Fluoride flux in the rabbit CCD: a pH-dependent event.

We measured fluoride flux (JF; pmol.min-1.mm-1) in the isolated rabbit cortical collecting duct (CCD) to investigate the determining factors of JF. The perfusate contained 100 microM fluoride and the bath was fluoride-free. Osmotically-induced lumen-to-bath water flux did not affect JF. When perfusate pH was reduced from 7.4 to 6.1 and from 6.1 to 5.0, JF increased from 0.008 +/- 0.002 to 0.027 +/- 0.007 (P less than 0.01) and from 0.018 +/- 0.003 to 0.040 +/- 0.005 (P less than 0.01), respectively. Acetazolamide at 10(-4) M in the bath reduced JF slightly though not statistically. The anion-transport inhibitor, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), at 10(-4) M in the perfusate did not affect JF. Substitution of luminal chloride with gluconate failed to affect JF in tubules from normal rabbits or from rabbits treated with deoxycorticosterone which stimulates chloride-bicarbonate exchange in the CCD. JF showed no correlation with transepithelial voltage which ranged from +4 to -104 mV. We conclude that the luminal pH represents the primary determining factor influencing JF in the rabbit CCD, and fluoride does not use a chloride-mediated or a DIDS-inhibitory transport pathway.     

Molecular homology and the luminal transport of Hg2+ in the renal proximal tubule

The aim of this study was to define mechanisms involved in the luminal uptake of inorganic mercury in the kidney using isolated perfused straight (S2) segments of the proximal tubule. When mercuric conjugates of glutathione (GSH), cysteinylglycine. or cysteine (containing 203Hg2+) were perfused through the lumen, the rates of luminal disappearance flux (JD) of inorganic mercury were approximately 39, 53, and 102 fmol/min per' min, respectively. Thus, the rates of luminal uptake of mercury are greater when the mercury is in the form of a mercuric conjugate of cysteine than in the form of a mercuric conjugate of cysteinylglycine or GSH. Addition of acivicin to the perfusate, to inhibit activity of the y-glutamyltransferase, caused significant reductions in the J,, for mercury in tubules perfused with mercuric conjugates of GSH. Addition of cilastatin, an inhibitor of dehydropeptidase- l (cysteinylglycinase) activity, caused significant reductions in the uptake of mercury in tubules perfused with mercuric conjugates of cysteinylglycine. These findings indicate that a significant amount of the luminal uptake of mercury, when mercuric conjugates of GSH are present in the lumen, is dependent on the activity of both y-glutamyltransferase and cysteinylglycinase. Finally, the JD for mercury in tubules perfused with mercuric conjugates of cysteine was reduced by approximately 50% when 3.0 mM L-lysine or 5.0 mM cycloleucine was added to the perfusate. It is concluded that these findings indicate that at least some of the luminal uptake of mercuric conjugates of cysteine occurs at the site of one or more amino acid transporters via a mechanism involving molecular homology.     

The role of preservation solution on acid-base regulation during machine perfusion of kidneys

To meet the current clinical organ demand, efficient preservation methods and solutions are needed to increase the number of viable kidneys for transplantation. In the present study, the influence of perfusion solution buffering strength on renal pH dynamics and regulation mechanisms during kidney ex vivo preservation was determined. Porcine kidneys were hypothermically machine perfused for 72 h with either Unisol-UHK or Belzer-Machine Perfusion solution, Belzer-MP solution. Renal perfusate samples were periodically collected and biochemically analyzed. The UHK solution, a Hepes-based solution (35 mM), provided a more efficient control of renal pH that, in turn, resulted in minor changes in the perfusate pH relative to baseline, in response to tissue CO2 and HCO3- production. In the perfusate of Belzer-MP kidney group a wider range of pH values were recorded and a pronounced pH reduction was seen in response to significant rises in pCO2 and HCO3- concentrations. The Belzer-MP solution, containing phosphate (25 mM) as its main buffer, and only 10 mM Hepes, had a greater buffering requirement to attenuate larger pH changes.     

Relationship between lactate and glutamine metabolism in vitro by the kidney: differences between dog and rat and importance of alanine synthesis in the dog

Interaction between lactate (1 or 5 mM) and glutamine (1 or 5 mM) metabolism was studied with renal cortical slices incubated at a pH of 7.0 and obtained from acidotic (ammonium chloride) dogs and rats. The effect of aminooxyacetate (0.2 mM), dichloroacetate (3 mM), and fluoroacetate (0.05 mM) was also studied. Significant differences were observed between dog and rat. In the dog, lactate had no effect on glutamine uptake and vice versa, but gluconeogenesis increased. Ammonia production, however, decreased by 13 to 21%, whereas a significant increase in alanine production was noted. In the rat, glutamine extraction and ammonia production dropped by 33% with 5 mM lactate. In contrast to the observation in the dog, no production of alanine was noted, but significant accumulation of glutamate took place. Amino-oxyacetate inhibited alanine production in the dog and reestablished ammoniagenesis, and it led to a marked decrement in the uptake of lactate and glucose production in both species. Dichloroacetate in the dog resulted in a reduction in pyruvate, alanine, glucose, and ammonia production while glutamate accumulation was observed. In both species, fluoroacetate stimulated glutamine uptake and ammonia production. With lactate alone, fluoroacetate decreased lactate uptake and glucose production. With both lactate and glutamine in the medium, fluoroacetate prevented any effect of lactate on ammoniagenesis. The present study demonstrates that lactate has a modest depressing effect on renal ammonia production by dog slices through increased synthesis of alanine and redistribution of nitrogen from glutamine. In the rat, the depressing effect of lactate on ammonia production in the alanine amino-transferase deficient kidney occurs through accumulation of glutamate. The data also reveal that oxidation of lactate to carbon dioxide is greater in the dog than it is in the rat, but that gluconeogenesis from lactate is more important in the rat.     

Effect of PCO2-adjusted pH on the neonatal heart during hypothermic perfusion and ischemia

The effect of pH regulation on the function of the isolated neonatal heart during continuous hypothermic perfusion and arrest was tested in 3- to 6-day-old piglet hearts. Three groups of hearts were perfused from an adult support pig, with pH varied by a carbon dioxide/oxygen gas exchanger and temperature controlled by a heat exchanger. After control function studies were obtained at normothermia with a pH of 7.4, the hearts were cooled over 15 minutes to 10 degrees C. Hypothermic perfusion was maintained for 1 hour, followed by rewarming to 37 degrees C. In group 1 (n = 5), the alpha stat (neutral) model, the blood perfusate was maintained at a pH of 7.4 (calculated at 37 degrees C). In group 2, the alkaline model, the pH was maintained at 7.9, and in group 3, the pH-stat (acid) model, the pH was maintained at 7.0. In addition, the effect of 1 hour of hypothermic ischemia after hypothermic perfusion at a pH of 7.0 was evaluated in five hearts (group 4). After rewarming no significant difference was noted in functional recovery (group 1 = 93% +/- 5%, group 2 = 92% +/- 6%, group 3 = 96% +/- 5%, and group 4 = 95% +/- 2%), oxygen consumption, coronary resistance, lactate extraction, and myocardial extravascular water content. We conclude that neonatal heart function is resistant within the range of this study to changes in pH caused by changes in carbon dioxide tension during hypothermic perfusion and ischemia.     

Elevation of arterial potassium during acute systemic hypoxia is abolished by alkalosis

BACKGROUND: Small elevations in plasma potassium evoke vasodilation in the peripheral circulation. Systemic hypoxia elevates arterial potassium and also modifies arterial pH. AIMS: We examined the interaction between pH and potassium in blood during systemic hypoxia and the effect of pH on the uptake/release of potassium in the peripheral tissues. METHODS: Anesthetized dogs were ventilated with air plus oxygen for normoxia or air plus nitrogen for hypoxia. Some animals received intravenous sodium bicarbonate to elevate pH by 0.1 units. Arterial plasma potassium concentration was measured in normoxia and hypoxia. A rat gracilis muscle was perfused with normoxic Krebs buffer and the potassium content of the venous outflow was compared during perfusion at pH 7.4, 6.8, or 7.8. RESULTS: In dogs with an arterial pH of 7.40-7.45, systemic hypoxia elevated the arterial potassium by 1 mmol/L. An arterial pH of 7.55 did not alter the basal potassium concentration, but it abolished the hypoxia-induced increase. In rat muscle, reduction of the perfusate pH from 7.4 to 6.8 reduced arterial perfusion pressure from 8.73 to 7.32 kPa and venous potassium from 6.6 to 5.2 mM. Elevation of perfusate pH to 7.8 decreased the arterial perfusion pressure from 8.44 to 6.95 kPa but did not affect venous potassium. CONCLUSIONS: The hypoxia-induced elevation of arterial potassium is abolished by increasing the pH to 7.55. This is not due to enhanced potassium uptake into peripheral tissues at high pH. Red blood cells are suggested as the most likely source of the potassium released in hypoxia.     

Taurodeoxycholate modulates the effects of pepsin and trypsin in experimental esophagitis

Pepsin and trypsin cause erosive, hemorrhagic lesions in our rabbit model of experimental esophagitis. Since the gastroduodenal contents of patients with reflux esophagitis may also contain bile salts, we used our model to determine the effect that a bile salt, taurodeoxycholate (TDC), would have on the esophageal mucosa when combined with either pepsin in an acid perfusate (pH 2) or trypsin in an alkaline perfusate (pH 7.5). Indexes of esophageal injury included gross appearance of the mucosa, microscopic examination, and mucosal barrier integrity as determined by permeability to hydrogen ion. We found that when 5 mM TDC was combined with pepsin (0.3 mg/ml), the gross and microscopic changes of esophagitis, as well as net hydrogen ion flux, were diminished when compared with those observed with pepsin exposure alone. When increasing concentrations of TDC (2 to 10 mM) were added to pepsin, the morphologic degree of injury as well as hydrogen ion flux decreased in a dose-dependent manner. In contrast, when 5 mM TDC was combined with trypsin (1000 U/ml) in the alkaline perfusate, the gross and microscopic changes of esophagitis and the net of hydrogen ion flux were increased when compared with either bile salt or trypsin alone. These effects were also dose dependent. These data demonstrate that bile salts present in the gastroduodenal contents of patients with reflux esophagitis have the capacity to modulate the effects of pepsin and trypsin on the esophageal mucosa.     

Technical aspects of isolation extremity perfusion: experimental studies and clinical experience

In experiments with dogs (n = 68), the influence of temperature, flow rate, perfusate, and perfusion duration on the hind leg subjected to an isolation perfusion was studied. The perfusion pressure, the blood gases, and the metabolic status in specimens of skeletal muscle obtained at the end of the perfusion period served as parameters. A perfusion of 1 and 2 h with whole blood, a flow rate of 10 mL/10 g/min, and temperatures of up to 42 degrees C did not result in alterations of the energy metabolism. When the flow rate was lowered to 5 mL/100 g/min, when other perfusates were used, or when the in temperature was raised to 43.5 degrees C, then tissue damage occurred. Knowledge gained in these experiments was utilized in the treatment of 371 patients with malignancies of the extremities, and an extremely low complication rate was observed.     

Pseudohypoaldosteronism type II: proximal renal tubular acidosis and dDAVP-sensitive renal hyperkalemia

The mechanisms of metabolic acidosis and hyperkalemia were investigated in a patient with chronic mineralocorticoid-resistant renal hyperkalemia (5.3-6.9 mmol/l), metabolic acidosis (arterial blood pH 7.27, total CO2 17 mmol/l), arterial hypertension, undetectable plasma renin activity (less than 0.10 ng/ml/h), high plasma aldosterone level (32-100 ng/dl), and normal glomerular filtration rate (131 ml/min/1.73 m2). During the hyperkalemic period, urine was highly acidic (pH 4.6-5.0), urinary NH4 excretion (10-13 microEq/min) and urinary net acid excretion (19-24 microEq/min) were not supernormal as expected from a chronic acid load. During NaHCO3 infusion, the maximal tubular HCO3 reabsorption was markedly diminished (19.8 mmol/l glomerular filtrate), and the fractional excretion of HCO3 (FE HCO3) when plasma HCO3 was normalized was 20%. Urine minus blood PCO2 increased normally during NaHCO3 infusion (31 mm Hg), and the urinary pH remained maximally low (less than 5.3) when the buffer urinary excretion sharply increased after NH4Cl load. When serum K was returned toward normal limits, metabolic acidosis disappeared, urinary NH4 excretion rose normally after short NH4Cl loading while the urinary pH remained maximally low (4.9-5.2), the maximal tubular HCO3 reabsorption returned to normal values (24.8 mmol/l glomerular filtrate), and FE HCO3 at normal plasma HCO3 was 1%. Nasal insufflation of 1-desamino-8-D-Arginine Vasopressin (dDAVP) resulted in an acute normalization of the renal handling of K and in an increase in net urinary acid excretion. We conclude that: the effect of dDAVP on renal handling of K may be explained by the reversal of the distal chloride shunt and/or an increase in luminal membrane conductance to K; the distal acidification seems to be normal which in the event of distal chloride shunt impairing distal hydrogen secretion might be explained by the presence of systemic acidosis which is a potent stimulus of hydrogen secretion, and metabolic acidosis in the steady state was accounted for by the diminution of bicarbonate reabsorption and ammonia production in the proximal tubule secondary to chronic hyperkalemia.     

Cl-/base exchange in rat mesangial cells: regulation of intracellular pH

The present study was designed to determine whether rat glomerular mesangial cells possess Cl- -dependent intracellular pH (pHi) regulatory processes. Rat glomerular mesangial cells were grown to confluence on glass coverslips. Intracellular pH (pHi) was measured with BCECF. Steady state pHi in HCO3- containing solutions was 7.08 +/- 0.03 (N = 13). When extracellular Cl- was acutely removed, pHi increased at a rate of 0.57 +/- 0.03 pH/min units (N = 8), P less than 0.001. DIDS (0.5 mM) significantly decreased the rate of increase in pHi to 0.34 +/- 0.04 pH/min, P less than 0.01. Na+ removal and amiloride (1 mM) did not alter the increase in pHi induced by Cl- removal. Steady state pHi in the absence of Cl- was significantly increased above control, 7.39 +/- 0.02 (N = 7), P less than 0.001. Following the acute alkalinization of pHi by CO2 removal, pHi recovered at a rate of 0.07 +/- 0.01 pH/min (N = 9). In the absence of Cl-, the pHi recovery rate was significantly decreased to 0.01 +/- 0.008 pH/min (N = 5), P less than 0.01. DIDS (0.5 mM) significantly decreased the rate of pHi recovery to 0.02 +/- 0.01 pH/min (N = 5), P less than 0.01. Na+ removal and amiloride (1 mM) had no effect on the rate of pHi recovery following acute alkaline loading.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of oxalate efflux and oxalate transport via anion exchange in rat renal brush border membrane vesicles

In order to study the characteristics of oxalate transport across the brush border membrane, we studied oxalate uptake and efflux by rat renal cortical brush border membrane vesicles (BBMV). The vesicles were prepared with MgCl2 precipitation method and oxalate uptake was measured by a modification of the rapid millipore filtration technique. In order to analyze efflux of oxalate from BBMV, the vesicles were pre-equilibrated with 100 microM [14-C]-oxalate for 10 min, at 30 degrees C. Temperature dependent and independent oxalate uptake were observed under the conditions of salt and anion free medium. Temperature dependent oxalate accumulation showed "over shoot", indicating carrier mediated oxalate flux. The stimulating effect of an inside alkaline pH gradient on oxalate uptake failed to show at intravesicular pH 8.5. The efflux of oxalate from pre-loaded BBMV showed three steps decreasing curve. Initial rapid efflux was inhibited by extravesicular 5 mM para-aminohippurate (PAH) and low temperature (5 degrees C). Extravesicular 100 microM PAH had no effect on oxalate efflux. These data suggested a carrier mediated oxalate transport system across the barrier from intracellular to luminal site. An outwardly directed chloride (Cl) gradient stimulated oxalate uptake in BBMV, DIDS, anion exchange inhibitor, inhibited this Cl-stimulated oxalate uptake completely. Harmaline, a Na-coupled cotransport inhibitor, had no effect and Probenecid, an organic anion transport inhibitor, caused 45% inhibitory effect on Cl-gradient stimulated oxalate transport system via anion exchange in the rat kidney BBMV.     

Acidosis impairs the coagulation: A thromboelastographic study

BACKGROUND: Coagulopathy is a contributor to and predictor of death in bleeding patients. Acidosis is known to be a predictor of worse outcome in trauma patients and to be a coexisting factor in coagulopathic patients, but it has not been studied if it is a causal factor in the development of coagulopathy. METHODS: We have adjusted the pH level in blood samples from healthy volunteers to different levels between 7.4 and 6.8 by the use of hydrochloric acid. Thereafter we have studied the coagulation at the different pH levels by the use of thromboelastography. RESULTS: We found a strong correlation between pH levels and impairment of the coagulation, where the clot strength is increased much slower at pH levels below 7.4 even though the initiation of the clotting seems to be normal. The clot formation time was increased by 168% at pH 6.8 as compared with pH 7.4 (p < 0.00001, r = 0.89) whereas the clotting time was left unaffected. CONCLUSIONS: Acidosis causes a strong impairment of the coagulation as measured with thromboelastography. The impairment found when lowering pH from 7.4 to 7.15 was almost identical to the impairment seen in another study by Kettner et al. when the temperature was lowered from 36 degrees C to 32 degrees C.     

Cytoplasmic pH regulation in canine renal proximal tubule cells

The precise mechanisms by which the mammalian kidney proximal tubule transports H+ and HCO3- and regulates cytosolic pH (pHi) remain in doubt, though both a H+-ATPase pump and Na+/H+ exchange at the luminal membrane are known to function in the export of protons. The mechanisms of HCO3- transport are less clear though recent reports suggest an important role for an electrogenic Na+/HCO3- symport in the basolateral membrane. The importance of chloride-dependent bicarbonate transport is unknown. In the present studies, the pH-sensitive fluorescent dye, bis-(carboxyethyl)-carboxyfluorescein (BCECF) has been used to study pHi changes in suspensions of canine proximal tubule cells following acidification or alkalinization of the cytosol. Cells were acid-loaded to pH 6.5 by exposure to the H+/K+ ionophore, nigericin. Following removal of nigericin, pHi returned to basal levels (pHi = 7.1) when the cells were resuspended in a buffer containing 100 mM Na+. This recovery was blocked by removal of Na+ or addition of 0.2 mM amiloride to the cell suspension. In the presence of 0.2 mM amiloride and Na+, partial excretion of the acid load occurred if the buffer also contained HCO3-/CO2, but this effect was blocked by the removal of Na+ or the addition of 1 mM 4-acetomido-4'-isothiocyano-2,2'-stilbene disulfonic acid (SITS). When cell membrane potential was monitored in these experiments using the potential-sensitive fluorescent dye, bis-(1,3-dibutylbarbiturate) trimethine oxonol, the increase in pHi seen in the presence of Na+ was found to be electroneutral, whereas when that occurred in the presence of Na+, amiloride and HCO3-/CO2 was associated with membrane hyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevation of Arterial Potassium During Acute Systemic Hypoxia Is Abolished by Alkalosis

Abstract Background: Small elevations in plasma potassium evoke vasodilation in the peripheral circulation. Systemic hypoxia elevates arterial potassium and also modifies arterial pH. Aims: We examined the interaction between pH and potassium in blood during systemic hypoxia and the effect of pH on the uptake/release of potassium in the peripheral tissues. Methods: Anesthetized dogs were ventilated with air plus oxygen for normoxia or air plus nitrogen for hypoxia. Some animals received intravenous sodium bicarbonate to elevate pH by 0.1 units. Arterial plasma potassium concentration was measured in normoxia and hypoxia. A rat gracilis muscle was perfused with normoxic Krebs buffer and the potassium content of the venous outflow was compared during perfusion at pH 7.4, 6.8, or 7.8. Results: In dogs with an arterial pH of 7.40–7.45, systemic hypoxia elevated the arterial potassium by 1 mmol/L. An arterial pH of 7.55 did not alter the basal potassium concentration, but it abolished the hypoxia‐induced increase. In rat muscle, reduction of the perfusate pH from 7.4 to 6.8 reduced arterial perfusion pressure from 8.73 to 7.32 kPa and venous potassium from 6.6 to 5.2 mM. Elevation of perfusate pH to 7.8 decreased the arterial perfusion pressure from 8.44 to 6.95 kPa but did not affect venous potassium. Conclusions: The hypoxia‐induced elevation of arterial potassium is abolished by increasing the pH to 7.55. This is not due to enhanced potassium uptake into peripheral tissues at high pH. Red blood cells are suggested as the most likely source of the potassium released in hypoxia.     

Cellular electrolyte and volume changes induced by acidosis in the rabbit proximal straight tubule.

Cellular acidosis induced either by high Pco2 or by low HCO3- concentrations has been shown to cause cell swelling in isolated, lumen-collapsed, S2 segments of the rabbit proximal tubule (Sullivan et al., Am J Physiol 1990; 258: F831-F839). The swelling is not followed by a volume regulatory response. The ionic basis of the swelling has been investigated by measurement of the cellular K+, Na+, and Cl- content (electron probe) and HCO3- concentration (pH-sensitive fluorescent dye). Cell content of K+, Na+, and Cl- was expressed as a ratio to P content. Exposure to 15% CO2 increased K/P from 0.98 to 1.16, Cl/P from 0.14 to 0.20, and Na/P from 0.09 to 0.11. Cell (HCO3-) increased from 22 to 32 mM. Reduction in bath (HCO3-) from 25 to 5 mM reduced cell (HCO3-) from 24 to 8 mM and increased K/P from 0.75 to 0.90. Na/P fell from 0.13 to 0.09, and Cl/P fell from 0.15 to 0.12. Thus, swelling resulting from acidosis induced by high CO2 was accompanied by an accumulation of K+, Cl-, and HCO3-; that resulting from acidosis induced by a fall in (HCO3-) was combined with an accumulation of K+ and an unidentified anion. To determine if the swelling induced by a fall in pH might be coupled with depolarization of the basolateral membrane, the effect of 1 mM barium was tested. Barium caused cell volume to increase 10.2%. Cell pH rose from 7.38 to 7.56, K/P increased from 0.63 to 0.73, Na/P did not change, and Cl/P rose from 0.17 to 0.20. Cell (HCO3-) increased 10.4 mM. When the pH of the barium-treated tissue was reduced to 7.02 by raising Pco2, additional cell swelling and accumulation of K+ occurred. The effect on cell volume of a reduction of bath (HCO3-) from 25 to 5 mM at constant bath pH was determined. Cell pH was not altered. Cell volume decreased 3% initially and then returned to the control level. When the bath (HCO3-) was restored to 25 mM, cell volume increased 3.9% and then returned to the baseline. Thus, volume regulation was not impaired. It was concluded that a fall in cell pH induces swelling, and this is coupled with an accumulation of K+. This is probably the result of a pH effect on barium-sensitive and barium-insensitive K+ conductance pathways. The nature of the anions that balance the gain in K+ depends on the means used to induce acidosis.     

Factors affecting inorganic mercury transport and toxicity in the isolated perfused proximal tubule.

The effects of cysteine (80 microM), glutathione (80 microM), rabbit albumin (100 microM), and an ultrafiltrate of rabbit plasma on the toxicity and transport of inorganic mercury (Hg2+; 18.4 microM) in isolated perfused S1, S2, and S3 segments of the renal proximal tubule from the rabbit were studied. Cellular and tubular injuries were assessed qualitatively by light microscopy observations and quantitatively by the tubular leak of the volume marker 3H-glucose. The lumen-to-bath transport of inorganic mercury was assessed by measuring both the rate of disappearance of inorganic mercury from the luminal fluid and the rate of appearance of inorganic mercury in the bath. When glutathione was added to the perfusate containing the inorganic mercury, no signs of epithelial cell necrosis or injury were detected in any of the three segments of the proximal tubule. There was also an absence of or a decrease in cellular injury in the epithelium of the same tubular segments when either cysteine or the ultrafiltrate was present in the perfusate. However, when rabbit albumin and inorganic mercury were present in the perfusate, severe degenerative and necrotic changes occurred very rapidly in the epithelium of all three segments of the proximal tubule. In almost every instance where glutathione, cysteine, or the plasma ultrafiltrate were present in the perfusate, the disappearance flux of inorganic mercury from the tubular lumen into the tubular epithelium was lowered. It was concluded that glutathione, cysteine, and the ultrafiltrate of rabbit plasma provide isolated perfused S1, S2, and S3 segments of the proximal tubule varying degrees of protection from the toxic effects of inorganic mercury. This protection appears to be related to a decrease in the movement of inorganic mercury across the luminal membrane of the tubular epithelial cells.     

Prostaglandin F2 alpha inhibits the ammoniagenic response to acute acidosis in LLC-PK1 cells

A kidney epithelial cell line, LLC-PK1, which does not synthesize prostaglandins, provides an ideal in vitro model system to investigate the effect of prostaglandins in the regulation of renal ammoniagenesis. Previous studies from our laboratory have demonstrated significant increases in glutamine-dependent ammonia and alanine production by rocked cultures of LLC-PK1 cells subjected to either acute metabolic or respiratory acidosis. In the study presented here, experiments were conducted to investigate the role of prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) in the response of ammonia metabolism to acute metabolic acidosis by LLC-PK1 cells. A low dose of PGF2 alpha (0.1 ng/mL) dramatically inhibited the stimulatory effect of a low pH (pH 6.8) on ammonia production. In contrast, the inhibition of cytosolically generated alanine was less dramatic and averaged only 20% of the effect on ammonia production. Furthermore, PGF2 alpha increased cellular alpha-ketoglutarate concentration, suggesting an increase in intramitochondrial pH. Thus, the cellular mechanism of PGF2 alpha action appears to involve either interference with the cytosolic pH signal or its translation to the intramitochondrial compartment. The inhibitory response of PGF2 alpha on pH-stimulated ammoniagenesis was progressively lost at higher concentrations. Both low-dose (0.1 ng/mL) and high-dose (10 ng/mL) PGF2 alpha had no significant effect on the basal rates of ammonia and alanine production at pH 7.4. PGE2, on the other hand, did not exhibit any significant response on ammonia or alanine production at either pH 6.8 or 7.4 when given in a wide range of doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of perfusate viscosity, RBC deformability and drag on the function of an isolated perfused rat kidney

There is increasing clinical interest in improving blood rheology to optimize organ function, but studies correlating the two are scarce. To study this, rat kidneys were perfused in vitro at 37 degrees C at a constant mean renal arterial pressure of 160 mm Hg. The perfusate consisted of an oxygenated Krebs HCO3 buffer containing 1 mg/ml glucose, 0.5 mg/ml creatinine, amino acids, [3H]inulin (marker for GFR), 2.5 g/dl albumin, and 10 or 20% hematocrit. In some experiments, RBC were made nondeformable by heating at 50 degrees C for 20 min. Deformability was measured by an ektacytometer. In other experiments, 0.001% of an anionic polyacrylamide (Separan), a drag-reducing agent, was added to the perfusate. Viscosity was measured with a cone and plate viscometer, [Na+] with a flame spectrophotometer, and perfusate flow with a Brooks in-line flowmeter. Other functional parameters, GFR, urine flow, RPF, and reabsorption of sodium and water, were also measured. The results indicate that (1) making RBC nondeformable or increasing viscosity by increasing hematocrit reduces renal function, and (2) addition of a drag-reducing agent improves renal function at 20% hematocrit. We conclude that rheologic manipulation of a perfusate solution can alter flow and renal function.     

Ammonia transport in the proximal tubule in vivo

Studies were performed to characterize the determinants of proximal tubule ammonia entry (and retention) in vivo. Rat proximal tubules were studied in vivo using in situ microperfusion. In both normal animals and animals with metabolic acidosis, increasing luminal flow rate significantly enhanced luminal ammonia entry. In contrast, luminal pH was not as important in determining ammonia entry. Analysis of the levels of luminal NH3 in these studies was not consistent with simple diffusion equilibrium of NH3. In animals with chronic metabolic acidosis, additional studies demonstrated that inhibition of the Na+-H+ exchanger had no direct effect on luminal ammonia entry. However, studies of ammonia efflux from tubules perfused with 10 mmol/L ammonia demonstrated significant transport of both NH3 and NH4+. Studies of luminal glutamine deamidation via gamma-glutamyltransferase in control conditions did not indicate a significant role for luminal ammoniagenesis in the superficial proximal tubule in vivo. These and other recent studies of proximal tubule ammonia transport significantly modify the traditional diffusion equilibrium (of NH3) model of ammonia transport. Luminal flow rate is an important determinant of luminal ammonia entry. Transport of NH4+, both into and out of the tubule lumen, represents a major component of total ammonia transport.