Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells

Binding of arginine-vasopressin (AVP) to its V2 receptor (V2R) in the basolateral membrane of principal cells induces Aquaporin-2-mediated water reabsorption in the kidney. To study the regulation of the V2R by dDAVP in a proper model, a polarized renal cell line stably-expressing V2R-GFP was generated. Labeled AVP-binding studies revealed an equal basolateral vs. apical membrane distribution for V2R-GFP and endogenous V2R. In these cells, GFP-V2R was expressed in its mature form and localized for 75% in the basolateral membrane and for 25% to late endosomes/lysosomes. dDAVP caused a dose- and time-dependent internalization of V2R-GFP, which was completed within 1 h with 100 nM dDAVP, was prevented by coincubation with a V2R antagonist, and which reduced its half-life from 11.5 to 2.8 h. Semiquantification of the V2R-GFP colocalization with E-cadherin (basolateral membrane), early endosomal antigen-1 (EEA-1) and lysosome-associated membrane protein-2 (LAMP-2) in time revealed that most dDAVP-bound V2R was internalized via early endosomes to late endosomes/lysosomes, where it was degraded. The dDAVP-internalized V2R did not recycle to the basolateral membrane. In conclusion, we established the itinerary of the V2R in a polarized cell model that likely resembles the in vivo V2R localization and regulation by AVP to a great extent.     

Stimulation of an N-ethylmaleimide-sensitive ATPase in the collecting duct segments of the rat nephron by metabolic acidosis

A plasma membrane ATPase sensitive to inhibition by N-ethylmaleimide (NEM) and insensitive to inhibition by oligomycin and ouabain has been shown to be involved in acidification of urine in the turtle bladder. The activity of this NEM-sensitive ATPase was determined in four types of distal nephron segments of normal rats and in rats treated with ammonium chloride. The enzyme activity was determined by a fluorometric micromethod in which ATP hydrolysis was coupled to NADH oxidation. Significant activities (10-35 pmol ADP X min-1 X mm-1) of NEM-sensitive ATPase were present in the distal convoluted tubule (DCT) and in the cortical and outer and inner medullary collecting duct segments of normal rats. In metabolic acidosis produced by ammonium chloride treatment (plasma CO2 content = 15.3 +/- 0.8 mequiv./L), the NEM-sensitive ATPase activity was increased significantly (60-100%) in the collecting duct segments without showing a significant change in the enzyme activity in the DCT. Our data are consistent with the hypothesis that a plasma membrane H+-ATPase (inhibited by NEM but not by oligomycin or ouabain) is involved in H+ secretion in the mammalian collecting duct.     

Regulation by vasopressin of NaCl absorption in the renal collecting duct

In the kidney, the fine control of NaCl absorption takes place in the distal nephron and is controlled by aldosterone and vasopressin. This review summarizes the effects of vasopressin on Na+ transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel in immortalized or primary cultured cortical collecting duct cells, expressing either the wild-type ENaC subunits, or mutations, or deletions of the PY domain of the beta- or gamma-ENaC subunits responsible for Liddle's syndrome, an inherited form of hypertension due to excessive salt absorption.     

Long term regulation of aquaporin-2 expression in vasopressin-responsive renal collecting duct principal cells.

Fine regulation of water reabsorption by the antidiuretic hormone [8-arginine]vasopressin (AVP) occurs in principal cells of the collecting duct and is largely dependent on regulation of the aquaporin-2 (AQP2) water channel. AVP-inducible long term AQP2 expression was investigated in immortalized mouse cortical collecting duct principal cells. Combined RNase protection assay, Western blot, and immunofluorescence analyses revealed that physiological concentrations of AVP added to the basal side, but not to the apical side, of cells grown on filters induced both AQP2 mRNA and apical protein expression. The stimulatory effect of AVP on AQP2 expression followed a V(2) receptor-dependent pathway because [deamino-8-d-arginine]vasopressin (dDAVP), a specific V(2) receptor agonist, produced the same effect as AVP, whereas the V(2) antagonist SR121463B antagonized action of both AVP and dDAVP. Moreover, forskolin and cyclic 8-bromo-AMP fully reproduced the effects of AVP on AQP2 expression. Analysis of protein degradation pathways showed that inhibition of proteasomal activity prevented synthesis of AVP-inducible AQP2 mRNA and protein. Once synthesized, AQP2 protein was quickly degraded, a process that involves both the proteasomal and lysosomal pathways. This is the first study that delineates induction and degradation mechanisms of AQP2 endogenously expressed by a renal collecting duct principal cell line.     

Volume regulation in cortical collecting duct cells: role of AQP2

BACKGROUND INFORMATION: The renal CCD (cortical collecting duct) plays a role in final volume and concentration of urine by a process that is regulated by the antidiuretic hormone, [arginine]vasopressin. This hormone induces an increase in water permeability due to the translocation of AQP2 (aquaporin 2) from the intracellular vesicles to the apical membrane of principal cells. During the transition from antidiuresis to diuresis, CCD cells are exposed to changes in environmental osmolality, and cell-volume regulation may be especially important for the maintenance of intracellular homoeostasis. Despite its importance, cell-volume regulation in CCD cells has not been widely investigated. Moreover, no studies have been carried out till date to evaluate the putative role of AQPs during this process in renal cells. RESULTS: In the present study, we have studied the regulatory cell-volume responses to hypo-osmotic or hyperosmotic challenges in two CCD cell lines: one not expressing AQPs and the other stably transfected with AQP2. We have used a fluorescent probe technique in which the acquisition of single-cell kinetic data can be simultaneously recorded with the intracellular pH. Experiments with hyperosmotic mannitol media demonstrated that, independent of AQP2 expression, CCD cells shrink but fail to show regulatory volume increase, at least under the studied conditions. In contrast, under hypo-osmotic shocks, regulatory volume decrease occurs and the activation of these mechanisms is more rapid in AQP2 transfected cells. This regulatory response takes place in parallel with intracellular acidification, which is faster in cells expressing AQP2. The acidification and the initial regulatory volume decrease response were inhibited by glibenclamide and BaCl2 only in AQP2 cells. CONCLUSIONS: These results suggest that increases in the osmotic water permeability due to the expression of AQP2 are critical for a rapid activation of regulatory volume decrease mechanisms, which would be linked to cystic fibrosis transmembrane conductance regulator and to barium-sensitive potassium channels.     

Large scale protein identification in intracellular aquaporin-2 vesicles from renal inner medullary collecting duct

Vasopressin acts on renal collecting duct cells to stimulate translocation of aquaporin-2 (AQP2)-containing membrane vesicles from throughout the cytoplasm to the apical region. The vesicles fuse with the plasma membrane to increase water permeability. To identify the intracellular membrane compartments that contain AQP2, we carried out LC-MS/MS-based proteomic analysis of immunoisolated AQP2-containing intracellular vesicles from rat inner medullary collecting duct. Immunogold electron microscopy and immunoblotting confirmed heavy AQP2 labeling of immunoisolated vesicles. Vesicle proteins were separated by SDS-PAGE followed by in-gel trypsin digestion in consecutive gel slices and identification by LC-MS/MS. Identification of Rab GTPases 4, 5, 18, and 21 (associated with early endosomes); Rab7 (late endosomes); and Rab11 and Rab25 (recycling endosomes) indicate that a substantial fraction of intracellular AQP2 is present in endosomal compartments. In addition, several endosome-associated SNARE proteins were identified including syntaxin-7, syntaxin-12, syntaxin-13, Vti1a, vesicle-associated membrane protein 2, and vesicle-associated membrane protein 3. Rab3 was not found, however, either by mass spectrometry or immunoblotting, suggesting a relative lack of AQP2 in secretory vesicles. Additionally, we identified markers of the trans-Golgi network, components of the exocyst complex, and several motor proteins including myosin 1C, non-muscle myosins IIA and IIB, myosin VI, and myosin IXB. Beyond this, identification of multiple endoplasmic reticulum-resident proteins and ribosomal proteins indicated that a substantial fraction of intracellular AQP2 is present in rough endoplasmic reticulum. These results show that AQP2-containing vesicles are heterogeneous and that intracellular AQP2 resides chiefly in endosomes, trans-Golgi network, and rough endoplasmic reticulum.     

Mitochondrial aquaporin-8 in renal proximal tubule cells: evidence for a role in the response to metabolic acidosis

Mitochondrial ammonia synthesis in proximal tubules and its urinary excretion are key components of the renal response to maintain acid-base balance during metabolic acidosis. Since aquaporin-8 (AQP8) facilitates transport of ammonia and is localized in inner mitochondrial membrane (IMM) of renal proximal cells, we hypothesized that AQP8-facilitated mitochondrial ammonia transport in these cells plays a role in the response to acidosis. We evaluated whether mitochondrial AQP8 (mtAQP8) knockdown by RNA interference is able to impair ammonia excretion in the human renal proximal tubule cell line, HK-2. By RT-PCR and immunoblotting, we found that AQP8 is expressed in these cells and is localized in IMM. HK-2 cells were transfected with short-interfering RNA targeting human AQP8. After 48 h, the levels of mtAQP8 protein decreased by 53% (P < 0.05). mtAQP8 knockdown decreased the rate of ammonia released into culture medium in cells grown at pH 7.4 (-31%, P < 0.05) as well as in cells exposed to acid (-90%, P < 0.05). We also evaluated mtAQP8 protein expression in HK-2 cells exposed to acidic medium. After 48 h, upregulation of mtAQP8 (+74%, P < 0.05) was observed, together with higher ammonia excretion rate (+73%, P < 0.05). In vivo studies in NH(4)Cl-loaded rats showed that mtAQP8 protein expression was also upregulated after 7 days of acidosis in renal cortex (+51%, P < 0.05). These data suggest that mtAQP8 plays an important role in the adaptive response of proximal tubule to acidosis possibly facilitating mitochondrial ammonia transport.     

Decrease in N-ethylmaleimide-sensitive ATPase activity in collecting duct by metabolic alkalosis

Changes in systemic acid-base balance are known to influence acidification in the collecting duct. The H+ secretion in the collecting duct has been shown to be an electrogenic process and it has been suggested that an H-ATPase sensitive to inhibition by N-ethylmaleimide (NEM) is responsible for H+ secretion. This study was designed to determine the effect of metabolic alkalosis on NEM-sensitive ATPase activity in the microdissected segments of the distal nephron. Metabolic alkalosis was produced by giving NaHCO3 to normal rats for 7 days. The plasma total CO2 concentration in the experimental group was 31.5 +/- 1.8 mM compared with 23.4 +/- 1.0 mM in the control group. NEM-sensitive ATPase activity was significantly lower in the cortical collecting duct and in the outer and inner medullary collecting ducts of alkali-loaded rats than those of control rats. There was no significant difference in the enzyme activity between the two groups of animals in the other nephron segments examined. Our results suggest that NEM-sensitive H-APTase activity in all three segments of the collecting duct is modulated by the acid-base status of the animal.     

Upregulation and protein trafficking of aquaporin-2 attenuate cold-induced osmotic damage during cryopreservation

Aquaporins (AQPs) are a recently discovered family of proteins that function as transmembrane water channels. These proteins regulate the delicate osmotic balance across the cell plasma membrane. Given that osmotic damage is the major contributing factor to cell death during freezing, we hypothesized that regulation of AQPs may have an unrealized role in protecting cells from osmotic damage during cryopreservation. Rat kidney inner medullar collecting duct (IMCD) cells were treated with arginine vasopressin (AVP) to increase the amount of AQP2 in the external plasma membrane before freezing in University of Wisconsin solution at -4 degrees C for 24 h. This resulted in a significant increase in cell viability on warming. Conversely, treatment of IMCD cells with AVP and W7 (which inhibits AQP2 protein trafficking to the plasma membrane) before freezing resulted in a 55% decrease in cell viability. These preliminary data indicate that regulation of AQP2 can attenuate cold-induced osmotic damage in rat kidney IMCD cells.     

Calciumdependent mechanisms in vasopressin regulation of osmotic water permeability in the mouse kidney collecting duct cells

In the study, the role of PKC and Ca++ in vasopressin regulation of the plasma membrane water permeability was studied in the cells of the mouse kidney collecting duct. Coefficient of osmotic water permeability of total cell surface (Pf) was calculated from the initial rate of cell swelling following the osmotic shock caused by changing the medium osmolarity from isotonic to hypotonic (300 mOsm to 200 mOsm). Desmopressin (dDAVP 1 nM) increased the Pf in hydrated mice from 168.4 +/- 11.8 microm/s up to 231.3 +/- 14.7 microm/s. The Ca++ chelator BAPTA prevented the desmopressin-induced increase in water permeability. Inhibition of PKC (Ro-31-8220 0.1 microM) also abolished the desmopressin-stimulated increase of plasma membrane water permeability, whereas inhibitor of PKC alone did not suppress the stimulation of the water permeability by db-cAMP. The PKC activity and calciumdependent second messengers seem to be important for regulation of water permeability by vasopressin.     

A role for collecting duct epithelial cells in renal antibacterial defences

Urinary tract infections (UTIs), which are mainly due to uropathogenic Escherichia coli (UPEC), occur via the retrograde ascent of the bacteria along the urinary tract system. The adhesion and invasion mechanisms of UPEC have been extensively studied in bladder epithelial cells, but less is known about the role of renal tubule epithelial cells (RTEC) in renal antibacterial defences. This review considers recent advances in the understanding of the role of RTECs in inducing an innate immune response mediated by Toll-like receptors (TLRs) in experimental UTI. Collecting duct cells are a preferential site of adhesion of UPEC colonizing the kidneys. Epithelial TLR4 activation induces an inflammatory response and the recruitment of lipid rafts to the plasma membrane, both of which facilitate the transcytosis of non-cytolytic UPEC strains across intact collecting duct cell layers to invade the renal interstitium. Arginine vasopressin, which regulates water absorption in the collecting duct, also acts as a potent modulator of the TLR4-mediated intrarenal innate response caused by UPEC. The role of epithelial TLR5 in renal host defences is also discussed. These findings highlight the role of RTECs in triggering the innate immune response in the context of ascending UTIs.     

Calpain-mediated AQP2 proteolysis in inner medullary collecting duct

Vitamin D-elicited hypercalcemia/hypercalciuria is associated with polyuria in humans and in animal models. In rats, dihydrotachysterol (DHT) induces AQP2 water channel downregulation despite unaltered AQP2 mRNA expression and thus we investigated the mechanism of AQP2 degradation. Incubation of AQP2-containing inner medullary collecting duct (IMCD) endosomes with Ca(2+) or calpain elicited AQP2 proteolysis, an effect abolished by leupeptin. This endogenous, Ca(2+)-sensitive protease activity exhibited a different proteolytic digest pattern from trypsin, which also degraded AQP2 in vitro. IMCDs contain abundant micro-calpain protein and functional calpain proteolytic activity as demonstrated by immunohistochemistry, immunoblotting, and gel zymography. Furthermore, by small particle flow cytometry we demonstrated that micro-calpain colocalizes with apical IMCD endosomes. DHT does not appear to elicit general proteolysis, however, in addition to AQP2 degradation, DHT treatment also diminished micro-calpain and calpastatin expression although whether these changes contributed to the AQP2 instability remains unclear. Together, these data show for the first time that AQP2 is a substrate for calpain-mediated proteolysis and that furthermore, micro-calpain, like AQP2, is both highly expressed in renal inner medulla and localized to apical IMCD endosomes.