The effects of varying acidity on Helicobacter pylori growth and the bactericidal efficacy of ampicillin.

BACKGROUND: Penicillins inhibit cell wall synthesis; therefore, Helicobacter pylori must be dividing for this class of antibiotics to be effective in eradication therapy. Identifying growth responses to varying medium pH may allow design of more effective treatment regimens. AIM: To determine the effects of acidity on bacterial growth and the bactericidal efficacy of ampicillin. METHODS: H. pylori were incubated in dialysis chambers suspended in 1.5-L of media at various pHs with 5 mM urea, with or without ampicillin, for 4, 8 or 16 h, thus mimicking unbuffered gastric juice. Changes in gene expression, viability and survival were determined. RESULTS: At pH 3.0, but not at pH 4.5 or 7.4, there was decreased expression of ~400 genes, including many cell envelope biosynthesis, cell division and penicillin-binding protein genes. Ampicillin was bactericidal at pH 4.5 and 7.4, but not at pH 3.0. CONCLUSIONS: Ampicillin is bactericidal at pH 4.5 and 7.4, but not at pH 3.0, due to decreased expression of cell envelope and division genes with loss of cell division at pH 3.0. Therefore, at pH 3.0, the likely pH at the gastric surface, the bacteria are nondividing and persist with ampicillin treatment. A more effective inhibitor of acid secretion that maintains gastric pH near neutrality for 24 h/day should enhance the efficacy of amoxicillin, improving triple therapy and likely even allowing dual amoxicillin-based therapy for H. pylori eradication.

Sulfur-containing amino acids decrease cisplatin cytotoxicity and uptake in renal tubule epithelial cell lines.

PURPOSE: Nephrotoxicity is one of the major dose-limiting side-effects of cisplatin (DDP). The disproportionate accumulation of cisplatin in kidney tissue may play an important role, however, therapeutic measures to prevent this prime cause of nephrotoxicity are not available. Because certain amino acids (AAs) have been reported to modulate DDP nephrotoxicity in vivo, we explored the potential of all 20 protein AAs, N-acetylcysteine and DL-homocysteine to reduce DDP cytotoxicity and uptake in S1, S3 (proximal tubule), and DCT (distal convoluted tubule) cell lines. METHODS: Immortalized but non-transformed renal tubule epithelial cell lines, derived from specific portions of the nephron of an SV40 transgenic mouse. were grown to confluency and exposed to various concentrations of DDP for 1 h with or without concurrent exposure to AAs in an otherwise AA-free Krebs-Ringer buffer (KRB). After 1 h, cell layers were washed and replenished with medium for cytotoxicity assays, or processed immediately for the determination of DDP accumulation. Cytotoxicity was assessed 48 h later by an MTT assay, and DDP uptake after 1 h was determined by atomic absorption spectroscopy. RESULTS: In an initial screening where the cells were concurrently incubated with 0.25 mM DDP and 1 mM AA for 1 h in KRB, only cysteine (Cys), methionine (Met), N-acetylcysteine and DL-homocysteine reduced DDP toxicity. This effect was enhanced at 5 mM AA and most potent for Cys, which reduced DDP cytotoxicity by 79 +/- 3% in S3 cells, by 78 +/- 12.2% in DCT cells, and by 19 +/- 3.6% in S1 cells (P < 0.05). Reduction of cytotoxicity was less for Met, DL-homocysteine, and N-acetylcysteine, in decreasing order. All four AAs also inhibited DDP uptake in renal cells, with Cys as the strongest inhibitor. Inhibition of DDP accumulation by 1 mM Cys after 1 h was 39% in S3 cells, 38% in DCT cells, and 28% in S1 cells. Again, reduction of uptake was less for the three other AAs. Pre-complexing of DDP with Cys for 16 h increased its uptake by 8- to 30-fold compared with native DDP, but markedly inhibited its toxicity. Thus, pre-complexing of DDP with Cys could not explain the reduced uptake of DDP, but could partly account for the reduction in cytotoxicity. Double-reciprocal Lineweaver-Burk plots of DDP concentration-versus-uptake rates at a constant concentration of Cys suggested that Cys competitively inhibited DDP uptake in S1 and DCT cells, and in a more complex fashion in S3 cells. CONCLUSIONS: We conclude that Cys, Met, N-acetylcysteine, and DL-homocysteine differentially inhibit DDP toxicity and uptake in cultured S1, S3, and DCT cells, and that the inhibition of uptake, as well as the complexation of DDP with Cys within the cell, may prevent toxicity. The structural element R-CH(NH2)-[CH2]1 2-S-R, which is common to all four molecules, may play a crucial role in blocking the transport of DDP, and could have future clinical applications.

Differential effects of cisplatin in proximal and distal renal tubule epithelial cell lines.

Pathological studies suggest that cisplatin injures different portions of the nephron to different extents. To investigate this issue further, we examined the cytotoxicity and uptake of cisplatin in cell lines derived from S1 and S3 proximal tubule and distal convoluted tubule segments isolated from a mouse carrying the SV40 large T-antigen transgene. S1 cells displayed the highest sensitivity to cisplatin cytotoxicity, followed by S3 and distal convoluted tubule (DCT) cells. These differences in cytotoxicity did not correlate with differences in cisplatin uptake. Cytotoxic concentrations of cisplatin triggered apoptosis in all three cell lines. Although BAX and BCL-2 expression was similar among the three cell lines, the expression of the anti-apoptotic protein, BCL-X(L), was significantly lower in S1 cells than in S3 and DCT cells, and this may have contributed to the heightened sensitivity of S1 cells. Cisplatin transport characteristics demonstrated a saturable component of cisplatin uptake and differences in apparent K(M) and Vmax values among the three cell lines. The three cell lines were 43- to 176-fold more sensitive to cisplatin than to carboplatin. This distinction between the two drugs could not be fully explained by differences in the uptake rates of carboplatin and cisplatin. We conclude that cells from different portions of the nephron display different sensitivities to cisplatin, different transport characteristics for cisplatin and different levels of expression of BCL-X(L). In addition, the relative resistance of renal cells to carboplatin vs cisplatin is mostly due to the differential effects that follow internalization.

Molecular cloning of the chicken oviduct ecto-ATP-diphosphohydrolase.

The chicken oviduct ecto-ATP diphosphohydrolase (ATPDase), a member of the ecto-ATPase family, was purified to homogeneity previously (Strobel, R. S., Nagy, A. K., Knowles, A. F., Buegel, J., and Rosenberg, M. O. (1996) J. Biol. Chem. 271, 16323-16331). It is an 80-kDa glycoprotein with high specific activity (approximately 1,000 micromol/min/mg with MgATP as the substrate) and hydrolyzes both nucleoside triphosphates and diphosphates. Using amino acid sequence information obtained from the purified enzyme, two partial cDNA clones were obtained using reverse transcriptase-polymerase chain reaction and library screening. This is the second ecto-ATPase family member and the first ecto-ATPDase to be cloned from information derived from purified proteins. The deduced primary sequence of the chicken oviduct ecto-ATPDase indicates a protein of 493 amino acid residues with a molecular mass of 54 kDa. The predicted orientation shows it to be anchored to the membrane by two transmembranous segments near the NH2 and COOH termini with very short intracytoplasmic peptides at either end. The bulk of the protein is extracellular and contains 12 potential N-glycosylation sites, several potential phosphorylation sites, and five sequences that are conserved in seven other related membrane proteins. Four of the conserved sequences, designated as apyrase conserved regions, are present in both ecto-ATPases and soluble E-type ATPases. The fifth conserved region, which occurs near the COOH terminus of the eight proteins, is observed only in the membrane-bound ecto-ATPases. Unexpectedly, sequence comparison revealed that the chicken oviduct ecto-ATPDase is equally distant from the two ecto-ATPases, which exhibit low activity toward ADP, and the four putative ecto-ATPDases, which are closely related to CD39.

Expression of a cut-related homeobox gene in developing and polycystic mouse kidney.

Cut is a diverged homeobox gene that is essential for normal development of the Malpighian tubules in Drosophila melanogaster. Homologues of Drosophila cut that encode transcriptional repressors have been identified in several mammalian species and cell lineages. We examined the expression of a murine cut homologue (named Cux-1) in the developing mouse using Northern blot analysis and in situ hybridization. At 12.5 d.p.c. and 13.5 d.p.c., Cux-1 was highly expressed in a subset of embryonic tissues, including the developing metanephros. Within the metanephros, Cux-1 was expressed in the nephrogenic zone including both mesenchymal cells (uninduced and condensed mesenchyme) and epithelial cells (ureteric buds, renal vesicles, S-shaped bodies). During later stages of nephrogenesis, Cux-1 was down-regulated such that there was minimal expression in mature glomeruli and tubules. In addition, Cux-1 was detected in the mesonephros, mesonephric duct, and bladder. Expression of Cux-1 was also examined in polycystic kidneys from C57BL/6J-cpk/ cpk mice. At 21 days of age, Cux-1 was highly expressed in cyst epithelium of polycystic kidneys but was minimally expressed in kidneys from phenotypically normal littermates. These results demonstrate that a cut-related homeobox gene is expressed in the developing kidney and urinary tract of the mouse. Expression of Cux-1 in the kidney is inversely related to degree of cellular differentiation. Cux-1 may encode a transcriptional repressor that inhibits terminally differentiated gene expression during early stages of nephrogenesis.

Cloning and kidney cell-specific activity of the promoter of the murine renal Na-K-C1 cotransporter gene.

The murine Nkcc2/Slcl2a1 gene encodes a bumetanide-sensitive Na-K-Cl cotransporter that is expressed exclusively in the kidney in the thick ascending limb of the loop of Henle. Nuclear run-off assays demonstrated that kidney-specific expression of Nkcc2 was due, at least in part, to kidney-specific gene transcription. To begin study of the gene promoter, a genomic clone that contained 13.5 kilobases of the 5'-flanking region of Nkcc2 was isolated. A single transcription initiation site was located 1330 base pairs (bp) upstream of the start codon. The sequence of the proximal 5'-flanking region contained typical eukaryotic promoter elements including a TATA box, two CCAAT boxes, and an initiator. A (G-A)28.(C-T)28 microsatellite and consensus binding sites for hepatocyte nuclear factor 1, cAMP-response element binding protein, CCAAT/enhancer-binding proteins, and basic helix-loop-helix proteins, were also identified. To functionally express the promoter, 2255 bp of the proximal 5'-flanking region was ligated to a luciferase reporter gene and transfected into thick ascending limb (TAL) cells, a stable cell line derived from microdissected loops of Henle of the Tg(SV40E)Bri7 mouse. TAL cells exhibited furosemide-sensitive Na-K((NH4)+)-Cl cotransport activity and endogenously expressed the 5.0-kilobase Nkcc2 transcript. Luciferase activity was 130-fold greater following transfection into TAL cells compared with transfection into cells that did not express Nkcc2 (NIH 3T3 fibroblasts). Deletion analysis revealed that promoter activity in TAL cells was similar in constructs extending from the transcription initiation site to -1529 to -469, whereas further deletion to -190 resulted in a 76% decrease in activity. We conclude that the Nkcc2 promoter exhibits kidney cell-specific activity. Regulatory elements required for maximal promoter activity are located in a 280-bp DNA segment that contains consensus binding sites for several transcription factors expressed in the kidney.

Effect of luminal angiotensin II on ammonia production and secretion by mouse proximal tubules.

Angiotensin II is an important regulator of acid-base and ammonia metabolism in the proximal tubule. Because angiotensin II receptors exist on the apical membrane and because luminal fluid angiotensin II concentrations may be substantial, the effects of luminal angiotensin II on ammonia production rates and net luminal total ammonia (tNH3) secretion rates were examined in dissected mouse S2 proximal tubule segments. Ammonia production rates reflected the total release of ammonia via the basolateral and luminal aspects of the tubule, whereas net luminal secretion rates reflected the rates at which ammonia left the tubule via the luminal fluid leaving the distal end of the perfused segment. The results demonstrated that 1) luminal angiotensin II affected tNH3 production in a concentration-dependent fashion, 2) luminal angiotensin II at concentrations that stimulated tNH3 production could counteract the effect of inhibitory basolateral concentrations of angiotensin II, 3) the stimulation of tNH3 production and the rise in intracellular calcium concentration induced by 10(-10) M luminal angiotensin II were blocked by the addition of an angiotensin II receptor inhibitor, saralasin, or the calcium channel blocker nifedipine to the luminal perfusion solution, and 4) in contrast to basolateral angiotensin II, which inhibited net luminal tNH3 secretion, luminal angiotensin II stimulated amiloride-sensitive net luminal tNH3 secretion in parallel with stimulation of luminal fluid acidification. Thus luminal angiotensin II at physiological and superphysiological concentrations has important effects on ammonia production and transport in the proximal tubule that in some ways differ from the effects of basolateral angiotensin II.

Inhibition of gentamicin uptake into cultured mouse proximal tubule epithelial cells by L-lysine.

Gentamicin uptake and toxicity was studied in a nontransformed cell line obtained from the S1 segment of the proximal tubule epithelium of a transgenic mouse. Cytotoxicity was assayed using the dye 3-(4,-5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Gentamicin uptake was assayed by a fluorescence polarization assay. No differences in toxicity were found among cells incubated for 4 hours in complete culture medium, enriched Kreb's buffer alone, or enriched Krebs' buffer with added 300 micrograms/mL gentamicin, 0.5 mmol/L L-lysine, or gentamicin plus L-lysine. Uptake of 300 micrograms/mL gentamicin was minimal at zero time and increased as a function of time. Uptake of gentamicin at 4 hours was positively correlated with medium gentamicin concentration. Addition of 0.5 mmol/L L-lysine inhibited uptake of 300 micrograms/mL gentamicin 38.9 +/- 10.2%. No other amino acid, including D-lysine or arginine, significantly changed gentamicin uptake. The authors conclude that gentamicin and L-lysine share a specific uptake mechanism located in the apical membrane of renal proximal tubule cells.

Effect of angiotensin II on ammonia production and secretion by mouse proximal tubules perfused in vitro.

The effects of angiotensin II on total ammonia (tNH3) production and net secretion were investigated using in vitro microperfused mouse S2 proximal tubule segments incubated in Krebs-Ringer bicarbonate buffer containing 0.5 mM L-glutamine. Basolateral exposure of mouse S2 segments to 10(-11), 10(-10), and 10(-9) M angiotensin II stimulated tNH3 production rates by 23, 52, and 49%, respectively. Addition of 10(-6) M angiotensin II inhibited the tNH3 production rate by 34%. 10(-10) M angiotensin II inhibited net luminal secretion of tNH3 in the presence of enhanced luminal acidification and in the absence of altered luminal tNH3 efflux rates. Measurements of intracellular pH (pHi) and intracellular calcium concentration [( Ca2+]i) suggested that the effects of angiotensin II on tNH3 production were not mediated by changes in pHi but by the stimulatory effect of angiotensin II correlated with increased [Ca2+]i. Inhibition of the calcium-calmodulin-dependent pathway with W-7 blocked the stimulatory effect of 10(-10) M angiotensin II on tNH3 production and luminal acidification. These results indicate that angiotensin II has concentration-dependent effects on tNH3 production; that its action to stimulate tNH3 production may be mediated by rises in [Ca2+]i and the calcium-calmodulin pathway; and that angiotensin II, at concentrations that stimulate tNH3 production, inhibits net luminal ammonia secretion by a mechanism that is not mediated by diminished luminal acidification or by changes in luminal ammonia efflux rates.

Platelet-activating factor attenuates the arginine vasopressin-induced fall of transepithelial resistance across inner medullary collecting duct monolayers.

Platelet-activating factor (PAF) is a vasoactive substance produced in the medulla which may alter Na excretion by the kidney. To examine a possible site and mechanism of action of PAF on the kidney, we evaluated the effects of PAF on transepithelial resistance and intracellular calcium concentration ([Ca2+]i) in cultured rat inner medullary collecting duct cells. Exposure of inner medullary collecting duct (IMCD) cell monolayers to PAF had no significant effect on basal transepithelial resistance. By contrast, incubation of IMCD cells with PAF reversibly blocked the fall in transepithelial resistance induced by arginine vasopressin (AVP): -11.1 +/- 1.4 omega.cm2 with AVP versus -0.02 +/- 1.6 omega.cm2 with PAF and AVP. Exposure of IMCD cells to PAF in Ca-replete medium caused a rise in intracellular calcium from 155 +/- 25 to 491 +/- 68 nM. By contrast, exposure of IMCD cells to PAF in Ca-free medium produced no change in [Ca2+]i. Because the rise in [Ca2+]i induced by PAF was absent in Ca-free medium, transepithelial resistance across IMCD monolayers was examined in calcium-free medium. The effect of PAF to block the fall in transepithelial resistance induced by AVP was maintained in Ca-free medium. These data suggest that PAF modulates the effect of AVP on conductive channels by a mechanism distinct from changes in intracellular calcium.

Low-density lipoprotein uptake and cholesterol accumulation by cultured renal cells.

The uptake of low-density lipoprotein (LDL) and the accumulation of cholesterol were assessed in opossum kidney (OK) and Madin-Darby canine kidney (MDCK) cells. OK and MDCK cells were grown to confluency on Millicell well inserts. The uptake of human LDL across the apical and basolateral surfaces of OK and MDCK cells was assessed by the degradation of internalized (125I)LDL to trichloroacetic acid-soluble products. LDL uptake via the apical surface of OK cells increased linearly with LDL concentration, indicating nonreceptor-mediated uptake. In contrast, LDL uptake via the basolateral surface of OK cells and both apical and basolateral surfaces of MDCK cells followed a saturable pattern. In addition, (125I)LDL bound to the apical membrane of MDCK cells, but not to the apical membrane of OK cells, was displaced by heparin and by excess of unlabeled LDL. Exposure to LDL (100 mg/mL) resulted in an increase in total cholesterol content of OK and MDCK cells (23 and 18%, respectively). Most of the increase in total cholesterol content with LDL exposure resulted from increased free cholesterol content in MDCK cells and esterified cholesterol in OK cells. The differences in cholesteryl ester formation were consistent with the slower rates of (14C) oleate incorporation into cholesteryl ester and lower cholesterol esterifying activity observed in MDCK cells compared with that in OK cells. These results demonstrate that LDL uptake can be receptor or nonreceptor mediated, depending upon the renal cell type and the surface exposed to LDL, and that LDL exposure leads to increased cholesterol content in OK and MDCK cells.

Effect of bath and luminal potassium concentration on ammonia production and secretion by mouse proximal tubules perfused in vitro.

To determine the effects of acute changes in K+ concentration in vitro on ammonia production and secretion by the proximal tubule, we studied mouse S2 segments perfused with and bathed in Krebs-Ringer bicarbonate buffers containing various K+ concentrations. All bath solutions contained L-glutamine as the ammoniagenic substrate. High bath and luminal K+ concentrations (8 mM), but not high luminal K+ concentration alone, inhibited total ammonia production rates by 26%, while low bath and luminal K+ concentrations (2 mM), but not low luminal K+ concentration alone, stimulated total ammonia production rates by 33%. The stimulation of ammonia production by low bath K+ concentration was not observed when L-glutamine was added to the luminal perfusion solution. On the other hand, high luminal K+ concentration stimulated, while low luminal K+ concentration inhibited, net luminal secretion of total ammonia in a way that was: (a) independent of total ammonia production rates, (b) independent of Na(+)-H+ exchange activity, and (c) not due to changes in transepithelial fluxes of total ammonia. These results suggest that luminal potassium concentration has a direct effect on cell-to-lumen transport of ammonia.

AVP reduces transepithelial resistance across IMCD cell monolayers.

The inner medullary collecting duct (IMCD) is an important site of action for arginine vasopressin (AVP). To examine the mode of action of AVP in this segment, we measured the change in transepithelial resistance of cultured rat IMCD cells grown to confluence on collagen-coated Millicell culture plate inserts in response to AVP. Resistance was measured by use of an EVOM voltage-ohm meter. AVP at 10(-11)-10(-8) M caused a fall in resistance of 6.9 +/- 1.3 to 14.0 +/- 1.4 omega.cm2 (P less than 0.05 to less than 0.01 vs. no AVP), which was reversed by removal of AVP or addition of 10(-6) M amiloride. Pretreating the apical surface of IMCD cells with trypsin had no effect on resistance but totally prevented the antidiuretic hormone-induced fall in resistance. Pretreating the apical surface with trypsin and amiloride did not prevent the fall in resistance to AVP. Addition of 10(-9) M AVP or 10(-6) M forskolin increased 2-min adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by 55 or 96%, respectively. Stimulation of endogenous cAMP accumulation by forskolin or the addition of exogenous 8-bromo-cAMP caused no change in resistance. To examine the relationship between intracellular calcium [( Ca2+]i) and AVP action, the response of [Ca2+]i to AVP was measured by use of fura-2. AVP induced no change in [Ca2+]i in IMCD cells in suspension, on glass cover slips, or on permeable supports. Ionomycin (25 nM) increased [Ca2+]i in IMCD cells and lowered resistance across monolayers, but the fall in resistance was not blocked by amiloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia production and secretion by isolated perfused proximal tubule segments.

The heterogeneous nature of renal ammonia production and transport necessitated the adaptation of the in vitro microperfusion technique to study ammonia production and transport by specific segments of the proximal tubule. Specific proximal tubule segments were dissected from mouse kidneys and microperfused in vitro with physiologic buffer solutions. This approach has demonstrated the important effects of luminal perfusion and luminal flow rate, in vitro and in vivo metabolic acidosis and in vitro potassium concentration on total ammonia production rates in proximal tubule segments. Studies examining net luminal ammonia secretion have suggested an important role of the Na(+)-H+ exchanger in the mechanism of ammonia secretion by the proximal tubule.

Ammonia production and secretion by the proximal tubule.

Ammonia production and secretion by the proximal tubule accounts for most of the ammonia that appears in the urine. Rates of ammonia production and net luminal ammonia secretion were measured in isolated perfused mouse proximal tubule segments. This approach combines the in vitro microperfusion technique with a sensitive bioluminescence assay for total ammonia and permits the determination of ammonia production and secretion rates in specific proximal tubule segments bathed and perfused with defined solutions. Luminal perfusion stimulates ammonia production by proximal tubule segments in a flow-related manner. The effect of perfusion is not dependent on intact Na+-H+ exchange. In contrast, the rate of net luminal secretion of ammonia is largely dependent on Na+-H+ exchange but not markedly dependent on an acid luminal fluid pH. These results suggest an important role of Na+-NH4+ exchange in the mechanism by which the Na+-H+ exchanger facilitates net ammonia secretion.

Effect of luminal perfusion on glucose production by isolated proximal tubules.

The effects of luminal perfusion on glucose production by the proximal tubule were examined by use of the technique of in vitro microperfusion with an ultramicroassay for glucose to measure the net glucose production rates in isolated mouse midproximal tubule segments. Tubules bathed in Krebs-Ringer bicarbonate (KRB) buffer containing L-glutamine and acetate and perfused with KRB buffer at a high flow rate produced glucose at a lower rate (0.12 +/- 0.02 pmol.min-1.mm-1) than unperfused segments (0.40 +/- 0.03) or segments perfused at a lower flow rate (0.24 +/- 0.03). In contrast, the estimated rates of glucose utilization were not affected by luminal perfusion. The inhibition of net fluid reabsorption by perfusion with a modified KRB buffer containing amiloride or by addition of ouabain to the bath medium raised glucose production rates to levels equaling or exceeding those observed in unperfused tubules. The inhibition of glucose production by luminal perfusion occurred in the presence of multiple substrates (i.e., glutamine, acetate, lactate, pyruvate, alanine, and valerate) or nonammoniagenic substrates (i.e., lactate and pyruvate) in the bath medium. Thus net glucose production is inhibited by luminal perfusion and the inhibitory effect is dependent on intact fluid reabsorption. The reduction in net glucose production observed with perfusion does not result from increased glucose utilization and is not dependent on the presence of specific substrates.

Luminal secretion of ammonia in the mouse proximal tubule perfused in vitro.

A major portion of the total ammonia (tNH3 = NH3 + NH+4) produced by the isolated perfused mouse proximal tubule is secreted into the luminal fluid. To assess the role of Na+-H+ exchange in net tNH3 secretion, rates of net tNH3 secretion and tNH3 production were measured in proximal tubule segments perfused with control pH 7.4 Krebs-Ringer bicarbonate (KRB) buffer or with modified KRB buffers containing 10 mM sodium and 0.1 mM amiloride. Net tNH3 secretion was inhibited by 90% in proximal tubule segments perfused with the pH 7.4 modified KRB buffer while tNH3 production remained unaffected. The inhibition of net tNH3 secretion by perfusion with the modified KRB buffer was only partially reversed by acidifying the modified KRB luminal perfusate from 7.4 to as low as 6.2. These data indicate that the Na+-H+ exchanger facilitates a major portion of net tNH3 secretion by the proximal tubule and that luminal acidification may play only a partial role in the mechanism by which the Na+-H+ exchanger mediates net tNH3 secretion.

Ammonia production by isolated mouse proximal tubules perfused in vitro. Effect of metabolic acidosis.

We examined the effects of metabolic acidosis in vivo and reduced bath and luminal pH in vitro on total NH3 (NH3 + NH+4) production rates by isolated mouse proximal tubule segments. Midproximal tubule segments were obtained from mice with NH4Cl-induced metabolic acidosis and from nonacidotic controls. The segments were perfused with modified Krebs-Ringer bicarbonate (KRB) buffer, incubated in KRB buffer containing 0.5 mM L-glutamine and 1.0 mM sodium acetate, and gassed with 95% O2 and 5% CO2. Isolated unperfused and perfused proximal tubules from acidotic mice produced total NH3 at higher rates than corresponding tubules from nonacidotic mice. Perfusion of the tubular lumen stimulated total NH3 production by tubules from both acidotic and nonacidotic mice. In contrast, lowering the bath pH to 7.0 by lowering the HCO3- concentration increased total NH3 production rates by tubules from nonacidotic mice but not by tubules from acidotic mice. Reducing the HCO3- concentration of the bath buffer to 10 mM while maintaining a pH of 7.4 had no significant effect on total NH3 production by tubules from nonacidotic mice. Lowering the luminal fluid pH by reducing the perfusate HCO-3 from 25 mM to 10, 5, or 1.2 mM while maintaining a bath pH of 7.4 lowered collected luminal fluid pH but had no effect on total NH3 production by proximal tubules from nonacidotic mice. These observations demonstrated that metabolic acidosis in vivo stimulated total NH3 production in isolated mouse proximal tubule segments and that low peritubular pH and HCO-3 stimulated total NH3 production by proximal tubule segments from nonacidotic mice in vitro.