Medullary Collecting Duct Segments Research Articles

The hyperpolarization-activated cyclic nucleotide-gated HCN2 channel transports ammonium in the distal nephron

Recent studies have identified Rhesus proteins as important molecules for ammonia transport in acid-secreting intercalated cells in the distal nephron. Here, we provide evidence for an additional molecule that can mediate NH3/NH4 excretion, the subtype 2 of the hyperpolarization-activated cyclic nucleotide-gated channel family (HCN2), in collecting ducts in rat renal cortex and medulla. Chronic metabolic acidosis in rats did not alter HCN2 protein expression but downregulated the relative abundance of HCN2 mRNA. Its cDNA was identical to the homolog from the brain and the protein was post-translationally modified by N-type glycosylation. Electrophysiological recordings in Xenopus oocytes injected with HCN2 cRNA found that potassium was transported better than ammonium, each of which was transported significantly better than sodium, criteria that are compatible with a role for HCN2 in ammonium transport. In microperfused rat outer medullary collecting duct segments, the initial rate of acidification, upon exposure to a basolateral ammonium chloride pulse, was higher in intercalated than in principal cells. A specific inhibitor of HCN2 (ZD7288) decreased acidification only in intercalated cells from control rats. In rats with chronic metabolic acidosis, the rate of acidification doubled in both intercalated and principal cells; however, ZD7288 had no significant inhibitory effect. Thus, HCN2 is a basolateral ammonium transport pathway of intercalated cells and may contribute to the renal regulation of body pH under basal conditions.

Dysregulation of ENaC in Animal Models of Nephrotic Syndrome and Liver Cirrhosis

Nephrotic syndrome and liver cirrhosis are common clinical manifestations, and are associated with avid sodium retention leading to the development of edema and ascites. However, the mechanism for the sodium retention is still incompletely understood and the molecular basis remains undefined. We examined the changes of sodium (co)transporters and epithelial sodium channels (ENaCs) in the kidneys of experimental nephrotic syndrome and liver cirrhosis. The results demonstrated that puromycin- or HgCl2-induced nephrotic syndrome was associated with 1) sodium retention, decreased urinary sodium excretion, development of ascites, and increased plasma aldosterone level; 2) increased apical targeting of ENaC subunits in connecting tubule and collecting duct segments; and 3) decreased protein abundance of type 2 11β-hydroxysteroid dehydrogenase (11βHSD2). Experimental liver cirrhosis was induced in rats by CCl4 treatment or common bile duct ligation. An increased apical targeting of alpha-, beta-, and gamma-ENaC subunits in connecting tubule, and cortical and medullary collecting duct segments in sodium retaining phase of liver cirhosis but not in escape phase of sodium retention. Immunolabeling intensity of 11βHSD2 in the connecting tubule and cortical collecting duct was significantly reduced in sodium retaining phase of liver cirrhosis, and this was confirmed by immunoblotting. These observations therefore strongly support the view that the renal sodium retention associated with nephrotic syndrome and liver cirrhosis is caused by increased sodium reabsorption in the aldosterone sensitive distal nephron including the connecting tubule and collecting duct, and increased apical targeting of ENaC subunits plays a role in the development of sodium retention in nephrotic syndrome and liver cirrhosis.

Increased Apical Targeting of Renal Epithelial Sodium Channel Subunits and Decreased Expression of Type 2 11β-Hydroxysteroid Dehydrogenase in Rats with CCl4-Induced Decompensated Liver Cirrhosis

It was hypothesized that dysregulation of renal epithelial sodium channel (ENaC) subunits and/or 11beta-hydroxysteroid dehydrogenase (11betaHSD2) may play a role in the increased sodium retention in liver cirrhosis (LC). Experimental LC was induced in rats by CCl(4) (1 ml/kg, intraperitoneally, twice a week) for 12 wk (protocol 1) or for 11 wk (protocol 2). In both protocols, one group of rats with cirrhosis showed significantly decreased urinary sodium excretion and urinary Na/K ratio (group A), whereas a second group exhibited normal urinary sodium excretion (group B) compared with controls, even though extensive ascites was seen in both groups of rats with cirrhosis. In group A, protein abundance of alpha-ENaC was unchanged, whereas beta-ENaC abundance was decreased in the cortex/outer stripe of outer medulla compared with controls. The gamma-ENaC underwent a complex change associated with increased abundance of the 70-kD band with a concomitant decrease in the main 85-kD band, corresponding to an aldosterone effect. In contrast, no changes in the abundance of ENaC subunit were observed in group B. Immunoperoxidase microscopy revealed an increased apical targeting of alpha-, beta-, and gamma-ENaC subunits in distal convoluted tubule (DCT2), connecting tubule (CNT), and cortical and medullary collecting duct segments in group A but not in group B. Immunolabeling intensity of 11betaHSD2 in the DCT2, CNT, and cortical collecting duct was significantly reduced in group A but not in group B, and this was confirmed by immunoblotting. In conclusion, increased apical targeting of ENaC subunits combined with diminished abundance of 11betaHSD2 in the DCT2, CNT, and cortical collecting duct is likely to play a role in the sodium retaining stage of liver cirrhosis.

Increased apical targeting of renal ENaC subunits and decreased expression of 11βHSD2 in HgCl2-induced nephrotic syndrome in rats

Nephrotic syndrome is often accompanied by sodium retention and generalized edema. We hypothesize that dysregulation of the epithelial sodium channel (ENaC) and/or of sodium (co)transporters may be responsible for the increased sodium retention associated with HgCl(2)-induced nephropathy. In addition, we examined the hypothesis that the expression of type 2 11beta-hydroxysteroid dehydrogenase (11betaHSD2) is reduced, contributing to the enhanced mineralocorticoid activity. Membranous nephropathy was induced in Brown Norway rats by repeated injections of HgCl(2) (1 mg/kg sc), whereas the control group received only vehicle. After 13 days of treatment, the abundance of ENaC subunits, sodium (co)transporters, and 11betaHSD2 in the kidney was examined by immunoblotting and immunohistochemistry. HgCl(2) treatment induced marked proteinuria, hypoalbuminemia, decreased urinary sodium excretion, and ascites. The protein abundance of alpha-ENaC was increased in the cortex/outer stripe of outer medulla (OSOM) and inner stripe of the outer medulla (ISOM). The protein abundances of beta-ENaC and gamma-ENaC were decreased in the cortex/OSOM while increased in the ISOM. Immunoperoxidase microscopy demonstrated increased targeting of ENaC subunits to the apical plasma membrane in the distal convoluted tubule, connecting tubule, and cortical and medullary collecting duct segments. Moreover, 11betaHSD2 abundance was decreased in cortex/OSOM and ISOM. The protein abundances of type 3 Na/H exchanger (NHE3), Na-K-2Cl cotransporter (NKCC2), and thiazide-sensitive Na-Cl cotransporter (NCC) were decreased. Moreover, the abundance of the alpha-1 subunit of the Na-K-ATPase was decreased in the cortex/OSOM and ISOM but remained unchanged in the inner medulla. These results suggest that increased apical targeting of ENaC subunits combined with diminished abundance of 11betaHSD2 may contribute to sodium retention associated with HgCl(2)-induced nephrotic syndrome. The decreased abundance of NHE3, NKCC2, NCC, and Na-K-ATPase may play a compensatory role in promoting sodium excretion.

Increased expression of ENaC subunits and increased apical targeting of AQP2 in the kidneys of spontaneously hypertensive rats

In models of genetic hypertension, renal tubular dysfunction could be involved in the increased sodium and water reabsorption. However, the molecular basis for the increased renal sodium and water retention remains largely undefined in spontaneously hypertensive rats (SHR). We hypothesized that dysregulation of renal epithelial sodium channels (ENaC), sodium (co)transporters, or aquaporin-2 (AQP2) could be involved in the pathogenesis of hypertension in SHR. Six-week-old or twelve-week-old SHR and corresponding age-matched Wistar-Kyoto control rats (WKY) were studied. In both SHR groups, systolic blood pressure was markedly increased, whereas urine output, creatinine clearance, and urinary sodium excretion were decreased compared with corresponding WKY. Moreover, urine osmolality and urine-to-plasma osmolality ratio were increased compared with WKY. Semiquantitative immunoblotting demonstrated that the protein abundance of beta- and gamma-subunits of ENaC was increased in the cortex and outer stripe of the outer medulla and inner stripe of the outer medulla (ISOM) in SHR, whereas alpha-ENaC abundance was increased in ISOM. Immunoperoxidase microscopy confirmed the increased labeling of beta-ENaC and gamma-ENaC subunits in the late distal convoluted tubule, connecting tubule, and cortical and outer medullary collecting duct segments. In contrast, subcellular localization of alpha-ENaC, beta-ENaC, and gamma-ENaC was not changed. Expression of sodium/hydrogen exchanger type 3, bumetanide-sensitive Na-K-2Cl cotransporter, and thiazide-sensitive Na-Cl cotransporter was not altered in SHR. AQP2 levels were increased in the ISOM in SHR, and immunoperoxidase microscopy demonstrated an increased apical labeling of AQP2 in the inner medullary collecting duct in SHR. These results suggest that the increased protein abundance of ENaC subunits as well as the increased apical targeting of AQP2 may contribute to renal sodium and water retention observed during the development of hypertension in SHR.

Angiotensin II Increases H + -ATPase B1 Subunit Expression in Medullary Collecting Ducts

Metabolic alkalosis is a common feature of hypokalemic hypertensive syndromes associated with angiotensin II excess. The alkalosis-generating effect of angiotensin II is usually ascribed to its stimulatory effect on aldosterone secretion, a hormone that upregulates collecting duct hydrogen ion secretion. We studied the effect of angiotensin II infusions on the expression of B1 and a4 protein, subunits of the renal H+-ATPase in adrenalectomized rats. Adrenalectomized rats were given either angiotensin II or vehicle for 7 days via osmotic mini-pumps. H+-ATPase B1 protein expression was evaluated by Western blot analysis in isolated medulla and cortex plasma membrane preparations from one kidney, whereas the contralateral kidney was used for immunostaining. By Western blotting, the relative abundance of B1 protein was 2-fold higher in renal medulla membranes from rats with intact adrenal glands (sham surgery) than from adrenalectomized rats (219+/-47%, n=12; P<0.05). In contrast to renal medulla, adrenalectomy did not significantly alter the relative abundance of B1 protein in renal cortex. Angiotensin II also did not significantly alter the relative levels of B1 protein in the cortex, but it increased it significantly in renal medullary membranes (231+/-56%, n=8; P<0.005). Moreover, enhanced H+-ATPase B1 subunit protein immunoreactivity was found in medullary collecting duct segments of rats infused with angiotensin II. In contrast to B1, expression of a4, another subunit of the H+-ATPase was not altered by adrenalectomy or angiotensin II. We conclude that adrenalectomy decreases whereas angiotensin II increases H+-ATPase B1 subunit expression in medullary, but not in cortical collecting ducts. By increasing the relative abundance of the B1 subunit of H+-ATPase in the collecting duct, angiotensin II excess may lead to increased hydrogen ion secretion and thus metabolic alkalosis-a common feature of hypertensive syndromes associated with angiotensin II overactivity.

Increased expression and apical targeting of renal ENaC subunits in puromycin aminonucleoside-induced nephrotic syndrome in rats.

Nephrotic syndrome is often accompanied by sodium retention and generalized edema. However, the molecular basis for the decreased renal sodium excretion remains undefined. We hypothesized that epithelial Na channel (ENaC) subunit dysregulation may be responsible for the increased sodium retention. An experimental group of rats was treated with puromycin aminonucleoside (PAN; 180 mg/kg iv), whereas the control group received only vehicle. After 7 days, PAN treatment induced significant proteinuria, hypoalbuminemia, decreased urinary sodium excretion, and extensive ascites. The protein abundance of alpha-ENaC and beta-ENaC was increased in the inner stripe of the outer medulla (ISOM) and in the inner medulla (IM) but was not altered in the cortex. gamma-ENaC abundance was increased in the cortex, ISOM, and IM. Immunoperoxidase brightfield- and laser-scanning confocal fluorescence microscopy demonstrated increased targeting of alpha-ENaC, beta-ENaC, and gamma-ENaC subunits to the apical plasma membrane in the distal convoluted tubule (DCT2), connecting tubule, and cortical and medullary collecting duct segments. Immunoelectron microscopy further revealed an increased labeling of alpha-ENaC in the apical plasma membrane of cortical collecting duct principal cells of PAN-treated rats, indicating enhanced apical targeting of alpha-ENaC subunits. In contrast, the protein abundances of Na(+)/H(+) exchanger type 3 (NHE3), Na(+)-K(+)-2Cl(-) cotransporter (BSC-1), and thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC) were decreased. Moreover, the abundance of the alpha(1)-subunit of the Na-K-ATPase was decreased in the cortex and ISOM, but it remained unchanged in the IM. In conclusion, the increased or sustained expression of ENaC subunits combined with increased apical targeting in the DCT2, connecting tubule, and collecting duct are likely to play a role in the sodium retention associated with PAN-induced nephrotic syndrome. The decreased abundance of NHE3, BSC-1, TSC, and Na-K-ATPase may play a compensatory role to promote sodium excretion.

Localization of prostaglandin E(2) EP2 and EP4 receptors in the rat kidney.

We investigated the localization of cAMP-coupled prostaglandin E(2) EP2 and EP4 receptor expression in the rat kidney. EP2 mRNA was restricted to the outer and inner medulla in rat kidney, as determined by RNase protection assay. RT-PCR analysis of microdissected resistance vessels and nephron segments showed EP2 expression in descending thin limb of Henle's loop (DTL) and in vasa recta of the outer medulla. The EP4 receptor was expressed in distal convoluted tubule (DCT) and cortical collecting duct (CCD) in preglomerular vessels, and in outer medullary vasa recta. Butaprost, an EP2 receptor-selective agonist, dose dependently raised cAMP levels in microdissected DTL and outer medullary vasa recta specimens but had no effect in EP2-negative outer medullary collecting duct segments. Dietary salt intake did not alter EP2 expression in the kidney medulla. These results suggest that PGE(2) may act in the resistance vessels and in the DTL and DCT-CCD segments as a paracrine, cAMP-dependent regulator of vascular resistance and tubular transport, respectively.

Pathways for HCO3– reabsorption in mouse medullary collecting duct segments

To determine pathways of HCO3– reabsorption in the collecting duct of the mouse kidney, the outer medullary collecting duct (OMCD) and the terminal inner medullary collecting duct (IMCDt) were dissected and perfused at 1 to 2 nL/min, and total CO2 was measured by microfluorometry. In the OMCD, net HCO3– flux (JtCO2) was 12.2 ± 0.7 pmol/min/mm tubule length and decreased to 6.9 ± 0.6 pmol/min/mm tubule length (n = 5) with 10 μmol/L Schering 28080 (SCH) in perfusate (P <.001) and to 7.7 ± 0.6 pmol/min/mm tubule length (P <.004; n = 4) with 50 μmol/L diethylstilbestrol (DES), an inhibitor of H+-adenosine triphosphatase; together they reduced JtCO2 to 3.7 ± 0.2 pmol/min/mm tubule length (P =.0002; n = 4). In IMCDt, JtCO2 was 10.9 ± 1.1 pmol/min/mm tubule length, and it decreased to 4.3 ± 0.9 pmol/min/mm tubule length (n = 4) with 10 μmol/L SCH in perfusate (P <.05) and to 7.0 ± 1.1 pmol/min/mm tubule length (P <.05; n = 4) with 50 μmol/L DES; together they decreased JtCO2 to 2.3 ± 0.3 pmol/min/mm tubule length (P <.002; n = 4). Ouabain (1 mmol/L), an inhibitor of colonic H-K-adenosine triphosphatase (cHKA), in perfusate had no effect on JtCO2 in either segment. Northern hybridization studies showed a high level of expression of gastric HKA (gHKA) in outer medulla and a low level in inner medulla; cHKA expression was undetectable. Thus, in normal mouse OMCD and IMCDt, HCO3– reabsorption is predominantly mediated by gHKA and H+-adenosine triphosphatase and not cHKA. A third isoform of HKA could be present in mouse IMCDt. (J Lab Clin Med 2000;136:218-23)

RECOVERY OF DISTAL NEPHRON ENZYME ACTIVITY AFTER RELEASE OF UNILATERAL URETERAL OBSTRUCTION

Unilateral ureteral obstruction (UUO) for 24 hours results in a severe compromise of distal tubular function. The acidification defect is believed to be localized in the collecting duct. To characterize distal tubular function recovery one month after junction release, clearance studies in whole animals and enzyme studies in microdissected segments were performed in an experimental model of unilateral ureteral obstruction. Following release of ureteral obstruction of 24 hours duration, a significant decrease of whole kidney glomerular filtration rate was observed in the postobstructed kidney (POK) with a marked increase in urinary pH, fractional excretion of bicarbonate (FEHCO3-) and decrease in urinary osmolality. By orthograde stop flow experiment, bicarbonate excretion rate (Fr:Ff HCO3-/Fr:Ff Inutest) increased in the first and second urine fractions of 120 microl. corresponding to the collecting segment in the POK, one day after release. Decrease in U-P pCO2 (p<0.01) suggested an impaired H+ secretion on distal nephron in POK. Recovery of inulin clearance and values of urinary pH, FEHCO3- and urinary osmolality near contralateral and control kidneys were observed thirty days following ureteral release. The decline in enzyme activity in the distal nephron due to structural damage from high intratubular pressure was evaluated. Bafilomycin sensitive H+ -ATPase activity measurement in the medullary collecting duct segments of the POK showed an important decrease (68%), with lightly reduced activity (20%) in the cortical collecting duct, 24 hours after obstruction release. Localized in the connecting tubule cells and secreted into the tubular fluid in the late distal nephron, renal kallikrein has been involved in bicarbonate transport at cortical collecting duct segments. The renal kallikrein-like activity was reduced in POK (p<0.01). Recovery of enzyme activity was shown thirty days after unilateral ureteral obstruction. Our results show severe functional damage of the collecting duct after 24 hours of unilateral ureteral obstruction. H+ -ATPase activity was markedly decreased on medullary collecting duct segments. A correlation between the functional impairment of distal H+ secretion and decreased distal nephron enzyme activity has been shown. Recovery of both the functional and the enzyme activity at the distal nephron was demonstrated thirty days after obstruction release.

Functional evidence for a Ca2+/polyvalent cation sensor in the mouse thick ascending limb.

The effects of extracellular polyvalent cations on the cytosolic free Ca2+ concentration ([Ca2+]i) of isolated segments of the mouse nephron were investigated using fura 2 microfluorometry. Extracellular Ca2+ concentration ([Ca2+]o), gadolinium (Gd3+), and neomycin (Neo) increased the [Ca2+]i in cortical thick ascending limb (CTAL) tubules with effective doses (ED50) of approximately 3.5 mM for Ca2+, 20 microM for Gd3+, and 40 microM for Neo. This effect was reproduced by Ba2+ but not by Mg2+. High [Ca2+]o inhibited the responses to Gd3+, Neo, and Ba2+. The Gd(3+)- and Neo-evoked [Ca2+]i transients persisted in the absence of external Ca2+ and were abolished by the depletion of internal Ca2+ stores with thapsigargin (TG). The responses to rises in [Ca2+]o were similarly inhibited by TG and slightly reduced by 20 microM La3+ but not by 10 microM nifedipine. Mn2+ also mobilized a TG-sensitive internal Ca2+ store and stimulated its own entry. External Ca2+, Gd3+, and Neo induced small but significant increases in [Ca2+]i in distal convoluted tubule, cortical collecting duct, and outer medullary collecting duct segments, transiently increased [Ca2+]i in some medullary TAL (MTAL) tubules, but had no effect on descending thin limb. We conclude that a Ca(2+)-mobilizing Ca2+/polyvalent cation sensor resembling that of the parathyroid gland cells is predominantly located in the mouse CTAL but also in the MTAL and, to a lesser extent, in more distal segments.

Kinetic model of water and urea permeability regulation by vasopressin in collecting duct

We present a mathematical model describing the kinetics of water-channel and urea-carrier regulation by vasopressin in the apical membrane of collecting duct cells. The rate of change of the number of activated channels or carriers in the apical membrane is modeled as a balance between the rate of activation (or exocytic insertion) and the rate of inactivation (or endocytic retrieval) of transporters. In a three-state version of the model, transporters are assumed to be partitioned into three physical states, i.e., an "activated" state that imparts a permeation pathway to the apical membrane, an "inactivated" state, and a "reserve" state. Both activation and inactivation are represented by first-order kinetic equations describing transition from reserve to activated transporters and from activated to inactivated transporters, respectively. A simplified two-state model is derived from the three-state model, with the assumption that the transformation from inactivated to reserve transporter occurs rapidly relative to the other state transitions. Simulated time courses obtained by solving model equations are compared with experimentally determined time courses to test whether the response to vasopressin in isolated inner medullary collecting duct segments can be explained by direct effects on the rate constants for activation or inactivation. The results indicate that, for both transporters, it must be assumed that vasopressin directly regulates rate constants for both activation (exocytosis) and inactivation (endocytosis) to account for the experimentally determined dynamic responses to vasopressin and its withdrawal. These studies provide a theoretical basis on which to design further experimental studies.

Distribution of mineralocorticoid and glucocorticoid receptor mRNA along the nephron

In the present study, a competitive polymerase chain reaction (PCR) technique was used to quantitate the relative levels of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA in microdissected nephron segments from the rat kidney and of MR mRNA from isolated principal and intercalated collecting duct cells from rabbit. RNA was isolated from cells and isolated tubules, cDNA was synthesized, and receptor cDNA was coamplified by PCR with a competitive control template. beta-Actin PCR products were also obtained from each nephron segment studied, to assess variations in RNA extraction and cDNA synthesis. MR mRNA, as determined by this competitive PCR technique, was 10-fold more abundant in cortical collecting duct (CCD), outer medullary collecting duct, and inner medullary collecting duct segments than in the proximal tubule and thick ascending limb segments (P < 0.05). Both principal and beta-intercalated cells of the CCD contained detectable levels of MR mRNA, although the levels in the principal cells were threefold higher (P < 0.01). GR mRNA was twofold more abundant in glomeruli, proximal tubule, and thick ascending limb segments than in the collecting duct segments (P < 0.05). In general, the distribution pattern of MR and GR mRNA is consistent with the distribution of adrenal corticosteroid function along the nephron.

Na+-K+-ATPase activities in renal tubule segments of rat inner medulla

Recent studies have revealed that both the thin segment of the long loop of Henle and the inner medullary collecting duct (IMCD) are morphologically and functionally nonuniform along their lengths. In the present study, we have carried out measurements of Na+-K+-ATPase activity in thin limb and IMCD segments microdissected from different positions along the axis of the rat inner medulla. Among collecting duct segments, in control rats, Na+-K+-ATPase activity was highest in the initial quarter of the IMCD. The Na+-K+-ATPase activity was lower and relatively uniform along the remaining three quarters of the IMCD. Na+-K+-ATPase activities in cortical and outer medullary collecting duct segments were similar to previously reported values. The Na+-K+-ATPase activity in the initial quarter of the IMCD was increased following in vivo deoxycorticosterone administration (by 140%) and following dietary NaCl restriction (by 100%). These procedures increased Na+-K+-ATPase activity by smaller degrees or not at all in the remaining three-quarters of the IMCD. Thin descending limbs and thin ascending limbs from all regions of the inner medulla had low but readily measurable values. Based on these results, we conclude that all inner medullary renal tubule segments have measurable Na+-K+-ATPase activities that could potentially drive solute active transport.

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.

Electrophysiological study of isolated perfused human collecting ducts: Ion dependency of the transepithelial potential difference.

Cortical and outer medullary collecting duct segments were dissected from human kidneys and perfused in vitro. The transepithelial potential difference was measured and found to be lumen positive +6.8 +/- 0.6 mV (n= 20). This lumen-positive potential difference was inhibited by ouabain and furosemide but not by acetazolamide. Replacement of chloride in bath and perfusion fluids caused a reversible decrease of the potential difference to near zero. We conclude from these studies: (a) the lumen-positive potential difference is dependent upon the presence of chloride ion suggesting the existence of an active electrogenic chloride reabsorptive process in the human collecting duct and (b) it is possible to examine human renal physiology directly using in vitro microperfusion of tubule segments.