No abstract

mpkCCDc14 cells, a polarized epithelial cell line derived from mouse kidney cortical collecting ducts, are known to express the vasopressin V2 receptor (V2R) and aquaporin-2 (AQP2) that are responsive to vasopressin. However, a low abundance of the endogenous AQP2 protein in the absence of vasopressin and heterogeneity of AQP2 protein abundance among the cultured cells may limit the further application of the cell line in AQP2 studies. To overcome the limitation, we aimed to establish mpkCCDc14 cells constitutively expressing V2R and AQP2 via CRISPR/Cas9-mediated genome engineering technology (i.e., V2R-AQP2 cells). 3′- and 5′-Junction PCR revealed that the V2R-AQP2 expression cassette with a long insert size (~2.2 kb) was correctly integrated. Immunoblotting revealed the expression of products of integrated Aqp2 genes. Cell proliferation rate and dDAVP-induced cAMP production were not affected by the knock-in of Avpr2 and Aqp2 genes. The AQP2 protein abundance was significantly higher in V2R-AQP2 cells compared with control mpkCCDc14 cells in the absence of dDAVP and the integrated AQP2 was detected. Immunocytochemistry demonstrated that V2R-AQP2 cells exhibited more homogenous and prominent AQP2 labeling intensity in the absence of dDAVP stimulation. Moreover, prominent AQP2 immunolabeling (both AQP2 and pS256-AQP2) in the apical domain of the genome-edited cells was observed in response to dDAVP stimulation, similar to that in the unedited control mpkCCDc14 cells. Taken together, mpkCCDc14 cells constitutively expressing V2R and AQP2 via genome engineering could be exploited for AQP2 studies.

Background and Aims Transcriptional coactivator with PDZ-binding motif (TAZ), a downstream effector of the hippo signaling pathway, regulates the expression of target genes by acting as a transcription cofactor. TAZ KO mice were known to display multicystic kidneys with polyuria. We aimed to study the role of TAZ in vasopressin-induced AQP2 regulation. Method 1) siRNA-mediated knockdown of TAZ in mpkCCDc11 cells; 2) RT-qPCR, semiquantitative immunoblotting, immunocytochemistry of AQP2; 3) Next Generation Sequencing (NGS) in mpkCCDc11 cells, the mouse collecting duct cell line. Results Endogenous AQP2 expression was induced in mpkCCDc11 cells by dDAVP (10−9M) treatment. After starvation for 24 h, dDAVP (10−9M) stimulation for 15 and 30 min induced a significant AQP2 translocation to the cell membrane. In contrast, the dDAVP-induced AQP2 translocation was markedly attenuated in the cells with siRNA-mediated TAZ knockdown (TAZ-KD), despite no changes in cAMP production. Phalloidin staining demonstrated the excessive stress fiber formation in the TAZ-KD. dDAVP (10−9 M) treatment for 24 h induced AQP2 mRNA (12,608 ± 177% of the control) and AQP2 protein abundance (270 ± 18%). In contrast, dDAVP-induced increase of AQP2 mRNA (273 ± 14% of the control) and protein abundance (99 ± 17%) was significantly attenuated in TAZ-KD. Unchanged TonEBP protein abundance was observed in TAZ-KD. NGS identified several potential AQP2 transcription factors (TF), and Klf6, Irf3, Cebpb, and Nr4a1 were selected based on previous in silico database. Among them, Nr4a1 was chosen for further studies due to a significant decrease in the mRNA expression levels in TAZ-KD, as demonstrated by RT-qPCR. Conclusion TAZ is likely to affect dDAVP-induced AQP2 trafficking. This is not mediated by the changes in cAMP/PKA pathway, but other non-canonical pathways are involved. TAZ could regulate AQP2 abundance, possibly via an interaction with several TF.

Background and Aims Poly(ADP-ribosy)lation (PARylation), which is mediated by poly(ADP-ribose) polymerases (PARPs), catalyzes the transfer of ADP-ribose from NAD+ molecules to acceptor proteins, regulates diverse cellular processes. Since PARP1 gene-deficient mice revealed an increase in urine volume, we aimed to examine the role of PARP1, the most abundant type of protein in the PARPs family, in the vasopressin-mediated AQP2 regulation. Method 1) Immunoblotting for PARP1 in mpkCCDc14 cells; 2) Pulldown assay of biotin-conjugated NAD+ and immunoprecipitation (IP) assay using poly(ADP-ribose) (PAR) antibody; 3) qRT-PCR and immunoblotting for AQP2; and 4) Bioinformatics for elucidating PARP1-interacting proteins in kidney collecting duct (CD) cells. Results Immunoblots showed that dDAVP treatment (10−9 M, 2 h, 6 h, 24 h, and 48 h) induced the cleavage of PARP1 (both 89 kDa and 25 kDa) in mpkCCDc14 cells. dDAVP treatment (10−9 M, 24 h) also increased the abundance of total PARylated proteins in biotin-NAD+ pulldown and IP assay of PAR in mpkCCDc14 cells. On the other hand, siRNA-mediated PARP1 knockdown significantly attenuated the dDAVP-induced mRNA and protein abundance, suggesting a role of PARP1 in AQP2 regulation. PARP1 cleavage induced by dDAVP was not changed under PARP1 knockdown, indicating that PARP1 cleavage is unlikely to be involved in AQP2 regulation. In contrast to PARP1 knockdown, PARP1 inhibitor (PJ34) did not reduce the dDAVP-induced AQP2 abundance, despite the significant decrease in the PARylation. The results suggest that dDAVP-regulated AQP2 expression is associated with PARP1 protein per se, but not with PARP1-mediated PARylation. Bioinformatics study revealed that 408 proteins interact with PARP1 in the kidney CD cells. Among them, 49 proteins were mapped on the vasopressin V2 receptor (V2R) signaling pathway. In particular, β-catenin, which is phosphorylated (S552) by dDAVP, was identified as the PARP1-interacting protein mapped on the V2R signaling. Immunoblotting demonstrated that siRNA-mediated knockdown of PARP1 was associated with decreased dDAVP-induced phosphorylation of β-catenin at S552 in mpkCCDc14 cells. Conclusion PARP1 is likely to play a role in vasopressin-mediated AQP2 regulation via the protein interaction with β-catenin rather than PARylation of proteins and/or PARP1 cleavage in the kidney CD cells.

This investigation was performed to determine the hydraulic conductivity coefficient and water holding capacity for a specified compaction forces which are the amount of mechanical energy applied to the porous granule (PG) volume. Most current specifications of minerals and perlite as growth media require to be compacted to a specified density, which in general is equivalent to a certain percentage of laboratory compaction. The water holding capacity of the saturated PG was very large at potential above -1 bar compared with perlite, but very little water remained below this value. The water holding capacity and hydraulic conductivity characteristics of graded PG amended with the ground coir less than 2 mm in diameter were also determined from pressure outflow data. The saturated hydraulic conductivity of the saturated and compacted PG was slightly lower by more than one tenth order of magnitude at equal matric potentials of perlite, but when expressed on the basis of equal water deficits, the conductivity of PG was higher at all but the smallest deficits than those of perlite.