Molecular insights into the role of basolateral Kir channels in controlling acid-base balance in salt-sensitive hypertension

Acid-base homeostasis is critical for normal physiological function. Chronic metabolic acidosis is an independent risk factor for the progression of chronic kidney diseases (CKD) and can contribute to increased morbidity and mortality in patients with CKD. Patients with KCNJ16 (the gene encodes the inwardly rectifying potassium channel Kir5.1) mutation display metabolic acidosis; however, the underlying mechanism is still not fully understood. The overarching goal of this study is to unmask the interaction between Kir5.1 and Kir4.2 channels and how such interaction regulates acid-base balance in the kidney. Mentored K99 Phase: Our preliminary data revealed that loss of Kir5.1 impairs renal ammonia metabolism in the proximal tubule. I hypothesize that loss of Kir5.1 depolarizes the proximal tubule cell membrane and inhibits the exit of HCO3- through NBCe1, resulting in increased intracellular pH (pHi) and thus, impairs the proximal ammonia metabolism. Specific Aim 1: Determine the role of Kir5.1 in regulating proximal tubule membrane potential and pHi .To test this hypothesis, I will use: 1) FluoVolt membrane potential kit and 2) pH-sensitive BCECF dye to measure the membrane potential and pHi of isolated proximal tubules from SSWT rats under the stimulation (VU206, agonist) and inhibition (VU720/VU992, antagonists) of Kir5.1 and compare the membrane potential and pHi of isolated proximal tubules from SSWT and SSKcnj16-/- rats. Independent R00 Phase: This phase of the project will develop an independent line of investigation into the physical and functional interactions between Kir5.1 and Kir4.2. Insights gained from these experiments will further explain the physiological mechanisms underlying inwardly rectifying potassium channels' regulation of acid-base balance. Previous studies suggest potential interactions between Kir5.1 and Kir4.2. However, the functional interactions between Kir5.1/4.2 in the kidneys still remain largely unknown. I hypothesize that Kir5.1 physically and functionally interacts with Kir4.2, and the Kir5.1/4.2 complexes determine the membrane potential of the proximal cell. Specific Aim 2: Investigate the physical and functional interactions of Kir5.1 and Kir4.2 in the proximal tubule. To test this hypothesis, I will: 1) use immunofluorescence and co-immunoprecipitation on isolated proximal tubules from SSWT rats to study the physical interaction between Kir5.1 and Kir4.2; 2) generate the Kir4.2 KO rat based on Dahl SS rat background (SSKcnj15-/-) to evaluate the effect of Kir4.2 on acid-base status, kidney function and blood pressure in SS hypertension; 3) use Western blot, immunofluorescence and patch clamp to study the expression and channel activity of Kir4.2 in SSKcnj16-/- rats and vice versa. The results of this proposal will unmask the molecular mechanisms of acid-base regulation by Kir5.1 and the physical and functional interactions of Kir5.1 and Kir4.2, which will not only help define the role of inwardly rectifying potassium channels in the proximal tubule but also contribute to developing new therapeutic strategies for metabolic acidosis.