Bayesian Identification of Candidate Transcription Factors for Regulation of Aqp2 Gene Expression

Aqp2 gene transcription is strongly regulated by vasopressin in the renal collecting duct. However, the transcription factors (TFs) responsible for regulation of expression of aquaporin-2 (Aqp2) remain largely unknown. We used Bayes' Theorem to integrate several -omics datasets to stratify the 1344 TFs present in the mouse genome with regard to probabilities of regulating Aqp2 gene transcription. Also, we carried out new RNA-Seq experiments mapping the time course of vasopressin-induced changes in the transcriptome of mpkCCD cells to identify TFs that change in tandem with Aqp2. The analysis identified 17 TFs out of 1344 that are most likely to be involved in regulation of Aqp2 gene transcription. These TFs included eight that have been proposed in prior studies to play a role in Aqp2 regulation, viz. Cebpb, Elf1, Elf3, Ets1, Jun, Junb, Nfkb1, and Sp1. The remaining nine represent new candidates for future studies (Atf1, Irf3, Klf5, Klf6, Mef2d, Nfyb, Nr2f6, Stat3, Nr4a1). Conspicuously absent is CREB (Creb1), which has been widely proposed to mediate vasopressin-induced regulation of Aqp2 gene transcription. Instead, another CREB-like TF, Atf1, ranked fourth among all transcription factors. RNA-Seq time course experiments showed a rapid increase in Aqp2 mRNA, within 3 hours of vasopressin exposure. This response was matched by an equally rapid increase in the abundance of the mRNA coding for Cebpb, which we have shown by ChIP-seq studies to bind downstream from the Aqp2 gene. The identified TFs provide a roadmap for future studies to understand regulation of Aqp2 gene expression.

Identification of β-catenin-interacting proteins in nuclear fractions of native rat collecting duct cells

The gene encoding the aquaporin-2 water channel is regulated transcriptionally in response to vasopressin. In the renal collecting duct, vasopressin stimulates the nuclear translocation and phosphorylation (at Ser552) of β-catenin, a multifunctional protein that acts as a transcriptional coregulator in the nucleus. The purpose of this study was to identify β-catenin-interacting proteins that might be involved in transcriptional regulation in rat inner medullary collecting duct (IMCD) cells, using experimental and computational approaches. We used a standard chromatin immunoprecipitation procedure coupled to mass spectrometry (ChIP-MS) in a nuclear fraction isolated from rat IMCD suspensions. Over four biological replicates, we reproducibly identified 43 β-catenin-binding proteins, including several known β-catenin-binding partners as well as novel interacting proteins. Multiple proteins involved in transcriptional regulation were identified (Taf1, Jup, Tdrd3, Cdh1, Cenpj, and several histones). Many of the identified β-catenin-binding partners were found in prior studies to translocate to the nucleus in response to vasopressin. There was only one DNA-binding transcription factor (TF), specifically Taf1, part of the RNA-polymerase II preinitiation complex. To identify sequence-specific TFs that might interact with β-catenin, Bayes’ theorem was used to integrate data from several information sources. The analysis identified several TFs with potential binding sites in the Aqp2 gene promoter that could interact with β-catenin in the regulation of Aqp2 gene transcription, specifically Jun, Junb, Jund, Atf1, Atf2, Mef2d, Usf1, Max, Pou2f1, and Rxra. The findings provide information necessary for modeling the transcriptional response to vasopressin.

Liver X receptor β increases aquaporin 2 protein level via a posttranscriptional mechanism in renal collecting ducts

Liver X receptors (LXRs) including LXRα and LXRβ are nuclear receptor transcription factors and play an important role in lipid and glucose metabolism. It has been previously reported that mice lacking LXRβ but not LXRα develop a severe urine concentrating defect, likely via a central mechanism. Here we provide evidence that LXRβ regulates water homeostasis through increasing aquaporin 2 (AQP2) protein levels in renal collecting ducts. LXRβ−/− mice exhibited a reduced response to desmopressin (dDAVP) stimulation, suggesting that the diabetes insipidus phenotype is of both central and nephrogenic origin. AQP2 protein abundance in the renal inner medulla was significantly reduced in LXRβ−/− mice but with little change in AQP2 mRNA levels. In vitro studies showed that AQP2 protein levels were elevated upon LXR agonist treatment in both primary cultured mouse inner medullary duct cells (mIMCD) and the mIMCD3 cell line with stably expressed AQP2. In addition, LXR agonists including TO901317 and GW3965 failed to induce AQP2 gene transcription but diminished its protein ubiquitination in primary cultured mIMCD cells, thereby inhibiting its degradation. Moreover, LXR activation-induced AQP2 protein expression was abolished by the protease inhibitor MG132 and the ubiquitination-deficient AQP2 (K270R). Taken together, the present study demonstrates that activation of LXRβ increases AQP2 protein levels in the renal collecting ducts via a posttranscriptional mechanism. As such, LXRβ represents a key regulator of body water homeostasis.

Molecular mechanisms regulating aquaporin-2 in kidney collecting duct

The kidney collecting duct is an important renal tubular segment for regulation of body water homeostasis and urine concentration. Water reabsorption in the collecting duct principal cells is controlled by vasopressin, a peptide hormone that induces the osmotic water transport across the collecting duct epithelia through regulation of water channel proteins aquaporin-2 (AQP2) and aquaporin-3 (AQP3). In particular, vasopressin induces both intracellular translocation of AQP2-bearing vesicles to the apical plasma membrane and transcription of the Aqp2 gene to increase AQP2 protein abundance. The signaling pathways, including AQP2 phosphorylation, RhoA phosphorylation, intracellular calcium mobilization, and actin depolymerization, play a key role in the translocation of AQP2. This review summarizes recent data demonstrating the regulation of AQP2 as the underlying molecular mechanism for the homeostasis of water balance in the body.