In vitro function of cyst epithelium from human polycystic kidney.

It is thought that cysts in polycystic kidneys originate from nephron segments and function in a manner similar to the segment or origin. The indirect evidence for this derives from studies of microanatomy and cyst fluid composition. Cysts with low Na+ have been classified as distal, whereas cysts with high Na+ have been classified as proximal. In order to directly determine the transport characteristics of cyst epithelium, cysts from a human polycystic kidney were studied in vitro using Ussing chamber techniques. Composition of cyst fluid was determined in parallel with these studies. Cysts with low Na+ (gradient cysts) demonstrate characteristics consistent with distal nephron origin including elevated potential difference (PD), short-circuit current (Isc), and low conductance. PD and Isc of gradient cysts were amiloride sensitive. Nongradient cysts, however, require additional characterization. At least two types of nongradient cysts were identified, one with characteristics consistent with proximal nephron origin and another apparently without function. These studies are the first direct evidence for active transport of cysts from human polycystic kidney and provide strong evidence to support the concept that cysts function in the same manner as the nephron segment of origin.

Purinoceptor P2U identification and function in human intrahepatic biliary epithelial cell lines.

The mechanisms that regulate ion and fluid transport by the human intrahepatic bile duct have not been well defined. Human intrahepatic biliary cell lines that we have developed were used to identify and characterize purinoceptors based on increases in intracellular calcium in response to ATP and other nucleotides. Intracellular free calcium was measured in cell suspensions using the fluorescent probe Fura-2 and a fluorescence spectrophotometer. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe SPQ. Intracellular calcium increases equivalently in response to ATP and UTP, peaking, then diminishing to a new, elevated baseline. The peak elevation of calcium is the result of both the release of intracellular stores of calcium and the influx of extracellular calcium. The purinoceptor P2U-subtype was identified based on the potency rank order of ATP-analogues. Halide efflux increases with P2U-purinoceptor stimulation which is consistent with the opening of a Ca(2+)-sensitive Cl- channel. The physiological significance of P2U-purinoceptor activation and its effect on the ionic content and flow rate of bile remains to be determined.

Biphasic 19-nor-deoxycorticosterone excretion after adrenal enucleation is associated with altered adrenal mitochondrial cytochrome P450 11 beta-, 18-, and 19-hydroxylase activities.

After adrenal enucleation (AE) rats avidly retain sodium (early phase), but after 7-10 days they lose this sodium avidity (late phase). Although increased production of a mineralocorticoid, 19-nor-deoxycorticosterone (19-Nor-DOC), has been implicated, 19-Nor-DOC levels during the early and late phases of AE have not been systematically measured. Furthermore it is not known why 19-Nor-DOC production should increase during a time when production of 11 beta- and 18-hydroxylated corticosteroids are decreased in AE. The purpose of this study was to examine the 11 beta, 18-, and 19-hydroxylase pathways in the early and late phases of AE. The results demonstrate increased urinary 19-Nor-DOC and decreased 18-OH-DOC and corticosterone excretion in the early phase of AE at a time when adrenal mitochondrial 11 beta- and 18-hydroxylase activities were decreased but 19-hydroxylase activity was unchanged. During the late phase of AE, urinary 19-Nor-DOC had decreased and 18-OH-DOC and corticosterone had increased to levels indistinguishable from those in sham controls. This reduction in 19-Nor-DOC was associated with a decrease in 19-hydroxylase activity in AE. Since the 11 beta, 18-, and 19-hydroxylases have a common substrate (DOC), it is possible that differential flux of DOC through these pathways could account for the changes in steroid production in AE. These data suggest that the increased 19-Nor-DOC excretion in AE may be due to alterations in enzyme activity leading to a shunting of DOC into the 19-Nor-DOC pathway. In addition, the synchronicity of 19-Nor-DOC with sodium excretion suggests that it has an important role in the pathogenesis of the sodium retention in AE.

Survival after end-stage renal disease in autosomal dominant polycystic kidney disease: contribution of extrarenal complications to mortality.

Autosomal dominant polycystic kidney disease (ADPKD) accounts for 8% to 10% of patients with end-stage renal disease (ESRD) in the United States and Europe. Progressive expansion of multiple bilateral renal cysts leads to massive enlargement of the kidneys and progressive renal failure. Extrarenal manifestations of ADPKD, such as liver cysts, intracranial aneurysms, cardiac valvular disease, and perhaps diverticulosis, have been documented extensively in cross-sectional studies, but little is known about their natural history. It is thought that extrarenal aspects of ADPKD contribute to increased mortality, yet survival on dialysis of the ADPKD population surpasses that of the general dialysis population. To address this issue, we analyzed the relative risk and causes of death after ESRD in ADPKD versus nondiabetic controls using data from the United States Renal Data System. Relative risk of death from any cause, including the major extrarenal manifestations of ADPKD, was determined as a function of ESRD treatment modality (dialysis or transplantation). We found a lower total mortality rate in ADPKD ESRD patients compared with nondiabetic control ESRD patients (relative risk of death in ADPKD = 0.57; P < 0.001). Mortality rates of extrarenal complications except for polycystic liver disease were similar or lower in ADPKD patients than in nondiabetic controls. Mortality secondary to extrarenal complications was substantially lower than that secondary to cardiovascular or cerebrovascular disease.

Cyst function in polycystic kidney disease: nongradient cysts.

Previous studies from this laboratory have demonstrated active sodium transport by cyst epithelia obtained from human polycystic kidneys. Cysts which maintained a steep sodium gradient between cyst fluid and plasma (gradient cysts) exhibited conductive amiloride sensitive sodium transport. Cysts which failed to maintain a sodium gradient between cyst fluid and plasma (nongradient cysts) were insufficiently characterized. In the present study, we report flux and electrical parameters of 23 nongradient cysts studied in vitro. Nongradient cysts exhibit low PD, low Isc, and high conductance. Unidirectional fluxes of sodium and chloride varied from approximately 14.4 to greater than 250 microEq.h-1.cm-2 and net flux was not significantly different from zero. There was no apparent effect on flux or electrical parameters of amiloride, ouabain, acetazolamide, or bumetanide. There was a very high correlation between unidirectional flux of sodium and chloride in individual cysts which was similar to the predicted relationship for diffusional fluxes. This correlation suggested that movement of sodium and chloride across cyst membranes was passive via an aqueous channel. Estimates of ionic permeability exceeded those determined for proximal nephron by almost one order of magnitude. We conclude that nongradient cysts are nonfunctional and, regardless of their origin, do not function analogously to the proximal nephron.

Aldosterone and PCO2 enhance rubidium absorption in rat distal colon.

Recent studies of rabbit colon have indicated the presence of a vanadate-sensitive K+-dependent proton pump, suggesting the existence of an H+-K+-ATPase. The participation of such a mechanism for colonic K+ absorption in the rat has not been determined. To this purpose, we attempted to detect the presence of pH-linked mechanisms for K+ absorption in rat distal colon using 86Rb as a marker for K+. We found that Rb+ absorption in Na-Ringer directly correlated with the in vitro partial pressure of CO2 (PCO2) in aldosterone-stimulated but not in control rats. Similar studies performed using Na-free Ringer demonstrated that PCO2 markedly augmented Rb+ absorption in both control and aldosterone-stimulated rat colon. Rb+ absorption was inhibited by orthovanadate, SCH28080, and mucosal ouabain in Na-free Ringer, but there was no effect of omeprazole, furosemide, or bumetanide. Barium applied to the serosa was also effective in inhibiting Rb+ absorption, suggesting that Rb+ exit from the cell was conductive. These findings are consistent with the presence of an active K+ pump that is activated by pretreatment with aldosterone and increased in vitro PCO2 and that is inhibited by orthovanadate, SCH28080, and mucosal ouabain. The constellation of findings suggests that participation of an ATPase that is not typical of either Na+-K+-ATPase or H+-K+-ATPase.

Aldosterone and PCO2 enhance K-dependent chloride absorption in rat distal colon.

We have previously demonstrated that, in the absence of Na+ in vitro, the rate of colonic K+ absorption is increased by increasing PCO2. Chronic secondary hyperaldosteronism induced by dietary Na-depletion further stimulated K+ absorption under these conditions. Because the observed increments in CO2-dependent K+ absorption were not accompanied by corresponding changes in short-circuit current, macroscopic electroneutrality must have been maintained either by anion absorption or by cation secretion. Colonic Cl- absorption is known to respond to increased PCO2 both in vivo and in vitro, but its response under Na-free conditions and the relationship to K+ absorption have not been examined. To determine the relationship of Cl- absorption to K+, we measured unidirectional fluxes of 36Cl and the response to PCO2 in voltage-clamped segments of rat distal colon. Our findings indicate that the rate of Cl- absorption is increased by increasing CO2, both in the presence and absence of Na+. Under Na-free conditions, Cl- absorption is inhibited by acetazolamide and by the absence of K+;K+ absorption (86Rb or 42K flux) is inhibited in a reciprocal fashion by the absence of Cl-. The rates of K+ and Cl- absorption are similar in controls and after secondary hyperaldosteronism due to a Na-deficient diet. These findings suggest that K- and Cl- absorption are closely coupled under Na-free conditions, most likely due to the operation of parallel, aldosterone-responsive H(+)-K+ and Cl(-)-HCO3- exchange pathways.

Suppression of coupled Na-Cl absorption by aldosterone and dexamethasone in rat distal colon in vitro.

Basal Na absorption in the rat colon is coupled to that of Cl in an electroneutral fashion. We previously determined that aldosterone or dexamethasone induces amiloride-sensitive mucosal-to-serosal Na flux approximately equal to the amiloride-sensitive short-circuit current in rat distal colon in vitro. However, the effect of these steroids on coupled Na-Cl absorption was not examined. For this purpose, we determined the unidirectional flux of Na and Cl in voltage-clamped distal colon segments from rats treated with aldosterone or dexamethasone. Amiloride was used as a probe for conductive Na absorption, and acetazolamide and Cl-free solutions were used as probes for coupled Na-Cl absorption. Our results indicate that the nature of colonic Na absorption is markedly changed after treatment with these steroids. In contrast to findings in the untreated rat, colonic Na absorption after treatment with aldosterone or dexamethasone was largely independent of the presence of Cl. Net Cl absorption and acetazolamide sensitivity were both greatly diminished. Thus, aldosterone and dexamethasone have multiple effects on Na transport in rat distal colon. In addition to the stimulation of conductive Na absorption by aldosterone, an effect well described in other epithelia, there is marked suppression of coupled Na-Cl absorption. Dexamethasone was less effective in suppressing Cl absorption but equally effective in stimulating conductive Na absorption. These steroid effects were greater in the terminal 1-2 cm of the rat colon.

Serum creatinine and renal function.

Serum creatinine is widely interpreted as a measure only of renal function; however, the serum level reflects not only renal excretion, but also the generation, intake, and metabolism of creatinine. In this review, we demonstrate that serum creatinine does not provide an adequate estimate of glomerular filtration rate (GFR), and contrary to recent teachings, that the slope of the reciprocal of serum creatinine vs time does not permit an accurate assessment of the rate of progression of renal disease. In clinical investigation, it is essential to utilize more accurate and sensitive measures of renal function to estimate GFR and progression. As effective treatments for progressive renal diseases are discovered, it will also be necessary to employ these measurements in clinical practice.

Serum creatinine as an index of renal function: new insights into old concepts.

The serum creatinine concentration is widely interpreted as a measure of the glomerular filtration rate (GFR) and is used as an index of renal function in clinical practice. Glomerular filtration of creatinine, however, is only one of the variables that determines its concentration in serum. Alterations in renal handling and metabolism of creatinine and methodological interferences in its measurement may have a profound impact on the serum concentration of creatinine. We review the fundamental principles of physiology, metabolism, and analytical chemistry that are necessary to correctly interpret the serum creatinine concentration. These principles are then applied to important clinical circumstances, including aging, pregnancy, diabetes mellitus, drug administration, and acute and chronic renal failure. Despite numerous limitations, serum creatinine remains a useful clinical tool, but more accurate measures of renal function are frequently necessary.

Secondary hyperaldosteronism stimulates acidification in rat distal colon.

Recent studies from this laboratory have determined that colonic K+ absorption is altered by the PCO2 and by secondary hyperaldosteronism. Partial inhibition by vanadate and mucosal ouabain suggested the operation of an H+/K+ exchange pump. To determine the mechanism of acidification in rat distal colon, we measured in vitro acidification using the pH-stat technique by voltage-clamped segments of colonic epithelium in controls and in the presence of secondary hyperaldosteronism, induced by a sodium-deficient diet. Chronic stimulation with aldosterone resulted in increased mucosal acidification in vitro for at least 2 h. This effect could not be accounted for by lactate production and was not altered by elimination of the aldosterone-induced increase in voltage and short-circuit current with 10 microM amiloride. Studies with inhibitors and ion substitution revealed that mucosal acidification resulted from both Na-dependent and Na-independent mechanisms. Na-dependent acidification was inhibited by ATPase inhibitors and was mediated in part by a luminal Na+/H+ exchanger in the presence of secondary hyperaldosteronism. Na-independent acidification was mediated by a pathway dependent on luminal K+ that was inhibited by vanadate and mucosal ouabain, consistent with the operation of an H+/K+ exchange pump.

Sodium retention after adrenal enucleation.

Adrenal enucleation (removal of the adrenal gland, leaving the capsule intact) results in regeneration of the adrenal cortex. During the first 1-2 wk of adrenal regeneration, marked renal sodium avidity and positive sodium balance are noted. This renal sodium avidity appears mediated via adrenocorticotropin-stimulated secretion of a potent mineralocorticoid by the regenerating adrenal cortex. In this review, we have examined relationships between the histology and ultrastructure of the regenerating adrenal cortex, renal sodium handling, and adrenal steroid production at various times after the initiation of adrenal regeneration. Plasma levels of known mineralocorticoids are subnormal during the period of most intense sodium avidity, while urinary excretion of a potent mineralocorticoid, 19-nordeoxycorticosterone, has been found to be increased in rats with regenerating adrenals during this period of most intense sodium avidity. This hormone, however, is not elevated in rats with regenerating adrenals after resolution of the period of sodium avidity. In this article, we review the experimental evidence regarding the potency of this mineralocorticoid and its likely role in the sodium retention after adrenal enucleation.

Rat kidney band 3 mRNA modulation in chronic respiratory acidosis.

Recent evidence indicates the existence of a protein related to the erythroid chloride-bicarbonate exchanger (band 3 protein) in the basolateral aspect of type A intercalated cells of the distal nephron. To probe the possible participation of this transporter in the renal adaptation to chronic hypercapnia, we examined the steady-state abundance of band 3 mRNA in the kidney during respiratory acidosis of variable duration. Total RNA was isolated from renal cortex and medulla of rats maintained in a 10% CO2 atmosphere for 2 or 5 days and from contemporaneous controls. The RNA was analyzed by Northern blot assay using cDNA probes for band 3 and beta-actin genes. Using a 3' cDNA probe encoding the membrane-associated domain of band 3 protein that is involved in anion exchange, we found a two- to threefold increase in steady-state mRNA levels (whether or not correction for the beta-actin signals was applied) in renal cortex and medulla at 5 days of hypercapnia. Similar, but less definitive, increases were observed at the 2-day time point. Using a 5' cDNA probe encoding an erythroid-protein segment absent from the kidney band 3 major transcript, we detected meager hybridization in renal tissue and no measurable variation during hypercapnia. Use of splenic RNA as a positive control for the 5' probe disclosed marked reduction of band 3 mRNA levels in hypercapnia, indicating organ specificity of band 3 gene expression. We conclude that steady-state levels of kidney band 3 mRNA increase in chronic respiratory acidosis as a result of transcriptional or posttranscriptional regulatory mechanisms. This adaptation might be involved in the augmentation of renal acidification characteristic of chronic hypercapnia.

Effect of aldosterone and dexamethasone pretreatment on sodium transport in rat distal colon in vitro.

Aldosterone and dexamethasone stimulate sodium absorption in the rat colon in vivo. In vitro, increased amiloride inhibitable short-circuit current (ISC) has been demonstrated following aldosterone or dexamethasone treatment. Since ISC bears no relationship to sodium flux (JNa) in the untreated rat colon, we measured JNa in partially stripped voltage clamped segments of rat distal colon. Our results demonstrate directly that continuous infusion of aldosterone or dexamethasone for 4-7 days stimulated amiloride inhibitable JNa by stimulating JNaM -S. The amiloride inhibitable portion of JNaM -S was highly correlated with and approximately equal to the amiloride inhibitable ISC. Amiloride had no effect in controls. We conclude that JNaM -S in the rat distal colon is only sensitive to mucosal amiloride after treatment with aldosterone or dexamethasone. The amiloride sensitive ISC in these treated tissues was a good measure of the amiloride sensitive JNa. Small differences between aldosterone and dexamethasone treatment were noted in the effect on transepithelial resistance, potential difference, and the ISC after amiloride.

Autosomal dominant polycystic kidney disease decreases anion exchanger activity.

Liver cysts, the most common extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD), derive from the intrahepatic biliary epithelium (IBE) and are found in 60-75% of ADPKD patients on dialysis. Secretin-induced secretion by the normal IBE is rich in HCO3-, whereas intact ADPKD liver cysts secrete primarily Cl- in response to secretin. To evaluate the mechanisms of decreased HCO3- secretion by ADPKD liver cysts, we utilized SV40 large T antigen-immortalized normal IBE and ADPKD liver cyst-derived epithelia (LCDE) cell lines that we created. These cell lines express biliary but not hepatocyte markers. Anion exchanger (AE) function was assessed by the response of intracellular pH (pHi) to acute Cl- removal. 2',7'-Bis(carboxyethyl)-5-(6)-carboxyfluorescein-loaded monolayers were continuously perfused with physiological HCO3- buffer containing Cl- or gluconate. In IBE cell line H75 (n = 6), acute Cl- removal alkalinized pHi at a rate of 0.04 +/- 0.01 min-1. AE function was significantly decreased in LCDE cell line CL3 (n = 6) to a rate of 0.01 +/- 0.01 min-1 after Cl- removal. Northern blot analysis demonstrated equivalent levels of AE2 mRNA in both cell lines. AE1 mRNA was undetectable. Immunoblot analysis demonstrated the AE2 polypeptide in both cell lines, but the level of mature glycosylated AE2 polypeptide was reduced in LCDE cells. Immunofluorescence microscopy demonstrated decreased membrane-localized AE2 in LCDE cells. These findings suggest that decreased plasmalemmal AE2 may account for decreased AE function in LCDE cells and suggest a possible explanation for decreased secretion of HCO3- by ADPKD liver cysts.

Mineralocorticoid activity of 19-nor-DOC and 19-OH-DOC in adrenalectomized rat.

Excess mineralocorticoid activity is thought to be responsible for the increased sodium reabsorption found after adrenal enucleation, but no known mineralocorticoid has been demonstrated in quantities sufficient to account for this antinatriuresis. 19-Hydroxydeoxycorticosterone (19-OH-DOC) has been synthesized by the incubated enucleate adrenal capsule and 19-nordeoxycorticosterone (19-nor-DOC), a possible metabolite, has been found in the urine of rats with regenerating adrenal glands. To evaluate the in vivo mineralocorticoid potency of these steroids, we studied glucocorticoid-replete adrenalectomized rats and measured the sodium and potassium excretion after administration of these steroids. Our results indicate that 19-nor-DOC has equipotent antinatriuretic activity compared to aldosterone but was less kaluretic. 19-OH-DOC had no significant antinatriuretic or kaluretic activity. We conclude that 19-nor-DOC is a potent mineralocorticoid and may be responsible for the enhanced sodium reabsorption found after adrenal enucleation.

Mineralocorticoid activity of 19-nor-DOC and 19-OH-DOC in toad bladder.

Adrenal enucleation is followed by a period of increased sodium reabsorption thought to be due to excess mineralocorticoid activity. However, it has not been demonstrated that increased production of any known mineralocorticoid accounts for this antinatriuresis. Recently, 19-hydroxydeoxycorticosterone (19-OH-DOC) was found in incubates of regenerating adrenal capsules 3-4 days postenucleation and 19-nordeoxycorticosterone (19-nor-DOC) was identified in the urine of rats with regenerating adrenals. Because it was possible that these hormones might play a role in the sodium retention after adrenal enucleation, we compared the mineralocorticoid activity of these steroids to aldosterone using the toad bladder. Using short-circuit current as a measure of sodium transport, we found that 19-OH-DOC (10(-8) M) had no significant effect on sodium transport. However, 19-nor-DOC (10(-8) M) increased sodium transport to a degree not different from aldosterone (10(-8) M). Furthermore, the onset of action, duration of activity, and inhibition of effect of 19-nor-DOC by spironolactone were not different from that of aldosterone. We conclude that 19-nor-DOC exhibits a significant effect on sodium transport and thus has the potential to play a role in the sodium retention following adrenal enucleation. Under the conditions of these studies, 19-OH-DOC exhibited no effect on sodium transport.

Aldosterone induces apical vesicles in rat distal colon.

Mineralocorticoid steroids markedly alter ion transport in responsive epithelia. Increases in absorption of Na+ and secretion of K+ and H+ are accompanied by increases in surface area of the basolateral membrane. The basolateral membrane changes are associated with increased Na(+)-K(+)-ATPase activity and increased numbers of Na(+)-K(+)-ATPase pump sites. It is thought that H+ secretion is mediated by H+ pumps contained in apical vesicles that are added to the luminal membrane in response to acidifying stimuli. Whether there are changes in the number or volume of apical vesicles in response to aldosterone has not been evaluated. To this purpose, we evaluated apical membrane morphology in rat distal colon, a mineralocorticoid-responsive epithelium. We found that aldosterone infused for 4-7 days by osmotic minipump significantly increased the number, surface density, and total volume of apical vesicles. Exposure of tissues to 5% CO2 for 15 min before fixation resulted in significant decreases in vesicle number, surface density, and volume in aldosterone-stimulated tissues. After CO2, apical vesicles in aldosterone-stimulated tissues tended to be closer to the luminal membrane; apical membrane surface density was increased but not to a significant degree. Fluorescence microscopy demonstrated acridine orange accumulation in discrete points under the lumen, suggesting the presence of acidic vesicles in this location. We propose that aldosterone increases the activity of a membrane shuttle system that is regulated by CO2 as found in other H(+)-secreting epithelia. This system may mediate aldosterone-induced changes in colonic H+ transport.

Diet, indicators of kidney disease, and later mortality among older persons in the NHANES I Epidemiologic Follow-Up Study.

OBJECTIVES: The purpose of this study was to determine whether diet adversely affected survival among 2572 older persons with indicators of kidney disease in a population-based cohort. Average follow-up time for survivors, of whom 1453 (57%) had died at analysis, was 14.5 years. METHODS: Kidney disease indicators were a "yes" response to "Has a doctor ever told you that you have kidney disease or renal stones?" and/or trace or greater amounts of protein in urine. Dietary protein intakes were calculated from 24-hour recalls. RESULTS: Cox proportional hazards models were used, stratified by sex, with age, body mass index, blood pressure, education, smoking status, total caloric intake, and diabetes mellitus as covariates. Relative risk of total mortality with an additional 15 g of protein per day was 1.25 (95% confidence interval [CI] = 1.09, 1.42) among White men with kidney disease indicators, vs 1.00 (95% CI = 0.95, 1.06) among those without them; relative risks of renal-related mortality were 1.32 (95% CI = 0.97, 1.79) and 0.95 (95% CI = 0.81, 1.11), respectively. No significant differences were found for White women. CONCLUSIONS: Once chronic renal disease is present, diet may be associated with earlier mortality in White males.