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.
Dysfunction of the proximal tubule (PT) is associated with variable degrees of solute wasting and low-molecular-weight proteinuria. We measured metabolic consequences and adaptation mechanisms in a model of inherited PT disorders using PT cells of ClC-5-deficient (Clcn5Y/-) mice, a well-established model of Dent's disease. Compared to cells taken from control mice, those from the mutant mice had increased expression of markers of proliferation (Ki67, proliferative cell nuclear antigen (PCNA), and cyclin E) and oxidative scavengers (superoxide dismutase I and thioredoxin). Transcriptome and protein analyses showed fourfold induction of type III carbonic anhydrase in a kidney-specific manner in the knockout mice located in scattered PT cells. Kidney-specific carbonic anhydrase type III (CAIII) upregulation was confirmed in other mice lacking the multiligand receptor megalin and in a patient with Dent's disease due to an inactivating CLCN5 mutation. The type III enzyme was specifically detected in the urine of mice lacking ClC-5 or megalin, patients with Dent's disease, and in PT cell lines exposed to oxidative stress. Our study shows that lack of PT ClC-5 in mice and men is associated with CAIII induction, increased cell proliferation, and oxidative stress.
BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL gene products and the mechanism that regulates splice shifting is incompletely understood. We identified and characterized a long non-coding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was 5- to 9-fold less abundant in tumor cell lines from kidney, liver, breast and prostate and in kidney tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA polymerase II, 5′-capped, nuclear enriched and binds Sam68 splicing-modulator. Three apoptosis-inducing agents increased INXS lncRNA endogenous expression in the 786-O kidney tumor cell line, increased BCL-XS/BCL-XL mRNA ratio and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing toward BCL-XS and activation of caspases, thus leading to apoptosis. BCL-XS protein accumulation was detected upon INXS overexpression. In a mouse xenograft model, intra-tumor injections of an INXS-expressing plasmid caused a marked reduction in tumor weight, and an increase in BCL-XS isoform, as determined in the excised tumors. We revealed an endogenous lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies.
Noninvasive analysis of renal function in conscious mice is necessary to optimize the use of mouse models. In this study, we evaluated whether single photon emission-computed tomography (SPECT) using specific radionuclear tracers can be used to analyze changes in renal proximal tubule functions. The tracers included 99m TC-dimercaptosuccinic acid ( 99m Tc-DMSA), which is used for cortex imaging; 99m Tc-mercaptoacetyltriglycine ( 99m Tc-MAG3), used for dynamic renography; and 123 I-2-microglobulin, which monitors receptor-mediated endocytosis.99m Tc-DMSA SPECT imaging was shown to delineate the functional renal cortex with a ϳ1-mm spatial resolution and accumulated in the cortex reaching a plateau 5 h after injection. The cortical uptake of 99m Tc-DMSA was abolished in Clcn5 knockout mice, a model of proximal tubule dysfunction. Dynamic renography with 99m Tc-MAG3 in conscious mice demonstrated rapid extraction from blood, renal accumulation, and subsequent tubular secretion. Anesthesia induced a significant delay in the 99m Tc-MAG3 clearance. The tubular reabsorption of 123 I-2-microglobulin was strongly impaired in the Clcn5 knockout mice, with defective tubular processing and loss of the native tracer in urine, reflecting proximal tubule dysfunction. Longitudinal studies in a model of cisplatin-induced acute tubular injury revealed a correlation between tubular recovery and 123 I-2-microglobulin uptake. These data show that SPECT imaging with well-validated radiotracers allows in vivo investigations of specific proximal tubule functions in conscious mice.receptor-mediated endocytosis; tubular secretion; ClC-5; Dent's disease; radionuclear tracers TRANSGENIC MOUSE STRAINS ARE widely used to investigate the structure and function of developing and mature kidneys and to model human disorders. Thus far, functional studies into disease mechanisms essentially require animal death and ex vivo investigations that are potentially limited by various factors including low fertility rates, gender effects (e.g., X-linked transmission), or limited life span in some models. These restrictions complicate the design of intervention studies and require the development of large colonies, increasing costs and raising ethical questions. To circumvent some of these limitations, efforts are being made to develop in vivo functional imaging techniques in mice. In particular, the possibility to perform quantitative and/or dynamic molecular imaging studies using high-resolution positron emission tomography (PET) or single photon emission-computed tomography (SPECT) has emerged recently (23).Among its numerous applications, SPECT may be useful for noninvasive investigation of renal function in the mouse by means of dynamic imaging, parenchymal scanning, and quantification of filtration, secretion, and reabsorption events (8). In particular, the possibility to use different radiotracers for SPECT imaging provides the opportunity to investigate various transport processes taking place in the proximal tubule (PT), a segment that plays a ...
The study shows that individuals with two UMOD mutations are viable, but they do have more severe disease on average than heterozygotes. This family sheds light on the possible disease mechanism in this disorder.
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