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.

Effect of oxidant stress on growth factor stimulation of proliferation in cultured human proximal tubule cells.

Restoring kidney function after injury involves cell migration and proliferation, processes that are yet to be precisely defined. Because epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) promote recovery from acute renal failure, we used SV-40 immortalized human proximal tubule cells to examine the effects of these growth factors on cell proliferation after peroxide injury (1.5 mM for 1 hour). ATP levels decreased to approximately 15% of control values immediately after injury but returned to nearly normal levels after 4 hours of recovery. Under control conditions, both EGF and IGF-1 stimulated proliferation and their effects were additive. However, 20-24 hours after injury, while IGF-1 stimulated proliferation, EGF was no longer effective nor was the combination of EGF and IGF-1. Although the EGF receptor was decreased 20 hours after injury, the lack of IGF-1 effect could not be explained by loss of the IGF-1 receptor, which remained unchanged after peroxide injury. Thus, the mechanism responsible for the blunting of the IGF-1 effect on proliferation following injury remains speculative. However, we conclude that the effects of growth factors under control conditions may not predict their effects after injury.

The actin cytoskeleton and integrin expression in the recovery of cell adhesion after oxidant stress to a proximal tubule cell line (JTC-12).

This study examines the role of the actin cytoskeleton and integrin expression in the recovery of cell adhesion in the proximal tubule cell line JTC-12 after peroxide injury. The cells were exposed to 10, 20, or 50 mM hydrogen peroxide for 10 min and then allowed to recover. Viability measurements by trypan blue exclusion confirmed that the injury was largely nonlethal with 85% viability at 1 h even at 50 mM peroxide. ATP levels fell immediately after the peroxide incubation in all groups to approximately 10% of normal, but already showed some recovery by 1 h and full recovery in the 10 and 20 mM groups by 24 h. Cell adhesion to extracellular matrix immediately after injury was depressed at 20 and 50 mM peroxide, but by 12 h was abnormal only at 50 mM peroxide and at 24 h was essentially normal at all peroxide concentrations. Immediately after exposure to 10 mM peroxide, there were subtle abnormalities in the actin cytoskeleton (thickening of fibrils) as assessed by phalloidin staining, with more pronounced effects at 20 and 50 mM. At 1 h, many cells showed collapse of the actin cytoskeleton to the periphery. There was some recovery at 4 h; by 12 h, the actin cytoskeleton showed further recovery, although was still abnormal (coarsened microfilaments), especially at 20 and 50 mM peroxide. By 24 h, the actin cytoskeleton showed only subtle coarsening. Integrin surface expression was assessed by flow cytometry. The alpha6 subunit on cells exposed to 20 mM peroxide was unchanged at 1 h and 4 h, but by 12 h had increased to 118.5+/-4.5% and by 24 h to 146+/-13.4% of control levels. The expression of the beta1 and alphaVbeta3 integrins remained unchanged. Thus, despite coarsening of the actin cytoskeleton and depressed ATP levels, cell adhesion recovered from oxidant stress. Abnormal cell adhesion after injury was not a consequence of a decrease in integrin expression, and recovery of cell adhesion was not a consequence of the modest and selective increase in integrin expression.

Evidence for acute renal cortical vasoconstriction after uninephrectomy.

The rate of progression of chronic renal failure (CRF) is similar for many diseases, suggesting a common, perhaps intrinsic, renal signal for its progression. The remnant nephron hypothesis of Bricker suggests that CRF may be the result of persistent compensatory renal growth (CRG). Normally, CRG after unilateral nephrectomy (uniNx) ceases within 1 week. Knowledge of the signals that initiate CRG may therefore shed light on the signals responsible for ongoing CRF. The signals responsible for the initiation of compensatory renal growth after uniNx are unknown. Hemodynamic changes in the remaining renal artery have been observed, but there are as yet no data for the main renal compartment which undergoes hypertrophy, the superficial renal cortex. The noninvasive technique of laser-Doppler flowmetry allows the continuous and independent monitoring of blood velocity and blood volume. The product of the two signals is proportional to tissue blood flow per unit volume of the tissue observed. Under controlled conditions in adult male Sprague-Dawley rats, renal cortical blood velocity increased by 22% within 5 min after uniNx and remained elevated at this level for 60 min. Renal cortical blood volume decreased throughout the experiment. Their product, renal cortical blood flow, increased briefly by 14% 5 min after uniNx but decreased over the time of observation in parallel with renal cortical blood volume. The simultaneous increase in blood velocity and decrease in blood volume in the superficial renal cortex acutely after uniNx suggest that vasoconstriction is an early event in compensatory renal growth.

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.

Characterization of integrins in cultured human renal cortical tubule epithelial cells.

We have characterized the integrins present on cultured tubule epithelial cells from human renal cortexes, enriched for proximal cells, using fluorescence microscopy, immunoprecipitation, and cell adhesion assays. By immunofluorescence, the alpha 3-integrin subunit stained most intensely and was present on all cells predominantly at cell-cell contacts. The alpha 6-subunit was present on all cells in a pattern consistent with extracellular matrix contacts. The alpha 5-subunit was present on most cells in a cell-matrix contact pattern; alpha V-subunit was weakly positive and occasionally seen in cell-matrix contacts. The alpha 2-subunit was present on clusters of distal tubule cells, predominantly at cell-cell contacts. Immunoprecipitation revealed the predominant integrin to be alpha 3 beta 1 with some alpha 2 beta 1, presumably contributed by distal cells. The alpha 5 beta 1-, alpha 6 beta 1-, alpha 6 beta 4-, and alpha V beta 3-integrins, as well as trace amounts of alpha 1 beta 1-integrins, were also present. The alpha 4 beta 1-integrin was not detected. Initial attachment to fibronectin was mediated by alpha V beta 3- and alpha 5 beta 1-integrins; initial attachment to laminin was mediated by the alpha 6 beta 1- and alpha 3 beta 1- integrins and, in some preparations, by an unidentified integrin; and initial attachment to collagen type IV was mediated by alpha V beta 3-integrin and an unidentified beta 1-integrin. After extensively immunodepleting membrane extracts with anti-alpha 1, -alpha 2, -alpha 3, -alpha 4, -alpha 5, -alpha 6, and -alpha V antibodies, an anti-beta 1 antibody still precipitated an integrin. Its electrophoretic mobility differs from the laminin-binding alpha 7 beta 1-integrin. Thus we have identified many of the integrins on cortical tubule cells and their role in mediating initial attachment to extracellular matrix. However, the cell adhesion assays and immunoprecipitations suggest the presence of an unidentified beta 1-integrin that may mediate renal tubule cell attachment to laminin and collagen.

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.

Integrin receptors in renal tubular epithelium: new insights into pathophysiology of acute renal failure.

This review summarizes the existing evidence implicating disordered adhesion of renal tubular epithelial cells to the basement membrane in the pathophysiology of acute renal failure. The following three major lines of investigation are discussed: 1) exfoliation of renal tubular epithelial cells as a potential mechanism of tubular obstruction, 2) normal distribution of integrin receptors along the tubular apparatus, and 3) redistribution of integrin receptors and remodeling of the cytoskeleton following acute injury to renal tubular epithelium. We advance the hypothesis that the loss of the basolateral expression of integrin receptors is responsible for the exfoliation of viable proximal epithelial cells and that the redistribution of integrin receptors from the basolateral to the apical surface of epithelial cells facilitates adhesion of detached cells to the in situ cells. These two processes culminate in tubular obstruction.

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.

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.

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.

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.

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.

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.

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.