Pathophysiology of distal renal tubular acidosis

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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.