Podocytes are critical in the maintenance of a healthy glomerular filter, however they have been difficult to study in the intact kidney due to technical limitations. Here we report the development of serial multiphoton microscopy (MPM) of the same glomeruli over several days to visualize the motility of podocytes and parietal epithelial cells (PEC) in vivo. In Podocin-GFP mice podocytes formed sporadic multi-cellular clusters after unilateral ureteral ligation (UUO) and migrated into the parietal Bowman’s capsule. The tracking of single cells in Podocin-confetti mice featuring cell-specific expression of CFP, GFP, YFP, or RFP revealed the simultaneous migration of multiple podocytes. In PEPCK-GFP mice serial MPM found PEC-to-podocyte migration and nanotubule connections. Our data support the highly dynamic rather than static nature of the glomerular environment and cellular composition. Future application of this new approach promises to advance our understanding of the mechanisms of glomerular injury and regeneration.

signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney.

P2Y12 receptor (P2Y12-R) signaling is mediated through Gi, ultimately reducing cellular cAMP levels. Because cAMP is a central modulator of arginine vasopressin (AVP)-induced water transport in the renal collecting duct (CD), we hypothesized that if expressed in the CD, P2Y12-R may play a role in renal handling of water in health and in nephrogenic diabetes insipidus. We found P2Y12-R mRNA expression in rat kidney, and immunolocalized its protein and aquaporin-2 (AQP2) in CD principal cells. Administration of clopidogrel bisulfate, an irreversible inhibitor of P2Y12-R, significantly increased urine concentration and AQP2 protein in the kidneys of Sprague-Dawley rats. Notably, clopidogrel did not alter urine concentration in Brattleboro rats that lack AVP. Clopidogrel administration also significantly ameliorated lithium-induced polyuria, improved urine concentrating ability and AQP2 protein abundance, and reversed the lithium-induced increase in free-water excretion, without decreasing blood or kidney tissue lithium levels. Clopidogrel administration also augmented the lithium-induced increase in urinary AVP excretion and suppressed the lithium-induced increase in urinary nitrates/nitrites (nitric oxide production) and 8-isoprostane (oxidative stress). Furthermore, selective blockade of P2Y12-R by the reversible antagonist PSB-0739 in primary cultures of rat inner medullary CD cells potentiated the expression of AQP2 and AQP3 mRNA, and cAMP production induced by dDAVP (desmopressin). In conclusion, pharmacologic blockade of renal P2Y12-R increases urinary concentrating ability by augmenting the effect of AVP on the kidney and ameliorates lithium-induced NDI by potentiating the action of AVP on the CD. This strategy may offer a novel and effective therapy for lithium-induced NDI.

The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure.

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Cells of the macula densa (MD) in the adult kidney are strategically localized in the distal tubule at the glomerular vascular entrance, which mirrors the position of nephron‐committed progenitor cells in the developing kidney, at the interface between the ureteric bud and the metanephric mesenchyme. Indirect evidence suggests that MD cells contribute to nephrogenesis. Here we aimed to assess the stem/progenitor identity of MD cells via in vivo lineage tracing. We developed MD‐Confetti mice with tamoxifen‐inducible expression of the confetti fluorescent reporter construct (CFP/GFP/YFP/RFP) specifically in MD cells by crossing nNOS‐CreERT2 and Confetti/fl mice. Confetti expression was induced by ip tamoxifen injections on postnatal days 2 and 3, and kidneys were harvested three weeks later. MD cells were identified by one of the four Confetti colors, expressing either membrane‐targeted CFP, nuclear‐GFP, cytosolic YFP or RFP. MD plaques usually contained individual MD cells of all four colors. However, in vivo multi‐color lineage tracing identified MD cells as a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb (TAL) of Henle’s loop. In addition to multicolor cell labeling in MD plaques, fluorescence image analysis revealed the presence of several, exclusively clonal (i.e. single‐color) multi‐cell tracing units in TALs. Our results highlight the new role and important contribution of individual MD stem/progenitor cells to nephron formation during kidney development.