Immune Landscape and Intercalated Cell Dysfunction in CA2 Knockout Kidneys Revealed by Single-Cell RNA Sequencing (scRNAseq) and Intravital Studies

Background: Carbonic anhydrase 2 (CA2) regulates acid-base balance. Our prior work showed that CA2 knockout (KO) mice lack intercalated cells (ICs), develop metabolic acidosis, and exhibit impaired bacterial clearance. ICs play a key role in innate immunity by phagocytosing bacteria. CA2 KO mice infected with CFT073 had higher kidney bacterial loads than WT mice, and correcting pH and bicarbonate levels did not reduce susceptibility, indicating other factors are involved. Methods: Intravital microscopy for phagocytosis, scRNAseq of CA2 KO mice kidney cells and biological pathway analysis on the transcriptomics data Results: ICs in CA2 KO mice were not only reduced in number but also showed loss of phagocytic ability on intravital imaging and impaired expression of the transcription factor Foxi1 and key V-ATPase subunits (Atp6v1b1, Atp6v1e1). Ingenuity Pathway Analysis (IPA) revealed disrupted calcium signaling, with significant downregulation of inositol 1,4,5-trisphosphate receptors (InsP3Rs), particularly ITPR2, which was confirmed to be reduced in ICs (Figure). Despite impaired IC function, CA2 KO mice exhibited elevated innate (macrophages, neutrophils) and adaptive (CD4+, CD8+ T cells) immune populations. These T cells expressed activation markers (CTLA4, ICOS) and proinflammatory cytokines (IFNγ, TNFα), though pathway analysis showed no major changes in T cell signaling. Conclusion: CA2 deficiency impairs IC development and function, particularly phagocytosis, through disrupted calcium signaling and transcriptional regulation. Despite compensatory immune cell activation, the loss of IC-mediated defense likely underlies the increased susceptibility to kidney infection in CA2 KO mice. Funding: NIDDK Support