scholarly journals Galectin-3 mediates oligomerization of secreted hensin using its carbohydrate-recognition domain

2013 ◽  
Vol 305 (1) ◽  
pp. F90-F99 ◽  
Author(s):  
Soundarapandian Vijayakumar ◽  
Hu Peng ◽  
George J. Schwartz
Keyword(s):  
Metabolic Acidosis ◽  
Collecting Duct ◽  
Critical Role ◽  
Rabbit Kidney ◽  
Intercalated Cells ◽  
Carbohydrate Recognition ◽  
Terminal Domain ◽  
Galectin 3 ◽  
Number Of Cells

A multidomain, multifunctional 230-kDa extracellular matrix (ECM) protein, hensin, regulates the adaptation of rabbit kidney to metabolic acidosis by remodeling collecting duct intercalated cells. Conditional deletion of hensin in intercalated cells of the mouse kidney leads to distal renal tubular acidosis and to a significant reduction in the number of cells expressing the basolateral chloride-bicarbonate exchanger kAE1, a characteristic marker of α-intercalated cells. Although hensin is secreted as a monomer, its polymerization and ECM assembly are essential for its role in the adaptation of the kidney to metabolic acidosis. Galectin-3, a unique lectin with specific affinity for β-galactoside glycoconjugates, directly interacts with hensin. Acidotic rabbits had a significant increase in the number of cells expressing galectin-3 in the collecting duct and exhibited colocalization of galectin-3 with hensin in the ECM of microdissected tubules. In this study, we confirmed the increased expression of galectin-3 in acidotic rabbit kidneys by real-time RT-PCR. Galectin-3 interacted with hensin in vitro via its carbohydrate-binding COOH-terminal domain, and the interaction was competitively inhibited by lactose, removal of the COOH-terminal domain of galectin-3, and deglycosylation of hensin. Galectin-9, a lectin with two carbohydrate-recognition domains, is also present in the rabbit kidney; galectin-9 partially oligomerized hensin in vitro. Our results demonstrate that galectin-3 plays a critical role in hensin ECM assembly by oligomerizing secreted monomeric hensin. Both the NH2-terminal and COOH-terminal domains are required for this function. We suggest that in the case of galectin-3-null mice galectin-9 may partially substitute for the function of galectin-3.

Galectin-3 expression is induced in renal β-intercalated cells during metabolic acidosis

2006 ◽  
Vol 290 (1) ◽  
pp. F148-F158 ◽  
Author(s):  
Andrew L. Schwaderer ◽  
Soundarapandian Vijayakumar ◽  
Qais Al-Awqati ◽  
George J. Schwartz
Keyword(s):  
Extracellular Matrix ◽  
Metabolic Acidosis ◽  
Collecting Duct ◽  
Rabbit Kidney ◽  
Cell Phenotype ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Outer Cortex ◽  
Galectin 3 ◽  
Inner Cortex

The adaptation of the cortical collecting duct (CCD) to metabolic acidosis requires the polymerization and deposition in the extracellular matrix of the novel protein hensin. HCO3−-secreting β-intercalated cells remove apical Cl−:HCO3− exchangers and may reverse functional polarity to secrete protons. Using intercalated cells in culture, we found that galectin-3 facilitated hensin polymerization, thereby causing their differentiation into the H+-secreting cell phenotype. We examined the expression of galectin-3 in the rabbit kidney and its relationship to hensin during metabolic acidosis. In control kidneys, galectin-3 was expressed in the cortical and medullary collecting ducts. In the outer cortex 26 ± 3% of CCD cells expressed galectin-3 compared with 64 ± 3% of the cells of the inner cortex. In the CCD, galectin-3 was rarely expressed in β-intercalated cells, being primarily present in α-intercalated and principal cells. During metabolic acidosis, the intensity of cellular staining for galectin-3 increased and more cells began to express it; the percentage of CCD cells expressing galectin-3 increased from 26 ± 3 to 66 ± 3% in the outer cortex and from 64 ± 3 to 78 ± 4% in the inner cortex. This was particularly evident in β-intercalated cells where expression was found in only 8 ± 2% in control animals but in 75 ± 2% during metabolic acidosis in the outer cortex and similarly for the inner cortex (26 ± 6 to 90 ± 7%). Importantly, both galectin-3 and hensin were found in the extracellular matrix of microdissected CCDs; and during metabolic acidosis, many more cells exhibited this extracellular colocalization. Thus galectin-3 may play several important roles in the CCD, including mediating the adaptation of β-intercalated cells during metabolic acidosis.

scholarly journals Intercalated cell-specific Rh B glycoprotein deletion diminishes renal ammonia excretion response to hypokalemia

2013 ◽  
Vol 304 (4) ◽  
pp. F422-F431 ◽  
Author(s):  
Jesse M. Bishop ◽  
Hyun-Wook Lee ◽  
Mary E. Handlogten ◽  
Ki-Hwan Han ◽  
Jill W. Verlander ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Collecting Duct ◽  
Ammonia Excretion ◽  
Ammonia Concentration ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Intercalated Cell ◽  
Transporter Family ◽  
Total Ammonia ◽  
Normal Increase

The ammonia transporter family member, Rh B Glycoprotein (Rhbg), is an ammonia-specific transporter heavily expressed in the kidney and is necessary for the normal increase in ammonia excretion in response to metabolic acidosis. Hypokalemia is a common clinical condition in which there is increased renal ammonia excretion despite the absence of metabolic acidosis. The purpose of this study was to examine Rhbg's role in this response through the use of mice with intercalated cell-specific Rhbg deletion (IC-Rhbg-KO). Hypokalemia induced by feeding a K+-free diet increased urinary ammonia excretion significantly. In mice with intact Rhbg expression, hypokalemia increased Rhbg protein expression in intercalated cells in the cortical collecting duct (CCD) and in the outer medullary collecting duct (OMCD). Deletion of Rhbg from intercalated cells inhibited hypokalemia-induced changes in urinary total ammonia excretion significantly and completely prevented hypokalemia-induced increases in urinary ammonia concentration, but did not alter urinary pH. We conclude that hypokalemia increases Rhbg expression in intercalated cells in the cortex and outer medulla and that intercalated cell Rhbg expression is necessary for the normal increase in renal ammonia excretion in response to hypokalemia.

scholarly journals ANP-stimulated Na+ secretion in the collecting duct prevents Na+ retention in the renal adaptation to acid load

2019 ◽  
Vol 317 (2) ◽  
pp. F435-F443
Author(s):  
Lydie Cheval ◽  
Naziha Bakouh ◽  
Christine Walter ◽  
Dignê Tembely ◽  
Luciana Morla ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Urinary Excretion ◽  
Secretory Pathway ◽  
Collecting Duct ◽  
Type A ◽  
Intercalated Cells ◽  
Membrane Expression ◽  
Acid Load ◽  
Anp Receptors ◽  
Stimulation Of

We have recently reported that type A intercalated cells of the collecting duct secrete Na+ by a mechanism coupling the basolateral type 1 Na+-K+-2Cl− cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+ exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+ retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+ secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopus oocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+ retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+ retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis.

Immunolocalization of the Na+/Ca2+ exchanger in rabbit kidney

1993 ◽  
Vol 265 (2) ◽  
pp. F327-F332 ◽  
Author(s):  
R. F. Reilly ◽  
C. A. Shugrue ◽  
D. Lattanzi ◽  
D. Biemesderfer
Keyword(s):  
Collecting Duct ◽  
Membrane Vesicle ◽  
Immunoblot Analysis ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Intracellular Loop ◽  
Nephron Segments ◽  
Vesicle Preparation

We recently isolated a cDNA encoding a Na+/Ca2+ exchanger from rabbit kidney that was highly similar to the canine cardiac sarcolemmal Na+/Ca2+ exchanger. In the present study, we used two different antibodies to the exchanger to identify the protein and establish its cellular and subcellular localization in the kidney. The first antibody was prepared against a fusion protein consisting of 190 amino acids of the large, presumably intracellular loop of the rabbit renal exchanger fused to the maltose-binding protein. The second was a monoclonal antibody generated against the isolated purified canine cardiac sarcolemmal exchanger. To identify the Na+/Ca2+ exchanger protein, we performed immunoblot analysis against a membrane vesicle preparation from rabbit kidney cortex. Both antibodies immunoblotted proteins of 120 and 70 kDa that are known to be associated with the exchanger. Indirect immunofluorescence revealed that both antisera labeled the basolateral surface of the majority of cells in the connecting tubule (CNT). Since the phase-dense (intercalated) cells in the CNT were not stained, this suggested that the labeled cells were CNT cells. No labeling was detected in other nephron segments with the exception of occasional faint staining of the majority cell population of the cortical collecting duct. The fact that we did not detect labeling in other nephron segments is consistent with either 1) the absence of expression of the Na+/Ca2+ exchanger in these segments, 2) the expression of the exchanger in levels below the threshold of detection of the two antibodies used in this study, or 3) the exchanger in these segments is represented by a different isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Conductive properties of the rabbit outer medullary collecting duct: inner stripe

1985 ◽  
Vol 248 (4) ◽  
pp. F500-F506 ◽  
Author(s):  
B. M. Koeppen
Keyword(s):  
Cell Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Rabbit Kidney ◽  
Apical Cell Membrane ◽  
Basolateral Cell Membrane ◽  
Medullary Collecting Duct ◽  
Conductive Properties

Segments of outer medullary collecting duct were dissected from the inner stripe of the rabbit kidney (OMCDi) and perfused in vitro. The conductive properties of the tubule epithelium and individual cell membranes were determined by means of cable analysis and intracellular voltage-recording microelectrodes. In 35 tubules the transepithelial voltage (VT) and resistance (RT) averaged 17.2 +/- 1.4 mV, lumen positive, and 58.6 +/- 5.3 k omega X cm, respectively. The basolateral membrane voltage, (Vbl) was -29.2 +/- 2.1 mV (n = 23). The apical cell membrane did not contain appreciable ion conductances, as evidenced by the high values of apical cell membrane fractional resistance (fRa = Ra/Ra + Rb), which approached unity (0.99 +/- 0.01; n = 23). Moreover, addition of amiloride or BaCl2 to the tubule lumen was without effect on the electrical characteristics of the cell, as was a twofold reduction in luminal [Cl-]. The conductive properties of the basolateral cell membrane were assessed with bath ion substitutions. A twofold reduction in bath [Cl-] depolarized Vbl by 14.7 +/- 0.4 mV (theoretical, 17 mV), while a 10-fold increase in bath [K+] resulted in only a 0.9 +/- 0.4 mV depolarization (theoretical, 61 mV). Substituting bath Na+ with tetramethylammonium (from 150 to 75 mM) was without effect. Reducing bath [HCO-3] from 25 to 5 mM (constant PCO2) resulted in a steady-state depolarization of Vbl of 8.4 +/- 0.4 mV that could not be attributed to conductive HCO-3 movement. Thus, the basolateral cell membrane is predominantly Cl- selective.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbonic anhydrase II mRNA is induced in rabbit kidney cortex during chronic metabolic acidosis

1993 ◽  
Vol 265 (6) ◽  
pp. F764-F772 ◽  
Author(s):  
G. J. Schwartz ◽  
C. A. Winkler ◽  
B. J. Zavilowitz ◽  
T. Bargiello
Keyword(s):  
Metabolic Acidosis ◽  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Carbonic Anhydrase Ii ◽  
Northern Analysis ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Rt Pcr ◽  
Chronic Metabolic Acidosis ◽  
Pcr Product

Carbonic anhydrase II (CA II), the predominant isoform of carbonic anhydrase in the kidney, is believed to be localized primarily in the cytoplasm of proximal tubule and collecting duct intercalated cells. Carbonic anhydrase facilitates H+ secretion by catalyzing the formation of HCO3- from OH- in the presence of CO2. We have shown that renal cortical CA II activity is stimulated during 4-6 days of chronic metabolic acidosis [L.P. Brion, B.J. Zavilowitz, O. Rosen, and G.J. Schwartz. Am. J. Physiol. 261 (Regulatory Integrative Comp. Physiol. 30): R1204-R1213, 1991]. The purpose of these studies was to examine under similar conditions the regulation of CA II mRNA. We obtained a major portion of the rabbit CA II cDNA by reverse transcription of total RNA from rabbit kidney followed by amplification using oligonucleotide primers prepared from conserved areas in the coding regions of human, mouse, and chick CA II cDNAs in a polymerase chain reaction (RT-PCR). The 696-bp RT-PCR product was sequenced and found to be 71-86% homologous to CA II cDNAs from the other three species. The deduced amino acid sequence agreed closely (> 97%) with a previous Edman analysis of rabbit erythrocyte CA II. Northern analysis showed expression of a approximately 1.4 kb RNA, with cortex > outer medulla > inner medulla. Steady-state mRNA expression from kidney cortex of acid-treated rabbits was about twice that from controls, when normalized to the expression of beta-actin or malate dehydrogenase. The stimulation of CA II mRNA was greater after 3 days than after 5-6 days of acid treatment. (ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation of the outer medullary collecting duct to metabolic acidosis in vitro

1998 ◽  
Vol 275 (6) ◽  
pp. F982-F990 ◽  
Author(s):  
Shuichi Tsuruoka ◽  
George J. Schwartz
Keyword(s):  
Protein Synthesis ◽  
Metabolic Acidosis ◽  
Collecting Duct ◽  
Low Ph ◽  
Acetoxymethyl Ester ◽  
Calmodulin Antagonist ◽  
Cell Calcium ◽  
Signal Transduction Inhibitors

Metabolic acidosis in vivo, as well as in vitro (1 h at pH 6.8 followed by 2 h at pH 7.4) stimulates H+-ATPase-dependent H+ secretion in outer medullary collecting ducts from the inner stripe (OMCDi) (S. Tsuruoka and G. J. Schwartz. J. Clin. Invest. 99: 1420–1431, 1997). Another group has shown that the adaptation to metabolic acidosis in vivo is mediated by an apical polarization of H+ pumps without an increase in total H+pump mRNA or protein (B. Bastani, H. Purcell, P. Hemken, D. Trigg, and S. Gluck. J. Clin. Invest. 88: 126–136, 1991). To further address the mechanism of adaptation, we measured net [Formula: see text] absorption before and after applying protein/RNA synthesis and signal transduction inhibitors during the 1 h of low pH and a cytoskeletal inhibitor during the entire 3-h incubation. Net [Formula: see text]transport, measured by microcalorimetry, increased ∼33% after in vitro acidosis. This increase was prevented by application during the first hour of anisomycin (10 μM) or actinomycin D (4 μM), but not by anisomycin applied during the 2-h incubation at pH 7.4. Similar results were obtained with the cell calcium chelator, 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM, 20 μM), the calmodulin antagonist, calmidazolium (30 nM), the endoplasmic reticulum Ca-ATPase inhibitor, thapsigargin (100 nM), and the protein kinase C (PKC) inhibitor, staurosporine (100 nM), applied during the 1 h at pH 6.8, but not with BAPTA-AM or thapsigargin used during the 2-h incubation at pH 7.4. Colchicine (10 μM) applied during the entire 3-h incubation also prevented this adaptive increase in H+ secretion, whereas lumicolchicine (10 μM, the inactive congener) did not. Colchicine also reversibly prevented any adaptive increases in transepithelial positive voltage. Thus the adaptation to acidosis in vitro required RNA and protein synthesis, changes in intracellular calcium and PKC activity, and intact microtubules. Time was required for the adaptation to occur, as the increase in [Formula: see text]transport was small after <3-h incubation. Protein synthesis and changes in cell calcium were critical during the initial period of low pH but not once the acid stimulus had been removed. Exocytosis of H+ pumps appears to occur continually during the entire 3-h incubation. These data would suggest that the synthesis and regulation of proteins involved in shuttling H+ pumps in cytoplasmic vesicles to the apical membrane via exocytosis are important for the OMCDi to adapt to low pH in vitro and probably to metabolic acidosis in vivo.

scholarly journals Carbonic anhydrase II and IV mRNA in rabbit nephron segments: stimulation during metabolic acidosis

1998 ◽  
Vol 274 (2) ◽  
pp. F259-F267 ◽  
Author(s):  
Shuichi Tsuruoka ◽  
Ann M. Kittelberger ◽  
George J. Schwartz
Keyword(s):  
Metabolic Acidosis ◽  
Carbonic Anhydrase ◽  
Mrna Expression ◽  
Collecting Duct ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Rt Pcr ◽  
Chronic Metabolic Acidosis ◽  
Nephron Segments ◽  
Medullary Collecting Duct

Carbonic anhydrase (CA) facilitates renal bicarbonate reabsorption and acid excretion. Cytosolic CA II catalyzes the buffering of intracellular hydroxyl ions by CO2, whereas membrane-bound CA IV catalyzes the dehydration of carbonic acid generated from the secretion of protons. Although CA II and IV are expressed in rabbit kidney, it is not entirely clear which segments express which isoforms. It was the purpose of this study to characterize the expression of CA II and CA IV mRNAs by specific segments of the nephron using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and to determine the effect of chronic metabolic acidosis on CA expression by those segments. Individual nephron segments (usually 1–2 mm) were isolated by microdissection and subjected to RT-PCR. Amplification was performed simultaneously for CA IV, CA II, and malate dehydrogenase (MDH), a housekeeping gene. The intensities of the PCR products were quantitated by densitometry. CA IV mRNA was expressed by S1 and S2 proximal tubules and by outer medullary collecting duct from inner stripe (OMCDi) and outer stripe and initial inner medullary collecting duct (IMCDi). CA II mRNA was expressed by S1, S2, and S3 proximal tubules, thin descending limb, connecting segment (CNT), and all collecting duct segments. Acid loading induced CA IV mRNA expression in S1 and S2 proximal tubules and in OMCDi and IMCDi. CA II mRNA was induced by acidosis in all three proximal segments and nearly all distal segments beginning with CNT. No upregulation of MDH mRNA expression occurred. These adaptive increases in CA II and IV mRNAs are potentially important in the kidney’s adaptation to chronic metabolic acidosis.

Renal expression of the ammonia transporters, Rhbg and Rhcg, in response to chronic metabolic acidosis

2006 ◽  
Vol 290 (2) ◽  
pp. F397-F408 ◽  
Author(s):  
Ramanathan M. Seshadri ◽  
Janet D. Klein ◽  
Shelley Kozlowski ◽  
Jeff M. Sands ◽  
Young-Hee Kim ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Protein Expression ◽  
Mrna Expression ◽  
Collecting Duct ◽  
Broad Band ◽  
Ammonia Excretion ◽  
Intercalated Cells ◽  
Outer Medulla ◽  
Chronic Metabolic Acidosis ◽  
Medullary Collecting Duct

Chronic metabolic acidosis induces dramatic increases in net acid excretion that are predominantly due to increases in urinary ammonia excretion. The current study examines whether this increase is associated with changes in the expression of the renal ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). Chronic metabolic acidosis was induced in Sprague-Dawley rats by HCl ingestion for 1 wk; control animals were pair-fed. After 1 wk, metabolic acidosis had developed, and urinary ammonia excretion increased significantly. Rhcg protein expression was increased in both the outer medulla and the base of the inner medulla. Intercalated cells in the outer medullary collecting duct (OMCD) and in the inner medullary collecting duct (IMCD) in acid-loaded animals protruded into the tubule lumen and had a sharp, discrete band of apical Rhcg immunoreactivity, compared with a flatter cell profile and a broad band of apical immunolabel in control kidneys. In addition, basolateral Rhcg immunoreactivity was observed in both control and acidotic kidneys. Cortical Rhcg protein expression and immunoreactivity were not detectably altered. Rhcg mRNA expression was not significantly altered in the cortex, outer medulla, or inner medulla by chronic metabolic acidosis. Rhbg protein and mRNA expression were unchanged in the cortex, outer and inner medulla, and no changes in Rhbg immunolabel were evident in these regions. We conclude that chronic metabolic acidosis increases Rhcg protein expression in intercalated cells in the OMCD and in the IMCD, where it is likely to mediate an important role in the increased urinary ammonia excretion.

scholarly journals Toward an understanding of the Cdc48/p97 ATPase

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1318 ◽  
Author(s):  
Nicholas Bodnar ◽  
Tom Rapoport
Keyword(s):  
Atp Hydrolysis ◽  
Critical Role ◽  
Ring Structure ◽  
Cellular Systems ◽  
Aaa Atpase ◽  
Double Ring ◽  
Terminal Domain ◽  
D2 Domain ◽  
Central Pore

A conserved AAA+ ATPase, called Cdc48 in yeast and p97 or VCP in metazoans, plays an essential role in many cellular processes by segregating polyubiquitinated proteins from complexes or membranes. For example, in endoplasmic reticulum (ER)-associated protein degradation (ERAD), Cdc48/p97 pulls polyubiquitinated, misfolded proteins out of the ER and transfers them to the proteasome. Cdc48/p97 consists of an N-terminal domain and two ATPase domains (D1 and D2). Six Cdc48 monomers form a double-ring structure surrounding a central pore. Cdc48/p97 cooperates with a number of different cofactors, which bind either to the N-terminal domain or to the C-terminal tail. The mechanism of Cdc48/p97 action is poorly understood, despite its critical role in many cellular systems. Recent in vitro experiments using yeast Cdc48 and its heterodimeric cofactor Ufd1/Npl4 (UN) have resulted in novel mechanistic insight. After interaction of the substrate-attached polyubiquitin chain with UN, Cdc48 uses ATP hydrolysis in the D2 domain to move the polypeptide through its central pore, thereby unfolding the substrate. ATP hydrolysis in the D1 domain is involved in substrate release from the Cdc48 complex, which requires the cooperation of the ATPase with a deubiquitinase (DUB). Surprisingly, the DUB does not completely remove all ubiquitin molecules; the remaining oligoubiquitin chain is also translocated through the pore. Cdc48 action bears similarities to the translocation mechanisms employed by bacterial AAA ATPases and the eukaryotic 19S subunit of the proteasome, but differs significantly from that of a related type II ATPase, the NEM-sensitive fusion protein (NSF). Many questions about Cdc48/p97 remain unanswered, including how it handles well-folded substrate proteins, how it passes substrates to the proteasome, and how various cofactors modify substrates and regulate its function.

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