Bellamkonda K. Kishore

Rat renal arcade segment expresses vasopressin-regulated water channel and vasopressin V2 receptor.

The arcades are long, branched renal tubules which connect deep and mid-cortical nephrons to cortical collecting ducts in the renal cortex. Because they are inaccessible by standard physiological techniques, their functions are poorly understood. In this paper, we demonstrate that the arcades are a site of expression of two proteins, aquaporin-2 (the vasopressin-regulated water channel) and the V2 vasopressin receptor, that are important to regulated water transport in the kidney. Using a peptide-derived polyclonal antibody to aquaporin-2, quantitative ELISA in microdissected segments showed that aquaporin-2 is highly expressed in arcades and that the expression is increased in response to restriction of fluid intake. Immunocytochemistry revealed abundant aquaporin-2 labeling of structures in the cortical labyrinth in a pattern similar to that of the Na(+)-Ca2+ exchanger and kallikrein, marker proteins expressed in arcades but not in cortical collecting ducts. RT-PCR experiments demonstrated substantial aquaporin-2 and V2 receptor mRNA in microdissected arcades. In situ hybridization, using 35S-labeled antisense cRNA probes for the V2 receptor demonstrated strong labeling of both arcades and cortical collecting ducts. Thus, these results indicate that the arcades contain the specific proteins associated with vasopressin-regulated water transport, and may be a heretofore unrecognized site of free water absorption.

Expression of syntaxins in rat kidney

Previously, we demonstrated that a putative vesicle-targeting protein, syntaxin-4, is expressed in renal collecting duct principal cells and is localized to the apical plasma membrane, suggesting a role in targeting aquaporin-2-containing vesicles to the apical plasma membrane. To investigate whether other syntaxin isoforms are present in the renal collecting duct, we determined the intrarenal localization of syntaxin-2 and -3. Reverse transcription-polymerase chain reaction (RT-PCR) experiments using total RNA extracted from kidney and various organs revealed that both syntaxin-2 and -3 are expressed in kidney cortex and medulla. RT-PCR experiments using microdissected tubules and vascular structures from the kidney revealed that syntaxin-3 mRNA, but not syntaxin-2, is expressed in collecting duct cells. Syntaxin-3 mRNA was also relatively abundant in the thick ascending limb of Henles loop and in vasa recta. Syntaxin-2 mRNA was found chiefly in glomeruli. To investigate the localization of syntaxin-3 protein, a peptide-derived polyclonal antibody was raised in rabbits. In immunoblotting experiments, this antibody labeled a 37-kDa protein in inner medulla that was most abundant in plasma membrane-enriched subcellular fractions. Immunoperoxidase labeling of thin cryosections combined with immunogold electron microscopy showed that, in contrast to the labeling seen for syntaxin-4, syntaxin-3 labeling in medullary collecting duct was predominantly in the basolateral plasma membrane of intercalated cells. These results suggest the possibility that syntaxin-3 may be involved in selective targeting of acid-base transporters and/or in basolateral membrane remodeling in response to systemic acid-base perturbations.

Lithium: a versatile tool for understanding renal physiology

By virtue of its unique interactions with kidney cells, lithium became an important research tool in renal physiology and pathophysiology. Investigators have uncovered the intricate relationships of lithium with the vasopressin and aldosterone systems, and the membrane channels or transporters regulated by them. While doing so, their work has also led to 1) questioning the role of adenylyl cyclase activity and prostaglandins in lithium-induced suppression of aquaporin-2 gene transcription; 2) unraveling the role of purinergic signaling in lithium-induced polyuria; and 3) highlighting the importance of the epithelial sodium channel (ENaC) in lithium-induced nephrogenic diabetes insipidus (NDI). Lithium-induced remodeling of the collecting duct has the potential to shed new light on collecting duct remodeling in disease conditions, such as diabetes insipidus. The finding that lithium inhibits glycogen synthase kinase-3β (GSK3β) has opened an avenue for studies on the role of GSK3β in urinary concentration, and GSK isoforms in renal development. Finally, proteomic and metabolomic profiling of the kidney and urine in rats treated with lithium is providing insights into how the kidney adapts its metabolism in conditions such as acquired NDI and the multifactorial nature of lithium-induced NDI. This review provides state-of-the-art knowledge of lithium as a versatile tool for understanding the molecular physiology of the kidney, and a comprehensive view of how this tool is challenging some of our long-standing concepts in renal physiology, often with paradigm shifts, and presenting paradoxical situations in renal pathophysiology. In addition, this review points to future directions in research where lithium can lead the renal community.

P2Y2 receptors and water transport in the kidney

The kidneys play a critical role in the maintenance of water homeostasis. This is achieved by the inherent architecture of the nephron along with the expression of various membrane transporters and channels that are responsible for the vectorial transport of salt and water. The collecting duct has become a focus of attention by virtue of its ability to transport water independent of solutes (free-water transport), and its apparent involvement in various water balance disorders. It was originally believed that the water transport capability of the collecting duct was solely under the influence of the circulating hormone, arginine vasopressin (AVP). However, during the past decade, locally produced autocrine and/or paracrine factors have emerged as potent modulators of transport of water by the collecting duct. Recently, much attention has been focused on the purinergic regulation of renal water transport. This review focuses on the role of the P2Y2 receptor, the predominant purinergic receptor expressed in the collecting duct, in the modulation of water transport in physiological and pathophysiological conditions, and its therapeutic potential as a drug target to treat water balance disorders in the clinic. Studies carried out by us and other investigators are unravelling potent interactions among AVP, prostanoid and purinergic systems in the medullary collecting duct, and the perturbations of these interactions in water balance disorders such as acquired nephrogenic diabetes insipidus. Future studies should address the potential therapeutic benefits of modulators of P2Y2 receptor signalling in water balance disorders, which are extremely prevalent in hospitalised patients irrespective of the underlying pathology.

Annexin A4 Reduces Water and Proton Permeability of Model Membranes but Does Not Alter Aquaporin 2–mediated Water Transport in Isolated Endosomes

Annexin A4 (Anx4) belongs to a ubiquitous family of Ca2+-dependent membrane-binding proteins thought to be involved in membrane trafficking and membrane organization within cells. Anx4 localizes to the apical region in epithelia; however, its physiological role is unclear. We show that Anx4 exhibited binding to liposomes (phosphatidylcholine:phosphatidylserine, 1:1) in the presence of Ca2+ and binding was reversible with EDTA. Anx4 binding resulted in liposome aggregation and a reduction in membrane water permeability of 29% (P < 0.001) at 25°C. These effects were not seen in the presence of Ca2+ or Anx4 alone and were reversible with EDTA. Measurements of membrane fluidity made by monitoring fluorescence anisotropy of 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-HPC) demonstrated that Anx4 binding rigidified the outer leaflet of the bilayer (P < 0.001), thus providing a molecular explanation for the inhibition of water flux. To determine whether Anx4 would produce similar effects on physiological membranes we constructed liposomes which recapitulated the lipid composition of the inner leaflet of the MDCK apical membrane. These membranes exhibited reductions to water permeability upon Anx4 binding (19.5% at 25°C, 31% at 37°C; P < 0.01 and P < 0.001, respectively) and to proton permeability (15% at 25°C, 19.5% at 37°C; P < 0.05). Since our in vitro experiments indicated an effect on membrane permeability, we examined localization of Anx4 in the kidney collecting duct, a region of the nephron responsible for concentrating urine through water reabsorbtion. Anx4 was shown to colocalize apically with aquaporin 2 (AQP2) in collecting duct epithelia. To test for the existence of a functional interaction between Anx4 and AQP2 we isolated AQP2-containing endosomes and exposed them to Anx4/Ca2+. Water flux rates were unchanged, indicating Anx4 does not directly regulate AQP2. We conclude that Anx4 can alter the physical properties of membranes by associating with them and regulate passive membrane permeability to water and protons. These properties represent important new functions for Anx4.

Potential role of purinergic signaling in urinary concentration in inner medulla: insights from P2Y2 receptor gene knockout mice

Osmotic reabsorption of water through aquaporin-2 (AQP2) in the inner medulla is largely dependent on the urea concentration gradients generated by urea transporter (UT) isoforms. Vasopressin (AVP) increases expression of both AQP2 and UT-A isoforms. Activation of the P2Y2 receptor (P2Y2-R) in the medullary collecting duct inhibits AVP-induced water flow. To gain further insights into the overarching effect of purinergic signaling on urinary concentration, we compared the protein abundances of AQP2 and UT-A isoforms between P2Y2-R knockout (KO) and wild-type (WT) mice under basal conditions and following AVP administration. Under basal conditions (a gel diet for 10 days), KO mice concentrated urine to a significantly higher degree, with 1.8-, 1.66-, and 1.29-fold higher protein abundances of AQP2, UT-A1, and UT-A2, respectively, compared with WT, despite comparable circulating AVP levels in both groups. Infusion of 1-desamino-8-d-arginine vasopressin (dDAVP; desmopressin; 1 ng/h sc) for 5 days resulted in 2.14-, 2.6-, and 2.22-fold higher protein abundances of AQP2, AQP3, and UT-A1, respectively, in the inner medullas of KO mice compared with WT mice. In response to acute (45 min) stimulation by AVP (0.2 unit/mouse sc), UT-A1 protein increased by 1.39- and 1.54-fold in WT and KO mice, respectively. These data suggest that genetic deletion of P2Y2-R results in increased abundances of key proteins involved in urinary concentration in the inner medulla, both under basal conditions and following AVP administration. Thus purinergic regulation may play a potential overarching role in balancing the effect of AVP on the urinary concentration mechanism.

Prolidase and prolinase activities in the erythrocytes of patients with chronic uremia

To elucidate the nature of the iminoacidopathy in uremia, the prolidase and prolinase activities in erythrocytes from 36 normal subjects, 28 patients with chronic uremia, and 21 patients on maintenance hemodialysis were determined. A statistically significant decrease in the activities of both prolidase and prolinase was found in the uremic patients and the hemodialysis group. No difference has been observed in the enzyme activities in the pre- and postdialysis samples of the hemodialysis patients. It is suggested that decreased activities of prolidase and prolinase may contribute to the iminoacidopathy in uremia, including high contents of serum iminoacid containing peptides.

Developmental expression of aquaporin 2 in the mouse inner ear

Objectives The maintenance of endolymph homeostasis is critical for the inner ear to perform its functions of hearing and maintaining balance. The identification and cloning of aquaporins (a family of water channel proteins) has allowed the study of a novel cellular mechanism potentially involved in endolymph homeostasis. The objective of the present study was to define the developmental temporal and spatial e‐pression pattern of aquaporin 2 (Aqp2) in the developing mouse inner ear.

Renal sodium transporter/channel expression and sodium excretion in P2Y2 receptor knockout mice fed a high-NaCl diet with/without aldosterone infusion.

The P2Y(2) receptor (P2Y2-R) antagonizes sodium reabsorption in the kidney. Apart from its effect in distal nephron, hypothetically, P2Y(2)-R may modulate activity/abundances of sodium transporters/channel subunits along the nephron via antagonism of aldosterone or vasopressin or interaction with mediators such as nitric oxide (NO), and prostaglandin E(2) (PGE(2)) or oxidative stress (OS). To determine the extent of the regulatory role of P2Y(2)-R in renal sodium reabsorption, in study 1, we fed P2Y(2)-R knockout (KO; n = 5) and wild-type (WT; n = 5) mice a high (3.15%)-sodium diet (HSD) for 14 days. Western blotting revealed significantly higher protein abundances for cortical and medullary bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), medullary α-1-subunit of Na-K-ATPase, and medullary α-subunit of the epithelial sodium channel (ENaC) in KO vs. WT mice. Molecular analysis of urine showed increased excretion of nitrates plus nitrites (NOx), PGE(2), and 8-isoprostane in the KO, relative to WT mice, supporting a putative role for these molecules in determining alterations of proteins involved in sodium transport along the nephron. To determine whether genotype differences in response to aldosterone might have played a role in these differences due to HSD, in study 2 aldosterone levels were clamped (by osmotic minipump infusion). Clamping aldosterone (with HSD) led to significantly impaired natriuresis with elevated Na/H exchanger isoform 3 in the cortex, and NKCC2 in the medulla, and modest but significantly lower levels of NKCC2, and α- and β-ENaC in the cortex of KO vs. WT mice. This was associated with significantly reduced urinary NOx in the KO, although PGE(2) and 8-isoprostane remained significantly elevated vs. WT mice. Taken together, our results suggest that P2Y(2)-R is an important regulator of sodium transporters along the nephron. Pre- or postreceptor differences in the response to aldosterone, perhaps mediated via prostaglandins or changes in NOS activity or OS, likely play a role.

Genetic deletion of the P2Y2 receptor offers significant resistance to development of lithium-induced polyuria accompanied by alterations in PGE2 signaling

Lithium (Li)-induced polyuria is due to resistance of the medullary collecting duct (mCD) to the action of arginine vasopressin (AVP), apparently mediated by increased production of PGE(2). We previously reported that the P2Y(2) receptor (P2Y(2)-R) antagonizes the action of AVP on the mCD and may play a role in Li-induced polyuria by enhancing the production of PGE(2) in mCD. Hence, we hypothesized that genetic deletion of P2Y(2)-R should ameliorate Li-induced polyuria. Wild-type (WT) or P2Y(2)-R knockout (KO) mice were fed normal or Li-added diets for 14 days and euthanized. Li-induced polyuria, and decreases in urine osmolality and AQP2 protein abundance in the renal medulla, were significantly less compared with WT mice despite the lack of differences in Li intake or terminal serum or inner medullary tissue Li levels. Li-induced increased urinary excretion of PGE(2) was not affected in KO mice. However, prostanoid EP(3) receptor (EP3-R) protein abundance in the renal medulla of KO mice was markedly lower vs. WT mice, irrespective of the dietary regimen. The protein abundances of other EP-Rs were not altered across the groups irrespective of the dietary regimen. Ex vivo stimulation of mCD with PGE(2) generated significantly more cAMP in Li-fed KO mice (130%) vs. Li-fed WT mice (100%). Taken together, these data suggest 1) genetic deletion of P2Y(2)-R offers significant resistance to the development of Li-induced polyuria; and 2) this resistance is apparently due to altered PGE(2) signaling mediated by a marked decrease in EP3-R protein abundance in the medulla, thus attenuating the EP3-mediated decrease in cAMP levels in mCD.