Sodium entry routes in principal and intercalated cells of the isolated perfused cortical collecting duct

Transmembrane sodium transport pathways were studied in principal and intercalated cells of the isolated perfused rabbit cortical collecting duct. Intracellular electrolyte concentrations in individual collecting duct cells were measured by electron microprobe analysis during blockage of basolateral Na-K-ATPase by ouabain and simultaneous inhibition of sodium entry across the apical and/or basolateral cell membrane. In principal cells the ouabain-induced rise in cell sodium concentration could only partially be blocked by amiloride (10^–4mol/l) in the perfusion fluid. Amiloride (10^–3mol/l) added to the bathing solution produced a further, significant reduction of sodium influx. In principal cells the ouabain-induced increase in sodium concentration was completely prevented by amiloride in the perfusion solution in combination with omission of sodium from the peritubular bathing solution. In intercalated cells ouabain caused a less pronounced increase in sodium concentration than in principal cells. Neither amiloride in the perfusate, nor amiloride in both bathing and perfusion solution, significantly reduced the ouabain-induced rise in intercalated cell sodium concentration. These results indicate that in principal cells amiloride-sensitive sodium channels constitute the predominant pathway for sodium entry across the apical cell membrane. In addition, substantial amounts of sodium enter principal cells across the basolateral cell membrane, probably via Na-H exchange. Finally, the data suggest that in intercalated cells sodium channels and the Na-H exchange are sparse or even absent.     

Ionic conductances of cultured principal cell epithelium of renal collecting duct

The ionic conductive properties were studied of epithelia of collecting duct principal cells which had been grown in primary tissue culture from renal cortex/capsule explants. When pretreated with aldosterone (10^–6 mol/l) and bathed on either surface with isotonic HCO ₃ ^– -free Ringer's solution, the transepithelial voltage,V _te, varied between –21 and –72 mV (apical surface negative) while the transepithelial resistance,R _te, ranged from 0.4 to 1.5 kcm². By 10:1 step-changes in Na⁺ concentration the apical cell membrane was shown to have a high conductivity for sodium, inhibitable by amiloride, 10^–6 mol/l. However, contrary to observations in natural collecting duct under control conditions, amiloride never reversed the polarity ofV _te even at 10^–4 mol/l. Both the apical and the basolateral cell membranes were conductive for potassium and both conductivities were inhibitable by Ba²⁺ (5 mmol/l). 10:1 reduction of apical Cl^– concentration strongly hyperpolarizedV _te with a monophasic time course suggesting the presence of a paracellular shunt conductance for Cl^–. In addition there may be a small Cl^– conductance present in the apical cell membrane since apical application of the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB) at 10^–7 mol/l produced a minute but significant hyperpolarization. On the other hand, 10:1 reduction of basolateral Cl^– concentration caused a biphasic change inV _te (initial depolarization, followed by repolarization) which indicates the presence of a large Cl^– conductance in the basolateral cell membrane. The latter was not inhibitable by 10^–7 mol/l NPPAB. Higher concentrations of this and of an other Cl^– channel blocker produced non-specific effects. In conclusion, our studies of a pure principal cell epithelium confirm findings described for the intact cortical collecting duct and add new information concerning chloride conductivity and related blocking agents.Dedicated to Prof. Dr. H. Sitte, Homburg, FRG, upon his 60th birthday.     

Electrical properties of renal collecting duct principal cell epithelium in tissue culture

Summary Whereas collecting duct epithelium in vivo is composed of principal and intercalated cells, we grew a pure principal cell epithelium using a new technique involving tissue culture. These principal cells were derived from collecting duct anlagen of newborn rabbits. We investigated the electrical properties of such epithelia in a newly designed lucite double-chamber with an inner opening of 0.08 cm². Our observations were: 1) mean transepithelial resistanceR _te was 0.83±0.2 kcm² at 37° C and after preincubation in aldosterone; 2) mean transepithelial potential differenceV _te was low and variable under standard conditions and at room temperature but increased to –59.5±4.4 mV (sign referring to polarity of apical surface) after preincubation in 10^–6 mol/l aldosterone and at 37° C; 3) 10^–6 mol/l amiloride added to the apical perfusion fluid largely abolished thisV _te while increasingR _te by 120%; 4) experiments with 5×10^–3 mol/l BaCl₂ in the apical perfusion fluid failed to changeR _te andV _te significantly. This principal cell epithelium therefore has characteristics of a tight epithelium with active sodium transport; however, its electrical properties differ from those of the isolated perfused collecting duct segment.Supported by grants from the Deutsche Forschungsgemeinschaft (Gr 605/3-1; SFB-90)     

Fluorescence activated cell sorting of principal and intercalated cells of the renal collecting duct

Summary A method is described for the isolation of homogeneous populations of principal and intercalated cells (PC and ICC) of the rabbit cortical collecting duct using fluorescence-activated cell sorting (FACS) in tandem with solid phase immunoadsorption. Three sorting strategies are described. In the first one the two cell types are separated based on the different intensities of their reaction with a monoclonal antibody. In the second and third strategies PC and ICC are tagged with cell-specific markers coupled to different fluorochromes and separated based on their green and red or green and blue fluorescence, respectively. These near homogeneous (∼99%) populations of PC and ICC can be obtained in numbers large enough to perform biochemical characterization, determination of hormonal sensitivity and initiation of cell cultures. This study was supported by grants DK 39523 and DK 41841 from the National Institutes of Health, Bethesda, MD, and by a Grant-in-Aid from the American Heart Association (89-985).     

High-conductance K+ channel in apical membranes of principal cells cultured from rabbit renal cortical collecting duct anlagen

Using the patch clamp technique, one type of K⁺ channel was identified in the apical cell membrane of cultured principal cells of rabbit renal collecting ducts in the cell-attached or excised-patch configuration. The channel was highly selective for K⁺ over Na⁺ (typically 30-70-fold) and had a conductance of 180, SD±39 pS (n=6), referred to a situation of 140 mmolar K⁺-Ringer solution present on either surface of the patch membrane. Channel activity was completely blocked by Ba²⁺ (5 mmol/l) and partially inhibited by Na⁺. The latter was evidenced by a deviation from Goldman rectification at high cytoplasm-positive membrane potentials, which was observed when Na⁺ competed with K⁺ for channel entrace from the cytoplasmic surface. Channel open probability depended strongly on membrane voltage and cytoplasmic Ca²⁺ concentration. Open-close kinetics exhibited double exponential behaviour, with a strong voltage dependence of the slow open time constant. Infrequently also a substate conductance level was identified. The voltage and calcium dependence suggest that the channel plays a role in adjusting K⁺ secretion to Na⁺ absorption in the fine regulation of cation excretion in renal collecting ducts.     

Centroacinar and intercalated duct cells as potential precursors of pancreatic endocrine cells in rats treated with streptozotocin

The present study examined the possibility for regeneration of pancreatic endocrine cells from centroacinar (CA) and intercalated duct (ICD) cells in rat pancreas after 5 days of continuous streptozotocin (STZ) administration. Nine rats were divided into 3 experimental groups: 1) Control group, 2) Short term recovery group; three days after STZ administration (STZ 3), and 3) Long term recovery group; ten days post-STZ administration (STZ 10). The CA and ICD cells in the STZ 3 group had swollen cytoplasm, and sometimes contained a vesicle within the core. An insulin positive signal was detected in and around the CA and ICD cells. In the STZ 3 group, cytokeratin 20 signals were co-localized with insulin signals in both CA and ICD cells. Electron microscopically, endocrine cells and small pancreatic islets were in close contact with CA and ICD cells. Systemic biophysical serum data reflected these immunohistological results. The present results suggest that CA and ICD cells are involved in the regeneration of pancreatic B cells in rats following a lesion produced by five consecutive days of STZ administration.     

Electrophysiological studies in principal cells of rat cortical collecting tubules ADH increases the apical membrane Na+-conductance

The mechanism of ion transport across principal cells of rat cortical collecting tubules (CCT) and its regulation by vasopressin (ADH) has been studied in the isolated perfused tubule. To amplify the response to ADH rats were treated with 5 mg I. M. desoxycorticosterone 4–9 days prior to the experiments. Addition of 2·10^–10 mol·1^–1 ADH increased the transepithelial voltage from –5.1 ±0.7 mV to –16.1±1.4 mV (n=37) and decreased the transepithelial resistance from 51±4 cm² to 39±2 cm² (n=33). Optical and functional differentiation of impalements of principal and intercalated cells was made and only data of principal cells are presented. ADH depolarized the apical membrane from 79±1 mV to 66±2 mV (n=26) and decreased the fractional resistance of the apical membrane from 0.76±0.04 to 0.70±0.04 (n=13). These ADH effects were prevented by 10^–5 or 10^–4 mol·1^–1 luminal amiloride which hyperpolarized the apical membrane when added in the presence or absence of ADH. Apical and basolateral membranes were dominated by large K⁺ conductances and addition of 3 mmol·1^–1 barium to bath or lumen perfusates increased transepithelial resistance almost two-fold, whereas luminal amiloride increased the transepithelial resistance only by 26–35%. Ouabain (0.5 mmol·1^–1, bath) depolarized the basolateral membrane and decreased its K⁺ conductance. These effects were prevented by the simultaneous presence of apical amiloride suggesting that the only route of Na⁺ entry into the principal cells occurred via the amiloride sensitive Na⁺ conductance. We conclude that ADH stimulates Na⁺ reabsorption and K⁺ secretion in the rat CCT primarily by increasing the Na⁺ conductance in the apical cell membrane.Parts of this study have been presented at the 19th ASN meeting in Washington, DC, USA 1986     

Cytologic findings of collecting duct carcinoma of the kidney

We report the cytologic features of eight fine-needle aspirations (FNA) and eight exfoliative specimens of collecting duct carcinoma (CDC) obtained from six patients. The four men and two women ranged in age from 27 to 69 years (mean = 45 yr) and all had advanced stage disease at presentation (one stage III, five stage IV). Five of the six patients died of widespread disease, and one is alive and well (mean survival, 28 mo; range, 11–48 mo). The smears of the FNA and exfoliative specimens were scantly to moderately cellular. Tumor cells showed moderate pleomorphism and were arranged primarily in cohesive groups that rarely had a papillary configuration. Nuclei had irregular nuclear contours, coarse chromatin, and one to three nucleoli. In the majority of cases the cytoplasm was finely vacuolated, and occasionally there were large intracytoplasmic vacuoles. Intracytoplasmic mucin was demonstrated in two aspirates. Psammoma bodies were present in four of the seven fluids. In two patients, the cytologic diagnosis was supported by positive immunostaining for high-molecular-weight keratin and Ulex europaeus agglutinin I lectin. Leu M-1 was focally positive in one case and negative in the other. The cytologic features of CDC were readily identified as malignant; however, they were not distinctive and overlapped with those of high-grade renal cell carcinoma with papillury features and transitional cell carcinoma. © 1995 Wiley-Liss, Inc.     

Urea handling by the medullary collecting duct of the rat kidney during hydropenia and urea infusion

Previous micropuncture studies of distal tubule fluid and ureteral urine have indicated a varying degree of urea reabsorption in the collecting duct. In the present experiments the microcatheterization technique was used to directly determine urea, Na, K, total solute and fluid reabsorption along the length of the medullary collecting duct in anaesthetized hydropenic rats and in rats given low dose urea infusion (P_urea 18.9 mM/l). In hydropenic rats, the remaining fraction of filtered urea did not change significantly along the collecting duct, as indicated both by regression analysis of all samples and by comparison of paired samples from the corticomedullary junction and papillary tip. During low dose urea infusion, urine osmolality increased in proportion to the increase in urea concentration and again there was no net urea reabsorption between the beginning and end of the duct. However, during urea infusion, analysis of samples from the beginning, mid-zone, and end of the collecting duct indicated that urea entry occurred in the proximal portion of the duct (beginning to mid-zone,P<0.01) and that urea reabsorption occurred in the distal portion (mid-zone to end,P<0.01). The lack of significant net urea reabsorption along the duct despite the excretion of moderately concentrated urine, has implicatios for the concept of medullary urea recycling and for models of the urinary concentrating mechanism. The finding of functional heterogeneity with respect to urea handling in the collecting duct in vivo, with both reabsorption and secretion being demonstrated, raises the possibility that internal recycling of urea in the medullary collecting duct itself may contribute to maintenance of a high papillary interstitial urea concentration.     

Fine-needle aspiration biopsy of renal collecting duct carcinoma

The fine-needle aspiration biopsy (FNA) findings in a case of collecting duct (ducts of Bellini) carcinoma of the kidney are described. The cytologic smears were modestly cellular and contained individual and small clusters of round to oval cells with small to moderate amounts of well-defined cytoplasm. The nuclei were large and hyperchromatic with prominent single nucleoli and focal chromatin clearing. The findings were distinct from those of the usual clear or granular cell renal-cell carcinomas and were more similar to adenocarcinomas arising in the breast, ovary, or pancreas. Because collecting duct carcinomas need to be distinguished from metastatic deposits and are believed to have a poorer prognosis than the majority of renal-cell carcinomas, preoperative diagnosis may have important therapeutic implications. The distinction of collecting duct carcinoma from the more common variants of renal-cell carcinoma and transitional-cell carcinoma is discussed. Diagn Cytopathol 1994; 11:74–78. © 1994 Wiley-Liss, Inc.     

Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties

Madin-Darby canine kidney (MDCK) cells originate from the renal collecting duct and consist of different cell subtypes. We cloned two MDCK cell subtypes denominated as C7 and C11 with different morphology and different function. The two clones maintained their functional differences after cloning. C7 monolayers exhibit a high transepithelial resistance (R _te=5648±206 · cm², n=20) and secrete K⁺ (K⁺=1.31±0.08 mmol/l, n=10) into the apical medium. C11 monolayers display a low R _te (330±52 · cm², n=20) and secrete Cl^– (Cl^–= 16.9±1.8 mmol/l, n=10) into the apical medium. Aldosterone (1 mol/l) stimulates K⁺ secretion (K⁺ of 3.58±0.11 mmol/l, n=7) in C7 cells and H⁺ secretion in C11 cells (pH=0.060±0.007, n=10). Aldosterone-induced stimulation of K⁺ secretion is inhibited by apical application of amiloride (1 mol/l). cAMP stimulates H⁺ secretion in C11 cells (pH= –0.068±0.004, n=10). Furthermore, C7 cells are peanut-lectin(PNA)-negative and exhibit an intracellular pH of 7.39±0.05 (n=7), whereas C11 cells maintain intracellular pH at 7.16±0.05 (n=8) and a major fraction of cells is PNA positive. We conclude that we have cloned two subtypes of MDCK cells which stably express different functional characteristics. The C7 subtype resembles principal cells (PC) of the renal collecting duct, whereas the C11 subtype resembles intercalated cells (ICC) of the renal collecting duct. The C11 subtype seems heterogenous and possibly consists of two subpopulations similar to the -ICC and -ICC of the renal collecting duct. The two cloned subtypes could serve as a valuable model to study PC-like cells and ICC-like cells independently.     

Intercalated duct cells in the rat parotid gland may behave as tissue stem cells

This study was conducted to determine whether intercalated duct cells in the rat parotid gland have the properties of tissue stem cells. After induction of cellular proliferation by repeated administration of isoproterenol (IPR), a β-adrenergic agonist, proliferation activity in acinar, intralobular, and intercalated ductal cells was quantified using Ki-67 immunohistochemistry. The total number of each type of component cell in a gland was also estimated in the course of IPR treatment. IPR was found to induce proliferation of acinar and intercalated duct cells, but not intralobular duct cells. The total number of acinar cells in a gland on day 5 of IPR treatment was 1.6 times of that at day 0 (baseline). In contrast, the total numbers of intercalated and intralobular duct cells did not change from baseline, indicating a high possibility that the proliferated intercalated duct cells differentiated into acinar cells. On days 2 to 3 of IPR treatment, intercalated duct cells with amylase-positive secretory granules were recognized in a region very close to the acini, and were suspected of being transitional cells from intercalated duct to acinar cells. This quantitative study indicates that intercalated duct cells may have the properties of tissue stem cells upon IPR stimulation.     

Transcellular sodium transport and basolateral rubidium uptake in the isolated perfused cortical collecting duct

The relation between transcellular Na⁺ absorption, intracellular Na⁺ concentration and Na⁺/K⁺-ATPase activity (the last estimated by the rubidium uptake across the basolateral cell membrane) was examined in the different cell types of the rabbit cortical collecting duct (CCD). Experiments were performed on isolated perfused CCD in which Na⁺ absorption was varied by perfusing the tubule with solutions containing different Na⁺ concentrations (nominally Na⁺-free, 30 mM and 144 mM). Experiments were terminated by shock-freezing the tubules during perfusion. Precisely 30 s before shock-freezing, the K⁺ in the bathing solution was exchanged for Rb⁺. Intracellular element concentrations, including Rb⁺, were determined in freeze-dried cryosections of the tubules using energy-dispersive X-ray analysis. Increasing Na⁺ concentration in the perfusion solution caused significant rises in intracellular Na⁺ concentration and Rb⁺ uptake of principal cells. Principal cell Na⁺ and Rb⁺ concentrations were 7.8±0.9 and 7.0±0.8 mmol/kg wet weight respectively, when the perfusion solution was Na⁺-free, 10.1±0.7 and 11.6±0.6 mmol/kg wet weight with 30 mM Na⁺ in the perfusion solution, and 14.5±1.5 and 14.9 ±0.9 mmol/kg wet weight with 144 mM Na⁺ in the perfusion solution. In contrast, a comparable relationship between lumen Na⁺ concentration, intracellular Na⁺ concentration and basolateral Rb⁺ uptake was not seen in intercalated cells. These results support the notion that principal, but not intercalated, cells are involved in transepithelial Na⁺ absorption. In addition, the data demonstrate that apical Na⁺ entry and basolateral Na⁺/K⁺-AT-Pase activity are closely coupled in principal cells of the rabbit CCD. A rise in lumen Na⁺ concentration leads to increased Na⁺ entry and augmented intracellular Na⁺ concentration, which then secondarily stimulates active basolateral Na⁺/K⁺(Rb⁺) exchange.     

Sorbitol metabolism in inner medullary collecting duct cells of diabetic rats

Intracellular accumulation of sorbitol, generated fromd-glucose via the aldose reductase pathway, is thought to play an important role in diabetic complications such as lens cataracts and neuropathy. In order to elucidate the effect of diabetes on the renal inner medulla, another sorbitol-rich tissue, male Wistar rats were treated with a single dose of streptozotocin (60 mg/kg body weight, i.p.). Six wecks later total inner medullary tissue (IM) or isolated inner medullary collecting duct (IMCD) cells were prepared. In diabetic IM tissue, sorbitol content was 1.8-fold higher than in control IM tissue (134±17 vs. 74±22 mol/g tissue protein). Sorbitol production in both normal and diabetic IMCD cells was strongly dependent on extracellulard-glucose concentration. In normal cells, for example, sorbitol production was 90±9 mol sorbitol/g protein x h at 45 mMd-glucose compared to 13±1 mol/g protein x h at 5 mM. At identicald-glucose concentrations sorbitol synthesis in diabetic IMCD cells was, however, always significantly higher than in control cells (122% of control at 15 mM and 126% of control at 45 mM). In addition, aldose reductase activity in diabetic IM was found to be augmented. The maximal velocity was 4.2 times higher (97±22 U/g protein vs. 23±7 U/g protein) while theK _m of the enzyme remained unchanged. Membrane permeability for sorbitol or the response to changes in extracellular osmolarity was not significantly different in diabetic IMCD cells and normal cells with correspondingly high intracellular sorbitol concentrations. Similarly the kinetic parameters ofd-glucose uptake were not altered by streptozotocin treatment. These results suggest that increased medullary sorbitol content in diabetic rats is a result of increased sorbitol synthesis due to a higher extracellulard-glucose concentration and augmented aldose reductase activity in face of an unaltered sorbitol permeability of the plasma membrane.     

Electrophysiological study of inner medullary collecting duct of hamsters

The electrophysiological properties of the hamster mid-inner medullary collecting duct (IMCD₂) cells were examined in isolated and perfused preparations by intracellular impalement with conventional 1 mol/l KCl microelectrodes and cable analysis. The transmural voltage (V _T) was not different from 0 mV, while the basolateral transmembrane voltage (V _B) was –81.7±0.91 mV (n=221). The transmural resistance (R _T) was 109 cm², indicating that the IMCD₂ is composed of tight epithelia. The fractional apical membrane resistance (fR _A) was 0.98±0.003 (n=10). Abrupt changes in the luminal concentration of Na⁺, K⁺ or Cl^– did not alter the apical membrane voltage (V _A) or V _T, and neither 2 mmol/l Ba²⁺ nor 10 mol/l amiloride in the lumen affected V _A and V _T. Moreover, pretreatment of hamsters with deoxycorticosterone acetate (5 mg/ kg, s.c.) for 10–14 days caused only a very small change in V _T in the negative direction. Amiloride in the lumen increased R _T and increased the voltage divider ratio very slightly. However, an abrupt increase in K⁺ concentration in the bath from 5 mmol/l to 50 mmol/l or addition of 2 mmol/l Ba²⁺ to the bath depolarized the basolateral membrane by 39 mV and 29 mV, respectively. In the presence of 2 mmol/l Ba²⁺ in the bath, a reduction of HCO₃ ^– concentration from 25 mmol/l to 2.5 mmol/l depolarized V _B by 20.4 mV. No Cl^– conductance was demonstrated in the basolateral membrane. Addition of ouabain to the bath or elimination of K⁺ from the bath caused only very small changes in V _B of the IMCD₂ as compared to the marked responses ovserved in the medullary thick ascending limb of the loop of Henle and in the upper portion of the descending limb of the long-looped nephron. These findings are compatible with the view that either a weak Na⁺-K⁺ pump or an ouabain-resistant pump in combination with high K⁺ conductance in the basolateral membrane mainly accounts for the maintenance of the intracellular concentration of Na⁺ and K⁺. The IMCD₂ may contribute little to the transmural transport of Na⁺ and K⁺. The physiological significance of HCO₃ ^– conductance in the basolateral membrane remains to be established.     

Intracellular sorbitol content in isolated rat inner medullary collecting duct cells

In order to study the mechanisms involved in the regulation of renal inner medullary sorbitol content, collecting duct cells were isolated from rat inner medulla and the effect of extracellular osmolarity on sorbitol synthesis and sorbitol content was investigated. Cells isolated at 300 mosmol/l and incubated up to 24 h as primary cultures in 300 mosmol/l media or in media made 600 mosmol/l by the addition of 150 mM NaCl showed no difference in total synthesis. Intracellular sorbitol content was, however, 2.3-fold higher in the cells kept in the higher osmotic medium. Cells isolated at 600 mosmol/l released sorbitol about 8 times faster when transferred into hypoosmotic medium (300 mosmol/l) than when transferred into isoosmotic (600 mosmol/l) media. Cells exposed to hyperosmotic media (900 mosmol/l with NaCl) maintained a higher intracellular sorbitol content than cells incubated in isoosmotic media. Changes of intracellular sorbitol content could not be attributed entirely to cell lysis — as demonstrated by determination of cellular content of lactate and lactate dehydrogenase. The alteration in sorbitol membrane permeability was reversible and was only observed when poorly permeable solutes (such as NaCl and sucrose) were used for the experiments, changes in urea elicited no effect. It is proposed that rapid changes in membrane permeability to sorbitol play an important role in the adjustment of intracellular sorbitol concentration in inner medullary collecting duct cells to changes in extracellular osmolarity.Supported by grant DFG Gr 916/1-1     

Osmotic work across inner medullary collecting duct accomplished by difference in reflection coefficients for urea and NaCl

To demonstrate that osmotic work can be accomplished across the inner medullary collecting duct (IMCD) by the difference in refelection coefficients for urea and NaCl, phenomenological coefficients for urea and NaCl transport were determined in isolated segments of the hamster IMCD perfused in vitro. Arginine vasopressin at 100 U/ml increased urea permeability from 11.5±2.9 to 31.7±4.2×10^–7 cm² s^–1 in the middle IMCD but not in the upper IMCD. Urea transport in the middle IMCD consisted of two components, transport with saturable kinetics and simple passive diffusion. Permeability to Na⁺ was very low (2×10^–7 cm² s^–1). Reflection coefficients as measured by the equiosmolality method, with raffinose being a reference solute, were 0.87±0.05 and 0.71±0.04 for urea and 1.03±0.07 and 0.91±0.04 for NaCl in the upper and the middle IMCD, respectively. Reflection coefficient for urea in the middle IMCD was 0.68 when determined by the zero volume flux method. When the middle IMCD was perfused with bicarbonate Krebs-Ringer (BKR) solution containing 200 mmol/l urea, the replacement of urea in the bathing fluid with equiosmolal NaCl caused large volume flux (3.81±0.45 nl mm^–1 min^–1) associated with dilatation of intercellular space. The existence of vasopressin in the bath was essential for this phenomenon. This effect was inhibited by 5×10^–4 M phloretin in the bath, suggesting that the vasoressin-stimulated urea transport is responsible for this phenomenon. From these observations, we conclude that transport parameters of the middle IMCD are appropriate for accomplishment of osmotic work across this segment in the absence of physicochemical osmotic gradients.     

小鼠肾集合管发育中主细胞的形态学变化及水通道蛋白-2和-3的表达

目的 观察小鼠肾集合管发育过程中主细胞的形态学变化及水通道蛋白(AQP)-2、-3的表达,探讨AQP-2、-3与小鼠肾集合管主细胞发育的关系及作用.方法 应用透射电镜、免疫组织化学、免疫印迹技术并结合体视学方法,观察并检测胚龄16、18 d 胎鼠及生后1、3、7、14、21 d仔鼠肾集合管发育过程中主细胞的形态学变化及...     

Role of [Ca2+]i in lethal oxidative injury in rat cultured inner medullary collecting duct cells

Reactive oxygen metabolites have been implicated in the pathogenesis of toxic, ischaemic and immunologically mediated renal injury. An increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) has been proposed as a mechanism of oxidative stress-induced cell injury. We used a fluorescence spectrometer and a fluorescence probe to measure the [Ca²⁺]_i and viability of rat primary cultured inner medullary collecting duct (IMCD) cells during oxidative stress induced by 5 mM tert-butyl hydroperoxide (TBHP). Initially, this oxidative stress evoked a small increase in [Ca²⁺]_i which was followed by a slower sustained increase from the resting level of 170.8±38.8 nM to 1490.5±301.7 nM after 60 min, and this preceded the loss of plasma membrane integrity, measured by the propidium iodide fluorescence method. The elimination of extracellular Ca²⁺ from the culture medium prevented the TBHP-induced [Ca²⁺]_i increase and improved cell viability. Restoration of extracellular Ca²⁺ resulted in an immediate and large increase in [Ca²⁺]_i and extensive cell death. Verapamil, a Ca²⁺ channel blocker, inhibited the [Ca⁺]_i increase and afforded significant protection against cellular injury following exposure to TBHPinduced oxidative stress. Extracellular acidosis also prevented the increase in [Ca²⁺]_i and cell death caused by this oxidative stress. These results are consistent with the hypothesis that oxidative stress-induced IMCD cellular injury may be the result of increased [Ca²⁺]_i caused, in part, by activation of voltage-dependent Ca²⁺ channels.     

奥美拉唑对家兔肾脏内髓集合管细胞H+/K+交换功能的影响

目的探讨奥美拉唑对家兔肾脏IMCD细胞H+/K+交换的影响,以及经洗涤处理是否解除这种影响.方法原代培养家兔肾脏IMCD细胞单层,在100 μmol/L奥美拉唑缓冲液中孵育25 min, 洗涤组则在奥美拉唑缓冲液孵育后,用不含奥美拉唑的缓冲液洗涤IMCD细胞;对照组在不含奥美拉唑的缓冲液孵育25 min;CECF/AM荧光探针法测定各组IMCD细胞H+/K+交换.结果奥美拉唑组的H+/K+交换为(0.016±0.006) dpHi/min(n=8);与对照组的(0.053±0.008) dpHi/min(n=8)相比,相差非常显著(P<0.001);洗涤组的H+/K+交换为(0.016±0.006) dpHi/min(n=6),与对照组(n=6)的(0.052±0.009)dpHi/min相比,相差非常显著(P<0.001).结论 100 mol/L的奥美拉唑对家兔IMCD细胞的H+/K+交换有显著影响,而且洗涤处理不能解除这种抑制.因而,肺心病合并上消化道出血患者使用奥美拉唑时,应综合考虑其利弊.