Transcription factor TFCP2L1 patterns cells in the mouse kidney collecting ducts

Although most nephron segments contain one type of epithelial cell, the collecting ducts consists of at least two: intercalated (IC) and principal (PC) cells, which regulate acid-base and salt-water homeostasis, respectively. In adult kidneys, these cells are organized in rosettes suggesting functional interactions. Genetic studies in mouse revealed that transcription factor Tfcp2l1 coordinates IC and PC development. Tfcp2l1 induces the expression of IC specific genes, including specific H(+)-ATPase subunits and Jag1. Jag1 in turn, initiates Notch signaling in PCs but inhibits Notch signaling in ICs. Tfcp2l1 inactivation deletes ICs, whereas Jag1 inactivation results in the forfeiture of discrete IC and PC identities. Thus, Tfcp2l1 is a critical regulator of IC-PC patterning, acting cell-autonomously in ICs, and non-cell-autonomously in PCs. As a result, Tfcp2l1 regulates the diversification of cell types which is the central characteristic of 'salt and pepper' epithelia and distinguishes the collecting duct from all other nephron segments

miRNA in situ hybridization in circulating tumor cells - MishCTC

irculating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype.JJDM thanks the Spanish Ministerio de Economía y Competitividad for a Ramón y Cajal Fellowship (Grant CTQ2012-34778). This research was partially supported by Marie Curie Career Integration Grants within the 7th European Community Framework Program (FP7-PEOPLE-2011-CIG-Project Number 294142 to RMSM and FP7-PEOPLE-2012-CIG-Project Number 322276 toJJDM). Thisresearch wasalso partially supported by Consejeria de Salud de la Junta de Andalucı ́ a (PI0294-2012)

Urine/Plasma Neutrophil Gelatinase Associated Lipocalin Ratio Is a Sensitive and Specific Marker of Subclinical Acute Kidney Injury in Mice

Background Detection of acute kidney injury (AKI) is still a challenge if conventional markers of kidney function are within reference range. We studied the sensitivity and specificity of NGAL as an AKI marker at different degrees of renal ischemia. Methods Male C57BL/6J mice were subjected to 10-, 20- or 30-min unilateral renal ischemia, to control operation or no operation, and AKI was evaluated 1 day later by histology, immunohistochemistry, BUN, creatinine, NGAL (plasma and urine) and renal NGAL mRNA expression. Results A short (10-min) ischemia did not alter BUN or kidney histology, but elevated plasma and urinary NGAL level and renal NGAL mRNA expression although to a much smaller extent than longer ischemia. Surprisingly, control operation elevated plasma NGAL and renal NGAL mRNA expression to a similar extent as 10-min ischemia. Further, the ratio of urine to plasma NGAL was the best parameter to differentiate a 10-min ischemic injury from control operation, while it was similar in the non and control-operated groups. Conclusions These results suggest that urinary NGAL excretion and especially ratio of urine to plasma NGAL are sensitive and specific markers of subclinical acute kidney injury in mice

Mutations in DSTYK and dominant urinary tract malformations.

ABSTRACT Introduction Congenital abnormalities of the kidney of the urinary tract are the most common cause of pediatric kidney failure. These disorders are highly heterogeneous, and their etiology is poorly understood. Methods We performed genome-wide linkage analysis and whole-exome sequencing in a family with autosomal dominant congenital abnormalities of the kidney of the urinary tract (7 affected family members). We also performed sequence analysis in 311 unrelated patients, as well as histologic and functional studies. Results Linkage analysis identified five regions of the genome that were shared among all affected family members. Exome sequencing identified a single rare deleterious variant within these linkage intervals, a heterozygous splice-site mutation in dual serine/threonine and tyrosine protein kinase (DSTYK). This variant, which resulted in aberrant gene product splicing, was present in all affected family members. Additional independent DSTYK mutations, including nonsense and splice-site mutations, were detected among 7/311 unrelated patients. DSTYK is highly expressed in the maturing epithelia of all major organs, localizing to cell membranes. Knockdown in zebrafish resulted in multi-organ developmental defects, resembling loss of fibroblast growth factor (FGF) signaling. Consistent with this finding, DSTYK colocalizes with FGF receptors in the ureteric bud and metanephric mesenchyme. Finally, DSTYK knockdown in human embryonic kidney cells inhibited FGF-stimulated ERK-phosphorylation, the principal signal downstream of receptor tyrosine kinases. Conclusions We detected DSTYK mutations in 2.2% of patients with congenital abnormalities of the kidney and urinary tract whom we studied, suggesting that DSTYK is a major determinant of human urinary tract development, downstream of FGF signaling

α-intercalated cells defend the urinary system from bacterial infection

{alpha}–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 Kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury

© 2020, The Author(s). Renal tubular epithelial cells (RTECs) perform the essential function of maintaining the constancy of body fluid composition and volume. Toxic, inflammatory, or hypoxic-insults to RTECs can cause systemic fluid imbalance, electrolyte abnormalities and metabolic waste accumulation- manifesting as acute kidney injury (AKI), a common disorder associated with adverse long-term sequelae and high mortality. Here we report the results of a kinome-wide RNAi screen for cellular pathways involved in AKI-associated RTEC-dysfunction and cell death. Our screen and validation studies reveal an essential role of Cdkl5-kinase in RTEC cell death. In mouse models, genetic or pharmacological Cdkl5 inhibition mitigates nephrotoxic and ischemia-associated AKI. We propose that Cdkl5 is a stress-responsive kinase that promotes renal injury in part through phosphorylation-dependent suppression of pro-survival transcription regulator Sox9. These findings reveal a surprising non-neuronal function of Cdkl5, identify a pathogenic Cdkl5-Sox9 axis in epithelial cell-death, and support CDKL5 antagonism as a therapeutic approach for AKI

A dexamethasone prodrug reduces the renal macrophage response and provides enhanced resolution of established murine lupus nephritis

We evaluated the ability of a macromolecular prodrug of dexamethasone (P-Dex) to treat lupus nephritis in (NZB × NZW)F1 mice. We also explored the mechanism underlying the anti-inflammatory effects of this prodrug. P-Dex eliminated albuminuria in most (NZB × NZW)F1 mice. Furthermore, P-Dex reduced the incidence of severe nephritis and extended lifespan in these mice. P-Dex treatment also prevented the development of lupus-associated hypertension and vasculitis. Although P-Dex did not reduce serum levels of anti-dsDNA antibodies or glomerular immune complexes, P-Dex reduced macrophage recruitment to the kidney and attenuated tubulointerstitial injury. In contrast to what was observed with free dexamethasone, P-Dex did not induce any deterioration of bone quality. However, P-Dex did lead to reduced peripheral white blood cell counts and adrenal gland atrophy. These results suggest that P-Dex is more effective and less toxic than free dexamethasone for the treatment of lupus nephritis in (NZB × NZW)F1 mice. Furthermore, the data suggest that P-Dex may treat nephritis by attenuating the renal inflammatory response to immune complexes, leading to decreased immune cell infiltration and diminished renal inflammation and injury