Aquaporin mediated water flux as a target for diuretic development.

Within the past decade an entire family of membrane proteins--aquaporins--which function as transmembrane water channels has been identified; they occur throughout the plant, animal, and bacterial kingdoms. Several family members permit glycerol and urea permeability. Most aquaporins are inhibited by mercury. Constitutively expressed aquaporin 1 is the major permeability channel of the proximal tubule, descending thin limb of the loop of Henle, and it is also found in vasa recta. Aquaporin 2 is expressed in the principal cells of the collecting duct where it shuttles between intracellular vesicles and the apical membrane in response to vasopressin. Aquaporin 2 mutations cause nephrogenic diabetes insipidus; increased aquaporin 2 activity is implicated in the pathophysiology of heart failure, cirrhosis, and nephrotic syndrome. Aquaporins 3 and 4 provide basolateral membrane water channels in the collecting duct. These 4 channels and 6 others are also found elsewhere throughout the body. The physiological importance of several of the channels remains unknown. Aquaporin 1 inhibitors might induce useful diuresis, but humans who lack aquaporin 1 have no significant clinical disease. Inhibition of aquaporin 2 activity by vasopressin receptor antagonists may be useful in heart failure, cirrhosis, nephrotic syndrome, and the syndrome of inappropriate antidiuretic hormone (ADH) release.

Acid-base disorders in medicine.

The practice of internal medicine involves daily exposure to abnormalities of acid-base balance. A wide variety of disease states either predispose patients to develop these conditions or lead to the use of medications that alter renal, gastrointestinal, or pulmonary function and secondarily alter acid-base balance. In addition, primary acid-base disease follows specific forms of renal tubular dysfunction (renal tubular acidosis). We review the acid-base physiologic functions of the kidney and gastrointestinal tract and the current understanding of acid-base pathophysiologic conditions. This includes a review of whole animal and renal tubular physiologic characteristics and a discussion of the current knowledge of the molecular biology of acid-base transport. We stress an approach to diagnosis that relies on knowledge of acid-base physiologic function, and we include discussion of the appropriate treatment of each disorder considered. Finally, we include a discussion of the effects of acidosis and alkalosis on human physiologic functions.

The renal adenosine triphosphatases: functional integration and clinical significance.

The ion-transporting ATPases determine the chemical composition of cells both directly and through their secondary effects. The Na,K-ATPase generates the transmembrane sodium gradient which provides the primary energy for uptake and extrusion of a wide variety of solutes by renal tubular epithelia. The H-ATPase and the H,K-ATPase acidify the urine, and also generates bicarbonate for excretion by the cortical collecting duct. Calcium ATPase regulates the intracellular calcium, which in turn impacts on the myriad of cellular functions for which calcium serves as an intracellular messenger. If one considers the impact of potential pump dysfunction in a purely speculative mode, the list of disorders which might be potentially ascribed to 'pump disease' would be enormous. This article reviews those disorders of renal transport already considered to be 'pump diseases'.

Diseases of renal adenosine triphosphatase.

Most renal transport is a primary or secondary result of the action of one of three membrane bound ion translocating ATPase pumps. The proximal tubule mechanisms for the reabsorption of salt, volume, organic compounds, phosphate, and most bicarbonate reabsorption depend upon the generation and maintenance of a low intracellular sodium concentration by the basolateral membrane Na-K-ATPase pump. The reabsorption of fluid and salt in the loop of Henle is similarly dependent on the energy provided by Na-K-ATPase activity. Some proximal tubule bicarbonate reabsorption and all distal nephron proton excretion is a product of one of two proton translocating ATPase pumps, either an electrogenic H-ATPase or an electroneutral H-K-ATPase. In this article, the authors review the biochemistry and physiology of pump activity and consider the pathophysiology of proximal and distal renal tubular acidosis, the Fanconi syndrome, and Bartter's syndrome as disorders of ATPase pump function.

Effect of respiratory acidosis and respiratory alkalosis on renal transport enzymes.

We studied the effect of respiratory acidosis and respiratory alkalosis on acid-base composition and on microdissected renal adenosinetriphosphatase (ATPase) enzymes. Rats were subjected to hypercapnia or hypocapnia of 6, 24, and 72 h duration. After 6 h of hypercapnia, collecting tubule (CT) ATPases were not changed. At 24 h, plasma bicarbonate was 35 +/- 1 meq/l (P < 0.01) and CT H-ATPase and H-K-ATPase activities were 90% greater than controls (P < 0.01). By 72 h, plasma bicarbonate was 37 +/- 1 meq/l (P < 0.005 vs. control) and CT enzyme activity had increased even more, averaging approximately 130% of control (P < 0.05). Significant increases in enzyme activities were also observed in the proximal convoluted tubule and medullary thick ascending limb. Plasma aldosterone was three to four times that of control at all three time periods. In hormone-replete adrenalectomized rats, acid-base parameters and ATPase activities were the same as those seen in adrenal intact animals. After 6 h of hypocapnia, plasma bicarbonate was not significantly changed, but H-ATPase and Na-K-ATPase activities were decreased by 35% along the entire nephron (P < 0.05). H-K-ATPase activity in CT also decreased by 35%. At 24 h, plasma bicarbonate was 20.5 +/- 0.5 meq/l (P < 0.05 vs. control) and CT H-ATPase and H-K-ATPase activities were 60% less than control (P < 0.01). By 72 h, plasma bicarbonate was 18.5 +/- 0.5 meq/l (P < 0.05); however, only CT H-ATPase activity continued to fall, averaging 75% less than control (P < 0.005). Hypocapnia had no effect on plasma aldosterone or potassium. These results demonstrate that chronic, but not acute, respiratory acidosis stimulates activity of both renal proton ATPases. By contrast, both acute and chronic respiratory alkalosis decrease the two renal proton pumps. The stimulatory effect of hypercapnia and the inhibitory effect of hypocapnia on the renal ATPases appear to be potassium and aldosterone independent. Although the precise mechanisms for these results are not known, a direct effect of PCO2, pH, or changes in bicarbonate delivery may be involved.

Characterization of the N-ethylmaleimide-sensitive ATPase in rat cortical and medullary collecting tubule.

Hydrogen ion secretion in the kidney is thought to be mediated in part by an N-ethylmaleimide (NEM)-sensitive proton-translocating adenosine triphosphatase (ATPase). This enzyme has been found throughout the nephron, but it has not been completely characterized enzymatically in the rat collecting duct. In the present study we characterized the NEM-sensitive ATPase from microdissected cortical (CCT) and medullary (MCT) collecting tubules of the rat nephron. At optimum conditions, NEM-sensitive ATPase activity was the same in both tubule segments: activity was 275.6 +/- 18.6 pmol/mm/h in the CCT and 280.3 +/- 35.2 pmol/mm/h in the MCT (n = 23, NS). ATP sensitivity was greater in CCT than in MCT, and in the former guanosine triphosphate was able to partially support enzyme activity. Maximal enzyme inhibition with NEM occurred at a lower concentration in CCT as compared to MCT. At pH 7.0 in MCT enzyme activity was approximately one half that seen at pH 7.4; in MCT and CCT, the pH optimum was 7.4. The temperature optimum in both segments was between 37 and 42 degrees C. Enzyme activity in CCT and MCT was linear to 30 min and proportional to tubule length. These results demonstrate that there are important differences in the NEM-sensitive ATPase isolated from two segments of rat collecting duct, and raise the possibility that enzyme heterogeneity may exist.

Microperfusion studies on the permeability of retinal vessels. A new model demonstrating organic anion transport and a reabsorptive fluid flux.

We developed an experimental model to study the permeability of individual retinal vessels in vitro using microperfusion techniques adapted from kidney tubule studies. The retinal vessels were isolated by freehand dissection and mounted on a microperfusion apparatus. When inulin was perfused luminally, it was diluted to 80.2 +/- 2.3% of its initial concentration. However, no radioactive leak into the bath side was observed, suggesting that the dilution was due to fluid flux from bath to lumen. The dilution of fluorescein (81.9 +/- 3.8%) was in the same range as that of inulin, the reference marker. The extremely low lumen-to-bath fluorescein flux, 0.5 +/- 0.9 X 10(-12) mol/min/mm, increased by 68% when probenecid was added to the perfusate and by 210% when probenecid was placed in the bath. The effect was concentration-dependent. When placed in the bath, fluorescein moved rapidly across the retinal vessel walls, accumulating in the lumen to concentrations 40 times higher than in the bath. This movement from bath to lumen, which was much higher (13.6 +/- 0.3 X 10(-12) mol/min/mm) than the lumen-to-bath fluorescein flux for the same fluorescein concentration, decreased by adding probenecid to the bath. The kinetics of this unidirectional movement of fluorescein were consistent with a saturable active transport process. The fluid flux from bath to lumen across the retinal vessels, which was 6.3 +/- 1.0 nl/min/mm for perfusion rates of 6.6 +/- 0.2 nl/min, was temperature-dependent and was coupled to the fluorescein transport. Fluorescein stimulated the fluid flux by 17% when added to the perfusate and by 60% when added to the bath, and this effect could be reversed by probenecid. Our results showed an active transport of fluorescein in the rabbit retinal vessels coupled with net fluid flux from outside the vessels into the lumen.

NEM-sensitive ATPase activity in rat nephron: effect of metabolic acidosis and alkalosis.

The present study was designed to quantitate the amount and to map the localization of N-ethylmaleimide (NEM)-sensitive adenosinetriphosphatase (ATPase) activity in microdissected segments of the rat nephron. After complete nephron mapping the effect of chronic metabolic acidosis and alkalosis on enzyme activity was determined. In control animals the highest enzyme activity was found in the early proximal convoluted tubule of juxtamedullary nephrons; superficial early proximal tubule as well as medullary and cortical thick ascending limbs and collecting ducts also contained substantial activity. Enzyme activity in the papillary collecting duct before entry into the ducts of Bellini was 329 +/- 93 pmol.mm-1.h-1 (n = 8); after entry, however, enzyme activity was approximately one-fourth that value (60 +/- 9 pmol.mm-1.h-1, n = 8, P less than 0.01). No NEM-sensitive ATPase activity was found in the thin limbs of the loop of Henle. Enzyme activity increased in both the medullary and cortical thick ascending limbs as well as in the cortical collecting tubule in response to NH4Cl-induced chronic metabolic acidosis; in the cortical collecting duct, metabolic acidosis increased maximum activity (Vmax) but did not change Michaelis-Menten constant (Km). In the proximal convoluted tubule, enzyme activity decreased with metabolic acidosis. Bicarbonate loading had no effect on enzyme activity except in the most distal portion of the collecting duct where it was stimulated. These results show that NEM-sensitive ATPase activity exists throughout much of the rat nephron. These data suggest that both the cortical collecting tubule and thick ascending limb are regulatory sites of distal urinary acidification during acid loading.

Collecting tubule adaptation to respiratory acidosis induced in vivo.

To examine the effects of respiratory acidosis in vivo on the adaptation of acidification in the collecting tubule, New Zealand White rabbits were exposed to a 6.7% CO2-93.3% O2 gas mixture in an environmental chamber for 0, 6, 24, or 48 h before obtaining collecting tubules for in vitro study. These collecting tubules were then perfused and bathed in vitro in identical Krebs-Ringer bicarbonate solutions. After 1 h equilibration total CO2 flux (JtCO2) was measured. The urine pH of the rabbits fell, whereas the blood bicarbonate rose as CO2 exposure time increased. In cortical collecting tubules, JtCO2 in vitro correlated with length of animal exposure to hypercarbia (y = 1.14174 + 0.1437x, r = 0.57, P = 0.002), and with the blood bicarbonate of the animal (y = 26.8471 + 0.0858x, r = 0.59, P less than 0.05). In vitro JtCO2 in medullary collecting tubules from rabbits that had been in hypercarbic atmosphere for 48 h (23.2 +/- 4.9 pmol.mm-1.min-1) did not differ from JtCO2 in control tubules (25.0 +/- 3.2 pmol.mm-1.min-1, not significant). Thus the cortical collecting tubule exhibits an adaptive increase in JtCO2 in response to hypercarbia, whereas the medullary collecting tubule does not.

Site of the acidification defect in the perfused postobstructed collecting tubule.

The effect of prior urinary tract obstruction on total CO2 flux in cortical and medullary collecting tubules was examined using an unilateral ureteral obstruction model and isolated tubule in vitro microperfusion. Tubules were obtained from the control and obstructed kidneys after 1, 2, and 4 days of obstruction. Paired comparison of function of cortical or medullary tubules and urine pH was made. Medullary collecting tubules showed decreased acidification after 2 days of obstruction (7.7 +/- 8.5 vs. 29.9 +/- 6.6 pmol/mm/min). Cortical collecting tubules from the obstructed kidney showed high rates of acidification at all time periods examined, and rates from the postobstructed cortical collecting tubules exceeded levels from control tubules at the 24-hour period while barely missing significance (p less than 0.06) at the 48- and 96-hour periods. These data are not consistent with the hypothesis that the acidification defect in the postobstructed kidney occurs in the cortical collecting tubule by a voltage-dependent mechanism.

Paraneoplastic syndromes in hypernephroma.

It is noted that while a wide variety of syndromes have been associated with hypernephroma in the clinical literature, there is clear understanding of the pathophysiology of these effects only in the cases of the endocrine disorders where direct tumor production of hormone can be demonstrated in vitro. Furthermore, this knowledge has done little to alter the care of patients with the disease, except for indications that indomethacin might be of benefit in some patients with hypercalcemia and that one might consider the use of converting enzyme inhibitors in patients with hypernephroma and hypertension. The overall approach to the disease is still surgical. Resection of the tumor also removes the paraneoplastic syndrome. Persistence or recurrence of a syndrome suggests the continued presence of the neoplasm, with the considerations for prognosis which that fact entails. To that degree, at least, these conditions are useful as tumor markers, but such use is limited because they are inconsistent. Further studies of pathophysiology of paraneoplastic syndromes will lead to better understanding of processes of cell differentiation and regulation, and possibly better ways to manage the patients in which they occur.

Total CO2 flux in isolated collecting tubules during carbonic anhydrase inhibition.

These studies evaluated the effects of sodium transport inhibitors on total CO2 flux (JtCO2) in the rabbit cortical collecting tubule after inhibition of carbonic anhydrase. When ouabain was added to tubules during carbonic anhydrase inhibition, reabsorptive JtCO2 rose and potential difference (PD) decreased. Amiloride added to perfusate after carbonic anhydrase inhibition decreased PD and did not alter JtCO2. If ouabain was added to an ethoxyzolamide-treated tubule with amiloride present in perfusate, no effects were detectable. Amiloride added to the bath of ethoxyzolamide-treated tubules in high concentration (10-3 M), decreased potential and increased JtCO2. If amiloride was added to the bath of cortical collecting tubules from fasted rabbits, JtCO2 rose. This response was not seen in cortical tubules from fed animals or in medullary collecting tubules. These data demonstrate the existence of carbonic anhydrase-independent acidification in this segment in vitro. The data from studies with amiloride in the presence of intact carbonic anhydrase are consistent with action on a basolateral sodium-proton exchange mechanism. A cellular model that postulates a basolateral sodium-proton exchanger in an acidifying cell is offered.

Juxtamedullary nephrons during acute metabolic alkalosis in the rat.

The renal handling of bicarbonate during acute metabolic alkalosis was examined in Munich-Wistar rats using micropuncture techniques. Group I received an acute bicarbonate load, and fractional delivery of total CO2 (tCO2) (FDtCO2) to the superficial late distal tubule (LD) was significantly lower than to the base of the papillary collecting duct (B) (18.4 +/- 1.7 vs. 22.9 +/- 1.5%; P less than 0.01), indicating net addition of bicarbonate between LD and B. When acutely bicarbonate-loaded rats had their deep nephrons destroyed with bromoethylamine hydrobromide (BEA) (group II), net addition of tCO2 between LD and B was abolished and net reabsorption uncovered (FDtCO2 LD: 28.0 +/- 3.6 vs. B: 17.5 +/- 2.5%; P less than 0.01). The infusion of amiloride (2.5 mg/kg body wt) to alkalotic rats treated with BEA (group III) completely inhibited distal bicarbonate reabsorption but did not reestablish addition (FDtCO2 LD: 27.6 +/- 1.6 vs. B: 26.1 +/- 3.7%; P = NS). The values obtained for sham-operated animals (group IV) were the same for group I. The patterns that were observed between LD and B were reproduced for the four groups of animals when FDtCO2 LD was compared with the fractional excretion of bicarbonate in the urine of the intact contralateral kidney. These studies suggest that juxtamedullary nephrons contribute a higher load of bicarbonate than superficial nephrons to the final urine during acute metabolic alkalosis in the rat.

Internephron heterogeneity for carbonic anhydrase-independent bicarbonate reabsorption in the rat.

The present experiments were designed to localize the sites of carbonic anhydrase-independent bicarbonate reabsorption in the rat kidney and to examine some of its mechanisms. Young Munich-Wistar rats were studied using standard cortical and papillary free-flow micropuncture techniques. Total CO2 (tCO2) was determined using microcalorimetry. In control rats both superficial and juxtamedullary proximal nephrons reabsorbed approximately 95% of the filtered load of bicarbonate. The administration of acetazolamide (20 mg/kg body weight [bw]/h) decreased proximal reabsorption to 65.6% of the filtered load in superficial nephrons (32% was reabsorbed by the proximal convoluted tubule while 31.7% was reabsorbed by the loop segment), and to 38.4% in juxtamedullary nephrons. Absolute reabsorption of bicarbonate was also significantly higher in superficial than in juxtamedullary nephrons after administration of acetazolamide (727 +/- 82 vs. 346 +/- 126 pmol/min; P less than 0.05). The infusion of amiloride (2.5 mg/kg bw/h) to acetazolamide-treated rats increased the fractional excretion of bicarbonate as compared with animals treated with acetazolamide alone (34.9 +/- 1.9 vs. 42.9 +/- 2.1%; P less than 0.01), and induced net addition of bicarbonate between the superficial early distal tubule and the final urine (34.8 +/- 3.0 vs. 42.9 +/- 2.1%; P less than 0.05). Amiloride at this dose did not affect proximal water or bicarbonate transport; our studies localize its site of action to the terminal nephron. Vasa recta (VR) plasma and loop of Henle (LH) tubular fluid tCO2 were determined in control and acetazolamide-treated rats in order to identify possible driving forces for carbonic anhydrase-independent bicarbonate reabsorption in the rat papilla. Control animals showed a tCO2 gradient favoring secretion (LH tCO2, 7.4 +/- 1.7 mM vs. VR tCO2, 19.1 +/- 2.3 mM; P less than 0.005). Acetazolamide administration reversed this chemical concentration gradient, inducing a driving force favoring reabsorption of bicarbonate (LH tCO2, 27.0 +/- 1.4 mM vs. VR tCO2, 20.4 +/- 1.0 mM; P less than 0.005). Our study shows that in addition to the superficial proximal convoluted tubule, the loop segment and the collecting duct show acetazolamide-insensitive bicarbonate reabsorption. No internephron heterogeneity for bicarbonate transport was found in controls. The infusion of acetazolamide, however, induced significant internephron heterogeneity for bicarbonate reabsorption, with superficial nephrons reabsorbing a higher fractional and absolute load of bicarbonate than juxtamedullary nephrons. We think that the net addition of bicarbonate induced by amiloride is secondary to inhibition of voltage-dependent, carbonic anhydrase-independent bicarbonate reabsorption at the level of the collecting duct, which uncovers a greater delivery of carbonate from deeper nephrons to the collecting duct. Finally, our results suggest that carbonic anhydrase-independent bicarbonate reabsorption is partly passive, driven by favorable chemical gradients in the papillary tubular structures, and partly voltage-dependent, in the collecting duct.

Characterization of acidification in the cortical and medullary collecting tubule of the rabbit.

Ouabain and lithium decrease acidification in open-circuited bladders by eliminating the electrical gradient favoring acidification. The effect of ouabain and lithium on acidification in cortical and medullary collecting tubules derived from starved New Zealand white rabbits was studied by using the techniques of isolated nephron microperfusion and microcalorimetric determination of total CO2 flux. Bath and perfusion solutions were symmetric throughout all studies, and solutions contained 25 meq of bicarbonate and were bubbled with 93.3% O2/6.7% CO2 gas mixtures. In cortical collecting tubules, ouabain (10(-8) M) addition to bath resulted in a decrease in both potential difference (PD), from -16.4 to -2.2 mV (P less than 0.001), and total CO2 flux (JTCO2), from +6.0 to 1.5 pmol/mm per min (P less than 0.005). In medullary collecting tubules neither PD nor JTCO2 changed with the addition of ouabain in either 10(-8) or 10(-4) M concentration. Replacement of 40 mM NaCl with 40 mM LiCl in both perfusate and bath in cortical collecting tubules resulted in decreases in both PD, from -11.6 to 0.4 mV (P less than 0.005), and JTCO2, from +10.8 to +4.2 pmol/mm per min (P less than 0.025). This substitution had no effect on medullary collecting tubules. When control flux rates were plotted against animal bladder urine pH, both medullary and cortical tubules showed good inverse correlation between these variables, with higher values of flux rate for the medullary tubules. The data support a role for transepithelial PD in acidification in the cortical collecting tubule and also suggest that both cortical and medullary segments of the collecting tubule participate when urinary acidification is increased during starvation in the rabbit.