Hmx2 and Dmrt2 Coordinate the Differentiation of Intercalated Cell Subtypes in Kidney

KEY POINTS: Hmx2 was required for type B intercalated cell differentiation but became dispensable in the absence of Dmrt2. Dmrt2 promoted type A differentiation of intercalated cells and repressed type B identity. Early divergence of Dmrt2 and Hmx2 marked subtype specification of intercalated cells. BACKGROUND: Intercalated cells in the kidney collecting ducts are essential for maintaining systemic acid-base homeostasis. Based on gene expression profiles and functional characteristics, intercalated cells are classified into type A, type B, non-A/non-B. Although several transcription factors have been reported to regulate intercalated cell differentiation, how the fates of intercalated cell subtypes are established remains unclear. METHODS: To investigate the roles of Dmrt2 and Hmx2 in intercalated cell subtype differentiation, we generated mice with single or double conditional deletion and knock-in of these transcription factors specifically in distal nephron segments and analyzed their effects on urine acidification. We also performed single-cell RNA sequencing analysis on mouse and human kidney datasets to trace intercalated cell progenitor fate trajectories. RESULTS: Loss of Hmx2 in the distal nephron prevented type B intercalated cell differentiation, whereas simultaneous deletion of Hmx2 and Dmrt2 compromised type A intercalated cell differentiation, with a concomitant increase in Hmx3 expression and type B intercalated cell differentiation. Dmrt2 knock-in mice exhibited a modest increase in type A intercalated cell differentiation and a marked reduction in type B intercalated cells. Notably, Dmrt2 knock-in did not rescue the intercalated cell differentiation defects observed in Foxp1-deficient mice. Analysis of mouse and human single-cell RNA sequencing data further confirmed the mutually exclusive expression patterns of Hmx2 and Dmrt2 in the kidney. CONCLUSIONS: Hmx2 and Dmrt2 were essential, mutually exclusive transcription factors that govern intercalated cell subtype differentiation in the kidney, with Hmx2 specifying type B intercalated cell fate and Dmrt2 promoting type A intercalated cell differentiation. Dmrt2 suppressed the expression of Hmx2 and Hmx3. In the absence of Dmrt2 and Hmx2, Hmx3 expression was activated to promote type B intercalated cell differentiation.