PTH Regulation of Renal Phosphate Homeostasis

PTH依赖的肾磷酸盐稳态调节仍然不完全清楚。PTH下调 两种肾脏特异性磷酸钠共转运蛋白NPT 2a和NPT 2c的活性。NPT2a 处理70-80%的磷酸盐重吸收，并通过PTH依赖性机制进行调节， 涉及PTH/PTHrP受体（PTHR 1）下游的cAMP/PKA信号传导。与这样一个 Gs_/cAMP/PKA通路在肾脏磷酸盐处理中的重要作用 的假性甲状旁腺功能减退症（PHP）发展为PTH抵抗性高磷血症，因为受损的Gs_ 表达或功能。NPT 2c似乎是通过PTHR 1介导的PLC/PKC信号转导来调节的。 NPT 2c仅处理20-30%的肾磷酸盐重吸收，但使纯合子/复合物失活 杂合突变导致遗传性低磷血症性佝偻病伴高钙尿症（HHRH）， 常染色体隐性遗传病因此，NPT 2a和NPT 2c可能在肾脏中具有非冗余作用 磷酸盐稳态PTHR 1表达于肾近端基底外侧膜（BLM）， 在肾小管细胞中，它通过cAMP/PKA途径调节NPT 2a;然而，尚不清楚是否 PTHR 1在BLM的IP 3/PKC依赖性信号转导参与NPT 2a和/或NPT 2c的调节 表情在刷状缘膜（BBM），PTHR 1似乎仅激活cAMP/PKA独立 信号通路，我们初步研究的证据表明，这些行动调节 NPT 2c表达。为了阐明PTH依赖性调节NPT 2a和NPT 2b的潜在机制， NPT 2c，我们建议使用表达PTHR 1突变体的小鼠，该突变体缺乏PLC信号传导， 对于NPT 2a为“空”的小鼠，以及在近端肾小管中缺乏Gs_表达的小鼠（Aim I）。而且我们 将使用基因修饰的近端肾小管LLC-PK 1细胞，其显示PTH依赖性下调 与NPT 2a或NPT 2c组合表达PTHR 1时的磷酸盐转运（目的II和 III）。我们的新型LLC-PK 1细胞模型将被操纵以表达不同的野生型和突变蛋白 并且用PTH和信号选择性PTH类似物治疗，将其施加到顶端或基底外侧表面，因此 为我们的调查提供了宝贵的工具。我们预测，这些在体内和体外的组合 这些方法将为磷酸盐稳态的调节提供重要的新见解， 导致与肾磷酸盐排泄受损相关的人类疾病的改善的治疗选择， 包括甲状旁腺功能减退症、肿瘤性钙质沉着症、PHP，特别是慢性肾病。

PTH-dependent regulation of renal phosphate homeostasis remains incompletely understood. PTH downregulates the activity of two kidney-specific sodium-phosphate co-transporters, NPT2a and NPT2c. NPT2a handles 70-80% of phosphate reabsorption and it is regulated through PTH-dependent mechanisms that involve cAMP/PKA signaling down-stream of the PTH/PTHrP receptor (PTHR1). Consistent with such an important role of the Gs_/cAMP/PKA pathway in renal phosphate handling, patients affected by some forms of pseudohypoparathyroidism (PHP) develop PTH-resistant hyperphosphatemia because of impaired Gs_ expression or function. NPT2c appears to be regulated through PLC/PKC-signaling mediated by the PTHR1. NPT2c handles only 20-30% of renal phosphate reabsorption, yet inactivating homozygous/compound heterozygous mutations lead to hereditary hypophosphatemic rickets with hypercalciuria (HHRH), an autosomal recessive disorder. NPT2a and NPT2c thus are likely to have non-redundant roles in renal phosphate homeostasis. The PTHR1 is expressed at the baso-lateral membrane (BLM) of renal proximal tubular cells where it regulates NPT2a via the cAMP/PKA pathway; it is, however, unknown whether IP3/PKC-dependent signaling of PTHR1 at the BLM is involved in the regulation of NPT2a and/or NPT2c expression. At the brush-border membrane (BBM), the PTHR1 appears to activate only cAMP/PKAindependent signaling pathways, and evidence from our preliminary studies suggest that these actions regulate NPT2c expression. To elucidate the mechanisms underlying the PTH-dependent regulation of NPT2a and NPT2c, we propose to use mice expressing a PTHR1 mutant that is deficient in PLC-signaling, mice that are "null" for NPT2a, and mice that lack Gs_ expression in the proximal renal tubules (Aim I). Furthermore, we will use genetically-modified proximal tubular LLC-PK1 cells that show PTH-dependent down-regulation of phosphate transport when expressing the PTHR1, in combination with either NPT2a or NPT2c (Aims II and III). Our novel LLC-PK1 cell model will be manipulated to express different wild-type and mutant proteins and treated with PTH and signal-selective PTH analogs applied to either apical or baso-lateral surfaces, thus presenting a valuable tool for our investigations. We predict that the combination of these in vivo and in vitro approaches will provide important new insights into the regulation of phosphate homeostasis and are likely to lead to improved treatment options for human disorders associated with impaired renal phosphate excretion, including hypoparathyroidism, tumoral calcinosis, PHP, and particularly, chronic kidney disease.