PURPOSE: Cell volume regulation is critical in kidney collecting duct cells which are subjected to large transepithelial osmotic gradients and stimulation of vasopressin. The present study aimed at validating the usefulness of the fluorescence quenching method to measure rapid changes in the cell volume of the kidney collecting duct cells in response to changes of extracellular osmolality and/or dDAVP (V2 receptor agonist) stimulation. METHODS: M-1 cell (a mouse cortical collecting duct cell line) was used and the data presented traces of cellular fluorescence in M-1 cells loaded with calcein collected over time as extracellular osmolality was repeatedly changed or dDAVP was treated. And the "initial relative rate of cell volume changes" was calculated. RESULTS: M-1 cells loaded with calcein revealed that fluorescence was increased when exposed to low extracellular osmolality (250 mOsm/KgH2O), whereas it was decreased by high extracellular osmolality (350 mOsm/KgH2O). This could reflect volume-dependent changes in fluorescence intensity in the range of quenching concentrations. The calculated "initial relative rate of cell volume changes" in M-1 cells during 1 sec was increased-7-fold by dDAVP treatment (10(-8)M, 2 min), compared with vehicle treatment when extracellular osmolality was changed from 350 to 250 mOsm/KgH2O. CONCLUSION: This study suggests that a fluorescence quenching method could be exploited for investigating an effect of dDAVP or other drugs/chemicals on the relative rate of cell volume changes in the kidney collecting duct cells.
Animals ; Aquaporins ; Cell Size ; Deamino Arginine Vasopressin ; Fluoresceins ; Fluorescence ; Kidney ; Kidney Tubules ; Kidney Tubules, Collecting ; Mice ; Osmolar Concentration ; Vasopressins

Ammonia metabolism is a fundamental process in the maintenance of life in all living organisms. Recent studies have identified ammonia transporter family proteins in yeast (Mep), plants (Amt), and mammals (Rh glycoproteins). In mammalian kidneys, where ammonia metabolism and transport are critically important for the regulation of systemic acid - base homeostasis, basolateral Rh B glycoprotein and apical/basolateral Rh C glycoprotein are expressed along the distal nephron segments. Data from experimental animal models and knockout mice suggest that the Rh glycoproteins appear to mediate important roles in urinary ammonia excretion.
Ammonia ; Animals ; Glycoproteins ; Homeostasis ; Humans ; Kidney ; Kidney Tubules, Collecting ; Mammals ; Mice ; Mice, Knockout ; Models, Animal ; Nephrons ; Proteins ; Yeasts

Renal ammonium metabolism is the primary component of net acid excretion and thereby is critical for acid - base homeostasis. Briefly, ammonium is produced from glutamine in the proximal tubule in a series of biochemical reactions that result in equimolar bicarbonate. Ammonium is predominantly secreted into the luminal fluid via the apical Na++xchanger, NHE3. The thick ascending limb of the loop of Henle reabsorbs luminal ammonium, predominantly by transport of NH4+y the apical Na++Cl - cotransporter, BSC1/NKCC2. This process results in renal interstitial ammonium accumulation. Finally, the collecting duct secretes ammonium from the renal interstitium into the luminal fluid. Although in past ammonium was believed to move across epithelia entirely by passive diffusion, an increasing number of studies demonstrated that specific proteins contribute to renal ammonium transport. Recent studies have yielded important new insights into the mechanisms of renal ammonium transport. In this review, we will discuss renal handling of ammonium, with particular emphasis on the transporters involved in this process.
Ammonia ; Diffusion ; Extremities ; Glutamine ; Handling (Psychology) ; Homeostasis ; Kidney ; Kidney Tubules, Collecting ; Loop of Henle ; Phenobarbital ; Proteins ; Quaternary Ammonium Compounds

Ubiquitination is known to be important for endocytosis and lysosomal degradation of aquaporin-2 (AQP2). Ubiquitin (Ub) is covalently attached to the lysine residue of the substrate proteins and activation and attachment of Ub to a target protein is mediated by the action of three enzymes (i.e., E1, E2, and E3). In particular, E3 Ub-protein ligases are known to have substrate specificity. This minireview will discuss the ubiquitination of AQP2 and identification of potential E3 Ub-protein ligases for 1-deamino-8-D-arginine vasopressin (dDAVP)-dependent AQP2 regulation.
Aquaporin 2 ; Deamino Arginine Vasopressin ; Endocytosis ; Kidney ; Kidney Tubules, Collecting ; Ligases ; Lysine ; Proteins ; Substrate Specificity ; Ubiquitin ; Ubiquitination ; Vasopressins

Phospholipase D (PLD) is an enzyme involved in signal transduction and widely distributed in mammalian cells. The signal transduction pathways and role for phospholipid metabolism during hormonal response in cortical collecting duct remain partly undefined. It has been reported that dexamethasone increases transepithelial transport in M-1 cells that are derived from the mouse cortical collecting duct. We investigated the expression and activity of PLD in M-1 cells. Basal PLD activity of M-1 cells cultured in the presence of dexamethasone (5 microM) was higher than in the absence of dexamethasone. Dexamethasone and ATP activated PLD in M-1 cells but phorbol ester did not stimulate PLD activity. Vasopressin, bradykinin, dibutyryl cyclic AMP, and ionomycin were ineffective in activating PLD of the cells. The PLD2 isotype was detected by immunoprecipitation but PLD1 was not detected in M-1 cells. Addition of GTPgammaS and ADP-ribosylation factor or phosphatidylinositiol 4,5-bisphosphate to digitonin-permeabilized cells did not augment PLD activity. In intact cells PLD activity was increased by sodium oleate but there was no significant change between dexamethasone treated- and untreated cells by oleate. These results suggest that at least two types of PLD are present in M-1 cells and PLD plays a role in the corticosteroid-mediated response of cortical collecting duct cells.
Animal ; Biological Transport/drug effects ; Dexamethasone/pharmacology* ; Dose-Response Relationship, Drug ; Drug Interactions ; Glycerophospholipids/analysis ; Isoenzymes/drug effects ; Kidney Cortex/cytology ; Kidney Tubules, Collecting/drug effects* ; Kidney Tubules, Collecting/cytology ; Mice ; Mice, Transgenic ; Oleic Acid/pharmacology ; Phospholipase D/drug effects*

OBJECTIVETo investigate the expression and function of PKD1 and PKD2 in different kidney tissues and cell lines.

METHODSImmunoprecipitation, Western blotting, In situ hybridization and immunohistochemical staining methods were used to observe the expression of PKD1 mRNA and PKD2 mRNA and their protein abundance in different kidney tissues and cell lines.

RESULTSCoordinate expressions of PKD1 and PKD2 were found in all kidney tissues and cell lines. Distribution of PKD1 mRNA and PKD2 mRNA and their protein polycystin-1 and polycystin-2 in normal human adult kidney tissue were mainly expressed in the medullary collecting ducts and distal tubules. Positive staining was also found in the majority of cyst-lining epithelial cells of PKD1 cystic kidney tissue, PKD1 cyst-lining epithelia cell line and LLC-PK1. The expression level of them in cystic epithelia of ADPKD kidney tissue was much higher than that in adult renal tubules (P < 0.01).

CONCLUSIONSSimilar expression pattern of PKD1 and PKD2 and their different tissue distribution in different kidney tissues show that the molecular mutuality of PC-1 and PC-2 might be the base of their functional correlation. Polycystins might play an important role in the maintenance of tubular architecture.

Adult ; Animals ; Cell Line ; Gene Expression ; Humans ; Kidney ; metabolism ; Kidney Tubules, Collecting ; metabolism ; Kidney Tubules, Distal ; metabolism ; Kidney Tubules, Proximal ; cytology ; Polycystic Kidney, Autosomal Dominant ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Swine ; TRPP Cation Channels ; metabolism

BACKGROUND/AIMS: Renal tubular acidosis (RTA) decreases the net acid excretion, predominantly due to a decrease in urinary ammonia excretion. This study examined whether this decrement is associated with changes in the renal expression of the ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), in rats with amiloride-induced RTA. METHODS: Male Sprague-Dawley rats were treated intraperitoneally with amiloride (3 mg/kg/day) for 6 days. Rhbg and Rhcg expression was evaluated by immunoblotting and immunohistochemistry. Cell height, total cellular expression, expression in the apical 25% of the cell, and apical expression as a percentage of total expression were quantified using immunohistochemistry with quantitative morphometric analysis. RESULTS: After amiloride treatment for 6 days, the serum bicarbonate level was decreased, and serum potassium was increased. The total urinary ammonia excretion and potassium excretion were decreased. The total Rhbg and Rhcg protein expression levels were not changed in the cortex or outer medulla of the kidney. Light microscopy and immunohistochemistry with quantitative morphometric analysis demonstrated that total Rhcg expression was decreased in the cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) in amiloride-induced RTA, whereas Rhbg immunoreactivity was unchanged. CONCLUSIONS: Rats with amiloride-induced RTA have decreased urinary ammonia excretion associated with decreased Rhcg expression in the CCD and OMCD, suggesting that the ammonia transporter Rhcg plays an important role in the pathogenesis of amiloride-induced RTA.
Acidosis, Renal Tubular ; Amiloride ; Ammonia ; Animals ; Glycoproteins ; Humans ; Immunoblotting ; Immunohistochemistry ; Kidney ; Kidney Tubules, Collecting ; Light ; Male ; Microscopy ; Potassium ; Rats ; Rats, Sprague-Dawley

Two examples of the rare case of complete duplicated collecting system with lower pole ureteropelvic junction obstruction are described. Ureteropelvic junction obstruction (UPJO) and duplicated collecting systems seldom occur in combination. Complete duplication of the ureter may be asymptomatic or recognized when complications develop as a result of reflux into the lower pole ureter or obstruction of the upper pole with an ectopic ureterocele. It is difficult to choose an optimal therapy due to the high variability in function, degree of obstruction, damage and potential for regeneration in growing kidneys. The diagnosis and management of UPJO of the lower pole in complete duplicated collecting systems are discussed.
Case Report ; Female ; Human ; Infant ; Infant, Newborn ; *Kidney Pelvis ; Kidney Tubules, Collecting/*abnormalities/surgery ; Male ; *Ureteral Obstruction/*complications/diagnosis/surgery ; Ureterostomy

The kidney collecting duct (CD) is a tubular segment of the kidney where the osmolality and final flow rate of urine are established, enabling urine concentration and body water homeostasis. Water reabsorption in the CD depends on the action of arginine vasopressin (AVP) and a transepithelial osmotic gradient between the luminal fluid and surrounding interstitium. AVP induces transcellular water reabsorption across CD principal cells through associated signaling pathways after binding to arginine vasopressin receptor 2 (AVPR2). This signaling cascade regulates the water channel protein aquaporin-2 (AQP2). AQP2 is exclusively localized in kidney connecting tubules and CDs. Specifically, AVP stimulates the intracellular translocation of AQP2-containing vesicles to the apical plasma membrane, increasing the osmotic water permeability of CD cells. Moreover, AVP induces transcription of the Aqp2 gene, increasing AQP2 protein abundance. This review provides new insights into the transcriptional regulation of the Aqp2 gene in the kidney CD with an overview of AVP and AQP2. It summarizes current therapeutic approaches for X-linked nephrogenic diabetes insipidus caused by AVPR2 gene mutations.
Aquaporin 2 ; Arginine Vasopressin ; Body Water ; Cell Membrane ; Diabetes Insipidus, Nephrogenic ; Gene Expression Regulation ; Homeostasis ; Kidney ; Kidney Tubules, Collecting ; Osmolar Concentration ; Permeability ; Phenobarbital ; Receptors, Vasopressin ; Water

Ammonia excretion in the renal collecting duct is critical in the regulation of the acid-base homeostasis. A novel family of ammonium transporter protein, Rh C Glycoprotein (RhCG) was recently identified in the mouse and rat kidney collecting duct. The purpose of this study was to examine the ultrastructural localization of RhCG in the collecting duct. Rat kidneys were processed for light and electron microscope immunocytochemistry using anti-RhCG rabbit polyclonal antibody. Strong RhCG immunolabeling was observed in the apical domain of AE1-positive type A intercalated cells in the collecting duct. Transmission electron microscopy revealed that a high level of RhCG was expressed in the apical plasma membrane and in subapical vesicles of type A intercalated cell. Interestingly, type A cells also expressed weak immunolabel in the basolateral plasma membrane. Type B cells had weak apical plasma membrane and subapical vesicle immunolabel, and very weak basolateral plasma membrane immunolabel. Principal cells had very weak apical plasma membrane immunolabel without detectable subapical or basolateral immunolabel These results demonstrate that RhCG is mainly expressed in the apical plasma membrane and in subapical vesicles of acid-secreting type A cells and may mediate ammonia excretion in the collecting duct.
Ammonia* ; Ammonium Compounds ; Animals ; B-Lymphocytes ; Cell Membrane ; Glycoproteins ; Homeostasis ; Humans ; Immunohistochemistry ; Kidney Tubules, Collecting* ; Kidney* ; Mice ; Microscopy, Electron, Transmission ; Rats*