An Acidic Environment Induces APOL1-Associated Mitochondrial Fragmentation.

BACKGROUND: Apolipoprotein L1 gene (APOL1) G1 and G2 kidney-risk variants (KRVs) cause CKD in African Americans, inducing mitochondrial dysfunction. Modifying factors are required, because a minority of individuals with APOL1 high-risk genotypes develop nephropathy. Given that APOL1 function is pH-sensitive and the pH of the kidney interstitium is <7, we hypothesized the acidic kidney interstitium may facilitate APOL1 KRV-induced mitochondrial dysfunction. METHODS: Human embryonic kidney (HEK293) cells conditionally expressing empty vector (EV), APOL1-reference G0, and G1 or G2 KRVs were incubated in media pH 6.8 or 7.4 for 4, 6, or 8 h. Genotype-specific pH effects on mitochondrial length (µm) were assessed using confocal microscopy in live cells and Fiji derivative of ImageJ software with MiNA plug-in. Lower mitochondrial length indicated fragmentation and early dysfunction. RESULTS: After 6 h doxycycline (Dox) induction in pH 6.8 media, G2-expressing cells had shorter mitochondria (6.54 ± 0.40) than cells expressing EV (7.65 ± 0.72, p = 0.02) or G0 (7.46 ± 0.31, p = 0.003). After 8 h Dox induction in pH 6.8 media, both G1- (6.21 ± 0.26) and G2-expressing cells had shorter mitochondria (6.46 ± 0.34) than cells expressing EV (7.13 ± 0.32, p = 0.002 and p = 0.008, respectively) or G0 (7.22 ± 0.45, p = 0.003 and p = 0.01, respectively). Mitochondrial length in cells incubated in pH 7.4 media were comparable after 8 h Dox induction regardless of genotype. APOL1 mRNA expression and cell viability were comparable regardless of pH or genotype after 8 h Dox induction. CONCLUSION: Acidic pH facilitates early mitochondrial dysfunction induced by APOL1 G1 and G2 KRVs in HEK293 cells. We propose that the acidic kidney interstitium may play a role in APOL1-mediated mitochondrial pathophysiology and nephropathy.

APOL1 Kidney-Risk Variants Induce Mitochondrial Fission.

INTRODUCTION: APOL1 G1 and G2 nephropathy-risk variants cause mitochondrial dysfunction and contribute to kidney disease. Analyses were performed to determine the genetic regulation of APOL1 and elucidate potential mechanisms in APOL1-nephropathy. METHODS: A global gene expression analysis was performed in human primary renal tubule cell lines derived from 50 African American individuals. Follow-up gene knock out, cell-based rescue, and microscopy experiments were performed. RESULTS: APOL1 genotypes did not alter APOL1 expression levels in the global gene expression analysis. Expression quantitative trait locus (eQTL) analysis in polyinosinic-polycytidylic acid (poly IC)-stimulated renal tubule cells revealed that single nucleotide polymorphism (SNP) rs513349 adjacent to BAK1 was a trans eQTL for APOL1 and a cis eQTL for BAK1; APOL1 and BAK1 were co-expressed in cells. BAK1 knockout in a human podocyte cell line resulted in diminished APOL1 protein, supporting a pivotal effect for BAK1 on APOL1 expression. Because BAK1 is involved in mitochondrial dynamics, mitochondrial morphology was examined in primary renal tubule cells and HEK293 Tet-on cells of various APOL1 genotypes. Mitochondria in APOL1 wild-type (G0G0) tubule cells maintained elongated morphology when stimulated by low-dose poly IC, whereas those with G1G1, G2G2, and G1G2 genotypes appeared to fragment. HEK293 Tet-on cells overexpressing APOL1 G0, G1, and G2 were created; G0 cells appeared to promote mitochondrial fusion, whereas G1 and G2 induced mitochondrial fission. The mitochondrial dynamic regulator Mdivi-1 significantly preserved cell viability and mitochondrial cristae structure and reversed mitochondrial fission induced by overexpression of G1 and G2. CONCLUSION: Results suggest the mitochondrial fusion/fission pathway may be a therapeutic target in APOL1-nephropathy.

Association of serum albumin levels with kidney function decline and incident chronic kidney disease in elders.

Background: Previous studies in HIV-infected individuals have demonstrated serum albumin to be strongly associated with kidney function decline, independent of urine albumin and inflammatory markers. Lower serum albumin concentrations may be an under-appreciated risk factor for kidney function decline in elders. Methods: We performed a cohort analysis in the Health Aging and Body Composition Study, a cohort of well-functioning, bi-racial, community-dwelling elders between the age of 70 and 79 years. We examined the associations of serum albumin concentration with longitudinal kidney function decline by estimated glomerular filtration rate (eGFR). Outcomes included linear eGFR decline, rapid kidney function decline defined as >30% decrease in eGFR, defined as a final eGFR <60 mL/min/1.73 m2 in those with an eGFR >60 mL/min/1.73 m2 at baseline. Cystatin C-based eGFR was calculated at baseline, Year 3 and Year 10. Results: Mean age was 74 years, and mean eGFR was 73 mL/min/1.73 m2 at baseline. The mean rate of eGFR change was 1.81 mL/min/1.73 m2 per year. After multivariate adjustment, lower serum albumin concentrations were strongly and independently associated with kidney function decline (-0.11 mL/min/1.73 m2 per year for each standard deviation decrease serum albumin; -0.01 to - 0.20) with no attenuation after adjustment for urine albumin and inflammatory markers (-0.12, -0.03 to - 0.22). When divided into quartiles, serum albumin levels ≤3.80 g/dL were associated with increased odds of rapid kidney function decline (odds ratio 1.59; 1.12-2.26) and increased risk of incident chronic kidney disease (incident rate ratio 1.29; 1.03-1.62) relative to levels >4.21g/dL. Urine albumin to creatinine ratio (ACR) was also significantly and independently associated with kidney function decline (-0.08 mL/min/1.73 m2 per year for urine ACR >30 mg/g; -0.82 to - 0.13). Conclusions: Lower serum albumin levels are strongly and independently associated with kidney function decline in elders, independent of clinical risk factors, urine albumin and measured inflammatory markers.

APOL1 Renal-Risk Variants Induce Mitochondrial Dysfunction.

APOL1 G1 and G2 variants facilitate kidney disease in blacks. To elucidate the pathways whereby these variants contribute to disease pathogenesis, we established HEK293 cell lines stably expressing doxycycline-inducible (Tet-on) reference APOL1 G0 or the G1 and G2 renal-risk variants, and used Illumina human HT-12 v4 arrays and Affymetrix HTA 2.0 arrays to generate global gene expression data with doxycycline induction. Significantly altered pathways identified through bioinformatics analyses involved mitochondrial function; results from immunoblotting, immunofluorescence, and functional assays validated these findings. Overexpression of APOL1 by doxycycline induction in HEK293 Tet-on G1 and G2 cells led to impaired mitochondrial function, with markedly reduced maximum respiration rate, reserve respiration capacity, and mitochondrial membrane potential. Impaired mitochondrial function occurred before intracellular potassium depletion or reduced cell viability occurred. Analysis of global gene expression profiles in nondiseased primary proximal tubule cells from black patients revealed that the nicotinate phosphoribosyltransferase gene, responsible for NAD biosynthesis, was among the top downregulated transcripts in cells with two APOL1 renal-risk variants compared with those without renal-risk variants; nicotinate phosphoribosyltransferase also displayed gene expression patterns linked to mitochondrial dysfunction in HEK293 Tet-on APOL1 cell pathway analyses. These results suggest a pivotal role for mitochondrial dysfunction in APOL1-associated kidney disease.

Deletion of proton-sensing receptor GPR4 associates with lower blood pressure and lower binding of angiotensin II receptor in SFO.

Diets rich in grains and meat and low in fruits and vegetables (acid-producing diets) associate with incident hypertension, whereas vegetarian diets associate with lower blood pressure (BP). However, the pathways that sense and mediate the effects of acid-producing diets on BP are unknown. Here, we examined the impact of the deletion of an acid sensor GPR4 on BP. GPR4 is a proton-sensing G protein-coupled receptor and an acid sensor in brain, kidney, and blood vessels. We found that GPR4 mRNA was higher in subfornical organ (SFO) than other brain regions. GPR4 protein was abundant in SFO and present in capillaries throughout the brain. Since SFO partakes in BP regulation through the renin-angiotensin system (RAS), we measured BP in GPR4-/- and GPR4+/+ mice and found that GPR4 deletion associated with lower systolic BP: 87 ± 1 mmHg in GPR4-/- (n = 35) vs. 99 ± 2 mmHg (n = 29) in GPR4+/+; P < 0.0001, irrespective of age and sex. Angiotensin II receptors detected by 125I-Sarthran binding were lower in GPR4-/- than GPR4+/+ mice in SFO and in paraventricular nucleus of hypothalamus. Circulating angiotensin peptides were comparable in GPR4-/- and GPR4+/+ mice, as were water intake and excretion, serum and urine osmolality, and fractional excretion of sodium, potassium, or chloride. A mild metabolic acidosis present in GPR4-/- mice did not associate with elevated BP, implying that deficiency of GPR4 may preclude the effect of chronic acidosis on BP. Collectively, these results posit the acid sensor GPR4 as a novel component of central BP control through interactions with the RAS.

Adaptation by the collecting duct to an exogenous acid load is blunted by deletion of the proton-sensing receptor GPR4.

We previously reported that the deletion of the pH sensor GPR4 causes a non-gap metabolic acidosis and defective net acid excretion (NAE) in the GPR4 knockout mouse (GPR4-/-) (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, and Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). Since the major regulatory site of NAE in the kidney is the collecting duct (CD), we examined acid-base transport proteins in intercalated cells (ICs) of the CD and found comparable mRNA expression of kidney anion exchanger 1 (kAE1), pendrin, and the a4 subunit of H(+)-ATPase in GPR4-/- vs. +/+. However, NH4Cl loading elicited adaptive doubling of AE1 mRNA in GPR4+/+, but a 50% less pronounced response in GPR4-/-. In GPR4+/+, NH4Cl loading evoked a cellular response characterized by an increase in AE1-labeled and a decrease in pendrin-labeled ICs similar to what was reported in rabbits and rats. This response did not occur in GPR4-/-. Microperfusion experiments demonstrated that the activity of the basolateral Cl(-)/HCO3(-) exchanger, kAE1, in CDs isolated from GPR4-/- failed to increase with NH4Cl loading, in contrast to the increase observed in GPR4+/+. Therefore, the deficiency of GPR4 blunted, but did not eliminate the adaptive response to an acid load, suggesting a compensatory response from other pH/CO2/bicarbonate sensors. Indeed, the expression of the calcium-sensing receptor (CaSR) was nearly doubled in GPR4-/- kidneys, in the absence of apparent disturbances of Ca(2+) homeostasis. In summary, the expression and activity of the key transport proteins in GPR4-/- mice are consistent with spontaneous metabolic acidosis, but the adaptive response to a superimposed exogenous acid load is blunted and might be partially compensated for by CaSR.

Localization of APOL1 protein and mRNA in the human kidney: nondiseased tissue, primary cells, and immortalized cell lines.

Although APOL1 gene variants are associated with nephropathy in African Americans, little is known about APOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examined APOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immunofluorescence microscopy performed on nondiseased nephrectomy cryosections from persons with normal kidney function revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin and Wilms' tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridization detected APOL1 mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenous synthesis in these cell types. When these analyses were extended to renal-derived cell lines, quantitative RT-PCR did not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were observed in proximal tubule cells and glomerular endothelial cells, with lower expression in podocytes. Western blot analysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancy between the marked abundance of APOL1 protein in kidney podocytes observed in cryosections versus the lesser abundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily by human podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, or proximal tubule cells. We hypothesize that the higher levels of APOL1 protein in human cryosectioned podocytes may reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate.

Activation of epidermal growth factor receptor is required for Chlamydia trachomatis development.

BACKGROUND: Chlamydia trachomatis (C. trachomatis) is a clinically significant human pathogen and one of the leading causative agents of sexually transmitted diseases. As obligate intracellular bacteria, C. trachomatis has evolved strategies to redirect the host's signaling and resources for its own survival and propagation. Despite the clinical notoriety of Chlamydia infections, the molecular interactions between C. trachomatis and its host cell proteins remain elusive. RESULTS: In this study, we focused on the involvement of the host cell epidermal growth factor receptor (EGFR) in C. trachomatis attachment and development. A combination of molecular approaches, pharmacological agents and cell lines were used to demonstrate distinct functional requirements of EGFR in C. trachomatis infection. We show that C. trachomatis increases the phosphorylation of EGFR and of its downstream effectors PLCγ1, Akt and STAT5. While both EGFR and platelet-derived growth factor receptor-ß (PDGFRß) are partially involved in bacterial attachment to the host cell surface, it is only the knockdown of EGFR and not PDGFRß that affects the formation of C. trachomatis inclusions in the host cells. Inhibition of EGFR results in small immature inclusions, and prevents C. trachomatis-induced intracellular calcium mobilization and the assembly of the characteristic F-actin ring at the inclusion periphery. By using complementary approaches, we demonstrate that the coordinated regulation of both calcium mobilization and F-actin assembly by EGFR are necessary for maturation of chlamydial inclusion within the host cells. A particularly important finding of this study is the co-localization of EGFR with the F-actin at the periphery of C. trachomatis inclusion where it may function to nucleate the assembly of signaling protein complexes for cytoskeletal remodeling required for C. trachomatis development. CONCLUSION: Cumulatively, the data reported here connect the function of EGFR to C. trachomatis attachment and development in the host cells, and this could lead to new venues for targeting C. trachomatis infections and associated diseases.

Pyk2 regulates H+-ATPase-mediated proton secretion in the outer medullary collecting duct via an ERK1/2 signaling pathway.

Acid-secreting intercalated cells respond to changes in systemic pH through regulation of apical H(+) transporters. Little is known about the mechanism by which these cells sense changes in extracellular pH (pH(o)). Pyk2 is a nonreceptor tyrosine kinase activated by autophosphorylation at Tyr402 by cell-specific stimuli, including decreased pH, and is involved in the regulation of MAPK signaling pathways and transporter activity. We examined whether the Pyk2 and MAPK signaling pathway mediates the response of transport proteins to decreased pH in outer medullary collecting duct cells. Immunoblot analysis of phosphorylated Pyk2 (Tyr402), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) was used to assay protein activation. To examine specificity of kinase activation and its effects, we used Pyk2 small interfering RNA to knockdown Pyk2 expression levels, the Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP 1) to inhibit Pyk2 phosphorylation, and the MEK inhibitor U0126 to inhibit ERK1/2 phosphorylation. The pH-sensitive fluorescent probe 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) was used to assay H(+) transporter activity. The activity of H(+) transporters was measured as the rate of intracellular pH (pH(i)) recovery after an NH(4)Cl prepulse. We show that Pyk2 is endogenously expressed and activated by acid pH in mouse-derived outer medullary collecting duct (mOMCD1) cells. Incubation of mOMCD1 cells in acid media [extracellular pH (pH(o)) 6.7] increased the phosphorylation of Pyk2, ERK1/2, and p38. Reduction in pH(i) induced by an NH(4)Cl prepulse also increased the phosphorylation of Pyk2, ERK1/2, and p38. Consistent with our previous studies, we found that mOMCD1 cells exhibit H(+)-ATPase and H(+),K(+)-ATPase activity. Pyk2 inhibition by Pyk2 siRNA and PP 1 prevented Pyk2 phosphorylation as well as H(+)-ATPase-mediated recovery in mOMCD1 cells. In addition, ERK1/2 inhibition by U0126 prevented acid-induced ERK1/2 phosphorylation and H(+)-ATPase-mediated pH(i) recovery but not phosphorylation of p38. We conclude that Pyk2 and ERK1/2 are required for increasing H(+)-ATPase, but not H(+),K(+)-ATPase, activity at decreased pH(i) in mOMCD1 cells.

pH-dependent regulation of the α-subunit of H+-K+-ATPase (HKα2).

The H(+)-K(+)-ATPase α-subunit (HKα(2)) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα(2) plasma membrane expression is regulated by PKA (Codina J, Liu J, Bleyer AJ, Penn RB, DuBose TD Jr. J Am Soc Nephrol 17: 1833-1840, 2006) and in a separate study demonstrated that genetic ablation of the proton-sensing G(s)-coupled receptor GPR4 results in spontaneous metabolic acidosis (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). In the present study, we investigated the ability of chronic acidosis and GPR4 to regulate HKα(2) expression in HEK-293 cells. Chronic acidosis was modeled in vitro by using multiple methods: reducing media pH by adjusting bicarbonate concentration, adding HCl, or by increasing the ambient concentration of CO(2). PKA activity and HKα(2) protein were monitored by immunoblot analysis, and HKα(2) mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα(2) mRNA; however, PKA activity and HKα(2) protein abundance increased when media pH decreased from 7.4 to 6.8. Furthermore, this increase was independent of the method used to create chronic acidosis. Heterologous expression of GPR4 was sufficient to increase both basal and acid-stimulated PKA activity and similarly increase basal and acid-stimulated HKα(2) expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα(2) protein by increasing PKA activity without altering HKα(2) mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα(2) and acid-base balance.

Deletion of the pH sensor GPR4 decreases renal acid excretion.

Proton receptors are G protein-coupled receptors that accept protons as ligands and function as pH sensors. One of the proton receptors, GPR4, is relatively abundant in the kidney, but its potential role in acid-base homeostasis is unknown. In this study, we examined the distribution of GPR4 in the kidney, its function in kidney epithelial cells, and the effects of its deletion on acid-base homeostasis. We observed GPR4 expression in the kidney cortex, in the outer and inner medulla, in isolated kidney collecting ducts, and in cultured outer and inner medullary collecting duct cells (mOMCD1 and mIMCD3). Cultured mOMCD1 cells exhibited pH-dependent accumulation of intracellular cAMP, characteristic of GPR4 activation; GPR4 knockdown attenuated this accumulation. In vivo, deletion of GPR4 decreased net acid secretion by the kidney and resulted in a nongap metabolic acidosis, indicating that GPR4 is required to maintain acid-base homeostasis. Collectively, these findings suggest that GPR4 is a pH sensor with an important role in regulating acid secretion in the kidney collecting duct.

Deletion of the anion exchanger Slc26a4 (pendrin) decreases apical Cl(-)/HCO3(-) exchanger activity and impairs bicarbonate secretion in kidney collecting duct.

The anion exchanger Pendrin, which is encoded by SLC26A4 (human)/Slc26a4 (mouse) gene, is localized on the apical membrane of non-acid-secreting intercalated (IC) cells in the kidney cortical collecting duct (CCD). To examine its role in the mediation of bicarbonate secretion in vivo and the apical Cl(-)/HCO(3)(-) exchanger in the kidney CCD, mice with genetic deletion of pendrin were generated. The mutant mice show the complete absence of pendrin expression in their kidneys as assessed by Northern blot hybridization, Western blot, and immunofluorescence labeling. Pendrin knockout (KO) mice display significantly acidic urine at baseline [pH 5.20 in KO vs. 6.01 in wild type (WT); P < 0.0001] along with elevated serum HCO(3)(-) concentration (27.4 vs. 24 meq/l in KO vs. WT, respectively; P < 0.02), consistent with decreased bicarbonate secretion in vivo. The urine chloride excretion was comparable in WT and KO mice. For functional studies, CCDs were microperfused and IC cells were identified by their ability to trap the pH fluorescent dye BCECF. The apical Cl(-)/HCO(3)(-) exchanger activity in B-IC and non-A, non-B-IC cells, as assessed by intracellular pH monitoring, was significantly reduced in pendrin-null mice. The basolateral Cl(-)/HCO(3)(-) exchanger activity in A-IC cells and in non-A, non-B-IC cells, was not different in pendrin KO mice relative to WT animals. Urine NH(4)(+) (ammonium) excretion increased significantly, consistent with increased trapping of NH(3) in the collecting duct in pendrin KO mice. We conclude that Slc26a4 (pendrin) deletion impairs the secretion of bicarbonate in vivo and reduces apical Cl(-)/HCO(3)(-) exchanger activity in B-IC and non-A, non-B-IC cells in CCD. Additional apical Cl(-)/HCO(3)(-) exchanger(s) is (are) present in the CCD.

Increased acid load and deletion of AE1 increase Slc26a7 expression.

BACKGROUND/AIMS: Slc26a7 is a member of a family of anion transport proteins, Solute-Linked Carrier 26 (Slc26). Slc26a7, which can mediate Cl-/HCO3- exchange, is expressed in the acid-secreting, A-intercalated cells of the kidney collecting duct. On the basolateral side of the A-intercalated cells, Slc26a7 co-localizes with the anion exchanger 1 (AE1), a Cl-/HCO3- exchanger that mediates bicarbonate reabsorption in the collecting duct. METHODS: To test if Slc26a7 is involved in acid-base regulation, as its localization and function suggest, we examined the effect of acid loading and deletion of AE1 on Slc26a7 expression with quantitative real-time RT-PCR and Western blotting. RESULTS: Four days of acid loading increased Slc26a7 mRNA expression in the kidney inner medulla by 57% (n = 6 acid loaded vs. n = 6 control rats; p < 0.001), whereas mRNA expression in the outer medulla and the cortex did not change. Western blotting analysis demonstrated increased Slc26a7 protein expression in both outer (140%) and inner medulla (50%) in acid-loaded animals (n = 3) compared to controls (n = 3; p < 0.05). The expression of Slc26a7 mRNA was increased by 66% in the kidneys of AE1 knockout mice (n = 5) compared to the wild types (n = 5, p < 0.001). The increase in Slc26a7 mRNA correlated with a twofold increase in protein expression (p < 0.05). CONCLUSION: We suggest that the increase in Slc26a7 expression caused by acid challenge and deletion of AE1 represents an adaptive response, indicating that Slc26a7 contributes to the regulation of acid-base balance by the kidney.

Decreased expression of Slc26a4 (Pendrin) and Slc26a7 in the kidneys of carbonic anhydrase II-deficient mice.

BACKGROUND/AIMS: Intercalated cells (ICs) of the kidney collecting duct are rich in carbonic anhydrase II (CAII), which facilitates proton and bicarbonate transport. Bicarbonate secretion is mediated via Pendrin (Slc26a4), which is expressed on the apical membrane of B-ICs and nonA-nonB ICs in the cortical collecting ducts (CCD). Bicarbonate absorption is mediated via anion exchanger 1 (AE1-Slc4a1) in the CCD and via AE1 and possibly Slc26a7 in the OMCD. Both exchangers are expressed on the basolateral membrane of A-ICs. The aim of this study was to examine the expression of pendrin, Slc26a7, and AE1 in the kidneys of CAII-deficient (CAR2-null) mice. METHODS: For the expression studies, we used real-time RT-PCR, Northern hybridization, immunolabeling, and immunoblotting. RESULTS: Pendrin mRNA expression was reduced 63% along with decreased pendrin immunolabeling in the cortex of CAR2-null mice present predominantly in nonA-nonB ICs. Slc26a7 mRNA expression was decreases by 73% and Slc26a7 immunolabeling, present in A-ICs, severely reduced in the outer medulla of CAR2-null mice. AE1 mRNA expression was decreased to a similar degree (62%) along with reduced AE1 immunolabeling. The expression of aquaporin 2 (AQP2) water channel, exclusively present in principal cells of the collecting duct, was comparable in the wild type and CAR2-null mice. CONCLUSION: CAII deficiency results in a significant decrease in the gene and protein expression of bicarbonate transport proteins from Slc26 gene family - Slc26a4 (pendrin) and Slc26a7. These results emphasize the critical role of CAII for the maintenance of the intercalated cell phenotype.

Slc26a6 (PAT1) deletion downregulates the apical Na+/H+ exchanger in the straight segment of the proximal tubule.

BACKGROUND/AIM: Slc26a6 (PAT1, CFEX) is a major chloride/base exchanger located on the apical membrane of the kidney proximal tubule. The purpose of the present study was to examine the effect of Slc26a6 deletion on the apical Na+/H+ exchanger 3 (NHE3) in the straight segment (S3) of the proximal tubule, which is the major site for the reabsorption of filtered chloride in the kidney. METHODS: The proximal tubule S3 segment was perfused and the intracellular pH and apical Na+/H+ exchanger activity and expression were measured. RESULTS: In the proximal tubule straight segments that were microperfused in vitro, baseline intracellular pH, measured by BCPCF-AM, was 7.10 +/- 0.02 in Slc26a6-/- and 7.33 +/- 0.02 in Slc26a6+/+ animals, a significant reduction in Slc26a6 mutant mice (p < 0.00001). The activity of the apical Na+/H+ exchanger was 0.49 +/- 0.02 pH units/min in Slc26a6+/+ and 0.26 +/- 0.03 pH units/min in Slc26a6-/- animals, a significant reduction in Slc26a6-/- mice (p < 0.0001). Formate-induced intracellular alkalinization, which is mediated via NHE3, was significantly blunted in Slc26a6-/- animals, with an alkalinization magnitude of 0.16 pH unit in Slc26a6-/- versus 0.37 in Slc26a6+/+ animals (p < 0.00001, n = 5 separate animals). Angiotensin II stimulation of NHE3 activity was intact in Slc26a6-/- animals. Buffering capacity was comparable in Slc26a6+/+ and Slc26a6-/- mice. Immunoblotting and immunofluorescent labeling demonstrated comparable NHE3 abundance and distribution in kidney proximal tubules of Slc26a6+/+ and Slc26a6-/- mice. CONCLUSION: In conclusion, Slc26a6 deletion downregulates the apical Na+/H+ exchanger activity in the straight segment of the proximal tubule. The absence of a significant renal sodium loss in Slc26a6-null mice, despite NHE3 downregulation in the in vitro perfused tubules, points to possible activation of signaling pathways that can stimulate the apical Na+/H+ exchanger in vivo.

Regulation of acid-base transporters by vasopressin in the kidney collecting duct of Brattleboro rat.

AIM: The objective of these studies was to examine the effects of long-term vasopressin treatment on acid-base transporters in the collecting duct of rat kidney. METHODS: Brattleboro rats were placed in metabolic cages and treated with daily injections of 1-desamino-8-D-arginine vasopressin (dDAVP), a selective V2-receptor agonist, or its vehicle (control) for up to 8 days. RESULTS: dDAVP treatment resulted in a significant reduction in serum bicarbonate concentration, and caused the upregulation of key ammoniagenesis enzymes, along with increased urinary NH4+ excretion. Northern hybridization and immunofluorescence labeling indicated a significant increase (+80%) in mRNA expression of the apical Cl-/HCO3- exchanger pendrin (PDS), along with a sharp increase in its protein abundance in B-type intercalated cells in the cortical collecting duct in dDAVP-treated rats. In the inner medullary collecting duct, the abundance of basolateral Cl-/HCO3- exchanger (AE1) and apical H+-ATPase was significantly reduced in dDAVP-treated rats. Kidney renin mRNA increased significantly and correlated with an increase in serum aldosterone levels in dDAVP-injected rats. Serum corticosterone levels were, however, reduced and correlated with increased mRNA levels of renal 11beta-hydroxysteroid dehydrogenase-2 (11beta-HSD2) and decreased mRNA expression of 11beta-hydroxylase in the adrenal gland of dDAVP-injected rats. CONCLUSION: Chronic administration of dDAVP to Brattleboro rats is associated with the upregulation of PDS and downregulation of H+-ATPase and AE1 in the collecting duct, along with increased ammoniagenesis. Stimulation of the renin-angiotensin-aldosterone system and/or decreased glucocorticoid levels likely plays a role in the transduction of these effects.

Chloride/bicarbonate exchanger SLC26A7 is localized in endosomes in medullary collecting duct cells and is targeted to the basolateral membrane in hypertonicity and potassium depletion.

SLC26A7 is a Cl(-)/HCO(3)(-) exchanger that is expressed on the basolateral membrane and in the cytoplasm of two distinct acid-secreting epithelial cells: The A-intercalated cells in the kidney outer medullary collecting duct and the gastric parietal cells. The intracellular localization of SLC26A7 suggests the possibility of trafficking between cell membrane and intracellular compartments. For testing this hypothesis, full-length human SLC26A7 cDNA was fused with green fluorescence protein and transiently expressed in MDCK epithelial cells. In monolayer cells in isotonic medium, SLC26A7 showed punctate distribution throughout the cytoplasm. However, in medium that was made hypertonic for 16 h, SLC26A7 was detected predominantly in the plasma membrane. The presence of mitogen-activated protein kinase inhibitors blocked the trafficking of SLC26A7 to the plasma membrane. Double-labeling studies demonstrated the localization of SLC26A7 to the transferrin receptor-positive endosomes. A chimera that was composed of the amino terminal fragment of SLC26A7 and the carboxyl terminal fragment of SLC26A1, and a C-terminal-truncated SLC26A7 were retained in the cytoplasm in hypertonicity. In separate studies, SLC26A7 showed predominant localization in plasma membrane in potassium-depleted isotonic medium (0.5 or 2 mEq/L KCl) versus cytoplasmic distribution in normal potassium isotonic medium (4 mEq/L). It is concluded that SLC26A7 is present in endosomes, and its targeting to the basolateral membrane is increased in hypertonicity and potassium depletion. The trafficking to the cell surface suggests novel functional upregulation of SLC26A7 in states that are associated with hypokalemia or increased medullary tonicity. Additional studies are needed to ascertain the role of SLC26A7 in enhanced bicarbonate absorption in outer medullary collecting duct in hypokalemia and in acid-base regulation in conditions that are associated with increased medullary tonicity.

Vasopressin induces expression of the Cl-/HCO3- exchanger SLC26A7 in kidney medullary collecting ducts of Brattleboro rats.

SLC26A7 is a newly identified basolateral Cl(-)/HCO(3)(-) exchanger specific to alpha-intercalated cells of the outer medullary collecting duct (OMCD). The purpose of the present experiments was to examine the expression of SLC26A7 in kidneys of vasopressin-deficient Brattleboro rats before and after treatment with desamino-Cys(1),d-Arg(8)-vasopressin (dDAVP). Brattleboro rats were treated with dDAVP, a vasopressin analog, for 8 days, and their kidneys were examined for the expression of SLC26A7. The expression of SLC26A7 protein, as examined by immunofluorescence, was undetectable in kidneys of Brattleboro rats. However, treatment with dDAVP induced expression of SLC26A7 protein, restoring it to levels observed in normal rats. These results were verified by Western blot analysis. The mRNA expression of SLC26A7 remained unchanged in response to dDAVP. Immunofluorescent labeling demonstrated abundant levels of anion exchanger type 1 in the OMCD of Brattleboro rats and a mild reduction in response to dDAVP. The abundance of H(+)-ATPase was not affected by dDAVP. The increased SLC26A7 expression directly correlated with enhanced aquaporin-2 expression, which is proportional to increased interstitial osmolarity in the medulla. In conclusion, vasopressin increases the expression of SLC26A7 protein through posttranscriptional mechanisms in the OMCD. The induction of SLC26A7 by vasopressin in OMCD cells of Brattleboro rats is likely an attempt by cells to regulate their cell volume and maintain HCO(3)(-) absorption in a state associated with increased interstitial medullary tonicity.

Distinct and sequential upregulation of genes regulating cell growth and cell cycle progression during hepatic ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) in liver and other organs is manifested as an injury phase followed by recovery and resolution. Control of cell growth and proliferation is essential for recovery from the injury. We examined the expression of three related regulators of cell cycle progression in liver IRI: spermidine/spermine N-acetyltransferase (SSAT), p21 (a cyclin-dependent kinase inhibitor), and stathmin. Mice were subjected to hepatic IRI, and liver tissues were harvested at timed intervals. The expression of SSAT, the rate-limiting enzyme in the polyamine catabolic pathway, had increased fivefold 6 h after IRI and correlated with increased putrescine levels in the liver, consistent with increased SSAT enzymatic activity in IRI. The expression of p21, which is transactivated by p53, was undetectable in sham-operated animals but was heavily induced at 12 and 24 h of reperfusion and declined to undetectable baseline levels at 72 h of reperfusion. The interaction of the polyamine pathway with the p53-p21 pathway was shown in vitro, where activation of SSAT with polyamine analog or the addition of putrescine to cultured hepatocytes induced the expression of p53 and p21 and decreased cell viability. The expression of stathmin, which is under negative transcriptional regulation by p21 and controls cell proliferation and progression through mitosis, remained undetectable at 6, 12, and 24 h of reperfusion and was progressively and heavily induced at 48 and 72 h of reperfusion. Double-immunofluorescence labeling with antibodies against stathmin and PCNA, a marker of cell proliferation, demonstrated colocalization of stathmin and PCNA at 48 and 72 h of reperfusion in hepatocytes, indicating the initiation of cell proliferation. The distinct and sequential upregulation of SSAT, p21, and stathmin, along with biochemical activation of the polyamine catabolic pathway in IRI in vivo and the demonstration of p53-p21 upregulation by SSAT and putrescine in vitro, points to the important role of regulators of cell growth and cell cycle progression in the pathophysiology and/or recovery in liver IRI. The data further suggest that SSAT may play a role in the initiation of injury, whereas p21 and stathmin may be involved in the resolution and recovery after liver IRI.