Risk factors for the occurrence of new and chronic cases of subclinical mastitis in dairy herds in southern Brazil.

The aim of this research was to evaluate the risk factors for new and chronic subclinical intramammary infections (IMI) using the monthly somatic cells count of dairy cows. The study took place at 30 dairy herds with approximately 1,700 cows in lactation. Data characterizing the dairy farms and their milking management were obtained from a survey questionnaire. The somatic cells count values from 2 consecutive months were used to classify cows as either healthy or with new or chronic infections. A chi-squared test was used in the analysis of subclinical IMI to evaluate associations between each independent variable, followed by logistic regression to estimate the risk of a new infection in healthy cows and of chronic infection in cows with new infections. Factors increasing the odds ratio of a cow developing a new case of subclinical mastitis were (1) cows with more than 3 lactations, (2) cows with a mean hyperkeratosis score above 3, (3) cows with the udder below the hock, (4) cows with very dirty udders, and (5) milking of infected animals before healthy cows. Factors increasing the risk of a subclinical chronic infection compared with new cases of subclinical mastitis were (1) a lack of regular maintenance of milking machinery, (2) cows over 100 d in lactation, and (3) cows with the udder on or below the hock. The risk factors identified in this study can be used in IMI control programs to reduce the frequency of new and chronic cases of subclinical mastitis.

Cytotoxicity of myeloma light chains in cultured human kidney proximal tubule cells.

We evaluated the effect of eight species of light chains on cultured human kidney proximal tubule cell proliferation. Exposure to light chains for 48 hours caused dose-dependent inhibition in tritium ((3)H)-thymidine incorporation by simian virus 40 immortalized human proximal tubule cells, although the effect was variable among different species of light chains. We studied cytotoxic effects of selected toxic light chains in further detail. Two of these light chains caused significant DNA degradation. A lambda-light chain caused lactate dehydrogenase release from exposed cells at 48 hours, but not at 24 hours. Cytomorphological and electron microscopic examination of cells exposed to light chains for 24 hours showed condensed nuclei, cell detachment, paucity of mitotic activity, and apoptosis, and at 48 hours of exposure, changes consistent with necrosis. Apoptosis assay by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method showed a sixfold increase in the number of apoptotic cells exposed to the same lambda-light chain for 24 hours. Rhodamine-phalloidin staining showed variable but significant disruptions in the actin cytoskeleton. These studies show that some myeloma light chains are toxic to cultured human proximal tubule cells and induce cytoskeletal injury and DNA damage consistent with apoptosis followed by secondary necrosis. Direct proximal tubule cell toxicity may be an important mechanism of renal involvement in multiple myeloma.

Rhabdomyolysis and acute renal failure in a cardiac transplant recipient due to multiple drug interactions.

BACKGROUND: The 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors lovastatin and simvastatin have been associated with rhabdomyolysis in cardiac transplant recipients. Herein, we report a case of a 52-year-old male recipient of a cardiac transplant who developed rhabdomyolysis and acute renal failure caused by simvastatin precipitated by multiple drug interactions. METHODS: The patient had a history of cardiac transplantation (5 years before) and presented with a 2-day history of dark urine preceded by 2 weeks of diffuse myalgias. He had been maintained on cyclosporine throughout the entire post-transplant period. Simvastatin was added and pravastatin was discontinued 2 months before admission. Two weeks before the onset of muscle symptoms, digoxin and verapamil were started for new-onset atrial fibrillation. Creatinine phosphokinase levels peaked at 950,000 IU with serum creatinine of 3.3 mg/dL (baseline, 1.8 mg/dL). RESULTS: Review of the medication history indicates a temporal association between the addition of 3 drugs (simvastatin, verapamil, and digoxin) to the medication regimen already containing cyclosporine and the episode of rhabdomyolysis. All of these drugs are cytochrome P450 3A4 and/or P-glycoprotein substrates that are known from previous pharmacokinetic studies to individually produce substantial increases in levels of simvastatin. CONCLUSION: We believe this case illustrates that avoiding the use of drugs that are cytochrome P450 3A4 and/or P-glycoprotein substrates reduces the risk of rhabdomyolysis caused by 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors.

Clinical predictors of HIV-1 infection among preschool children in Dar es Salaam, Tanzania.

Seroprevalence of HIV-1 infection was determined in children aged between eighteen months and five years, attending maternal and child health (MCH) clinics in Dar es Salaam, Tanzania. A total of 889 children were eligible for the study, however seven children could not be enrolled because their mothers/guardians absconded and blood could not be drawn from 21 children due to refusal of mothers/guardians and from another 12 children due to technical reasons. Therefore, the participation rate was 95.5%. Of the 849 children screened, 14 (1.65%) were found to have IgG anti HIV-1 antibodies in their sera. The main clinical features found in children with symptomatic HIV-1 disease were weight loss, generalized lymphadenopathy, recurrent fevers, and prolonged diarrhoea. The utility of clinical features suggestive of HIV-1 infection (according to CDC classification) in identifying HIV-1 infection in children was evaluated and found to have high sensitivity (100%), specificity (96.9%) and negative predictive value (100%), but a low positive predictive value (35%). Marked variations in progression to symptomatic phase were noted, whereby some relatively young children had progressed to symptomatic phase (CDC class P-2A), while some older children were still in the asymptomatic stage (CDC class P-1 C). None of the symptomatic HIV-1 infected children presented with neurological disease, severe opportunistic infections, or malignancies. Although reduced mid-upper arm circumference and weight-for-age were associated with HIV seropositivity, these clinical parameters had low positive predictive values compared to the CDC classification.

PIP: Serologic testing of 849 children 18 months-5 years of age attending the Lugalo and Mwananyamala maternal-child health clinics in Dar es Salaam, Tanzania, during May-August 1994 identified 14 cases (1.65%) of HIV infection. The sample represented 95.5% of children making mandatory monthly clinic visits during the 3-month study period. The main clinical symptoms in HIV-infected children were weight loss, generalized lymphadenopathy, recurrent fever, and prolonged diarrhea, all included in the Centers for Disease Control and Prevention (CDC) classification scheme. Children with symptomatic HIV infection were younger than those with asymptomatic infection. None of the children with symptomatic HIV infection presented with neurologic impairment, severe opportunistic infections, or cancers. The clinical features included in the CDC classification for HIV had a 100% sensitivity, a 96.9% specificity, and a 100% negative predictive value, but the positive predictive value was only 35%. Although the model with the best fit included mid-upper arm circumference less than 14 cm and a reduced weight-for-age (odds ratios, 3.8 and 1.9, respectively), the positive predictive values for these two factors were only 4.3% and 4.1%, respectively. The 1.65% HIV seroprevalence rate recorded in this community-based study is lower than the 2.4% rate estimated among newborns in Dar es Salaam, presumably because of high infant mortality and hospitalization among HIV-infected newborns. Use of a simplified version of the CDC classification of HIV infection in children is recommended for routine clinical use in Tanzania.

Potential role of integrins in acute renal failure.

Many cell adhesion molecules probably contribute to the pathogenesis of acute renal failure. This review focuses on the potential importance of integrins. Integrins are a family of molecules that mediate cell-matrix and cell-cell adhesion, and are found on almost all cells. After acute renal injury, tubule epithelial cell-cell contacts (mediated by many molecules including uvomorulin and possibly integrins) are disrupted, as is the cytoskeleton. Detachment of viable tubule epithelial cells from the basement membrane has also been documented following acute renal injury. Such detachment would lead to back-leak of glomerular filtrate and could also be important in the production of cellular casts. As the attachment of the cytoskeleton to integrin is critical to cell-matrix adhesion, it is likely that cytoskeletal disruption is one of the mechanisms contributing to cell detachment. Integrins are also likely to be important in repair, as integrins will probably participate in migration along the basement membrane and may also influence cell function during the redifferentiation process. Finally, leukocyte integrins will probably play a role in the migration of leukocytes into areas of injury and also be critically important to their function.

Roles and mechanisms of urinary buffer excretion.

Excretion of acid (or generation of bicarbonate) by the kidneys is necessary for acid-base homeostasis. Most of this acid is excreted in the form of ammonia and titratable acid, the latter representing the amount of acid required to titrate the urine buffers from the plasma pH to urine pH. The transport of ammonia in the kidney is now recognized to entail more than simple nonionic diffusion of NH3 and trapping of NH4+. NH4+ transport in the kidney probably occurs by passive diffusion and by transport on the Na+-H+ exchanger, the Na+-K+-2Cl- transporter and on Na+-K+-ATPase. NH3 diffusion is stimulated by an acid disequilibrium pH in various nephron segments. Also, diffusion equilibrium of NH3 in various regions of the kidney has now been disproved. These various mechanisms of ammonia transport are considered in terms of their possible changes with acid-base disturbances. Phosphate is the most predominant urine buffer; its urinary excretion increases with acidosis. The mechanisms probably involve a decrease in the preferentially transported species, HPO4(2-), and a direct effect of pH on proximal tubule apical phosphate transport. With chronic acidosis, changes in the activity of the apical Na+-phosphate transporter occur. These effects of systemic acid-base balance interact with parathyroid hormone and dietary phosphate status to alter phosphate reabsorption. Citrate transport in the kidney is analyzed because of its sensitivity to systemic pH and because of the possible influence on systemic acid-base status in certain circumstances. Alterations in citrate excretion with acid-base disturbances depend on changes in the concentration of the transported species, citrate2-, and on changes in renal metabolism.

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.

Extracellular matrix receptors in the kidney cortex.

Extracellular matrix (ECM) receptors anchor cells to substratum and impart positional information to cells. Within the group of ECM receptors known as integrins, alpha-subunits of these alpha beta heterodimers define ligand specificity, whereas beta-subunits define the subclass. We used immunofluorescence with anti-ECM receptor antibodies to examine distribution within human kidney cortex of all known alpha-subunits in the beta 1 subclass of integrins as well as a non-integrin 67-kDa elastin/lamin receptor. The alpha 1-subunit (alpha 1 beta 1 defines a collagen receptor) was present in mesangium and base of all tubule epithelial cells; alpha 2 (collagen) was present in mesangium and in distal but not proximal tubule cells; alpha 3 (collagen, laminin, fibronectin) was diffusely distributed within glomeruli but tubule staining was less intense; alpha 4 (fibronectin) was absent; alpha 5 (fibronectin) was present in blood vessels; and alpha 6 (laminin) was present along basolateral aspect of all tubule cells but absent in glomeruli. The elastin/laminin receptor was present in all tubule epithelial cells, but staining was heavier in distal tubules, especially intercalated cells. Thus striking heterogeneity in ECM receptor distribution was noted. For collagen receptors, differences in tubule staining were pronounced. Despite the presence of laminin within both glomeruli and tubules, laminin receptors also showed marked differences in staining between these structures. Both differences in ECM structure and intrinsic differences among different cells may underlie these differences in ECM receptor distribution.

Ammonia entry along rat proximal tubule in vivo: effects of luminal pH and flow rate.

The roles of luminal pH and flow rate in determining ammonia entry along the rat proximal tubule were examined using in vivo microperfusion. With perfusion rate constant at 15 nl/min, perfusate bicarbonate concentration was varied. Collected fluid ammonia concentration correlated with collected fluid bicarbonate concentration, consistent with nonionic diffusion (r = 0.726; P less than 0.001). Hence ammonia entry was dependent on luminal pH. With perfusate bicarbonate constant at 5 or 25 mM, perfusion rate was varied. In all groups, there was little change in collected fluid ammonia concentration with flow rate. Thus ammonia entry was also highly dependent on flow rate. With paired collections using a 25 mM bicarbonate perfusate, collected fluid bicarbonate was higher at a 30 nl/min perfusion rate than at 15 nl/min (16.8 +/- 1.1 vs. 10.3 +/- 1.1 mM), whereas total ammonia concentrations were similar (0.54 +/- 0.1 and 0.55 +/- 0.1). Thus the NH3 concentration was higher at 30 than at 15 nl/min (6.1 +/- 1.2 vs. 3.4 +/- 0.5 microM; P less than 0.025), a result not predicted by simple nonionic diffusion. Thus these studies demonstrate the importance of nonionic diffusion in determining ammonia entry along the proximal tubule. However, the results also demonstrate that flow rate importantly determines ammonia entry in vivo in a manner not predicted by simple nonionic diffusion of NH3. This augmentation of ammonia entry with increasing flow rate may involve flow-dependent alterations in ammonia synthesis or transport of NH+4.

Ammonia transport in the proximal tubule.

The transport of ammonia in the proximal tubule is a complex interaction of a number of processes. Ammonia transport in the proximal tubule is clearly bidirectional; ammonia is secreted into the early proximal tubule lumen, but later in the proximal tubule, efflux out of the lumen may result in net ammonia reabsorption. Two mechanisms of ammonia transport have clearly been established: NH3 diffusion and NH4+ transport on the Na(+)-H+ exchanger. The relative contribution of these pathways to ammonia transport is still unsettled. Other pathways for ammonia transport, particularly NH4+ efflux out of the lumen, may be important as well. A variety of factors may modulate ammonia transport: plasma, cell and luminal pH, luminal flow rate, luminal potassium, and angiotensin II. Each of these factors also alters ammonia production rates and in most circumstances, ammonia transport appears to follow ammonia production rates.

Contribution of the distal tubule to potassium excretion in experimental glomerulonephritis.

The renal adaptations that maintain potassium homeostasis in diffuse forms of glomerular disease are not well defined. Thus, handling of potassium by superficial nephron segments was examined in a rat model of antiglomerular basement membrane nephritis. Sampling the same nephron successively from the end and beginning of the distal tubule and the end of the proximal tubule allowed a segmental analysis. Despite a 40% reduction in GFR, potassium excretion in the glomerulonephritis animals was normal due to an increase in FEK. The proximal tubule and loop segment did not contribute to the enhanced FEK seen in these animals. In contrast, potassium entry along the distal tubule was significantly greater in the experimental group averaging 13.7 +/- 4.3 pmol/min compared to 1.2 +/- 1.7 pmol/min in controls (P less than 0.01). Multiple linear regression analysis showed that distal tubule potassium entry at any level of flow was enhanced in glomerulonephritis compared to controls (P less than 0.0001). Plasma aldosterone levels were similar in both groups of animals. Thus, the adaptation to potassium excretion seen in glomerulonephritis is partly achieved by the distal tubule through flow-rate independent mechanisms and appears to be independent of plasma aldosterone levels.

Contribution of superficial nephron segments to sodium excretion in experimental glomerulonephritis.

The present study examined the contribution of individual superficial nephron segments to sodium excretion in antiglomerular basement membrane nephritis in the rat by sampling the same nephron successively from the end and beginning of the distal tubule and end of the proximal tubule. Whole kidney GFR in glomerulonephritic rats was reduced by approximately 40% from controls; absolute sodium excretion was about 25% of normal. Metabolic balance studies in the awake state had suggested that the animals were in sodium balance. Plasma renin levels before and during micropuncture were similar to controls. These findings suggest that the defect in sodium handling is intrinsic to the kidney. Glomerulotubular balance was maintained along the proximal tubule. Sodium reabsorption in the loop of Henle was reduced in absolute terms but was proportional to the load delivered. Due to the decreased absolute sodium reabsorption in the preceding segments, sodium delivery to the beginning of the distal tubule was comparable in the two groups of animals. Along the distal tubule sodium reabsorption was comparable to control animals. Therefore, the avid urinary sodium retention seen during micropuncture was due to increased sodium reabsorption by segments past the superficial proximal tubule and/or by deep nephrons.

Micropuncture localization of the natriuretic effect of interleukin 1.

Interleukin 1 (IL-1) has been demonstrated to elicit an increase in renal sodium excretion. This effect occurs in the absence of any increase in the filtered load of sodium, raising the possibility of an IL-1-mediated decrease in tubule sodium reabsorption. To localize the nephron segment(s) responsible for the natriuretic effect of IL-1, we performed micropuncture experiments on rats. Intravenous IL-1 administration caused a marked increase in sodium excretion that was not accompanied by changes in glomerular filtration rate or systemic blood pressure. Single-nephron glomerular filtration rate and fractional and absolute delivery of sodium to the late proximal and mid-distal tubule were not affected by IL-1. Fractional delivery of sodium to the early and late papillary collecting duct, however, was significantly enhanced by IL-1 administration. Sodium reabsorption was inhibited along the papillary collecting duct. These findings demonstrate that the natriuretic effect of IL-1 is due, at least in part, to inhibition of collecting duct sodium reabsorption.

Ammonia loss from rat proximal tubule in vivo: effects of luminal pH and flow rate.

The roles of luminal pH and flow rate in determining ammonia loss from proximal tubules perfused with solutions containing 10 mM NH4Cl were examined using in vivo microperfusion. Perfusate bicarbonate concentration was varied between 5, 25, and 40 mM in tubules perfused at 50 nl/min. As expected, ammonia loss from the 25 or 40 mM bicarbonate-containing perfusates was greater than from that containing 5 mM bicarbonate. Furthermore, there was a correlation between ammonia loss and the log mean luminal bicarbonate concentration (r = 0.39, P less than 0.01). From the collected fluid ammonia and bicarbonate concentrations, the transtubular gradients for NH+4 and NH3 were estimated, allowing a calculation of the apparent permeability coefficients for NH3 (PNH3) and NH+4 (PNH+4). The calculated PNH3 of 2.2 +/- 0.5 X 10(-2) cm/s was similar to previous estimates in the rabbit; the calculated PNH+4 of 5.5 +/- 0.8 X 10(-4) cm/s was approximately 10 times that previously found in the rabbit proximal straight tubule in vitro. Next, flow rate was varied between 25 and 50 nl/min using the 5 mM bicarbonate perfusate. Ammonia loss was significantly higher from the latter. Thus these studies suggest that NH+4 loss from the proximal tubule may be an important determinant of ammonia movement along this segment. Ammonia loss is flow-rate dependent, similar to ammonia entry in previous studies.

Contribution of luminal ammoniagenesis to proximal tubule ammonia appearance in the rat.

The contribution of luminal ammoniagenesis in the late proximal convolute tubule (PCT) via phosphate-independent glutaminase [gamma-glutamyltransferase (gamma-GT)] remains controversial. If this pathway is important, it must rely on glutamine secretion, because filtered glutamine is reabsorbed in the early PCT. The contribution of gamma-GT to luminal ammoniagenesis was tested by use of in vivo microperfusion in conjunction with a new microfluorometric assay for glutamate. We first confirmed that aspartate completely blocked glutamate uptake in the PCT. Furthermore, the gamma-GT inhibitor acivicin completely eliminated glutamate entry, showing that passive glutamate entry was negligible. Thus the accumulation of glutamate can be used as an estimate of luminal glutamine deamidation. L-Phenylalanine was used to inhibit glutamine loss, and hippurate was used to stimulate gamma-GT activity; therefore luminal glutamine conversion to glutamate was promoted. Perfusing the tubule at 30 nl/min with a solution containing 10 mM each of hippurate, phenylalanine, and aspartate resulted in a glutamate delivery of 1.08 +/- 0.12 pmol.min-1.mm-1. Ammonia appearance was 10-fold higher, averaging 11.5 +/- 1.3 pmol.min-1.mm-1 under these same conditions. Thus the luminal conversion of glutamine to glutamate via gamma-GT is a small component of total ammoniagenesis in this segment.

Effects of barium and 5-(N-ethyl-N-isopropyl)-amiloride on proximal tubule ammonia transport.

Ionic NH+4 transport is an important mode of ammonia transport in the proximal convoluted tubule (PCT). NH+4 transport via the Na-H exchanger has been previously demonstrated. Potassium channels are present in the proximal tubule, but their role in ammonia transport has not been evaluated. We studied rat PCTs perfused in situ at 30 nl/min with solutions containing 5 mM HCO3; ammonia was measured with a fluorometric method. When perfused with the potent amiloride analogue 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 500 microM) or with BaCl2 (10 mM), ammonia entry (14.3 +/- 1.7 and 11.8 +/- 1.5 pmol.min-1.mm-1, respectively) was unaffected compared with control (12.8 +/- 1.0 pmol.min-1.mm-1). However, the combination of EIPA and barium inhibited entry (7.4 +/- 1.0 pmol.min-1.mm-1, P less than 0.02 vs. other groups). Also, when perfused with 10 mM ammonia, neither EIPA nor BaCl alone blocked ammonia loss (70.5 +/- 9.1 and 60.5 +/- 5.9 pmol.min-1.mm-1, respectively) compared with control (53.4 +/- 6.0 pmol.min-1.mm-1). However, the combination inhibited ammonia loss (29.2 +/- 6.3 pmol.min-1.mm-1, P less than 0.025 vs. other groups). Thus blocking both the Na-H exchanger and potassium channels decreases PCT ammonia transport. As the combination was required, this implies that multiple pathways exist for NH+4 transport in the PCT. This is the first demonstration that a mode of NH+4 transport other than via the Na-H exchanger is important in this segment, and the data are most consistent with transport of ammonia via potassium channels.

Sodium handling by deep nephrons and the terminal collecting duct in glomerulonephritis.

The present study was designed to characterize the effects of anti-glomerular basement membrane (anti-GBM) glomerulonephritis (GN) on sodium handling by surface nephrons, deep nephrons and the terminal collecting duct segment. Studies were performed in rats during hydropenia and volume expansion. In hydropenia, the glomerular filtration rate (GFR) and sodium excretion tended to be lower in rats with GN than in controls. However, the major differences between the control and GN animals were seen in volume expansion. In the volume expanded groups fractional excretion of sodium was greater in controls (3.20 +/- 0.51%) than in GN (1.20 +/- 0.36%, P less than 0.01). Despite this, delivery to end proximal sites was similar in the two groups in absolute terms and higher in the expanded GN group compared to the expanded controls. Absolute sodium delivery to the bend of the loop of Henle in the expanded GN rats was decreased in absolute terms but increased in fractional terms compared to expanded controls. However, fractional delivery of sodium to the base of the terminal collecting duct was less in GN (3.71 +/- 1.39%) than in controls (7.19 +/- 0.96%, P less than 0.002). In both groups, fractional delivery between tip of the collecting duct fell compared to base (P less than 0.05) but delivery to the tip was again greater in controls (5.49 +/- 1.08%) than in GN (1.51 +/- 0.47%). In GN 62.6 +/- 5.0% of delivered sodium was reabsorbed between collecting duct sites, nearly twofold that of controls (28.8 +/- 9.4%, P less than 0.01). Thus, fractional sodium reabsorption in the collecting duct was enhanced by GN.

Determinants of ammonia entry along the rat proximal tubule during chronic metabolic acidosis.

The technique of in vivo microperfusion was used to examine the determinants of ammonia entry along the rat proximal tubule under conditions of chronic metabolic acidosis (CMA). When perfused with a 5 mM bicarbonate-containing perfusate, collected fluid ammonia concentrations remained constant with increasing flow rate and thus ammonia entry was highly flow-rate dependent. Ammonia entry was also flow-rate dependent using a 25 mM bicarbonate perfusate but entry reached a plateau as perfusion rate increased. Also, ammonia entry tended to be lower at all perfusion rates with the 25 mM perfusate compared with the 5 mM bicarbonate perfusate, but this was most evident at the highest perfusion rate (45 nl/min). The decline in ammonia entry was associated with increasing collected fluid bicarbonate concentrations, suggesting that there was inhibition of diffusion trapping of ammonia. The effects of Na+-H+ exchange inhibition on ammonia entry were examined using the amiloride analogue, 5-(N-ethyl-N-isopropyl)amiloride. With a 25 mM bicarbonate-containing perfusate, the amiloride analogue caused a significant decrease in bicarbonate reabsorption but a nonsignificant decrease in ammonia entry associated with a significant rise in collected fluid bicarbonate concentration. When the potential effects of decreased diffusion trapping of ammonia were eliminated with 12 and 5 mM bicarbonate-containing perfusates, the analogue had no effect on ammonia entry despite significant inhibition of bicarbonate reabsorption. Thus ammonia entry in CMA is moderately affected by tubule fluid pH but is highly flow-rate dependent. There were no effects of inhibition of Na+-H+ exchange above those expected from inhibition of diffusion trapping of ammonia.