Molecular Physiology of Renal K-Cl Cotransporters

描述（由申请人提供）：偶联K+- ci -共转运由KCC蛋白介导，由slc12阳离子-氯共转运基因家族的四个成员编码。遗传学、生理学和基因敲除小鼠的特征表明，KCCs与多种疾病的发病机制有关，如高血压、癫痫、肾小管酸中毒、神经性疼痛和镰状细胞性贫血。在肾近端小管中，尖钠吸收引起的等渗肿胀激活了KCC3和KCC4介导的基底侧K+ - cl -共转运，表明这些转运体参与了过滤后的Na+- cl -和其他溶质的近端再吸收。事实上，除了液体输送减少外，KCC3缺陷小鼠在碳酸氢盐吸收方面也表现出缺陷，这表明KCC3的缺失导致了广泛性近端肾小管缺陷。此外，考虑到KCC3在调节体积减少（RVD）、氧化应激反应和严重神经退行性综合征中的作用，我们认为KCC3在应对缺血性体积增加（“IVI”）时需要维持细胞完整性，因此KCC3的缺失易导致缺血性小管坏死。因此，近端小管中K+- cl -共转运的作用和调节是这种竞争性更新的焦点。在Aim 1中，我们建议完成现有的KCC3 （Slc12a6）基因种系缺失小鼠株的肾脏表型表征，此外，我们还创建了KCC3基因缺失仅限于肾近端小管的小鼠株并对其进行了表征。这些动物研究将包括免疫组织化学、肾脏生理学和对肾缺血/再灌注损伤反应的评估。

DESCRIPTION (provided by applicant): Coupled K+-CI- co-transport is mediated by the KCC proteins, encoded by four members of the SLC 12 cation-chloride cotransporter gene family. Genetics, physiology, and the characterization of knockout mice have implicated the KCCs in the pathogenesis of disorders as diverse as hypertension, epilepsy, renal tubular acidosis, neuropathic pain, and sickle cell anemia. In the renal proximal tubule, isotonic swelling induced by apical Na absorption activates basolateral K+ -Cl- cotransport mediated by KCC3 and KCC4, implicating these transporters in proximal re-absorption of filtered Na+-Cl- and other solutes. Indeed, in addition to reduced fluid transport, KCC3-deficient mice exhibit defects in the absorption of bicarbonate, suggesting that loss of KCC3 causes generalized proximal tubular defects. Furthermore, given the demonstrated role of KCC3 in regulatory volume decrease (RVD), the response to oxidant stress, and a severe neurodegenerative syndrome, we propose that KCC3 is required to maintain cellular integrity in response to ischemic volume increase ("IVI"), such that loss of KCC3 predisposes to ischemic tubular necrosis. The role and regulation of K+-Cl - cotransport in the proximal tubule is thus the focus of this competing renewal. We propose in Aim 1 to finish characterizing the renal phenotype of our existing mouse strain with germline deletion of the KCC3 (Slc12a6) gene, in addition to creating and characterizing a mouse strain with KCC3 deletion that is limited to the renal proximal tubule. These animal studies will encompass immunohistochemistry, renal physiology, and assessment of the response to renal ischemia/reperfusion injury. Whereas neuronal-specific KCC2 is unique in mediating constitutive K+-Cl - cotransport, the other three KCCs are quiescent in the absence of cell swelling. Using a chimeric approach, we have localized the molecular determinants of constitutive isotonic activity to a KCC2-specific expansion in the C-terminal cytoplasmic domain. Given the role of swelling-activated K +-Cl- cotransport in proximal tubular salt and solute transport we will characterize the molecular determinants of swelling activation, using chimeras between K CC4 and t he s welling-inhibited N a-K-2Cl cotransporter N KCC2. T he effect o f c ell volume o n phosphorylation status and membrane trafficking will also be studied in Aim 2, using KCC4 and several N-terminal variants of KCC3. These studies will begin in Xenopus oocytes but will ultimately be extended to the opossum kidney (OK) cell line and/or other epithelial cell lines. In Aim 3 we will focus on the role of conserved cysteines in the mechanism and regulation of K+-Cl - cotransport, building on data from both activating and inactivating mutations of conserved transmembrane cysteines. Various cysteine-depleted mutants will also be used to distinguish the cytoplasmic and membrane-associated mechanisms in the activation and inactivation of the KCCs by nitric oxide (NO) and related cysteine-reactive compounds.