Snapshots of nascent RNA reveal cell- and stimulus-specific responses to acute kidney injury.

The current strategy to detect acute injury of kidney tubular cells relies on changes in serum levels of creatinine. Yet serum creatinine (sCr) is a marker of both functional and pathological processes and does not adequately assay tubular injury. In addition, sCr may require days to reach diagnostic thresholds, yet tubular cells respond with programs of damage and repair within minutes or hours. To detect acute responses to clinically relevant stimuli, we created mice expressing Rosa26-floxed-stop uracil phosphoribosyltransferase (Uprt) and inoculated 4-thiouracil (4-TU) to tag nascent RNA at selected time points. Cre-driven 4-TU-tagged RNA was isolated from intact kidneys and demonstrated that volume depletion and ischemia induced different genetic programs in collecting ducts and intercalated cells. Even lineage-related cell types expressed different genes in response to the 2 stressors. TU tagging also demonstrated the transient nature of the responses. Because we placed Uprt in the ubiquitously active Rosa26 locus, nascent RNAs from many cell types can be tagged in vivo and their roles interrogated under various conditions. In short, 4-TU labeling identifies stimulus-specific, cell-specific, and time-dependent acute responses that are otherwise difficult to detect with other technologies and are entirely obscured when sCr is the sole metric of kidney damage.

MicroRNA-21 deficiency suppresses prostate cancer progression through downregulation of the IRS1-SREBP-1 signaling pathway.

Sterol regulatory element-binding protein 1 (SREBP-1), a master transcription factor in lipogenesis and lipid metabolism, is critical for disease progression and associated with poor outcomes in prostate cancer (PCa) patients. However, the mechanism of SREBP-1 regulation in PCa remains elusive. Here, we report that SREBP-1 is transcriptionally regulated by microRNA-21 (miR-21) in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of SREBP-1, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) in Pten/Trp53 double-null mouse embryonic fibroblasts (MEFs) and Pten/Trp53 double-null mutant mice. Strikingly, miR-21 loss significantly reduced cell proliferation and suppressed the prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, miR-21 inactivation decreased the levels of SREBP-1, FASN, and ACC in human PCa cells through downregulation of insulin receptor substrate 1 (IRS1)-mediated transcription and induction of cellular senescence. Conversely, miR-21 overexpression increased cell proliferation and migration; as well as the levels of IRS1, SREBP-1, FASN, and ACC in human PCa cells. Our findings reveal that miR-21 promotes PCa progression by activating the IRS1/SREBP-1 axis, and targeting miR-21/SREBP-1 signaling pathway can be a novel strategy for controlling PCa malignancy.

A Subpopulation of Label-Retaining Cells of the Kidney Papilla Regenerates Injured Kidney Medullary Tubules.

To determine whether adult kidney papillary label-retaining cells (pLRCs) are specialized precursors, we analyzed their transcription profile. Among genes overexpressed in pLRCs, we selected candidate genes to perform qPCR and immunodetection of their encoded proteins. We found that Zfyve27, which encodes protrudin, identified a subpopulation of pLRCs. With Zfyve27-CreERT2 transgenic and reporter mice we generated bitransgenic animals and performed cell-lineage analysis. Post tamoxifen, Zfyve27-CreERT2 marked cells preferentially located in the upper part of the papilla. These cells were low cycling and did not generate progeny even after long-term observation, thus they did not appear to contribute to kidney homeostasis. However, after kidney injury, but only if severe, they activated a program of proliferation, migration, and morphogenesis generating multiple and long tubular segments. Remarkably these regenerated tubules were located preferentially in the kidney medulla, indicating that repair of injury in the kidney is regionally specified. These results suggest that different parts of the kidney have different progenitor cell pools.

α-Intercalated cells defend the urinary system from bacterial infection.

α-Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC-dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.

A BAC-based transgenic mouse specifically expresses an inducible Cre in the urothelium.

Cre-loxp mediated conditional knockout strategy has played critical roles for revealing functions of many genes essential for development, as well as the causal relationships between gene mutations and diseases in the postnatal adult mice. One key factor of this strategy is the availability of mice with tissue- or cell type-specific Cre expression. However, the success of the traditional molecular cloning approach to generate mice with tissue specific Cre expression often depends on luck. Here we provide a better alternative by using bacterial artificial chromosome (BAC)-based recombineering to insert iCreERT2 cDNA at the ATG start of the Upk2 gene. The BAC-based transgenic mice express the inducible Cre specifically in the urothelium as demonstrated by mRNA expression and staining for LacZ expression after crossing with a Rosa26 reporter mouse. Taking into consideration the size of the gene of interest and neighboring genes included in a BAC, this method should be widely applicable for generation of mice with tissue specific gene expression or deletions in a more specific manner than previously reported.

Distinct expression profiles of p63 variants during urothelial development and bladder cancer progression.

The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform-specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors.

Inactivation of p53 and Pten promotes invasive bladder cancer.

Although bladder cancer represents a serious health problem worldwide, relevant mouse models for investigating disease progression or therapeutic targets have been lacking. We show that combined deletion of p53 and Pten in bladder epithelium leads to invasive cancer in a novel mouse model. Inactivation of p53 and PTEN promotes tumorigenesis in human bladder cells and is correlated with poor survival in human tumors. Furthermore, the synergistic effects of p53 and Pten deletion are mediated by deregulation of mammalian target of rapamycin (mTOR) signaling, consistent with the ability of rapamycin to block bladder tumorigenesis in preclinical studies. Our integrated analyses of mouse and human bladder cancer provide a rationale for investigating mTOR inhibition for treatment of patients with invasive disease.

The mechanisms of PML-nuclear body formation.

PML nuclear bodies (NBs) are nuclear structures that have been implicated in processes such as transcriptional regulation, genome stability, response to viral infection, apoptosis, and tumor suppression. PML has been found to be essential for the formation of the NBs, as these structures do not form in Pml null cells, although PML add back fully rescues their formation. However, the basis for such a structural role of PML is unknown. We demonstrate that PML contains a SUMO binding motif that is independent of its SUMOylation sites and is surprisingly necessary for PML-NB formation. We demonstrate that the PML RING domain is critical for PML SUMOylation and PML-NB formation. We propose a model for PML-NB formation whereby PML SUMOylation and noncovalent binding of PML to SUMOylated PML through the SUMO binding motif constitutes the nucleation event for subsequent recruitment of SUMOylated proteins and/or proteins containing SUMO binding motifs to the PML NBs.