Pathophysiology of distal renal tubular acidosis

肾脏在酸碱平衡中起着至关重要的作用。在远端肾小管酸中毒（dRTA）中，尿液不能充分酸化，可导致低钾血症、肾钙质沉着症、肾结石、骨脱矿和肾功能衰竭。在远端小管中，A插层细胞（ic）通过顶端细胞极的v型atp酶分泌H+，并通过Cl-/ HCO3*交换剂AE1在基底侧重新吸收HCO3-。b - ic主要分泌HCO3*，并通过v型atp酶在基底侧重吸收H+。假设两种类型的ic可以根据代谢需求相互转换。v型atp酶的b1或a4亚基以及AE1的突变都与dRTA有关。为了更好地解决潜在的病理生理问题，我们之前建立了a4亚基敲除小鼠模型和AE1中最常见的突变R589H敲入小鼠模型。a4基因敲除小鼠表现出严重的dRTA并伴有早期死亡，而Ae1基因敲入小鼠表现出不完全dRTA。令人惊讶的是，Ae1变体正确地靶向于基底外侧膜，而v型atp酶的靶向性发生了改变。由于Ae1敲入蛋白小鼠的a - ic积累p62阳性囊泡，我们假设v型ATPase靶向缺陷也可能损害溶酶体降解。为了解决细胞病理问题，我们建议用FACS从转基因小鼠中分离a - ic和我们在第一个资助期产生的a - ic特异性creline。这将允许比较两种基因型之间的a - ic特异性转录组和溶酶体蛋白质组。作为原理证明，WT a - ic的RNAseq证实了已知标记基因的表达，并鉴定了转录因子Dmrt2在a - ic中具有高特异性表达。我们将融合A-IC衍生的细胞系，以敲除Ae1， Dmrt2或敲入R607H变体，并在细胞功能水平上研究由此产生的后果。如果Dmrt2被证明对a - ic的分化起作用，我们将产生肾脏特异性Dmrt2敲除。由于我们进一步获得了b - ic特定的creline，我们将扩展我们对b - ic的分析。两个creline结合一个报告将进一步使我们能够评估a -和b - ic是否确实可以相互转换。

The kidney plays an essential role for acid-base homeostasis. A failure to acidify the urine adequately is observed in distal renal tubular acidosis (dRTA) and can result in hypokalemia, nephrocalcinosis, nephrolithiasis, bone demineralization and renal failure. In the distal tubule, A intercalated cells (ICs) secrete H+ via the V-type ATPase at the apical cell pole and reabsorb HCO3- basolaterally via the Cl-/ HCO3* exchanger AE1. B-ICs secrete HCO3* apically and reabsorb H+ basolaterally via the V-type ATPase. It is assumed that both types of ICs can interconvert depending on metabolic demands. Mutations in either the b1 or the a4 subunit of the V-type ATPase and in AE1 are associated with dRTA. To better resolve the underlying pathophysiology we previously generated a knockout mouse model for the a4 subunit and a knockin mouse model of the most common mutation in AE1, i.e. R589H. While a4 knockout mice displayed severe dRTA with early mortality, Ae1 knock-in mice showed incomplete dRTA. Surprisingly, the Ae1 variant was correctly targeted to the basolateral membrane, while V-type ATPase targeting was altered. Since A-ICs of Ae1 knockin mice accumulate p62-positive vesicles, we hypothesize that the V-type ATPase targeting defect may also impair lysosomal degradation. To resolve the cellular pathology we propose to isolate A-ICs by FACS from mice transgenic for a reporter and our A-IC-specific Cre-line that we had generated in the first funding period. This will allow comparing the A-IC-specific transcriptome and lysosomal proteome between both genotypes. As prove of principle RNAseq of WT A-ICs confirmed the expression of known marker genes and in addition led to the identification of the transcription factor Dmrt2 to be highly and specifically expressed in A-ICs. We will fuse an A-IC derived cell line to knockdown Ae1, Dmrt2 or to knockin the R607H variant and study the resulting consequences at the cellular functional level. If Dmrt2 proves to play a role for the differentiation of A-ICs, we will generate a kidney specific Dmrt2 knockout. Since we further have access to a B-IC-specific Cre-line, we will expand our analysis for B-ICs. Both Cre-lines in combination with a reporter will further allow us to assess whether A- and B-ICs can indeed interconvert.