Rat Inner Medullary Collecting Duct Research Articles

Phosphatases Decrease Water and Urea Permeability in Rat Inner Medullary Collecting Ducts.

We previously showed that the phosphatases PP1/PP2A and PP2B dephosphorylate the water channel, AQP2, suggesting their role in water reabsorption. In this study, we investigated whether protein phosphatase 2A (PP2A) and protein phosphatase 2B (PP2B or calcineurin), which are present in the inner medullary collecting duct (IMCD), are regulators of urea and water permeability. Inhibition of calcineurin by tacrolimus increased both basal and vasopressin-stimulated osmotic water permeability in perfused rat IMCDs. However, tacrolimus did not affect osmotic water permeability in the presence of aldosterone. Inhibition of PP2A by calyculin increased both basal and vasopressin-stimulated osmotic water permeability, and aldosterone reversed the increase by calyculin. Previous studies showed that adrenomedullin (ADM) activates PP2A and decreases osmotic water permeability. Inhibition of PP2A by calyculin prevented the ADM-induced decrease in water reabsorption. ADM reduced the phosphorylation of AQP2 at serine 269 (pSer269 AQP2). Urea is linked to water reabsorption by building up hyperosmolality in the inner medullary interstitium. Calyculin increased urea permeability and phosphorylated UT-A1. Our results indicate that phosphatases regulate water reabsorption. Aldosterone and adrenomedullin decrease urea or osmotic water permeability by acting through calcineurin and PP2A, respectively. PP2A may regulate water reabsorption by dephosphorylating pSer269, AQP2, and UT-A1.

Psychotropic drugs upregulate aquaporin-2 via vasopressin-2 receptor/cAMP/protein kinase A signaling in inner medullary collecting duct cells.

Psychotropic drugs may be associated with hyponatremia, but an understanding of how they induce water retention in the kidney remains elusive. Previous studies have postulated that they may increase vasopressin production in the hypothalamus without supporting evidence. In this study, we investigated the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis using haloperidol, sertraline, and carbamazepine. Haloperidol, sertraline, or carbamazepine were treated in inner medullary collecting duct (IMCD) suspensions and primary cultured IMCD cells prepared from male Sprague-Dawley rats. The responses of intracellular cAMP production, aquaporin-2 (AQP2) protein expression and localization, vasopressin-2 receptor (V2R) and AQP2 mRNA, and cAMP-responsive element-binding protein (CREB) were tested with and without tolvaptan and the protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. In IMCD suspensions, cAMP production was increased by haloperidol, sertraline, or carbamazepine and was relieved by tolvaptan cotreatment. In primary cultured IMCD cells, haloperidol, sertraline, or carbamazepine treatment increased total AQP2 and decreased phosphorylated Ser261-AQP2 protein expression. Notably, these responses were reversed by cotreatment with tolvaptan or a PKA inhibitor. AQP2 membrane trafficking was induced by haloperidol, sertraline, or carbamazepine and was also blocked by cotreatment with tolvaptan or a PKA inhibitor. Furthermore, upregulation of V2R and AQP2 mRNA and phosphorylated CREB was induced by haloperidol, sertraline, or carbamazepine and was blocked by tolvaptan cotreatment. We conclude that, in the rat IMCD, psychotropic drugs upregulate AQP2 via V2R-cAMP-PKA signaling in the absence of vasopressin stimulation. The vasopressin-like action on the kidney appears to accelerate AQP2 transcription and dephosphorylate AQP2 at Ser261.NEW & NOTEWORTHY It is unclear whether antipsychotic drugs can retain water in the kidney in the absence of vasopressin. This study demonstrates that haloperidol, sertraline, and carbamazepine can produce nephrogenic syndrome of inappropriate antidiuresis because they directly upregulate vasopressin-2 receptor and aquaporin-2 (AQP2) via cAMP/PKA signaling. We showed that, in addition to AQP2 trafficking, AQP2 protein abundance was rapidly increased by treatment with antipsychotic drugs in association with dephosphorylation of AQP2 at Ser261 and accelerated AQP2 transcription.

Adrenomedullin Inhibits Osmotic Water Permeability in Rat Inner Medullary Collecting Ducts.

Adrenomedullin (ADM) is a vasodilator that causes natriuresis and diuresis. However, the direct effect of ADM on osmotic water permeability in the rat inner medullary collecting duct (IMCD) has not been tested. We investigated whether ADM and its ADM receptor components (CRLR, RAMP2, and 3) are expressed in rat inner medulla (IM) and whether ADM regulates osmotic water permeability in isolated perfused rat IMCDs. The mRNAs of ADM, CRLR, and RAMP2 and 3 were detected in rat IM. Abundant protein of CRLR and RAMP3 were also seen but RAMP2 protein level was extremely low. Adding ADM (100 nM) to the bath significantly decreased osmotic water permeability. ADM significantly decreased aquaporin-2 (AQP2) phosphorylation at Serine 256 (pS256) and increased it at Serine 261 (pS261). ADM significantly increased cAMP levels in IM. However, inhibition of cAMP by SQ22536 further decreased ADM-attenuated osmotic water permeability. Stimulation of cAMP by roflumilast increased ADM-attenuated osmotic water permeability. Previous studies show that ADM also stimulates phospholipase C (PLC) pathways including protein kinase C (PKC) and cGMP. We tested whether PLC pathways regulate ADM-attenuated osmotic water permeability. Blockade of either PLC by U73122 or PKC by rottlerin significantly augmented the ADM-attenuated osmotic water permeability and promoted pS256-AQP2 but did change pS261-AQP2. Inhibition of cGMP by L-NAME did not change AQP2 phosphorylation. In conclusion, ADM primarily binds to the CRLR-RAMP3 receptor to initiate signaling pathways in the IM. ADM reduced water reabsorption through a PLC-pathway involving PKC. ADM-attenuated water reabsorption may be related to decreased trafficking of AQP2 to the plasma membrane. cAMP is not involved in ADM-attenuated osmotic water permeability.

Aldosterone Decreases Vasopressin-Stimulated Water Reabsorption in Rat Inner Medullary Collecting Ducts.

Aldosterone indirectly regulates water reabsorption in the distal tubule by regulating sodium reabsorption. However, the direct effect of aldosterone on vasopressin-regulated water and urea permeability in the rat inner medullary collecting duct (IMCD) has not been tested. We investigated whether aldosterone regulates osmotic water permeability in isolated perfused rat IMCDs. Adding aldosterone (500 nM) to the bath significantly decreased osmotic water permeability in the presence of vasopressin (50 pM) in both male and female rat IMCDs. Aldosterone significantly decreased aquaporin-2 (AQP2) phosphorylation at S256 but did not change it at S261. Previous studies show that aldosterone can act both genomically and non-genomically. We tested the mechanism by which aldosterone attenuates osmotic water permeability. Blockade of gene transcription with actinomycin D did not reverse aldosterone-attenuated osmotic water permeability. In addition to AQP2, the urea transporter UT-A1 contributes to vasopressin-regulated urine concentrating ability. We tested aldosterone-regulated urea permeability in vasopressin-treated IMCDs. Blockade of gene transcription did not reverse aldosterone-attenuated urea permeability. In conclusion, aldosterone directly regulates water reabsorption through a non-genomic mechanism. Aldosterone-attenuated water reabsorption may be related to decreased trafficking of AQP2 to the plasma membrane. There may be a sex difference apparent in the inhibitory effect of aldosterone on water reabsorption in the inner medullary collecting duct. This study is the first to show a direct effect of aldosterone to inhibit vasopressin-stimulated osmotic water permeability and urea permeability in perfused rat IMCDs.

Mechanical Stimulation of Piezo1 Induced Protein Expression and Intracellular Trafficking of Aquaporin‐2 in IMCD Cells

Pressure and shear stress‐induced stretch of tubular epithelial cells is relevant to both physiological and diseased kidney. Piezo1 is a mechanically‐activated cation channel stimulated by membrane tension and stretch; it is highly expressed in the inner medulla of the kidney. Aquaporin‐2 (AQP2) is located in the principal cells of the collecting ducts in the kidney, which is a key aquaporin determining final urine output. The current study aims to investigate whether activation of Piezo1 induces AQP2 expression in the collecting duct principal cells. In human kidney biopsy specimen, Piezo1 was found expressed mainly in the collecting ducts where it co‐localized with AQP2. Upon treating primary cultured rat inner medullary collecting duct (IMCD) cells with Piezo1 activator Yoda1 (0.01μM to 1μM), the protein expression of AQP2 was markedly increased. Activation of Piezo1 by Yoda1 was also associated with intracellular F‐actin depolymerization and dramatically increased accumulation of AQP2 on the apical plasma membrane of IMCD cells. Upregulation of AQP2 protein was also observed when Piezo1 was stimulated with mechanical cyclic tensile strain at 5% elongation, 1Hz for 3hour in IMCD cells, which was markedly inhibited by GsMTx4 (0.1μM to 5μM), a Piezo1 antagonist. Both stretch and Yoda1 treatment induced elevation of intracellular calcium levels and upregulation of calpain2 protein expression (calcium‐activated non‐lysosomal thiol‐proteases) as well as phosphorylation of Akt protein in rat IMCD cells. Dehydration for 24h (which is presumably leading to the swelling of principal cells, Chou CL, Am J Physiol Renal Physiol, 2008) in mice was associated with increased levels of piezo1 mRNA and protein expression in the kidney inner medulla. In conclusion, activation of Piezo1 by mechanical stretch or activators increased protein expression and intracellular trafficking of AQP2 in rat IMCD cells, which was associated with F‐actin depolymerizing and elevated intracellular calcium levels. These studies indicate that piezo1 may play a functional role in the regulation of urinary osmolarity in pressure‐induced stretch of principal cells relevant to both physiological and pathophysiological conditions.

Claudin 19 Is Regulated by Extracellular Osmolality in Rat Kidney Inner Medullary Collecting Duct Cells.

The inner medullary collecting duct (IMCD) is subject to severe changes in ambient osmolality and must either allow water transport or be able to seal the lumen against a very high osmotic pressure. We postulate that the tight junction protein claudin-19 is expressed in IMCD and that it takes part in epithelial adaptation to changing osmolality at different functional states. Presence of claudin-19 in rat IMCD was investigated by Western blotting and immunofluorescence. Primary cell culture of rat IMCD cells on permeable filter supports was performed under different osmotic culture conditions and after stimulation by antidiuretic hormone (AVP). Electrogenic transepithelial transport properties were measured in Ussing chambers. IMCD cells cultivated at 300 mosm/kg showed high transepithelial resistance, a cation selective paracellular pathway and claudin-19 was mainly located in the tight junction. Treatment by AVP increased cation selectivity but did not alter transepithelial resistance or claudin-19 subcellular localization. In contrast, IMCD cells cultivated at 900 mosm/kg had low transepithelial resistance, anion selectivity, and claudin-19 was relocated from the tight junctions to intracellular vesicles. The data shows osmolality-dependent transformation of IMCD epithelium from tight and sodium-transporting to leaky, with claudin-19 expression in the tight junction associated to tightness and cation selectivity under low osmolality.

Bile Acid G Protein-Coupled Membrane Receptor TGR5 Modulates Aquaporin 2-Mediated Water Homeostasis.

The bile acid-activated receptors, including the membrane G protein-coupled receptor TGR5 and nuclear farnesoid X receptor (FXR), have roles in kidney diseases. In this study, we investigated the role of TGR5 in renal water handling and the underlying molecular mechanisms. We used tubule suspensions of inner medullary collecting duct (IMCD) cells from rat kidneys to investigate the effect of TGR5 signaling on aquaporin-2 (AQP2) expression, and examined the in vivo effects of TGR5 in mice with lithium-induced nephrogenic diabetes insipidus (NDI) and Tgr5 knockout (Tgr5-/-) mice. Activation of TGR5 by lithocholic acid (LCA), an endogenous TGR5 ligand, or INT-777, a synthetic TGR5-specific agonist, induced AQP2 expression and intracellular trafficking in rat IMCD cells via a cAMP-protein kinase A signaling pathway. In mice with NDI, dietary supplementation with LCA markedly decreased urine output and increased urine osmolality, which was associated with significantly upregulated AQP2 expression in the kidney inner medulla. Supplementation with endogenous FXR agonist had no effect. In primary IMCD suspensions from lithium-treated rats, treatment with INT-767 (FXR and TGR5 dual agonist) or INT-777, but not INT-747 (FXR agonist), increased AQP2 expression. Tgr5-/- mice exhibited an attenuated ability to concentrate urine in response to dehydration, which was associated with decreased AQP2 expression in the kidney inner medulla. In lithium-treated Tgr5-/- mice, LCA treatment failed to prevent reduction of AQP2 expression. TGR5 stimulation increases renal AQP2 expression and improves impaired urinary concentration in lithium-induced NDI. TGR5 is thus involved in regulating water metabolism in the kidney.

Proteomic determination of the lysine acetylome and phosphoproteome in the rat native inner medullary collecting duct.

Phosphorylation and lysine (K)-acetylation are dynamic posttranslational modifications of proteins. Previous proteomic studies have identified over 170,000 phosphorylation sites and 15,000 K-acetylation sites in mammals. We recently reported that the inner medullary collecting duct (IMCD), which functions in the regulation of water-reabsorption, via the actions of vasopressin, expresses many of the enzymes that can modulated K-acetylation. The purpose of this study was to determine the K-acetylated or phosphorylated proteins expressed in IMCD cells. Second we questioned whether vasopressin V2 receptor activation significantly affects the IMCD acetylome or phosphoproteome? K-acetylated or serine-, threonine-, or tyrosine-phosphorylated peptides were identified from native rat IMCDs by proteomic analysis with four different enzymes (trypsin, chymotrypsin, ASP-N, or Glu-C) to generate a high-resolution proteome. K-acetylation was identified in 431 unique proteins, and 64% of the K-acetylated sites were novel. The acetylated proteins were expressed in all compartments of the cell and were enriched in pathways including glycolysis and vasopressin-regulated water reabsorption. In the vasopressin-regulated water reabsorption pathway, eight proteins were acetylated, including the novel identification of the basolateral water channel, AQP3, acetylated at K282; 215 proteins were phosphorylated in this IMCD cohort, including AQP2 peptides that were phosphorylated at four serines: 256, 261, 264, and 269. Acute dDAVP did not significantly affect the IMCD acetylome; however, it did significantly affect previously known vasopressin-regulated phosphorylation sites. In conclusion, presence of K-acetylated proteins involved in metabolism, ion, and water transport in the IMCD points to multiple roles of K-acetylation beyond its canonical role in transcriptional regulation.

Proteomic Determination of the Rat Native Inner Medullary Collecting Duct Lysine Acetylome and Phosphoproteome

Phosphorylation and lysine (K)‐acetylation are dynamic post‐translational modifications of proteins. K‐acetylation of histone proteins is classically regarded as a critical regulatory pathway for “opening” chromatin structure and promoting transcription. Previous proteomic studies have identified over 170,000 phosphorylation sites, and greater than 15,000 K‐acetylation sites of mammalian proteins. We recently reported that the inner medullary collecting duct (IMCD), which functions in the regulation of vasopressin‐dependent water‐reabsorption, expresses many of the enzymes (acetyltransferases and deacetylases) that can modulate K‐acetylation. The purpose of this study was to determine the K‐acetylated or phosphorylated proteins expressed in IMCD cells. Second, we asked whether vasopressin V2 receptor activation significantly affects the IMCD acetylome or phosphoproteome? K‐acetylated or serine‐, threonine‐, or tyrosine‐phosphorylated peptides were identified from native rat IMCDs using proteomic analysis with four different enzymes (trypsin, chymotrypsin, ASP‐N, or Glu‐C) to generate a high‐resolution proteome. We identified 709 K‐acetylated sites that mapped to 431 unique proteins. Of these K‐acetylated sites 583 (64%) were novel. The K‐acetylated proteins were expressed in all compartments of the cell, and were enriched in pathways including glycolysis (12 proteins), the TCA cycle (8 proteins), and vasopressin‐regulated water reabsorption (8 proteins). In these same IMCD cells, we detected 312 phosphorylated sites that mapped to 215 proteins, including AQP2 peptides that were phosphorylated at 4 serines: 256, 261, 264, and 269. Moreover, we identified 155 novel phosphorylation sites. Acute dDAVP did not significantly affect the IMCD acetylome; however it did significantly affect previously known vasopressin‐regulated phosphorylation sites. In conclusion, presence of K‐acetylated proteins involved in metabolism, ion and water transport in the IMCD points to multiple roles of K‐acetylation beyond its canonical role in transcriptional regulation.Support or Funding InformationK01‐DK‐105038 to K. A. H., projects ZIA‐HL‐001285 and ZIA‐HL‐006129, M. A. K.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of UT-A1- and AQP2-interacting proteins in rat inner medullary collecting duct.

The urea channel UT-A1 and the water channel aquaporin-2 (AQP2) mediate vasopressin-regulated transport in the renal inner medullary collecting duct (IMCD). To identify the proteins that interact with UT-A1 and AQP2 in native rat IMCD cells, we carried out chemical cross-linking followed by detergent solubilization, immunoprecipitation, and LC-MS/MS analysis of the immunoprecipitated material. The analyses revealed 133 UT-A1-interacting proteins and 139 AQP2-interacting proteins, each identified in multiple replicates. Fifty-three proteins that were present in both the UT-A1 and the AQP2 interactomes can be considered as mediators of housekeeping interactions, likely common to all plasma membrane proteins. Among proteins unique to the UT-A1 list were those involved in posttranslational modifications: phosphorylation (protein kinases Cdc42bpb, Phkb, Camk2d, and Mtor), ubiquitylation/deubiquitylation (Uba1, Usp9x), and neddylation (Nae1 and Uba3). Among the proteins unique to the AQP2 list were several Rab proteins (Rab1a, Rab2a, Rab5b, Rab5c, Rab7a, Rab11a, Rab11b, Rab14, Rab17) involved in membrane trafficking. UT-A1 was found to interact with UT-A3, although quantitative proteomics revealed that most UT-A1 molecules in the cell are not bound to UT-A3. In vitro incubation of UT-A1 peptides with the protein kinases identified in the UT-A1 interactome revealed that all except Mtor were capable of phosphorylating known sites in UT-A1. Overall, the UT-A1 and AQP2 interactomes provide a snapshot of a dynamic process in which UT-A1 and AQP2 are produced in the rough endoplasmic reticulum, processed through the Golgi apparatus, delivered to endosomes that move into and out of the plasma membrane, and are regulated in the plasma membrane.

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.

ET‐1 and Nitric Oxide Stimulate Trans‐Epithelial Electrical Resistance (TER) in Inner Medullary Collecting Duct Cells

In the renal collecting duct, it is the epithelial Na channel (ENaC) that is the rate‐limiting step for Na reabsorption. We recently reported, that collecting duct nitric oxide synthase‐1 (NOS1) is necessary for endothelin‐1 (ET‐1) dependent inhibition of ENaC activity. Loss of either ET‐1 or NOS1 expression specifically in the collecting duct generates a salt‐sensitive blood pressure phenotype. In the kidney, it is the IMCD that expresses the highest NOS activity in the kidney, and produces the most ET‐1 throughout the body. Previous studies demonstrating ENaC regulation in the collecting duct have utilized patch‐clamp electro‐physiology in the cortical collecting duct, however the inner medullary collecting duct (IMCD) is technically difficult to patch clamp. Thus, the purpose of this study was to utilize TER, which is inversely related to ion transport, to determine the effect of ET‐1 and NOS inhibition on IMCD ENaC activity. TER was measured with a volt‐ohmmeter and STX2 electrode. Transwell cell culture inserts were used, allowing mouse or rat IMCD cells to polarize. Both rat primary IMCD and mouse mIMCD‐3 exhibited amiloride‐sensitive ENaC inhibition. Amiloride (1 uM) increased TER (decreased ENaC activity) in rat IMCD cells (+5.27 ± 0.63%) and mIMCD‐3 cells (+9.53 ± 1.33%) compared to vehicle controls (+0.29 ±0.19%). ET‐1 (100 nM) stimulated a significant increase in TER for rat IMCD (+2.66‐‐ ± 0.23%) and mouse mIMCD‐3 cells (+3.25 ± 0.62%−) compared to vehicle, indicating an inhibition of ion transport. NOS inhibition (5 mM L‐NAME) also stimulated a significant decrease in TER in rat IMCD (−3.50 ± 0.5%) and mouse mIMCD‐3 cells (−8.35 ± 1.20%). The decrease in resistance, and therefore increase in ion transport, with NOS inhibition could not be reversed with the addition of ET‐1, indicating that the NOS system contributes to the tonic inhibition of ion transport in the IMCD. In conclusion, these data support the hypothesis that the ET‐1 and NOS systems regulate ion transport, most likely ENaC, in the IMCD of rats and mice.Support or Funding InformationNIHP01HL95499 and The University of Alabama at Birmingham Preparation for Graduate and Medical Education Program (PARADiGM) R25 HL120883

Proteomic profiling of nuclear fractions from native renal inner medullary collecting duct cells

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.

Dynamin‐2 is a negative regulator of NOS1β in the collecting duct

Nitric oxide synthase 1 (NOS1)/NO pathway in the inner medullary collecting duct (IMCD) promotes natriuresis. Rat IMCD express NOS1α and NOS1β splice variants, while mouse and human IMCD expresses predominantly NOS1β. We reported dynamin 2 (DNM2) activates NO production via direct interaction with NOS1α; however it is undetermined if DNM2 also activates NOS1β. Accordingly, we hypothesized that DNM2 is an activator of NOS1β. To test this hypothesis, we used COS7 cells, which lack endogenous NOS, and mIMCD‐3 immortalized cells that express NOS1β. By immunoprecipitation and western blot, we confirmed that NOS1β interacts with DNM2. Nitrite production (an index of NO production) was assessed by HPLC, and we determined that overexpression of DNM2 in mIMCD‐3 cells resulted in a significant reduction in nitrite production (323 ± 25 pmol/mg pr/h vs. 1734 ± 35, N = 3‐4, P = 0.016). Using a variety of pharmacological inhibitors and control agents targeting different domains of DNM2, we determined that inhibition of DNM2 resulted in a significant increase in mIMCD‐3 nitrite production (ranged from 1410 ± 419 to 1910±105 pmol/mg pr/h) compared to controls (ranged from 665 ± 57 to 839 ± 120 pmol/mg pr/h; n = 4, P &lt; 0.05). Finally, we confirmed our pharmacological experiments by decreasing DNM2 expression with siRNA. DNM2 siRNA resulted in a 95% reduction in mIMCD‐3 DNM2 expression and produced a significant increase in nitrite production (scramble siRNA 112 ± 15 vs DNM2 siRNA 249 ± 21 pmol/ mg pr/ h). In conclusion, these data suggest that DNM2 is a novel negative regulator of NOS1β.

Dynamin 2 is a Ca2+‐dependent regulator of NOS1α and a possible negative regulator of NOS1β

Nitric oxide synthase 1 (NOS1)/NO pathway in the inner medullary collecting duct (IMCD) promotes natriuresis. Rat IMCD express NOS1α and NOS1β splice variants, while mouse IMCD express NOS1β. We reported dynamin 2 (DNM2) activates NO production via direct interaction with NOS1α. We hypothesized that 1) intracellular Ca2+ regulates DNM2/NOS1 interaction, and 2) knocking down or inhibiting DNM2 decreases NO production in IMCD. Reducing intracellular Ca2+ with BAPTA decreased the NOS1α/DNM2 interaction as determined by immunoprecipitation in NOS1α + DNM2 transfected COS7 cells. While increasing intracellular Ca2+ with ionomycin, enhanced the NOS1α/DNM2 interaction. Thus, NOS1α/DNM2 interactions are Ca2+‐dependent. We confirmed that NOS1β also interacts with DNM2 in mouse IMCD3 cells. NO production, assessed by HPLC, was similar in control and DNM2 siRNA (95% knockdown confirmed) groups (P&gt;;0.05). NO production was significantly increased with DNM2 inhibitors (ranged from 1410 ± 419 to 1910±105 pmol/mg pr/h) compared to controls (ranged from 665 ± 57 to 839 ± 120 pmol/mg pr/h; n = 4, P &lt; 0.05). Reducing expression or inhibition of DNM2 in mouse IMCD, which express NOS1β only, either has no effect or increases NO production. These data suggest that DNM2 acts as a Ca2+‐dependent positive regulator of NOS1α and a possible negative effector of NOS1β. Funding support: APS to SKHM; AHA to KAH; NIH to JSP.

Distinct regulation of inner medullary collecting duct nitric oxide production from mice and rats

Nitric oxide (NO) and NO synthase 1 (NOS1) maintain sodium and water homeostasis. The NOS1α and NOS1β splice variants are expressed in the rat inner medulla, but only NOS1β is expressed in the mouse. Collecting duct NOS1 is necessary for blood pressure control. We hypothesized that NOS1 splice variant expression and NO production in the inner medullary collecting duct (IMCD) are regulated differently in mice and rats by high dietary sodium. Male C57blk/J6 mice and Sprague-Dawley rats were fed a 0.4% (normal salt; NS), or 4% (high salt; HS) NaCl diet for 2 or 7 days. Mean arterial pressure was not altered by HS, whereas urinary sodium excretion in mice and rats was increased significantly. Urinary excretion of nitrate/nitrite (NO(x)) and IMCD nitrite production were significantly greater in mice compared with rats on the HS diet. Western blotting indicated that only NOS1β and NOS3 were expressed in the mouse IMCD and that expression was unaffected by the HS diet at either time point. In contrast, NOS1α was detected in the IMCD of rats, in addition to NOS1β and NOS3. Feeding of the HS diet for 2 days increased NOS1α and NOS1β expression in the rat IMCD and 7 day feeding of the HS diet further increased NOS1β expression. Expression of NOS3 was unchanged by the HS diet at either time point. In conclusion, IMCD NO production in mice and rats is distinctly regulated under both NS and HS conditions, including expression of NOS1 splice variants.

Urine concentration in the diabetic mouse requires both urea and water transporters

The regulation of the inner medullary collecting duct (IMCD) urea transporters (UT-A1, UT-A3) and aquaporin-2 (AQP2) and their interactions in diabetic animals is unknown. We investigated whether the urine concentrating defect in diabetic animals was a function of AQP2, the UT-As, or both transporters. UT-A1/UT-A3 knockout (UT-A1/A3 KO) mice produce dilute urine. We gave wild-type (WT) and UT-A1/A3 KO mice vasopressin via minipump for 7 days. In WT mice, vasopressin increased urine osmolality from 3,000 to 4,550 mosmol/kgH(2)O. In contrast, urine osmolality was low (800 mosmol/kgH(2)O) in the UT-A1/A3 KOs and remained low following vasopressin. Surprisingly, AQP2 protein abundance increased in UT-A1/A3 KO (114%) and WT (92%) mice. To define the role of UT-A1 and UT-A3 in the diabetic responses, WT and UT-A1/A3 KO mice were injected with streptozotocin (STZ). UT-A1/A3 KO mice showed only 40% survival at 7 days post-STZ injection compared with 70% in WT. AQP2 did not increase in the diabetic UT-A1/A3 KO mice compared with a 133% increase in WT diabetic mice. Biotinylation studies in rat IMCDs showed that membrane accumulation of UT-A1 increased by 68% in response to vasopressin in control rats but was unchanged by vasopressin in diabetic rat IMCDs. We conclude that, even with increased AQP2, UT-A1/UT-A3 is essential to optimal urine concentration. Furthermore, UT-A1 may be maximally membrane associated in diabetic rat inner medulla, making additional stimulation by vasopressin ineffective.

Protein kinase C-α comes to the rescue of aquaporin-2

the article by Thai et al. ([12][1]) in an issue of the American Journal of Physiology-Renal Physiology brings to the forefront a previously unrecognized role of PKC-α in preventing downregulation of aquaporin (AQP)2 by ANG II. Regulated transport of water, sodium, and urea in the renal medulla is

Dynamin activates NO production in rat renal inner medullary collecting ducts via protein-protein interaction with NOS1

We hypothesized that nitric oxide synthase (NOS) isoforms may be regulated by dynamin (DNM) in the inner medullary collecting duct (IMCD). The aims of this study were to determine which DNM isoforms (DNM1, DNM2, DNM3) are expressed in renal IMCDs, whether DNM interacts with NOS, whether a high-salt diet alters the interaction of DNM and NOS, and whether DNM activates NO production. DNM2 and DNM3 are highly expressed in the rat IMCD, while DNM1 is localized outside of the IMCD. We found that DNM1 interacts with NOS1α, NOS1β, and NOS3 in the inner medulla of male Sprague-Dawley rats on a 0.4% salt diet. DNM2 interacts with NOS1α, while DNM3 interacts with both NOS1α and NOS1β. DNM2 and DNM3 do not interact with NOS3 in the rat inner medulla. We did not observe any change in the DNM/NOS interactions with rats on a 4% salt diet after 7 days. Furthermore, NOS1α interacts with DNM2 in mIMCD3 and COS7 cells transfected with NOS1α and DNM2-GFP constructs and the NOS1 reductase domain is necessary for the interaction. Finally, COS7 cells expressing NOS1α or NOS1α/DNM2-GFP had significantly higher nitrite production compared with DNM2-GFP only. Nitrite production was blocked by the DNM inhibitor dynasore or the dominant negative DNM2K44A. Ionomycin stimulation further increased nitrite production in the NOS1α/DNM2-GFP cells compared with NOS1α only. In conclusion, DNM and NOS1 interact in the rat renal IMCD and this interaction leads to increased NO production, which may influence NO production in the renal medulla.

Dynamin interacts with NOS1 in the renal collecting duct and activates NO production

Within the kidney, the inner medullary collecting duct (IMCD) has the highest total NOS activity. We hypothesized that NOS isoforms may be regulated by dynamin (DNM) in the IMCD. Thus, the aims of this study were to determine which DNM isoforms (DNM1, DNM2, DNM3) are expressed in IMCDs, whether DNM interacts with NOS in the renal inner medulla (IM) and IMCD, and finally whether DNM activates NO production from NOS1. DNM2 and DNM3 are highly expressed in the rat IMCD, while DNM1 is localized outside of the IMCD. We performed immunoprecipitation experiments to probe for DNM/NOS interactions in the IM and IMCD of male Sprague Dawley rats. We found that DNM1 interacts with NOS1α, NOS1β, and NOS3 in the IM. DNM2 interacts with NOS1α in the IMCD, while DNM3 interacts with both NOS1α and NOS1β in the IMCD. DNM2 and DNM3 do not interact with NOS3 in the rat IM. Furthermore, we transfected mIMCD3 and COS7 cells with NOS1α and DNM2-GFP constructs and determined that NOS1α interacts with DNM2. Finally, nitrite production in COS7 cells (N=6) expressing NOS1α (193 ± 20 pmol nitrite/mg protein/hr) or NOS1α/DNM2-GFP (390 ± 65 pmol/mg protein/hr) was significantly higher compared to DNM2-GFP only transfected cells (not detectable; p <0.001). Upon stimulation with ionomycin, nitrite production further increased in the NOS1α/DNM2-GFP transfected cells (2122 ± 592 pmol nitrite/mg protein/hr; N=4) compared to NOS1α only (574 ± 34 pmol nitrite/mg protein/hr; N=5). In conclusion, these data demonstrate that DNM interacts with NOS1 to activate NO production in the IMCD.