Snezana Petrovic

Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin

Metabolic acidosis causes a reversal of polarity of HCO(3)(-) flux in the cortical collecting duct (CCD). In CCDs incubated in vitro in acid media, beta-intercalated (HCO(3)(-)-secreting) cells are remodeled to functionally resemble alpha-intercalated (H(+)-secreting) cells. A similar remodeling of beta-intercalated cells, in which the polarity of H(+) pumps and Cl(-)/HCO(3)(-) exchangers is reversed, occurs in cell culture and requires the deposition of polymerized hensin in the ECM. CCDs maintained 3 h at low pH ex vivo display a reversal of HCO(3)(-) flux that is quantitatively similar to an effect previously observed in acid-treated rabbits in vivo. We followed intracellular pH in the same beta-intercalated cells before and after acid incubation and found that apical Cl/HCO(3) exchange was abolished following acid incubation. Some cells also developed basolateral Cl(-)/HCO(3)(-) exchange, indicating a reversal of intercalated cell polarity. This adaptation required intact microtubules and microfilaments, as well as new protein synthesis, and was associated with decreased size of the apical surface of beta-intercalated cells. Addition of anti-hensin antibodies prevented the acid-induced changes in apical and basolateral Cl(-)/HCO(3)(-) exchange observed in the same cells and the corresponding suppression of HCO(3)(-) secretion. Acid loading also promoted hensin deposition in the ECM underneath adapting beta-intercalated cells. Hence, the adaptive conversion of beta-intercalated cells to alpha-intercalated cells during acid incubation depends upon ECM-associated hensin.

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.

Identification of a carboxyl-terminal motif essential for the targeting of Na+-HCO-3 cotransporter NBC1 to the basolateral membrane.

The Na+-\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HC}\mathrm{O}_{3}^{-}\) \end{document} cotransporter NBC1 is located exclusively on the basolateral membrane and mediates vectorial transport of bicarbonate in a number of epithelia, including kidney and pancreas. To identify the motifs that direct the targeting of kidney NBC1 to basolateral membrane, wild type and various carboxyl-terminally truncated kidney NBC1 mutants were generated, fused translationally in-frame to GFP, and transiently expressed in kidney epithelial cells. GFP was linked to the NH2 terminus of NBC1, and labeling was examined by confocal microscopy. Full-length (1035 aa) and mutants with the deletion of 3 or 20 amino acids from the COOH-terminal end of NBC1 (lengths 1032 and 1015 aa, respectively) showed strong and exclusive targeting on the basolateral membrane. However, the deletion of 26 amino acid residues from the COOH-terminal end (length 1010 aa) resulted in retargeting of NBC1 to the apical membrane. Expression studies in oocytes demonstrated that the NBC1 mutant with the deletion of 26 amino acid residues from the COOH-terminal end is functional. Additionally, the deletion of the last 23 amino acids or mutation in the conserved residue Phe at position 1013 on the COOH-terminal end demonstrated retargeting to the apical membrane. We propose that a carboxyl-terminal motif with the sequence QQPFLS, which spans amino acid residues 1010-1015, and specifically the amino acid residue Phe (position 1013) are essential for the exclusive targeting of NBC1 to the basolateral membrane.

Differential Regulation of Basolateral Cl−/HCO3− Exchangers SLC26A7 and AE1 in Kidney Outer Medullary Collecting Duct

SLC26A7 is a recently identified Cl(-)/HCO(3)(-) exchanger that co-localizes with AE1 on the basolateral membrane of Alpha intercalated cells (A-IC) in outer medullary collecting duct (OMCD). The purpose of these studies was to determine whether AE1 and SLC26A7 are differentially regulated in OMCD in pathophysiologic states. Toward this end, the expression and regulation of AE1 and SLC26A7 was examined in water deprivation, a condition known to increase the osmolality of the medulla. Rats were subjected to 3 d of water deprivation while having free access to food. Northern hybridizations demonstrated that in the outer medulla, the mRNA expression of SLC26A7 increased by approximately 300% (P < 0.01 versus control; n = 3), whereas the expression of AE1 decreased by approximately 50% (P < 0.05 versus control, n = 3) in water-deprived rats. Immunoblot analysis studies demonstrated that in the outer medulla, SLC26A7 abundance increased by approximately 3.5-fold (P < 0.02 versus control; n = 3), whereas the AE1 abundance decreased by approximately 55% (P < 0.05 versus control) in water deprivation. The expression of SLC26A7 remained unchanged in the kidney cortex and stomach in water deprivation, indicating the specificity of SLC26A7 upregulation in outer medulla. In situ hybridization indicated the exclusive expression of SLC26A7 in the outer medulla and double immunofluorescence labeling confirmed the co-localization of AE1 and SLC26A7 on the basolateral membrane of A-IC cells in OMCD. It is concluded that AE1 and SLC26A7 are differentially regulated in OMCD in water deprivation. On the basis of these results and previous functional studies indicating the activation of SLC26A7 activity by high osmolality, it is proposed that SLC26A7 may play an important role in bicarbonate reabsorption and or cell volume regulation in OMCD (specifically under hypertonic conditions).

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.

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.

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.

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 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2) and decreased mRNA expression of 11β-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.

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

BackgroundChlamydia 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.ResultsIn 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-β (PDGFRβ) are partially involved in bacterial attachment to the host cell surface, it is only the knockdown of EGFR and not PDGFRβ 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.ConclusionCumulatively, 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.