New insights into the natural history of bronchopulmonary dysplasia from proteomics and multiplexed immunohistochemistry.

Bronchopulmonary dysplasia (BPD) is a disease of prematurity related to the arrest of normal lung development. The objective of this study was to better understand how proteome modulation and cell-type shifts are noted in BPD pathology. Pediatric human donors aged 1-3 yr were classified based on history of prematurity and histopathology consistent with "healed" BPD (hBPD, n = 3) and "established" BPD (eBPD, n = 3) compared with respective full-term born (n = 6) age-matched term controls. Proteins were quantified by tandem mass spectroscopy with selected Western blot validations. Multiplexed immunofluorescence (MxIF) microscopy was performed on lung sections to enumerate cell types. Protein abundances and MxIF cell frequencies were compared among groups using ANOVA. Cell type and ontology enrichment were performed using an in-house tool and/or EnrichR. Proteomics detected 5,746 unique proteins, 186 upregulated and 534 downregulated, in eBPD versus control with fewer proteins differentially abundant in hBPD as compared with age-matched term controls. Cell-type enrichment suggested a loss of alveolar type I, alveolar type II, endothelial/capillary, and lymphatics, and an increase in smooth muscle and fibroblasts consistent with MxIF. Histochemistry and Western analysis also supported predictions of upregulated ferroptosis in eBPD versus control. Finally, several extracellular matrix components mapping to angiogenesis signaling pathways were altered in eBPD. Despite clear parsing by protein abundance, comparative MxIF analysis confirms phenotypic variability in BPD. This work provides the first demonstration of tandem mass spectrometry and multiplexed molecular analysis of human lung tissue for critical elucidation of BPD trajectory-defining factors into early childhood.NEW & NOTEWORTHY We provide new insights into the natural history of bronchopulmonary dysplasia in donor human lungs after the neonatal intensive care unit hospitalization. This study provides new insights into how the proteome and histopathology of BPD changes in early childhood, uncovering novel pathways for future study.

Ammonium chloride-induced acidosis exacerbates cystitis and pyelonephritis caused by uropathogenic E. coli.

Acute pyelonephritis caused by uropathogenic E. coli (UPEC) can cause renal scarring and lead to development of chronic kidney disease. Prevention of kidney injury requires an understanding of host factors and/or UPEC adaptive responses that are permissive for UPEC colonization of the urinary tract. Although some studies have suggested urine acidification limits UPEC growth in culture, other studies have described acid-resistance mechanisms (AR) in E. coli such as the CadC/CadBA module that promotes adaptation to acid and nitrosative stress. Herein we confirm and extend our previous study by demonstrating that despite urine acidification, metabolic acidosis induced by dietary ammonium chloride (NH4 Cl-A) exacerbates cystitis and pyelonephritis in innate immune competent (C3H-HeN) mice characterized by: (1) markedly elevated UPEC burden and increased chemokine/cytokine and NOS2 mRNA expression, (2) accumulation of intravesicular debris noninvasively detected by Power Doppler Ultrasound (PDUS), and (3) collecting duct (CD) dysfunction that manifests as a urine concentration defect. Bladder debris and CD dysfunction were due to the inflammatory response, as neither was observed in Tlr4-deficient (C3H-HeJ) mice. The effect of NH4 Cl-A was unrelated to acidosis as dietary administration of hydrochloric acid (HCl-A) yielded a comparable acid-base status yet did not increase UPEC burden. NH4 Cl-A increased polyamines and decreased nitric oxide (NO) metabolites in urine indicating that excess dietary ammonium shifts arginine metabolism toward polyamines at the expense of NO synthesis. Furthermore, despite increased expression of NOS2, NO production post UPEC infection was attenuated in NH4 Cl-A mice compared to controls. Thus, in addition to induction of metabolic acidosis and urine acidification, excess dietary ammonium alters the polyamine:NO balance and thereby compromises NOS2-mediated innate immune defense.

Lipopolysaccharide directly inhibits bicarbonate absorption by the renal outer medullary collecting duct.

Acidosis is associated with E. coli induced pyelonephritis but whether bacterial cell wall constituents inhibit HCO3 transport in the outer medullary collecting duct from the inner stripe (OMCDi) is not known. We examined the effect of lipopolysaccharide (LPS), on HCO3 absorption in isolated perfused rabbit OMCDi. LPS caused a ~ 40% decrease in HCO3 absorption, providing a mechanism for E. coli pyelonephritis-induced acidosis. Monophosphoryl lipid A (MPLA), a detoxified TLR4 agonist, and Wortmannin, a phosphoinositide 3-kinase inhibitor, prevented the LPS-mediated decrease, demonstrating the role of TLR4-PI3-kinase signaling and providing proof-of-concept for therapeutic interventions aimed at ameliorating OMCDi dysfunction and pyelonephritis-induced acidosis.

Metabolic acidosis exacerbates pyelonephritis in mice prone to vesicoureteral reflux.

Acute pyelonephritis is a common, serious bacterial infection in children. The prevalence of acute pyelonephritis is due at least in part to vesicoureteral reflux (VUR). Although an association between abnormalities in electrolyte and acid-base balance and pyelonephritis is common in young children, the impact of metabolic acidosis (MA) on progression of acute pyelonephritis is not fully understood. In this study, the effect of MA on pyelonephritis was studied in C3H mouse strains prone to VUR. MA induced by ammonium chloride supplementation in food specifically impaired clearance of urinary tract infection with uropathogenic Escherichia. coli (UPEC-UTI) in innate immune competent C3H strains (HeOuJ, HeN), whereas kidney UPEC burden in Tlr-4-deficient HeJ mice was unaffected. Antibody-mediated depletion of myeloid cells (monocytes, neutrophil) markedly increased UPEC burden in the bladder and kidney confirming the pivotal role of neutrophils and tissue-resident macrophages in clearance of UPEC-UTI. MA concurrent with UPEC-UTI markedly increased expression of cytokine (TNFα, IL-1ß, IL-6) and chemokine (CXCL 1, 2, and 5) mRNA in isolated kidney CD cells and kidney neutrophil infiltrates were increased four- to fivefold compared to normal, UPEC-infected mice. Thus, MA intensified pyelonephritis and increased the risk of kidney injury by impairing clearance of UPEC-UTI and potentiating renal inflammation characterized by an elevated kidney neutrophil infiltrate.

Acidosis induces antimicrobial peptide expression and resistance to uropathogenic E. coli infection in kidney collecting duct cells via HIF-1α.

Acute pyelonephritis is frequently associated with metabolic acidosis. We previously reported that metabolic acidosis stimulates expression of hypoxia-inducible factor (HIF)-1α-induced target genes such as stromal derived factor-1 and cathelicidin, an antimicrobial peptide. Since the collecting duct (CD) plays a pivotal role in regulating acid-base homeostasis and is the first nephron segment encountered by an ascending microbial infection, we examined the contribution of HIF-1α to innate immune responses elicited by acid loading of an M-1 immortalized mouse CD cell line. Acid loading of confluent M-1 cells was achieved by culture in pH 6.8 medium supplemented with 5-(N-ethyl-N-isopropyl)-amiloride to block Na+/H+ exchange activity for 24 h. Acid loading induced antimicrobial peptide [cathelicidin and ß-defensin (Defb2 and Defb26)] mRNA expression and M-1 cell resistance to uropathogenic Escherichia coli infection to an extent similar to that obtained by inhibition of HIF prolyl hydroxylases, which promote HIF-1α protein degradation. The effect of acid loading on M-1 cell resistance to uropathogenic E. coli infection was reduced by inhibition of HIF-1α (PX-478), and, in combination with prolyl hydroxylase inhibitors, acidosis did not confer additional resistance. Thus, metabolic stress of acidosis triggers HIF-1α-dependent innate immune responses in CD (M-1) cells. Whether pharmacological stabilization of HIF prevents or ameliorates pyelonephritis in vivo warrants further investigation.

Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct.

The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanism by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells that consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H+-ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cre+tdT+ mice to fluorescently label ICs and AQP2-cre+tdT+ mice to fluorescently label PCs, and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure antimicrobial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC) challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge, this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.

Metabolic acidosis stimulates the production of the antimicrobial peptide cathelicidin in rabbit urine.

Intercalated cells of the collecting duct (CD) are critical for acid-base homeostasis and innate immune defense of the kidney. Little is known about the impact of acidosis on innate immune defense in the distal nephron. Urinary tract infections are mainly due to Escherichia coli and are an important risk factor for development of chronic kidney disease. While the effect of urinary pH on growth of E. coli is well established, in this study, we demonstrate that acidosis increases urine antimicrobial activity due, at least in part, to induction of cathelicidin expression within the CD. Acidosis was induced in rabbits by adding NH4Cl to the drinking water and reducing food intake over 3 days or by casein supplementation. Microdissected CDs were examined for cathelicidin mRNA expression and antimicrobial activity, and cathelicidin protein levels in rabbit urine were measured. Cathelicidin expression in CD cells was detected in kidney sections. CDs from acidotic rabbits expressed three times more cathelicidin mRNA than those isolated from normal rabbits. Urine from acidotic rabbits had significantly more antimicrobial activity (vs. E. coli) than normal urine, and most of this increased activity was blocked by cathelicidin antibody. The antibody had little effect on antimicrobial activity of normal urine. Urine from acidotic rabbits had at least twice the amount of cathelicidin protein as did normal urine. We conclude that metabolic acidosis not only stimulates CD acid secretion but also induces expression of cathelicidin and, thereby, enhances innate immune defense against urinary tract infections via induction of antimicrobial peptide expression.

SDF1 induction by acidosis from principal cells regulates intercalated cell subtype distribution.

The nephron cortical collecting duct (CCD) is composed of principal cells, which mediate Na, K, and water transport, and intercalated cells (ICs), which are specialized for acid-base transport. There are two canonical IC forms: acid-secreting α-ICs and HCO3-secreting ß-ICs. Chronic acidosis increases α-ICs at the expense of ß-ICs, thereby increasing net acid secretion by the CCD. We found by growth factor quantitative PCR array that acidosis increases expression of mRNA encoding SDF1 (or CXCL12) in kidney cortex and isolated CCDs from mouse and rabbit kidney cortex. Exogenous SDF1 or pH 6.8 media increased H+ secretion and decreased HCO3 secretion in isolated perfused rabbit CCDs. Acid-dependent changes in H+ and HCO3 secretion were largely blunted by AMD3100, which selectively blocks the SDF1 receptor CXCR4. In mice, diet-induced chronic acidosis increased α-ICs and decreased ß-ICs. Additionally, IC-specific Cxcr4 deletion prevented IC subtype alterations and magnified metabolic acidosis. SDF1 was transcriptionally regulated and a target of the hypoxia-sensing transcription factor HIF1α. IC-specific deletion of Hif1a produced no effect on mice fed an acid diet, as α-ICs increased and ß-ICs decreased similarly to that observed in WT littermates. However, Hif1a deletion in all CCD cells prevented acidosis-induced IC subtype distribution, resulting in more severe acidosis. Cultured principal cells exhibited an HIF1α-dependent increase of Sdf1 transcription in response to media acidification. Thus, our results indicate that principal cells respond to acid by producing SDF1, which then acts on adjacent ICs.

Distinct α-intercalated cell morphology and its modification by acidosis define regions of the collecting duct.

During metabolic acidosis, the cortical collecting duct (CCD) of the rabbit reverses the polarity of bicarbonate flux from net secretion to net absorption, and this is accomplished by increasing the proton secretory rate by α-intercalated cells (ICs) and decreasing bicarbonate secretion by ß-ICs. To better characterize dynamic changes in H(+)-secreting α-ICs, we examined their morphology in collecting ducts microdissected from kidneys of normal, acidotic, and recovering rabbits. α-ICs in defined axial regions varied in number and basolateral anion exchanger (AE)1 morphology, which likely reflects their relative activity and function along the collecting duct. Upon transition from CCD to outer medullary collecting duct from the outer stripe to the inner stripe, the number of α-ICs increases from 11.0 ± 1.2 to 15.4 ± 1.11 and to 32.0 ± 1.3 cells/200 µm, respectively. In the CCD, the basolateral structure defined by AE1 typically exhibited a pyramidal or conical shape, whereas in the medulla the morphology was elongated and shallow, resulting in a more rectangular shape. Furthermore, acidosis reversibly induced α-ICs in the CCD to acquire a more rectangular morphology concomitant with a transition from diffusely cytoplasmic to increased basolateral surface distribution of AE1 and apical polarization of B1-V-ATPase. The latter results are consistent with the supposition that morphological adaptation from the pyramidal to rectangular shape reflects a transition toward a more "active" configuration. In addition, α-ICs in the outer medullary collecting duct from the outer stripe exhibited cellular morphology strikingly similar to dendritic cells that may reflect a newly defined ancillary function in immune defense of the kidney.

Insights into acidosis-induced regulation of SLC26A4 (pendrin) and SLC4A9 (AE4) transporters using three-dimensional morphometric analysis of ß-intercalated cells.

The purpose of this study was to examine the three-dimensional (3-D) expression and distribution of anion transporters pendrin (SLC26A4) and anion exchanger (AE)4 (SLC4A9) in ß-intercalated cells (ß-ICs) of the rabbit cortical collecting duct (CCD) to better characterize the adaptation to acid-base disturbances. Confocal analysis and 3-D reconstruction of ß-ICs, using identifiers of the nucleus and zona occludens, permitted the specific orientation of cells from normal, acidotic, and recovering rabbits, so that adaptive changes could be quantified and compared. The pendrin cap likely mediates apical Cl(-)/HCO3 (-) exchange, but it was also found beneath the zona occludens and in early endosomes, some of which may recycle back to the apical membrane via Rab11a(+) vesicles. Acidosis reduced the size of the pendrin cap, observed as a large decrease in cap volume above and below the zona occludens, and the volume of the Rab11a(+) apical recycling compartment. Correction of the acidosis over 12-18 h reversed these changes. Consistent with its proposed function in the basolateral exit of Na(+) via Na(+)-HCO3 (-) cotransport, AE4 was expressed as a barrel-like structure in the lateral membrane of ß-ICs. Acidosis reduced AE4 expression in ß-ICs, but this was rapidly reversed during the recovery from acidosis. The coordinate regulation of pendrin and AE4 during acidosis and recovery is likely to affect the magnitude of acid-base and possibly Na(+) transport across the CCD. In conclusion, acidosis induces a downregulation of AE expression in ß-ICs and a diminished presence of pendrin in apical recycling endosomes.

Renal intercalated cells are rather energized by a proton than a sodium pump.

The Na(+) concentration of the intracellular milieu is very low compared with the extracellular medium. Transport of Na(+) along this gradient is used to fuel secondary transport of many solutes, and thus plays a major role for most cell functions including the control of cell volume and resting membrane potential. Because of a continuous leak, Na(+) has to be permanently removed from the intracellular milieu, a process that is thought to be exclusively mediated by the Na(+)/K(+)-ATPase in animal cells. Here, we show that intercalated cells of the mouse kidney are an exception to this general rule. By an approach combining two-photon imaging of isolated renal tubules, physiological studies, and genetically engineered animals, we demonstrate that inhibition of the H(+) vacuolar-type ATPase (V-ATPase) caused drastic cell swelling and depolarization, and also inhibited the NaCl absorption pathway that we recently discovered in intercalated cells. In contrast, pharmacological blockade of the Na(+)/K(+)-ATPase had no effects. Basolateral NaCl exit from ß-intercalated cells was independent of the Na(+)/K(+)-ATPase but critically relied on the presence of the basolateral ion transporter anion exchanger 4. We conclude that not all animal cells critically rely on the sodium pump as the unique bioenergizer, but can be replaced by the H(+) V-ATPase in renal intercalated cells. This concept is likely to apply to other animal cell types characterized by plasma membrane expression of the H(+) V-ATPase.

Adaptation to metabolic acidosis and its recovery are associated with changes in anion exchanger distribution and expression in the cortical collecting duct.

It is well known that acid/base disturbances modulate proton/bicarbonate transport in the cortical collecting duct. To study the adaptation further we measured the effect of three days of acidosis followed by the rapid recovery from this acidosis on the number and type of intercalated cells in the rabbit cortical collecting duct. Immunofluorescence was used to determine the expression of apical pendrin in ß-intercalated cells and the basolateral anion exchanger (AE1) in α-intercalated cells. Acidosis resulted in decreased bicarbonate and increased proton secretion, which correlated with reduced pendrin expression and the number of pendrin-positive cells, as well as decreased pendrin mRNA and protein abundance in this nephron segment. There was a concomitant increase of basolateral AE1 and α-cell number. Intercalated cell proliferation did not seem to play a role in the adaptation to acidosis. Alkali loading for 6-20 h after acidosis doubled the bicarbonate secretory flux and reduced proton secretion. Pendrin and AE1 expression patterns returned to control levels, demonstrating that adaptive changes by intercalated cells are rapidly reversible. Thus, regulation of intercalated cell anion exchanger expression and distribution plays a key role in adaptation of the cortical collecting duct to perturbations of acid/base.

Secreted cyclophilin A, a peptidylprolyl cis-trans isomerase, mediates matrix assembly of hensin, a protein implicated in epithelial differentiation.

Hensin is a rabbit ortholog of DMBT1, a multifunctional, multidomain protein implicated in the regulation of epithelial differentiation, innate immunity, and tumorigenesis. Hensin in the extracellular matrix (ECM) induced morphological changes characteristic of terminal differentiation in a clonal cell line (clone C) of rabbit kidney intercalated cells. Although hensin is secreted in monomeric and various oligomeric forms, only the polymerized ECM form is able to induce these phenotypic changes. Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase (PPIase) inhibitors cyclosporin A (CsA) and a derivative of cyclosporin A with modifications in the d-Ser side chain (Cs9) but not by the calcineurin pathway inhibitor FK506. PPIase inhibition led to failure of hensin polymerization in the medium and ECM, plus the loss of apical cytoskeleton, apical microvilli, and the columnar epithelial shape of clone C cells. Cyclophilin A was produced and secreted into the media to a much greater extent than cyclophilins B and C. Our results also identified the direct CsA-sensitive interaction of cyclophilin A with hensin, suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin. These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation.

Endothelin and nitric oxide mediate adaptation of the cortical collecting duct to metabolic acidosis.

Endothelin (ET) and nitric oxide (NO) modulate ion transport in the kidney. In this study, we defined the function of ET receptor subtypes and the NO guanylate cyclase signaling pathway in mediating the adaptation of the rabbit cortical collecting duct (CCD) to metabolic acidosis. CCDs were perfused in vitro and incubated for 3 h at pH 6.8, and bicarbonate transport or cell pH was measured before and after acid incubation. Luminal chloride was reversibly removed to isolate H(+) and HCO(3)(-) secretory fluxes and to raise the pH of beta-intercalated cells. Acid incubation caused reversal of polarity of net HCO(3)(-) transport from secretion to absorption, comprised of a 40% increase in H(+) secretion and a 75% decrease in HCO(3)(-) secretion. The ET(B) receptor antagonist BQ-788, as well as the NO synthase inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME), attenuated the adaptive decrease in HCO(3)(-) secretion by 40%, but only BQ-788 inhibited the adaptive increase in H(+) secretion. There was no effect of inactive d-NAME or the ET(A) receptor antagonist BQ-123. Both BQ-788 and l-NAME inhibited the acid-induced inactivation (endocytosis) of the apical Cl(-)/HCO(3)(-) exchanger. The guanylate cyclase inhibitor LY-83583 and cGMP-dependent protein kinase inhibitor KT-5823 affected HCO(3)(-) transport similarly to l-NAME. These data indicate that signaling via the ET(B) receptor regulates the adaptation of the CCD to metabolic acidosis and that the NO guanylate cyclase component of ET(B) receptor signaling mediates downregulation of Cl(-)/HCO(3)(-) exchange and HCO(3)(-) secretion.

The TRAF6, but not the TRAF2/3, binding domain of CD40 is required for cytokine production in human lung fibroblasts.

Fibroblasts are key effector cells in inciting inflammation, wound healing, and scarring. CD40, a member of the TNF receptor superfamily, mediates intercellular communication between fibroblasts and cells that express CD154 (CD40L), including T lymphocytes and platelets. To better understand the mechanisms by which CD40 regulates fibroblast function in inflammation and scarring, we examined the ability of CD40 cytoplasmic tail regions (CD40ct) containing the TRAF6 or the TRAF2/3 binding domains to regulate cytokine and chemokine expression by primary human lung fibroblasts. The full-length human CD40ct, the first 35 amino acids of the CD40ct encompassing the TRAF6 binding site (1-35), and amino acids 35-53 containing the TRAF2/TRAF3 binding domain were expressed in human lung fibroblasts as fusion proteins with the extracellular domain of human CD8alpha by retroviral transduction. The TRAF6, but not the TRAF2/3, binding domain was found to regulate IL-8 and IL-6 production, and induce activation of NF-kappaB and Jun kinase in lung fibroblasts, demonstrating for the first time that CD40ct domains can function independently to regulate pro-inflammatory responses of primary human fibroblasts. Thus, targeting TRAF6 function through pharmacological intervention may represent a viable strategy for modulating localized inflammation.

Expression of membrane-associated carbonic anhydrase isoforms IV, IX, XII, and XIV in the rabbit: induction of CA IV and IX during maturation.

Several carbonic anhydrase (CA) isoforms are associated with plasma membranes. It is probable that these enzymes interact with anion transporters to facilitate the movement of HCO3- into or out of the cell. A better knowledge of CA isoform expression in a given tissue would facilitate a systematic examination of any associations with such transporters. We examined the expression of CAs IV, IX, XII, and XIV mRNAs in rabbit tissues, including kidney, heart, lung, skeletal muscle, liver, pancreas, gall bladder, stomach, small intestine, colon, and spleen, using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). CA IV mRNA was mainly in kidney, heart, lung, colon, and gall bladder. CA IX mRNA was restricted to stomach, gall bladder, duodenum, and early jejunum. CA XII mRNA was found in kidney and colon. CA XIV mRNA was localized to heart, lung, skeletal muscle, and liver. The data indicate that there are different patterns of CA expression in various tissues: CA IX was expressed in the proximal gastrointestinal tract, whereas CA XII and CA IV were more distal. CA IV and CA XII are important kidney isoforms. CA XIV was abundant in metabolically active tissues such as liver, heart, lung, and skeletal muscle. Some significant species differences were noted in the expression of some of these isoforms; for example, CA XIV is not expressed in rabbit kidney, despite being abundant in mouse kidney. Maturational studies showed that the expression of CA IX mRNA and protein increased markedly with weaning ( approximately 3-4 postnatal wk) and was well correlated with the maturational expression of the alpha-subunit of the gastric H+,K+-ATPase, suggesting that function of CA IX and the gastric H+ pump might be linked in the digestion of adult foodstuffs. The unique pattern of membrane-bound CA isoforms suggests different functional associations with transporters, depending on the physiological demands on the tissue.

Nitric oxide production modulates cyclosporin A-induced distal renal tubular acidosis in the rat.

Cyclosporine A (CsA) causes distal renal tubular acidosis (dRTA) in humans and rodents. Because mice deficient in nitric-oxide (NO) synthase develop acidosis, we examined how NO production modulated H+ excretion during acid loading and CsA treatment in a rat model. Rats received CsA, L-arginine (L-Arg), or N omega-nitro-L-arginine methyl ester (L-NAME), or combinations of CsA and L-NAME or L-Arg, followed by NH4Cl (acute acid load). In vehicle-treated rats, NH4Cl loading reduced serum and urine (HCO3-) and urine pH, which was associated with increases in serum [K+] and [Cl-] and urine NH3 excretion. Similar to CsA (7.5 mg/kg), L-NAME impaired H+ excretion of NH4Cl-loaded animals. The combination CsA and L-NAME reduced H+ excretion to a larger extent than either drug alone. In contrast, administration of L-Arg ameliorated the effect of CsA on H+ excretion. Urine pH after NH4Cl was 5.80 +/- 0.09, 6.11 +/- 0.13*, 6.37 +/- 0.16*, and 5.77 +/- 0.09 in the vehicle, CsA, CsA + L-NAME and CsA + L-Arg groups, respectively (*P < 0.05). The effect of CsA and alteration of NO synthesis were mediated at least in part by changes in bicarbonate absorption in perfused cortical collecting ducts. CsA or L-NAME reduced net HCO3- absorption, and, when combined, completely inhibited it. CsA + L-Arg restored HCO3- absorption to near control levels. Administration of CsA along with L-NAME reduced NO production to below levels observed with either drug alone. These results suggest that CsA causes dRTA by inhibiting H+ pumps in the distal nephron. Inhibition of NO synthesis may be one of the mechanisms underlying the CsA effect.