Project description:To study the effects of aldosterone on miRNA in the kidney cortical collecting duct, polarized mpkCCDc14 cell were treated with aldosterone or vehicle. A putative target for miR-466g was identified, the effect of modulating miR-466g levels on SGK1 levels was examined
Project description:Vasopressin/cAMP/protein kinase A (PKA) signaling phosphorylates AQP2 water channels in renal collecting ducts to reabsorb water from urine for the prevention of further water loss. Lipopolysaccharide-responsive and beige-like anchor protein (LRBA) mediates vasopressin-induced AQP2 phosphorylation; therefore LRBA is essential for urinary concentration. LRBA is identified as the PKA substrates in a mouse cortical collecting duct principal cell line (mpkCCDcl4) whose phosphorylation levels are nearly perfectly correlated with those of AQP2. Although mouse LRBA contains several consensus PKA phosphorylation sites, their phosphorylation status in response to vasopressin remain unknown. Post-translational modification analysis revealed that RRDS1607 and RRIS2189 were phosphorylated by vasopressin.
Project description:Purpose: The goal of this study is to identify vasopressin-regulated genes in mouse kidney collecting duct cell line mpkCCD. To explore dynamic regulation of vasoressin-mediated transcriptional regulation, transcriptome of vasopressin-treated cells at four different time points (3h, 6h, 12h, and 24h) were profiled and analyzed. Methods: Total RNAs were isolated from vasopressin-treated mpkCCD cells at different time points (3h, 6h, 12h, and 24h). Three replicates for vehicle- or vasopressin-treated group were generated at each tested time point. cDNA libraries were prepared using a Nextera DNA library preparation protocol. The sequence reads from Illumina HiSeq3000 platform were qualified and quantified at the transcript level using Salmon (0.14.1). Differential expression analysis were performed using edgeR. Results: mRNA profiles of mouse kidney collecting duct mpkCCD cells treated with vasopressin analog (dDAVP) for four different time potins (3h, 6h, 12h, and 24h) were generated using an optimized RNA-Seq workflow. Transcript level quantification using a pseudo-alignment quantification method (Salmon) was performed to calculate transcript abundance in each sample. Then differential expression analysis identified the differentially expressed genes including Aqp2 gene at each time point comparison (dDAVP vs vehicle, FDR < 0.05). In addition, several new vasopressin-responsive genes that have not been elucidated before were identified. Conclusions: This study revealed dynamic changes of vasopressin-responsive gene expression within 24 hours in mouse kidney collecting duct cells. The results from time-course transcriptome profiling identified the known vasopressin-responsive genes and several novel gene that are regulated temporally.
Project description:miRNA plays a role as post-transcriptional regulator. However, miRNAs in the kidney collecting duct cell have not been well understood. So we aimed to profile miRNAs in the kidney inner medullary collecting duct (IMCD) cells, and to identify the vasopressin-responsive miRNAs in the kidney IMCD cells. The microarray assay revealed that relative expression of miRNAs in the kidney IMCD cells was changed by desmopressin (dDAVP) stimulation.
Project description:Murine inner medullary collecting duct cells were treated for 1 hour with vehicle (control) or aldosterone. Total RNA was isolated and used as template to generate the eventual cRNA target. The experiment was repeated a total of three times. Six cRNA samples, three control and three treated, were generated and used in a total of six hybridizations. The mineralocorticoid aldosterone is a major regulator of Na+ and acid-base balance and control of blood pressure. Although the long-term effects of aldosterone have been extensively studied, the early aldosterone-responsive genes remain largely unknown. Using DNA array technology, we have characterized changes in gene expression after 1 h of exposure to aldosterone in a mouse inner medullary collecting duct cell line, mIMCD-3. Results from three independent microarray experiments revealed that the expression of many transcripts was affected by aldosterone treatment. Northern blot analysis confirmed the upregulation of four distinct transcripts identified by the microarray analysis, namely, the serum and glucose-regulated kinase sgk, connective tissue growth factor, period homolog, and preproendothelin. Immunoblot analysis for preproendothelin demonstrated increased protein expression. Following the levels of the four transcripts over time showed that each had a unique pattern of expression, suggesting that the cellular response to aldosterone is complex. The results presented here represent a novel list of early aldosterone-responsive transcripts and provide new avenues for elucidating the mechanism of acute aldosterone action in the kidney.
Project description:Maximizing the potential of human kidney organoids for drug testing, regenerative medicine and to model development and disease requires addressing cell immaturity, the lack of a branching collecting system and the off target cell types. Here we establish methods to independently generate the two kidney progenitor cell populations – metanephric mesenchyme and ureteric bud. Combining these two progenitor cell types results in organoids with an improved branched collecting system. We also identified the hormones aldosterone and arginine vasopressin as critical to promote maturation of collecting duct cell types. The resulting organoids contain the full range of epithelial cells in the nephron, including principal and intercalated cells. By scRNA-seq, we demonstrate superior proximal tubule maturation and reduced off-target cell populations using this protocol.
Project description:Purpose: PKA plays a crucial role in vasopressin signaling of renal collecting duct cells. To understand regulation of mRNA expression mediated by vasopressin/PKA signaling, mRNA expression was profiled by RNA-Seq in double knockout cells (both PKA catalytic genes) generated from mouse cortical collecting duct mpkCCD cell line versus control lines with intact PKA expression. Methods: PKA double knockout (dKO) cell lines were generated from mouse cortical collecting duct mpkCCDc11 cells by CRISPR/Cas-9 genome editing method. For mRNA profiling using RNA-Seq analysis, three biological replicates of control (not mutated in PKA two catalytic subunits) cell lines and PKA double knockout cell lines were used. The reads uniquely mapped on GENCODE mouse gene set were analyzed with HOMER (v4.8) and edgeR (v3.10.5). Results and conclusion: About 40-50 million sequence reads per sample were sucessfully mapped in the mouse genome (GENCODE, GPCm38.p5). Among total transcripts of the mouse genome, 10,190 transcripts (cutoff: Counts Per Million > 4 by edgeR) were considered as genes expressed in the cell lines. In differential expression analysis by standard edgeR analysis, 354 transcripts were differentially expressed between control cell lines and PKA dKO cell lines (FDR < 0.05). We also identified nine genes that were markedly decreased in PKA dKO cell lines (log2 PKA dKO/Control < -2, FDR < 0.05) including aquaporin-2 (Aqp2) and two genes that were markedly increased in PKA dKO cell lines (log2 PKA dKO/Control > 2, FDR < 0.05). These results suggest PKA signaling is important for regulation of expression of a very limited number of genes in vasopressin-responsive renal collecting duct cells.
Project description:The Ca2+/CaM-dependent protein kinase 2-delta (CAMK2D) has been proposed to be involved in vasopressin signaling in the renal collecting duct, which controls water and salt excretion by the kidney. RNA sequancing and quantitative proteomics analyses identified the expression of multiple CAMK2 isoforms, with CAMK2D being the most abundant in collecting duct cells. To investigate the role of CAMK2D in regulating RNA expression in response to vasopressin signaling, the transcriptome of CRISPR/Cas9-mediated Camk2d knock-out mpkCCD cells was profiled using RNA-Seq in the presence of vasopressin analogue dDAVP.