The mechanism of bicarbonate reabsorption in the proximal and distal tubules of the kidney. 1965.

The mechanism of HCO3- reabsorption in proximal and distal tubules was examined in rats undergoing NaHCO3 diuresis. The steady-state intratubular pH was measured with pH-sensitive glass microelectrodes and compared with the equilibrium pH calculated from the HCO3- concentration of the tubular fluid (measured with quinhydrone electrodes) and plasma Pco2. In the proximal tubule the intratubular pH and the equilibrium pH were identical, indicating no accumulation of excess H2CO3. After inhibition of carbonic anhydrase, however, intratubular pH was significantly lower (0.85 pH U) than the equilibrium pH. It was concluded that HCO3- reabsorption in the proximal tubule was mediated by H+ secretion, but that carbonic anhydrase located in the luminal membrane of the cell prevented H2CO3 from accumulating in the tubular fluid. In the distal tubule the intratubular pH was 0.85 U lower than the equilibrium pH. This difference could be obliterated by an intravenous injection of carbonic anhydrase. It was concluded that HCO3- reabsorption in this segment was also accomplished by H+ secretion. The accumulation of excess H2CO3 in the tubular fluid indicated that, in contrast to the proximal tubule, carbonic anhydrase was not located in the luminal membrane of distal tubular cells.

The Scottish tonsillectomy audit. Audit Sub-Committee of the Scottish Otolaryngological Society.

Regional specialist societies offer a valuable mechanism for the conduct of medical audit. The experience of the audit sub-committee of The Scottish Otolaryngological Society in conducting an audit on laryngeal cancer encouraged us to undertake a larger audit of tonsillectomy practice in Scotland. Although the number of tonsillectomies performed has declined over the last 10 years, they still account for about 20 per cent of all operations performed by otolaryngologists and as such are a major consumer of resources (Personal communication--Directorate of Information Services, Information and Statistics Division. NHS in Scotland, Management Executive, Edinburgh). The Scottish tonsillectomy audit was devised to define current practice, review indications for surgery and recommend such modifications in practice as may be necessary to optimise patient care and the use of resources. Funding was obtained from the Clinical Resource and Audit Group (CRAG) of the Scottish Home and Health Department. Data on current practice was collected during the period February 1992 to January 1993. Proformas were completed by medical, administrative and secretarial staff in all participating hospitals, collected by an audit secretary and passed to the relevant data collection centre. Data was then entered into a specially designed database before being forwarded to the audit co-ordinator based in Dundee for collation. Six and 12 months following surgery, all inpatients were sent a questionnaire to obtain data on the efficacy of the operation. Data were obtained from a total of 9,773 patients. Two thousand and seventy-nine of these were seen as both outpatients and inpatients, 4,309 were outpatients only and 3,385 were inpatients only. Four thousand, one hundred and one patients returned at least one follow-up questionnaire. The topics audited included source and reason for referral, indications for surgery, grade of staff involved, type of surgery and length of stay in hospital. In agreement with previous studies (H.M.S.O., 1989), differences were found in the rates of tonsillectomy performed in different Health Boards. Although the highest referral and operation rates were found in the Highland region, referral and operation rates did not correlate in all other areas. Recurrent tonsillitis was the most frequent principal reason for the decision to operate although there were differences between Health Boards for other indications including obstructive symptoms. Most patients had symptoms for two to three years although some patients had been affected for 40 years prior to being listed for tonsillectomy. Some area ENT services were consultant-based while others involved more junior staff. The grade of staff involved did not appear to affect the decision made at the Outpatient Department (OPD) or the outcome of the operation. Ninety-eight per cent of patients who returned the questionnaire were glad that the operation had been performed. Recommendations regarding changes in tonsillectomy practice are given.

The Scottish tonsillectomy audit. The Audit Sub-Committee of the Scottish Otolaryngological Society.

Regional specialist societies offer a valuable mechanism for the conduct of medical audit. The experience of the audit sub-committee of The Scottish Otolaryngological Society in conducting an audit on laryngeal cancer encouraged us to undertake a larger audit of tonsillectomy practice in Scotland. Although the number of tonsillectomies performed has declined over the last 10 years, they still account for about 20 per cent of all operations performed by otolaryngologists and as such are a major consumer of resources (Personal communication-Directorate of Information Services, Information and Statistics Division. NHS in Scotland, Management Executive, Edinburgh). The Scottish tonsillectomy audit was devised to define current practice, review indications for surgery and recommend such modifications in practice as may be necessary to optimise patient care and the use of resources. Funding was obtained from the Clinical Resource and Audit Group (CRAG) of the Scottish Home and Health Department. Data on current practice was collected during the period February 1992 to January 1993. Proformas were completed by medical, administrative and secretarial staff in all participating hospitals, collected by an audit secretary and passed to the relevant data collection centre. Data was then entered into a specially designed database before being forwarded to the audit co-ordinator based in Dundee for collation. Six and 12 months following surgery, all inpatients were sent a questionnaire to obtain data on the efficacy of the operation. Data were obtained from a total of 9,773 patients. Two thousand and seventy-nine of these were seen as both outpatients and inpatients, 4,309 were outpatients only and 3,385 were inpatients only. Four thousand, one hundred and one patients returned at least one follow-up questionnaire. The topics audited included source and reason for referral, indications for surgery, grade of staff involved, type of surgery and length of stay in hospital. In agreement with previous studies (H.M.S.O., 1989), differences were found in the rates of tonsillectomy performed in different Health Boards. Although the highest referral and operation rates were found in the Highland region, referral and operation rates did not correlate in all other areas. Recurrent tonsillitis was the most frequent principal reason for the decision to operate although there were differences between Health Boards for other indications including obstructive symptoms. Most patients had symptoms for two to three years although some patients had been affected for 40 years prior to being listed for tonsillectomy. Some are ENT services were consultant-based while others involved more junior staff. The grade of staff involved did not appear to affect the decision made at the Outpatient Department (OPD) or the outcome of the operation. Ninety-eight per cent of patients who returned the questionnaire were glad that the operation had been performed. Recommendations regarding changes in tonsillectomy practice are given.

Microelectrode of the Thomas type using a liquid membrane electrode.

By replacing the glass-based pH electrode (L. R. Pucacco, S. K. Corona, H. R. Jacobson, and N. W. Carter (1986) Anal. Biochem. 153, 251-261) with a liquid membrane-based pH electrode, a relatively easy-to-manufacture modified Thomas electrode has been developed. The liquid membrane-based modified Thomas electrode can be manufactured without the special equipment (forge) and materials (glass) required to make the glass membrane pH microelectrode (L. R. Pucacco and N. W. Carter (1976) Anal. Biochem. 73, 501-512). The sensitivity (57.4 +/- 0.22 mV/pH unit), response time (20.0 +/- 2.67 s), and electrical resistance (3.48 +/- 0.67 X 10(11) ohm) of this electrode are similar to those of the glass-based version.

Sodium-sensitive glass microelectrode: modified Thomas recessed-tip configuration.

Using a glass-membrane, sodium-sensitive microelectrode, a modified Thomas sodium-sensitive electrode has been developed. The modified Thomas electrode possesses a high sensitivity (57.9 mV/log aNa), a high selectivity (KpotNa,K less than 0.005), a relatively low electrical resistance (7.65 X 10(11) ohms), a small sensing chamber (10 microns3), and can be made in the double-barreled configuration. The modified Thomas electrode is designed to directly measure the intracellular sodium concentration of epithelial cells.

pH microelectrode: modified Thomas recessed-tip configuration.

Through the use of a glass-membrane pH electrode and a water-tight seal a modified Thomas pH microelectrode has been developed. The modified Thomas electrode has a relatively low electrical resistance (10(11) omega), a small sensing chamber (10 microns3), and a rapid response time (10 s) and can be manufactured in both single- and double-barreled configurations. The modified Thomas electrode is designed to measure the intracellular pH of small cells such as those found in the mammalian kidney tubule.

Muscle cell electrical hyperpolarization and reduced exercise hyperkalemia in physically conditioned dogs.

Contracting muscle cells release K ions into their surrounding interstitial fluid, and some of these ions, in turn, enter venous plasma. Thereby, intense or exhaustive exercise may result in hyperkalemia and potentially dangerous cardiotoxicity. Training not only reduces hyperkalemia produced by exercise but in addition, highly conditioned, long-distance runners may show resting hypokalemia that is not caused by K deficiency. To examine the factors underlying these changes, dogs were studied before and after 6 wk of training induced by running on the treadmill. Resting serum [K] fell from 4.2 +/- 0.2 to 3.9 +/- 0.3 meq/liter (P less than 0.001), muscle intracellular [K] rose from 139 +/- 7 to 148 +/- 14 meq/liter (P less than 0.001), and directly measured muscle cell membrane potential (Em) in vivo rose from -92 +/- 5 to -103 +/- 5 mV (P less than 0.001). Before training, resting Em of isolated intercostal muscle in vitro was -87 +/- 5 mV, and after incubation in 10(-4) M ouabain, Em fell to -78 +/- 5 mV. After training, resting Em of intercostal muscle rose to -95 +/- 4, but fell to -62 +/- 4 mV during incubation in 10(-4) M ouabain. The measured value for the Em was not completely explained by the increased ratio of intracellular to extracellular [K] or by the potassium diffusion potential. Skeletal muscle sarcolemmal Na,K-ATPase activity (microM inorganic phosphate mg-1 protein h-1) increased from 0.189 +/- 0.028 to 0.500 +/- 0.076 (P less than 0.05) after training, whereas activities of Mg2+ -dependent ATPase and 5'nucleotidase did not change. In untrained dogs, exercise to the point of exhaustion elevated serum [K] from 4.4 +/- 0.5 to 6.0 +/- 1.0 meq/liter (P less than 0.05). In trained dogs, exhaustive exercise was associated with elevation of serum [K] from 3.8 +/- 0.3 to 4.2 +/- 0.4 (NS). The different response of serum [K] to exercise after training was not explainable by blood pH. Basal insulin levels rose from 7.0 +/- 0.7 microU/ml in the untrained dogs to 9.9 +/- 1.0 microU/ml (P less than 0.05) after training. Although insulin might have played a role in the acquired electrical hyperpolarization, the reduced exercise-produced hyperkalemia after training was not reversed by blockade of insulin release with somatostatin. Although the fundamental mechanisms underlying the cellular hyperpolarization were not resolved, our observations suggest that increased Na-K exchange across the sarcolemmal membrane, the increase of Na,K-ATPase activity and possibly increased electrogenicity of the sodium pump may all play a role in the changes induced by training.

In situ PCO2 in the renal cortex, liver, muscle, and brain of the New Zealand white rabbit.

Recent studies have shown that in situ PCO2 in rat renal cortical structures far exceeds systemic arterial PCO2. These results were opposite to previous assumptions that renal proximal tubule fluid PCO2 approximated arterial PCO2. The present studies examined the species and organ specificity of the elevated PCO2 in 39 New Zealand White rabbits studied under normal acid-base conditions. In situ PCO2 was measured in renal cortex, superficial hepatic parenchyma, skeletal muscle, superficial cerebral cortex, and femoral nerve, artery, and vein. The results showed rabbit renal cortical PCO2 (57.2 +/- 1.2 mmHg) to be higher than both systemic arterial (39.1 +/- 2.0 mmHg) and venous PCO2 (45.4 +/- 2.1 mmHg). Similarly, liver PCO2 (64.1 +/- 3.5 mmHg) was found to be significantly higher than systemic arterial and venous PCO2 and also higher than portal and hepatic vein PCO2. Skeletal muscle, cerebral cortex, and femoral nerve PCO2 levels were usually greater than systemic arterial PCO2 but less than systemic venous PCO2. These observations show that in situ PCO2 is significantly elevated above afferent and efferent blood PCO2 in the kidney and liver but not in muscle or brain. A possible explanation for these findings in the former two organs may be high CO2 production and/or trapping of CO2 by their vascular systems.

Derangements of muscle composition, ion transport, and oxygen consumption in chronically alcoholic dogs.

Muscle ion composition, Na-K-ATPase activity, tissue respiration, and transmembrane potential differences were measured after 28 and 56 days of ethanol consumption (6.2 g X kg-1 X day-1) or an isocaloric amount of glucose in 12 and 4 dogs, respectively. Ethanol and glucose were given as supplements to an otherwise nutritious diet. After 28 and 56 days of alcohol consumption, skeletal muscle contents of phosphorus, magnesium, and potassium were significantly reduced as compared with either the control values or those in glucose-fed animals. In alcohol-fed animals, muscle sodium chloride, and calcium were significantly elevated. Ethanol consumption also resulted in hyperpolarization of the resting transmembrane potential of skeletal muscle fibers and a significant increase in Na-K-ATPase activity. No change was noted in Mg-ATPase activity. The increase in Na-K-ATPase activity was accompanied by increased sodium transport-dependent respiration. These results indicate that a subclinical myopathy may be induced by alcohol in the dog. Malnutrition did not appear to be a factor in this study, and thus the changes observed are believed to be due to ethanol per se. The magnitude and direction of these changes are similar to those observed in the skeletal muscle of chronically alcoholic humans. The changes in Na-K-ATPase activity and sodium transport-dependent respiration may represent adaptive responses of the muscle cell to ion transport or membrane disorders induced by ethanol.

Aldosterone effects on papillary collecting duct pH profile of the rat.

A number of recent studies have suggested that the medullary and papillary collecting ducts possess a high capacity for bicarbonate reabsorption. Because 3H binding studies have suggested the presence of mineralocorticoid receptors along the papillary collecting duct and because clearance studies have suggested that aldosterone affects urinary acidification, we designed a series of clearance and micropuncture studies to examine the role of the papillary collecting duct in final urinary acidification. The studies were conducted in intact and adrenalectomized rats receiving either replacement glucocorticoids alone or glucocorticoids with acute infusion of physiological doses of aldosterone. The results show that under normal acid-base status all rats had a pH profile along the papillary collecting duct with more terminal portions being more acidic than the proximal portions. This decreasing pH profile was diminished in acidotic adrenalectomized rats but returned to normal with acute administration of aldosterone. In all cases the luminal potential difference was slightly positive and did not change with adrenalectomy or administration of aldosterone. These results demonstrate that the papillary collecting duct participates in the generation of maximal urinary pH gradient and that this process is partly influenced by aldosterone.

Serum and muscle potassium in experimental alcoholic myopathy.

We investigated the possible role of potassium deficiency, a recognized cause of myopathy, in the pathogenesis of experimental acute alcoholic myopathy (EAM) in the rat. Alcohol-treated animals receiving the same dietary potassium as controls developed mild hypokalemia; however, muscle potassium was preserved or elevated even in the presence of scattered muscle fiber necrosis. Increasing dietary potassium raised serum potassium but did not prevent EAM. These data indicate that hypokalemia in EAM is due to potassium redistribution between intra- and extracellular compartments rather than potassium deficiency, and confirm clinical observations that alcohol-induced muscle injury occurs independently of potassium deficiency.

Comparison of acidification parameters in superficial and deep nephrons of the rat.

The purpose of this study was to determine and compare pH, PCO2, and fractional bicarbonate delivery in both superficial and juxtamedullary nephrons by microelectrode techniques and microcalorimetry in the rat in vivo in order to define more clearly the role of deeper nephron segments in urinary acidification. Values for pH and total CO2 concentration ([tCO2]) at the bend of Henle's loop (LOH) (7.39 +/- 0.04 units and 20.5 +/- 1.5 mM) were significantly greater and the PCO2 was significantly less (36.6 +/- 1.5 mmHg) than values for these same parameters in the superficial late proximal tubule (LPT) (6.78 +/- 0.03 units, 8.1 +/- 1.2 mM, and 63.2 +/- 1.0 mmHg, P less than 0.001). The fraction of filtered bicarbonate delivered to the LPT and LOH did not differ, however (12.2 +/- 2.5 vs. 9.0 +/- 0.8%). The pH and PCO2 values in the late distal tubule (6.59 +/- 0.04 units and 64.0 +/- 1.3 mmHg) were significantly greater than at the base (6.24 +/- 0.07 units and 34.5 +/- 1.5 mmHg) and tip (6.12 +/- 0.03 units and 35.2 +/- 1.2 mmHg) of the papillary collecting duct. The [tCO2] in the LOH and an adjacent vasa recta was compared and did not differ significantly (20.5 +/- 1.5 vs. 21.2 +/- 1.3 mM, P greater than 0.05). In summary, we have demonstrated significant alkalinization of tubule fluid in the deep LOH as a result of water abstraction and CO2 diffusion from the nephron. Our results suggest that a spontaneous disequilibrium pH may not exist in the LOH. Furthermore, similar values for [tCO2] in vasa recta and the LOH suggest that passive HCO-3 reabsorption in the thin ascending limb of Henle would be unlikely and does not contribute to the "loop" component of bicarbonate reabsorption.

Evaluation of bicarbonate transport in rat distal tubule: effects of acid-base status.

Previous micropuncture studies utilizing indirect methods to estimate bicarbonate transport in the rat superficial distal tubule have indicated that the distal bicarbonate reabsorptive process normally operates well below the saturation level. Recent studies from our laboratory failed to demonstrate a spontaneous acid disequilibrium pH in this segment, implying that the bicarbonate reabsorptive rate was less than previously estimated. The purpose of the present experiments were 1) to measure the rate of absolute bicarbonate reabsorption by the rat superficial distal tubule while controlling bicarbonate delivery, and 2) to examine the effects of alterations in acid-base status on the rate of bicarbonate reabsorption. Five groups of rats in different states of acid-base balance were studied. No significant bicarbonate reabsorption was detected in the control hydropenic, combined respiratory acidosis-metabolic alkalosis, acute respiratory acidosis, or acute metabolic acidosis groups. In contrast, metabolic acidosis of 3 days duration resulted in a significant bicarbonate reabsorptive rate of 52.6 +/- 13.9 pmol . mm-1 . min-1. The observation of significant bicarbonate reabsorption in the distal tubule only during chronic metabolic acidosis of 3 days duration is compatible with adaptation of this normally low-capacity segment to chronic changes in systemic acid-base states.

Direct evaluation of the permeability of the rat proximal convoluted tubule to CO2.

Conflicting data exist regarding the ability of the rat proximal convoluted tubule to maintain a transepithelial gradient for CO2 and the effects of carbonic anhydrase on CO2 permeability. The present in vivo microperfusion experiments were designed to assess the ability of the rat proximal tubule to sustain a CO2 gradient between tubule lumen and peritubular blood. Tubules were perfused at rates ranging from 10 to 100 nl/min with isotonic sodium chloride containing no CO2. Peritubular capillary and intraluminal PCO2 was measured during microperfusion with PCO2 microelectrodes to allow determination of the transepithelial CO2 gradient. The mean PCO2 measured in peritubular capillaries of control rats was 60.6 +/- 1.9 mmHg. Since the perfusion solution initially contained no CO2, a gradient of 60 mmHg was imposed across the tubule epithelium. Intraluminal PCO2 rapidly approached that of the surrounding capillaries. At a tubule perfusion rate of 20 nl/min, the gradient between lumen and blood decreased to 0.9 mmHg, a value not significantly greater than zero. The calculated CO2 permeability coefficient (KCO2) was 3.69 X 10(-5) cm2/s. Addition of either 10(-4) M acetazolamide or benzolamide did not prolong the rapid dissipation of the imposed CO2 gradient. The KCO2 during carbonic anhydrase inhibition was not significantly different from control values. It is concluded that the rat proximal tubule does not present a physiologically significant diffusion barrier to CO2 either in the presence or absence of carbonic anhydrase activity. The previously demonstrated acid disequilibrium pH in the proximal tubule during inhibition of carbonic anhydrase represents an intraluminal accumulation of carbonic acid rather than of carbon dioxide gas.

Determination of disequilibrium pH in the rat kidney in vivo: evidence of hydrogen secretion.

The recent demonstration of elevated PCO2 in structures of the rat renal cortex indicated that previous determinations of disequilibrium pH (pHDq), and thus the differentiation of H+ secretion from bicarbonate reabsorption per se, required further evaluation. A new aspiration pH electrode was developed to allow tubule fluid to achieve chemical equilibrium at the PCO2 prevailing in vivo. In control and bicarbonate-loaded rats a pHDq was not observed in either proximal or distal tubules. After intravenous benzolamide a significant acid pHDq was observed in the proximal (but not the distal) nephron, and increased further during metabolic alkalosis. During combined metabolic alkalosis and respiratory acidosis a significant pHDq was present in the distal but not in the proximal tubule. Aldosterone administration to bicarbonate-loaded, hypercapnic rats did not alter the distal pHDq further. When present, the pHDq in the distal tubule was obliterated by carbonic anhydrase infusion. We conclude that proximal but not distal tubule fluid is in functional contact with carbonic anhydrase; the enzyme is in excess in the proximal lumen and H2CO3 did not accumulate even during conditions associated with increased H+ secretion; the basal rate of H+ secretion in the distal nephron accessible to cortical micropuncture is less than previously assumed. The data support the view that H+ secretion is the major mechanism of renal bicarbonate reabsorption.

Multiple on-line data collection and processing for radioimmunoassy using a micro-computer system.

A micro-computer system is described which has been designed to perform on-line data capture from up to seven radioisotope counters of different types in parallel with interactive results processing and subsequent transmission to a laboratory computer-based data management system.