The overarching theme of my research is the epithelial ion transport in human organs with a goal to unveil novel physiological regulation, disease pathogenesis, and potential treatments. Specifically, I focus on ClC-K chloride channels, Kir potassium channels, and with-no-lysine (WNK) kinase signaling cascade, which are associated with human Bartter syndrome, thyrotoxic periodic paralysis, aldosterone-producing adenoma, and pseudohypoaldosteronism type II, respectively. My laboratory uses disease mouse models and applied technique-demanding microdissection of renal tubules for ex vivo microperfusion, transcriptome analysis, and metabolomics; patch clamp experiments for functional validation of mutated ion channels; state-of-the-art 3D imaging of optically cleared kidneys for renal tubule development and remodeling. Lately, we have developed a new functional assay to study mitochondrial respiration and energy metabolism in isolated renal tubules, which allows us to explore mitochondrial metabolism in the specific renal segment. In conjunction with molecular biological, biochemical, and physiological approaches, these powerful tools grant us the privilege of investigating the pathogenesis of renal tubulopathies and ion channelopathies.
Current Projects
Classic Bartter syndrome is a congenital renal tubulopathy caused by ClC-Kb mutation, resulting in excessive urinary salt excretion, hypokalemia, and metabolic alkalosis. We identified novel missense ClC-Kb mutations and functionally characterized them using patch-clamp experiments. Our results demonstrate a genotype-phenotype association in classic Bartter syndrome. We further created Clc-k2 (mouse ortholog of ClC-Kb) knockout mice to explore early pathogenesis. Two-week-old Clc-k2 knockout mice had hypoplasic renal medulla compared to wild-type controls. To quantitatively measure the length of the thick ascending limb (TALs), we optically cleared one-week-old mouse kidneys and stained TALs using anti-NKCC2 antibody. Advanced 3-D imaging using light-sheet fluorescent microscopy demonstrated that TALs in Clc-k2 knockout mice were shorter than wild-type controls, contributing to medulla hypoplasia, water diuresis, and excessive urinary salt-wasting, recapitulating a severe form of Bartter syndrome. Inducible deletion of Clc-k2 beginning after medulla maturation produces only mild salt wasting, mimicking a milder variant of Bartter syndrome called Gitelman syndrome. Therefore, we proposed an updated view of the pathogenesis of classic Bartter syndrome, suggesting that developmental defects in the renal medulla directly contribute to the phenotype of BS.
The kidney contains at least 16 types of renal epithelial cells featuring highly variable transport activities and metabolic features. Traditional assays using kidney slices or cultured cells do not provide spatial resolution for TALs or DCTs. Hence, we invented a new assay using freshly isolated renal tubules for extracellular flux analysis (EFA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to reflect mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, respectively. We proved that EFA detected small metabolic changes in millimeter-length renal tubules with a strong linear correlation between OCR/ECAR readouts and sample sizes. Compared to cultured cells, isolated renal tubules have a much higher OCR to ECAR ratio and mitochondrial-dependent ATP production rate, suggesting robust oxidative phosphorylation (OXPHOS) in renal tubules. Besides, the new EFA assay can study substrate oxidation in renal tubules. There has been a long debate regarding the relative importance of energy substrates to specific renal segments. Previous studies using kidney slices or homogenates elude zonal metabolism in the renal cortex or medulla. We revisited this issue by using isolated tubules for EFA combined with the sequential injections of energy pathway inhibitors. The results suggest that TALs and DCTs primarily utilize glucose/pyruvate for OXPHOS in the basal state and shift to glycolysis when mitochondrial respiration is shut off. In contrast, PTs primarily use fatty acids for OXPHOS and have minimal compensatory glycolysis. This new EFA assay is a reliable method to study mitochondrial respiration and energy metabolism in renal tubules.
We have proven intracellular chloride is an endogenous inhibitor of with-no-lysine kinase 4 and regulate sodium chloride cotransporter in the distal convoluted tubule. Several chloride channels/cotransporters regulate intracellular chloride concentration. Genetic deletion of ClC-Kb impaired but did not abolish salt reabsorption in the thick ascending limb and distal convoluted tubules, suggesting other chloride channels or cotransporters may partially compensate for transepithelial chloride flux. Potassium chloride cotransporter 1 and 4 (KCC1 & KCC4) are highly expressed in the distal nephron. However, the physiological function of KCC1 and KCC4 is still unknown. Whether KCC1 and KCC4 functionally compensate for ClC-Kb remains unknown. We have created KCC1 and KCC4 single-knockout and double-knockout mice to investigate the in vivo function of these cotransporters. By cross-breeding with Clc-k2 knockout mice, we will be able to answer these questions.