Identification of Transcription Factors Driving Proliferative Cell State Transitions in The Distal Convoluted Tubule

The distal tubule structure is highly plastic, undergoing proliferative and hypertrophic cell state transitions when transport is activated. Although the remodeling process helps the distal convoluted tubule (DCT) maintain blood pressure and electrolyte homeostasis, maladaptive DCT remodeling can diminish the therapeutic effects of loop diuretics and can contribute to disease. Here, we sought to identify the transcriptional regulatory axes that induce proliferative cell state transitions and maintain DCT cell identity. RNA sequencing (RNA-Seq) was performed in microdissected DCTs in mice treated with loop-diuretic and in kidneys of mice genetically engineered to constrictively activate the thiazide-sensitive sodium chloride cotransporter, NCC, both of which exhibit DCT proliferative and hypertrophic cell state transitions. Multimodal-bioinformatic analyses with molecular and biochemical validation in Wild-type (WT) and Knockout (KO) were used to identify core transcriptional regulatory axes in DCT cells. Time-series transcriptomic analysis of microdissected DCTs in mice treated with furosemide (12-96 h) revealed the rapid induction of a cell proliferation gene set associated with hypoxia-induced responses, including activation of hypoxia-responsive transcription factors (TFs, HIF1A, STAT3 and RELA) and metabolic gene expression. This hypoxia-induced response and proliferative cell state transition were paralleled by activation of the NCC-SPAK-WNK pathway in the DCT, suggesting that oxygen consuming salt transport activates proliferative cell transition through hypoxic stress. Consistent with this, analysis of transcriptomic responses in human and mouse acute kidney injury exhibited activation of Stat3 in distal tubule cells. In the model of chronic NCC-SPAK-WNK activation by DCT-specific CA-SPAK expression, RNA-Seq and bioinformatic analysis identified Spalt-like transcription factor 3 (Sall3) as a key DCT TF that was significantly induced by chronic NCC-SPAK activation in the DCT. A similar response was observed in WT mice when WNK-SPAK was physiologically activated with low K diet. Induction of DCT-specific Sall3 KO significantly reduced the expression of DCT-specific genes, indicating that Sall3 is necessary to define and maintain DCT identity. In summary, we identified transcriptional regulatory networks of hypoxia-induced cell state transition that drive DCT proliferation and DCT cell identity determination through expression of Sall3. NIDDK, LeDucq Foundation This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.