kinase dependent. Thus these two acid-mediated effects are associated, yet independent processes.

A consensus sequence in the endothelin-B receptor second intracellular loop is required for NHE3 activation by endothelin-1.

Endothelin-1 (ET-1) increases the activity of Na(+)/H(+) exchanger 3 (NHE3), the major proximal tubule apical membrane Na(+)/H(+) antiporter. This effect is seen in opossum kidney (OKP) cells expressing the endothelin-B (ET(B)) and not in cells expressing the endothelin-A (ET(A)) receptor. However, ET-1 causes similar patterns of protein tyrosine phosphorylation, adenylyl cyclase inhibition, and increases in cell [Ca(2+)] in ET(A)- and ET(B)-expressing OKP cells, implying that an additional mechanism is required for NHE3 stimulation by the ET(B) receptor. The present studies used ET(A) and ET(B) receptor chimeras and site-directed mutagenesis to identify the ET receptor domains that mediate ET-1 regulation of NHE3 activity. We found that binding of ET-1 to the ET(A) receptor inhibits NHE3 activity, an effect for which the COOH-terminal tail is necessary and sufficient. ET-1 stimulation of NHE3 activity requires the COOH-terminal tail and the second intracellular loop of the ET(B) receptor. Within the second intracellular loop, a consensus sequence was identified, KXXXVPKXXXV, that is required for ET-1 stimulation of NHE3 activity. This sequence suggests binding of a homodimeric protein that mediates NHE3 stimulation.

Pyk2 activation is integral to acid stimulation of sodium/hydrogen exchanger 3.

The present study examines the role of Pyk2 in acid regulation of sodium/hydrogen exchanger 3 (NHE3) activity in OKP cells, a kidney proximal tubule epithelial cell line. Incubation of OKP cells in acid media caused a transient increase in Pyk2 phosphorylation that peaked at 30 seconds and increased Pyk2/c-Src binding at 90 seconds. Pyk2 isolated by immunoprecipitation and studied in a cell-free system was activated and phosphorylated at acidic pH. Acid activation of Pyk2 (a) was specific for Pyk2 in that acid did not activate focal adhesion kinase, (b) required calcium, and (c) was associated with increased affinity for ATP. Transfection of OKP cells with dominant-negative pyk2(K457A) or small interfering pyk2 duplex RNA blocked acid activation of NHE3, while neither had an effect on glucocorticoid activation of NHE3. In addition, pyk2(K457A) blocked acid activation of c-Src kinase, which is also required for acid regulation of NHE3. The present results demonstrate that Pyk2 is directly activated by acidic pH and that Pyk2 activation is required for acid activation of c-Src kinase and NHE3. Given that partially purified Pyk2 can be activated by acid in a cell-free system, Pyk2 may serve as the pH sensor that initiates the acid-regulated signaling cascade involved in NHE3 regulation.

An autocrine role for endothelin-1 in the regulation of proximal tubule NHE3.

BACKGROUND: Chronic metabolic acidosis leads to an increase in NHE3 activity that is mediated by endothelin-1 (ET-1) expression and activation of the proximal tubule endothelin B receptor. Chronic metabolic acidosis increases preproET-1 mRNA abundance in kidney cortex, but the cell responsible has not been identified. METHODS: PreproET-1 mRNA abundance was quantified by competitive reverse transcription-polymerase chain reaction (RT-PCR) on tissue harvested from control rats or rats in which chronic metabolic acidosis was induced by addition of NH(4)Cl to the drinking water. RESULTS: Chronic metabolic acidosis leads to an increase in preproET-1 mRNA expression in kidney cortex, proximal tubules, and glomeruli. The increase in preproET-1 expression correlates with the decrease in blood [HCO3(-)]. ET-1 expression is also increased by acidosis in abdominal aorta, but not in cardiac muscle. CONCLUSION: In the renal proximal tubule, chronic metabolic acidosis induces an increase in preproET-1 expression, providing a mechanism for autocrine regulation of proximal tubule NHE3 activity. This response is not unique to the proximal tubule cell, but is also not ubiquitous.

OKP cells express the Na-dicarboxylate cotransporter NaDC-1.

Urinary citrate concentration, a major factor in the formation of kidney stones, is primarily determined by its rate of reabsorption in the proximal tubule. Citrate reabsorption is mediated by the Na-dicarboxylate cotransporter-1 (NaDC-1). The opossum kidney (OKP) cell line possesses many characteristics of the renal proximal tubule. The OKP NaDC-1 (oNaDC-1) cDNA was cloned and encodes a 2.4-kb mRNA. When injected into Xenopus oocytes, the cotransporter is expressed and demonstrates Na-coupled citrate transport with a stoichiometry of >or=3 Na:1 citrate, specificity for di- and tricarboxylates, pH-dependent citrate transport, and pH-independent succinate transport, all characteristics of the other NaDC-1 orthologs. Chronic metabolic acidosis increases proximal tubule citrate reabsorption, leading to profound hypocitraturia and an increased risk for stone formation. Under the conditions studied, endogenous OKP NaDC-1 mRNA abundance is not regulated by changes in media pH. In OKP cells transfected with a green fluorescent protein-oNaDC-1 construct, however, media acidification increases Na-dependent citrate uptake, demonstrating posttranscriptional acid regulation of NaDC-1 activity.

Dietary acid, endothelins, and sleep.

Acid addition to the body activates a series of homeostatic responses, one example of which is activation of NHE3, the proximal tubule Na(+)/H(+) antiporter. Feeding acid to rats increases apical membrane NHE3 abundance. Similarly, addition of acid to the media of OKP cells, a proximal tubule cell line, leads to an increase in apical membrane NHE3 activity that is due to increased trafficking of NHE3 to the apical membrane, and increased NHE3 mRNA and protein expression. Endothelins also increase NHE3 activity by inducing trafficking of NHE3 to the apical membrane, an effect mediated by the ET(B), but not the ET(A) receptor. Receptor specificity resides in the C-terminal loop and the second intracellular loop of the ET(B) receptor. In addition, the ET(B) receptor is required for acid signaling. An acid-induced signaling cascade has been defined that includes Pyk2, c-Src, ERK, c-fos, c-jun, and endothelin expression.

Bicarbonate reabsorption and NHE-3 expression: abundance and activity are increased in Henle's loop of remnant rats.

BACKGROUND: The bulk of bicarbonate reabsorption along the loop of Henle (LOH) is localized at the level of the thick ascending limb (TAL) and is mainly dependent on the presence of luminal Na+-H+ exchanger (NHE-3). We investigated whether the reduction of renal mass is associated with alterations in LOH bicarbonate transport coupled to changes in NHE-3 gene expression and in vivo activity. METHODS: Sham-operated and remnant rats (4/6 nephrectomy) were studied 15 days after the surgery. To measure net bicarbonate reabsorption (JHCO3-) superficial loops were perfused by in vivo micropuncture. Perfusate was an end-like proximal solution containing 3H-methoxy-inulin. NHE-3 gene expression was quantified by competitive PCR using an internal standard of cDNA that differed from the wild-type NHE-3 by a deletion of 76 bp. Western blot experiments were performed on TAL suspension using anti-NHE-3 antibodies. RESULTS: At various LOH bicarbonate loads, JHCO3- was constantly larger in remnant rats as compared to sham-operated animals. NHE-3 mRNA abundance was estimated to be 0.339 +/- 0.031 attomoles (amol)/ng-1 total RNA in sham-operated (N = 5) and it increased to 0.465 +/- 0.023 in remnant rats (N = 5, P < 0.01). Western blot experiments showed a significant increase of NHE-3 protein abundance in TAL of remnant rats as compared to sham-operated animals. Finally, by means of a specific NHE-3 inhibitor, S-3226, in vivo microperfusion experiments demonstrated that NHE-3 in vivo activity along the LOH was substantially increased in remnant rats in addition to the non-NHE-3 bicarbonate transport. CONCLUSIONS: These data indicate that the reduction of renal mass increases mRNA, protein abundance and in vivo activity of NHE-3 along the TAL. This may explain, at least in part, the augmented transepithelial bicarbonate transport along the LOH. Such an effect will counterbalance the increased glomerular bicarbonate load, thus preventing urinary bicarbonate loss and mitigating the ensuing metabolic acidosis.