Adrian Caplanusi

ZONAB Promotes Proliferation and Represses Differentiation of Proximal Tubule Epithelial Cells

Epithelial polarization modulates gene expression. The transcription factor zonula occludens 1 (ZO-1)-associated nucleic acid binding protein (ZONAB) can shuttle between tight junctions and nuclei, promoting cell proliferation and expression of cyclin D1 and proliferating cell nuclear antigen (PCNA), but whether it also represses epithelial differentiation is unknown. Here, during mouse kidney ontogeny and polarization of proximal tubular cells (OK cells), ZONAB and PCNA levels decreased in parallel and inversely correlated with increasing apical differentiation, reflected by expression of megalin/cubilin, maturation of the brush border, and extension of the primary cilium. Conversely, ZONAB reexpression and loss of apical differentiation markers provided a signature for renal clear cell carcinoma. In confluent OK cells, ZONAB overexpression increased proliferation and PCNA while repressing megalin/cubilin expression and impairing differentiation of the brush border and primary cilium. Reporter and chromatin immunoprecipitation assays demonstrated that megalin and cubilin are ZONAB target genes. Sparsely plated OK cells formed small islands composed of distinct populations: Cells on the periphery, which lacked external tight junctions, strongly expressed nuclear ZONAB, proliferated, and failed to differentiate; central cells, surrounded by continuous junctions, lost nuclear ZONAB, stopped proliferating, and engaged in apical differentiation. Taken together, these data suggest that ZONAB is an important component of the mechanisms that sense epithelial density and participates in the complex transcriptional networks that regulate the switch between proliferation and differentiation.

Role of Mitochondrial Na Concentration, Measured by CoroNa Red, in the Protection of Metabolically Inhibited MDCK Cells

In ischemic or hypoxic tissues, elevated cytosolic calcium levels can induce lethal processes. Mitochondria, besides the endoplasmic reticulum, play a key role in clearing excessive cytosolic Ca2+. In a previous study, it was suggested that the clearance of cytosolic Ca2+, after approximately 18 min of metabolic inhibition (MI) in renal epithelial cells, occurs via the reverse action of the mitochondrial Na+/Ca2+ exchanger (NCX). For further investigating the underlying mechanism, changes in the mitochondrial Na+ concentration ([Na+](m)) were monitored in metabolically inhibited MDCK cells. CoroNa Red, a sodium-sensitive fluorescence probe, was used to monitor [Na+]m. In the first 15 min of MI, a twofold increase of [Na+]m was observed reaching 113 +/- 7 mM, whereas the cytosolic Na+ concentration ([Na+]c) elevated threefold, to a level of 65 +/- 6 mM. In the next 45 min of MI, [Na+]m dropped to 91 +/- 7 mM, whereas [Na+]c further increased to 91 +/- 4 mM. The striking rise in [Na+]m is likely sufficient to sustain the driving force for mitochondrial Ca2+ uptake via the NCX. Furthermore, when CGP-37157, a specific inhibitor of the mitochondrial NCX, was applied during MI, the second-phase drop of [Na+]m was completely abolished. The obtained results support the hypothesis that the mitochondrial NCX reverses after approximately 15 min of MI. Moreover, because the cellular homeostasis can recover after MI, the mitochondria likely protect MDCK cells from injury during MI by the reversal of the mitochondrial NCX. This study is the first to report [Na+]m measurements in nonpermeabilized living cells.

Intravital multi-photon microscopy reveals several levels of heterogeneity in endocytic uptake by mouse renal proximal tubules.

Understanding renal function requires one to integrate the structural complexity of kidney nephrons and the dynamic nature of their cellular processes. Multi‐photon fluorescence microscopy is a state‐of‐the‐art imaging technique for in vivo analysis of kidney tubules structure and function in real time. This study presents visual evidence for several levels of heterogeneity of proximal tubular endocytic uptake in the superficial renal mouse cortex and illustrates the potential of multi‐photon microscopy for providing a comprehensive and dynamic portrayal of renal function.