Vasopressin (AVP) and the V2R specific agonist dDAVP 7 day infusion into Aquaporin 1 knockout animals
ABSTRACT: Vasopressin, the antidiuretic hormone, acts on the renal collecting duct. In this experiment both vasopressin (AVP) and the V2R specific agonist dDAVP were infused into Aquaporin 1 knockout animals for 7 days. The aim of the experiment was to identify genes increased by vasopressin receptors in the renal medullary collecting ducts, in the absence of an increase in renal medullary osmolarity (the AQP1 knockouts are concentrating mechanism knockouts). All experiments used inner medulla tissue for the RNA isolation. Hybridizations were performed that compared kidney inner medulla total RNA from three control mice against kidney medulla total RNA from 3 mice infused with either arginine vasopressin (AVP) or des-amino-D-arginine vasopressin (dDAVP).
Project description:Activation of V2 receptors (V2R) during antidiuresis increases the permeability of the inner medullary collecting duct to urea and water. Extracellular osmolality is elevated as the concentrating capacity of the kidney increases. Osmolality is known to contribute to the regulation of collecting duct water (aquaporin-2; AQP2) and urea transporter (UT-A1, UT-A3) regulation. AQP1KO mice are a concentrating mechanism knockout, a defect attributed to the loss of high interstitial osmolality. A V2R-specific agonist, deamino-8-D-arginine vasopressin (dDAVP), was infused into wild-type and AQP1KO mice for 7 days. UT-A1 mRNA and protein abundance were significantly increased in the medullas of wild-type and AQP1KO mice following dDAVP infusion. The mRNA and protein abundance of UT-A3, the basolateral urea transporter, was significantly increased by dDAVP in both wild-type and AQP1KO mice. Semiquantitative immunoblots revealed that dDAVP infusion induced a significant increase in the medullary expression of the endoplasmic reticulum (ER) chaperone GRP78. Immunofluorescence studies demonstrated that GRP78 expression colocalized with AQP2 in principal cells of the papillary tip of the renal medulla. Using immunohistochemistry and immunogold electron microscopy, we demonstrate that vasopressin induced a marked apical targeting of GRP78 in medullary principal cells. Urea-sensitive genes, GADD153 and ATF4 (components of the ER stress pathway), were significantly increased in AQP1KO mice by dDAVP infusion. These findings strongly support an important role of vasopressin in the activation of an ER stress response in renal collecting duct cells, in addition to its role in activating an increase in UT-A1 and UT-A3 abundance.
Project description:Arginine vasopressin (AVP) enhances water reabsorption in the renal collecting duct by vasopressin V? receptor (V?R)-mediated activation of adenylyl cyclase (AC), cAMP-promoted phosphorylation of aquaporin-2 (AQP2), and increased abundance of AQP2 on the apical membrane. Multiple isoforms of adenylate cyclase exist, and the roles of individual AC isoforms in water homeostasis are not well understood. Here, we found that levels of AC6 mRNA, the most highly expressed AC isoform in the inner medulla, inversely correlate with fluid intake. Moreover, mice lacking AC6 had lower levels of inner medullary cAMP, reduced abundance of phosphorylated AQP2 (at both serine-256 and serine-269), and lower urine osmolality than wild-type mice. Water deprivation or administration of the V?R agonist dDAVP did not increase urine osmolality of AC6-deficient mice to the levels of wild-type mice. Furthermore, AC6-deficient mice lacked dDAVP-promoted inner medullary cAMP formation and phosphorylation of serine-269 and had attenuated increases in both phosphorylation of serine-256 and apical membrane AQP2 trafficking. In summary, AC6 expression determines inner medullary cAMP formation and AQP2 phosphorylation and trafficking, the absence of which causes nephrogenic diabetes insipidus.
Project description:The action of vasopressin in rodent collecting ducts to regulate water permeability depends in part on increases in phosphorylation of the water channel aquaporin-2 (AQP2) at three sites: Ser256, Ser264, and Ser269. Previous studies of AQP2 phosphorylation have depended largely on qualitative data using protein mass spectrometry and phospho-specific antibodies. Here, we use a new method employing phospho-specific antibodies to determine the percentage of total AQP2 phosphorylated at each site in the presence and absence of the V2-receptor-selective vasopressin analog dDAVP in rat renal inner medullary collecting duct (IMCD) and cultured mpkCCD cells. Phosphorylation of Ser269, a site previously implicated in plasma membrane retention, was found to increase from 3 to 26% of total AQP2 in rat IMCD cells following dDAVP. Quantification of immunogold labeling of the opposite kidneys from the same rats estimated that 11% of total AQP2 is present in the apical plasma membrane (APM) without injection of dDAVP and 25% is present in the APM after dDAVP. Surprisingly, the baseline level of Ser256 phosphorylation was constitutively high, and there was no increase with dDAVP (confirmed in 2 more sets of rats). In general, Ser264 phosphorylation remained below 5% of total. The pattern of response was similar in cultured mpkCCD cells (large increase in Ser269 phosphorylation following dDAVP, but constitutively high levels of Ser256 phosphorylation). We suggest from these studies that Ser269 phosphorylation may be a more consistent indicator of vasopressin action and AQP2 membrane abundance than is Ser256 phosphorylation.
Project description:Pax2 is a transcription factor that is crucial for kidney development, and it is also expressed in the normal adult kidney, where its physiological function is unknown. In the present study, we find by cDNA microarray analysis that Pax2 expression in second-passage mouse inner-medullary epithelial cells is increased by a high NaCl concentration, which is significant because NaCl levels are normally high in the inner medulla in vivo, and varies with urinary concentration. Furthermore, a high NaCl concentration increases Pax2 mRNA and protein expression in mouse inner medullary collecting duct (mIMCD3) cells, and its transcriptional activity. Pax2 mRNA and protein expression is high in normal adult mouse renal inner medulla but much lower in renal cortex. Pax2 protein is present in collecting duct cells in both renal medulla and cortex and in thin descending limbs of Henle's loop in inner medulla. Treating Brattleboro rats with desamino-Cys-1,d-Arg-8 vasopressin, which increases inner-medullary NaCl concentration, causes a 4-fold increase in inner-medullary Pax2 protein. Treatment with furosemide, which decreases inner-medullary NaCl, reduces inner-medullary Pax2 mRNA and protein. Pax2-specific short interfering RNA increases high NaCl concentration-induced activation of caspase-3 and apoptotic bodies in mIMCD3 cells. We thus conclude that (i) Pax2 is expressed in normal renal medulla, (ii) its expression is regulated there by the normally high and variable NaCl concentration, and (iii) it protects renal medullary cells from high NaCl concentration-induced apoptosis.
Project description:AIM: Aquaporin-2 (AQP2) is a vasopressin-regulated water channel located in the collecting tubule and collecting duct cells of mammalian kidney. The aim of this study is to investigate whether PKC? plays a role in vasopressin-induced AQP2 trafficking in mouse inner medullary collecting duct 3 (mIMCD3) cells. METHODS: AQP2-mIMCD3 stable cell line was constructed by transfection of mouse inner medullary collecting duct 3 (mIMCD3) cells with AQP2-GFP construct. Then the cells were transfected with PKC? shRNA, PKC? A/25E, or PKC? scrambled shRNA. The expression levels of PKC?, AQP2, and phospho-S256-AQP2 were analyzed using Western blot. The interaction between AQP2 and PKC? was examined using immunoprecipitation. The distribution of AQP2 and microtubules was studied using immunocytochemistry. The AQP2 trafficking was examined using the biotinylation of surface membranes. RESULTS: Treatment of AQP2-mIMCD3 cells with 100 ?mol/L of 1-desamino-8-D-arginine vasopressin (DdAVP) for 30 min stimulated the translocation of AQP2 from the cytoplasm to plasma membrane through influencing the microtubule assembly. Upregulation of active PKC? by transfection with PKC? A/25E plasmids resulted in de-polymerization of ?-tubulin and redistributed AQP2 in the cytoplasm. Down-regulation of PKC? by PKC? shRNA partially inhibited DdAVP-stimulated AQP2 trafficking without altering ?-tubulin distribution. Although 100 ?mol/L of DdAVP increased AQP2 phosphorylation at serine 256, down-regulation of PKC? by PKC? shRNA did not influence DdAVP-induced AQP2 phosphorylation, suggesting that AQP2 phosphorylation at serine 256 was independent of PKC?. Moreover, PKC? did not physically interact with AQP2 in the presence or absence of DdAVP. CONCLUSION: Our results suggested that PKC? regulates AQP2 trafficking induced by DdAVP via microtubule assembly.
Project description:The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.
Project description:Classic methods for delivery of agents to specific organs are technically challenging and causes superfluous stress. The current study describes a method using programmable, implantable peristaltic pumps to chronically deliver drugs in vivo, while allowing animals to remain undisturbed for accurate physiological measurements. In this study, two protocols were used to demonstrate accurate drug delivery to the renal medulla. First, the vasopressin receptor-2 agonist, dDAVP, was delivered to the renal medulla resulting in a significant increase in water retention, urine osmolality and aquaporin-2 expression and phosphorylation. Second, in a separate group of rats, the histone deacetylase (HDAC) inhibitor, MS275, was delivered to the renal medulla. HDAC inhibition resulted in a significant increase in histone H3-acetylation, the hallmark for histone deacetylase inhibition. However, this was confined to the medulla, as the histone H3-acetylation was similar in the cortex of vehicle and MS275 infused rats, suggesting targeted drug delivery without systemic spillover. Thus, implantable, peristaltic pumps provide a number of benefits compared to externalized chronic catheters and confer specific delivery to target organs.
Project description:Vasopressin regulates water excretion through effects on the renal collecting duct. Vasopressin signaling in the inner medullary collecting duct (IMCD) is mediated by V2 receptor occupation coupled to the generation of cyclic AMP. Here, we employ a "systems" approach to analysis of vasopressin signaling. The objective is to investigate roles of activation of the Akt and ERK1/2 MAP kinase pathways, as well as Ca2+ mobilization, in IMCD cells isolated from rat kidney. The V2 receptor-selective vasopressin analog dDAVP increased the state of Akt activation (increased phosphorylation at T308 and S473) and decreased the state of ERK1/2 activation (decreased phosphorylation at T202 and Y204). Akt activation was blocked by an inhibitor of PI3K, LY294002. In microdissected IMCD segments, nonperiodic spike-like increases in intracellular Ca2+ (FLUO-4) were accelerated by vasopressin. Chelation of Ca2+ or calmodulin inhibition markedly decreased Akt phosphorylation. Decreased ERK1/2 phosphorylation was associated with a decrease in MEK1/2 phosphorylation and an increase in c-Raf phosphorylation at S259 (an inhibitory site). Based on the current findings integrated with previous findings in the IMCD, we now report a 33-node vasopressin signaling network involved in vasopressin regulation of IMCD function.
Project description:The control of renal water excretion occurs in part by regulation of transcription in response to vasopressin in cells of the collecting duct. A systems biology-based approach to understanding transcriptional control in renal collecting duct cells depends on knowledge of what transcription factors and other regulatory proteins are present in the cells' nuclei. The goal of this article is to report comprehensive proteomic profiling of cellular fractions enriched in nuclear proteins from native inner medullary collecting duct (IMCD) cells of the rat. Multidimensional separation procedures and state-of-the art protein mass spectrometry produced 18 GB of spectral data that allowed the high-stringency identification of 5,048 proteins in nuclear pellet (NP) and nuclear extract (NE) fractions of biochemically isolated rat IMCD cells (URL: https://helixweb.nih.gov/ESBL/Database/IMCD_Nucleus/). The analysis identified 369 transcription factor proteins out of the 1,371 transcription factors coded by the rat genome. The analysis added 1,511 proteins to the recognized proteome of rat IMCD cells, now amounting to 8,290 unique proteins. Analysis of samples treated with the vasopressin analog dDAVP (1 nM for 30 min) or its vehicle revealed 99 proteins in the NP fraction and 88 proteins in the NE fraction with significant changes in spectral counts (Fisher exact test, P < 0.005). Among those altered by vasopressin were seven distinct histone proteins, all of which showed decreased abundance in the NP fraction, consistent with a possible effect of vasopressin to induce chromatin remodeling. The results provide a data resource for future studies of vasopressin-mediated transcriptional regulation in the renal collecting duct.
Project description:In angiotensin II (Ang II)-dependent hypertensive rats there is an increased expression of proximal tubule angiotensinogen (AGT), collecting duct renin and angiotensin converting enzyme (ACE), which contributes to intratubular Ang II formation. Ang II acts on Ang II type 1 receptors promoting sodium retention and vasoconstriction. However concurrently, the ACE2-Ang-(1-7) axis and the expression of kallikrein and medullary prostaglandins counteract the effects of Ang II, promoting natriuresis and vasodilation. Human studies demonstrate that dietary potassium (K+) intake lowers blood pressure. In this report we evaluate the expression of AGT, ACE, medullary prorenin/renin, ACE2, kallikrein and cyclooxygenase-2 (COX-2) in Ang II-infused rats fed with high K+ diet (2%) for 14 days. Dietary K+ enhances diuresis in non-infused and in Ang II-infused rats. The rise in systolic blood pressure in Ang II-infused rats was attenuated by dietary K+. Ang II-infused rats showed increased renal protein levels of AGT, ACE and medullary prorenin and renin. This effect was attenuated in the Ang II + K+ group. Ang II infusion decreased ACE2 compared to the control group; however, K+ diet prevented this effect in the renal medulla. Furthermore, medullary COX-2 was dramatically induced by K+ diet in non-infused and in Ang II infused rats. Dietary K+ greatly increased kallikrein immunostaining in normotensive rats and in Ang II-hypertensive rats. These results indicate that a high K+ diet attenuates Ang II-dependent hypertension by preventing the induction of ACE, AGT and collecting duct renin and by enhancing medullary COX-2 and ACE2 protein expression in the kidney.