scholarly journals Cloning, functional analysis and cell localization of a kidney proximal tubule water transporter homologous to CHIP28.

1993 ◽  
Vol 120 (2) ◽  
pp. 359-369 ◽  
Author(s):  
R Zhang ◽  
W Skach ◽  
H Hasegawa ◽  
A N van Hoek ◽  
A S Verkman
Keyword(s):  
Proximal Tubule ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Membrane Vesicles ◽  
High Water ◽  
Amino Acid Identity ◽  
In Vitro Translation ◽  
Kidney Cortex ◽  
Kidney Proximal Tubule

The localization and transporting properties of a kidney protein homologous to human erythrocyte protein CHIP28 was evaluated. The cDNA encoding rat kidney protein CHIP28k was isolated from a rat renal cortex cDNA library. A 2.8-kb cDNA was identified which contained an 807 bp open reading frame encoding a 28.8 kD protein with 94% amino acid identity to CHIP28. in vitro translation of CHIP28k cDNA in rabbit reticulocyte lysate generated a 28-kD protein; addition of ER-derived microsomes gave a 32-kD transmembrane glycoprotein. Translation of truncated RNA demonstrated glycosylation of residue Asn42 which is predicted to lie between the first and second transmembrane domains. Expression of in vitro transcribed mRNA encoding CHIP28k in Xenopus oocytes increased oocyte osmotic water permeability (Pf) from (4 +/- 1) x 10(-4) to (33 +/- 4) x 10(-4) cm/s at 10 degrees C; the increase in oocyte Pf was weakly temperature dependent and inhibited by HgCl2. Two-electrode voltage clamp measurements indicated that CHIP28k was not permeable to ions. Oocyte Pf also increased with expression of total mRNA from kidney cortex and papilla; the increase in Pf with mRNA from cortex, but not kidney papilla, was blocked by coinjection with excess antisense CHIP28k cRNA. In situ hybridization of a 150 base cRNA antisense probe to tissue sections from rat kidney showed selective CHIP28k localization to epithelial cells in proximal tubule and thin descending limb of Henle. Pf in purified apical membrane vesicles from rat and human proximal tubule, and in proteoliposomes reconstituted with purified protein, was very high and inhibited by HgCl2; stripping of apical vesicles with N-lauroylsarcosine enriched a 28-kD protein by 25-fold and yielded a vesicle population with high water, but low urea and proton permeabilities. CHIP28k identity was confirmed by NH2-terminus sequence analysis. These results indicate that CHIP28k is a major and highly selective water transporting protein in the kidney proximal tubule and thin descending limb of Henle, but not collecting duct.

Identification of an apical \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger in rat kidney proximal tubule

2003 ◽  
Vol 285 (3) ◽  
pp. C608-C617 ◽  
Author(s):  
Snezana Petrovic ◽  
Liyun Ma ◽  
Zhaohui Wang ◽  
Manoocher Soleimani
Keyword(s):  
Proximal Tubule ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Immunocytochemical Staining ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Kidney Proximal Tubule ◽  
Anion Transporter

SLC26A6 (or putative anion transporter 1, PAT1) is located on the apical membrane of mouse kidney proximal tubule and mediates [Formula: see text] exchange in in vitro expression systems. We hypothesized that PAT1 along with a [Formula: see text] exchange is present in apical membranes of rat kidney proximal tubules. Northern hybridizations indicated the exclusive expression of SLC26A6 (PAT1 or CFEX) in rat kidney cortex, and immunocytochemical staining localized SLC26A6 on the apical membrane of proximal tubules, with complete prevention of the labeling with the preadsorbed serum. To examine the functional presence of apical [Formula: see text] exchanger, proximal tubules were isolated, microperfused, loaded with the pH-sensitive dye BCPCF-AM, and examined by digital ratiometric imaging. The pH of the perfusate and bath was kept at 7.4. Buffering capacity was measured, and transport rates were calculated as equivalent base flux. The results showed that in the presence of basolateral DIDS (to inhibit [Formula: see text] cotransporter 1) and apical EIPA (to inhibit Na+/H+ exchanger 3), the magnitude of cell acidification in response to addition of luminal Cl– was ∼5.0-fold higher in the presence than in the absence of [Formula: see text]. The Cl–-dependent base transport was inhibited by ∼61% in the presence of 0.5 mM luminal DIDS. The presence of physiological concentrations of oxalate in the lumen (200 μM) did not affect the [Formula: see text] exchange activity. These results are consistent with the presence of SLC26A6 (PAT1) and [Formula: see text] exchanger activity in the apical membrane of rat kidney proximal tubule. We propose that SLC26A6 is likely responsible for the apical [Formula: see text] (and Cl–/OH–) exchanger activities in kidney proximal tubule.

scholarly journals Estrogen directly and specifically downregulates NaPi-IIa through the activation of both estrogen receptor isoforms (ERα and ERβ) in rat kidney proximal tubule

2015 ◽  
Vol 308 (6) ◽  
pp. F522-F534 ◽  
Author(s):  
Dara Burris ◽  
Rose Webster ◽  
Sulaiman Sheriff ◽  
Rashma Faroqui ◽  
Moshe Levi ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Ovariectomized Rats ◽  
Kidney Cortex ◽  
Kidney Proximal Tubule ◽  
Receptor Isoforms ◽  
Ovx Rats ◽  
Estrogen Receptor Isoforms

We have previously demonstrated that estrogen (E2) downregulates phosphate transporter NaPi-IIa and causes phosphaturia and hypophosphatemia in ovariectomized rats. In the present study, we examined whether E2directly targets NaPi-IIa in the proximal tubule (PT) and studied the respective roles of estrogen receptor isoforms (ERα and ERβ) in the downregulation of NaPi-IIa using both in vivo and an in vitro expression systems. We found that estrogen specifically downregulates NaPi-IIa but not NaPi-IIc or Pit2 in the kidney cortex. Proximal tubules incubated in a “shake” suspension with E2for 24 h exhibited a dose-dependent decrease in NaPi-IIa protein abundance. Results from OVX rats treated with specific agonists for either ERα [4,4′,4″;-(4-propyl-[1 H]-pyrazole-1,3,5-triyl) trisphenol, PPT] or ERβ [4,4′,4″-(4-propyl-[1 H]-pyrazole-1,3,5-triyl) trisphenol, DPN] or both (PPT + DPN), indicated that only the latter caused a sharp downregulation of NaPi-IIa, along with significant phosphaturia and hypophosphatemia. Lastly, heterologous expression studies demonstrated that estrogen downregulated NaPi-IIa only in U20S cells expressing both ERα and ERβ, but not in cells expressing either receptor alone. In conclusion, these studies demonstrate that rat PT cells express both ERα and ERβ and that E2induces phosphaturia by directly and specifically targeting NaPi-IIa in the PT cells. This effect is mediated via a mechanism involving coactivation of both ERα and ERβ, which likely form a functional heterodimer complex in the rat kidney proximal tubule.

Pendrin: an apical Cl−/OH−/HCO3 −exchanger in the kidney cortex

2001 ◽  
Vol 280 (2) ◽  
pp. F356-F364 ◽  
Author(s):  
Manoocher Soleimani ◽  
Tracey Greeley ◽  
Snezana Petrovic ◽  
Zhaohui Wang ◽  
Hassane Amlal ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Immunoblot Analysis ◽  
Kidney Cortex ◽  
Cortical Collecting Duct ◽  
Hek 293 ◽  
Kidney Proximal Tubule ◽  
293 Cells ◽  
Nephron Segment

The identities of the apical Cl−/base exchangers in kidney proximal tubule and cortical collecting duct (CCD) cells remain unknown. Pendrin (PDS), which is expressed at high levels in the thyroid and its mutation causes Pendred's syndrome, is shown to be an anion exchanger. We investigated the renal distribution of PDS and its function. Our results demonstrate that pendrin mRNA expression in the rat kidney is abundant and limited to the cortex. Proximal tubule suspensions isolated from kidney cortex were highly enriched in pendrin mRNA. Immunoblot analysis studies localized pendrin to cortical brush-border membranes. Nephron segment RT-PCR localized pendrin mRNA to proximal tubule and CCD. Expression studies in HEK-293 cells demonstrated that pendrin functions in the Cl−/OH−, Cl−/HCO3 −, and Cl−/formate exchange modes. The conclusion is that pendrin is an apical Cl−/base exchanger in the kidney proximal tubule and CCD and mediates Cl−/OH−, Cl−/HCO3 −, and Cl−/formate exchange.

Internalization and apical-to-basolateral transport of folate in rat kidney proximal tubule

1997 ◽  
Vol 272 (1) ◽  
pp. F70-F78 ◽  
Author(s):  
H. Birn ◽  
S. Nielsen ◽  
E. I. Christensen
Keyword(s):  
Light Microscopy ◽  
Proximal Tubule ◽  
Binding Sites ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Proximal Tubule Cells ◽  
Gold Particles ◽  
Kidney Proximal Tubule ◽  
Tubule Cells

Folate derivatives are filtered in the glomeruli and reabsorbed within the nephron. The amount filtered largely exceeds the minimum daily requirements. Thus folate reabsorbed within the kidney must be returned to the circulation. To establish whether renal proximal tubule can accomplish this by transport, of [3H]folate across the cell, microperfusion of rabbit, proximal tubule with [3H]folate and [14C]inulin was performed. Transtubular transport of [3H]folate was 5 +/- 1% (0.25 +/- 0.07 fmol/min) of perfused amount/mm tubule and remained constant during a 2-h perfusion period. An accumulation of 15 +/- 4% (0.8 +/- 0.3 fmol/min) of perfused amount/mm tubule was observed during the same period. Furthermore, to determine whether endocytosis may be involved in the initial process of folate uptake in proximal tubule cells, we performed light microscopy autoradiography on cryosections of rat kidney cortex incubated with [3H]folate. Folate binding sites were located apically as well as intracellularly similar to the location of [3H]folate when injected into the abdominal aorta and visualized by light microscopy autoradiography. Thus folate binding sites as well as internalized folate is localized both apically and intracellularly. Micropuncture of rat proximal tubules with folate-coupled collodial gold particles showed significantly increased endocytosis of folate gold when evaluated quantitatively and compared with controls injected with noncoupled gold particles (0.22 +/- 0.08 vs. 0.03 +/- 0.01 gold particles/micron 2 tubule cell). The results show that kidney proximal tubule cells are capable of transcellular transport of [3H]folate with limited capacity. Folate gold particle uptake suggests that folate can be internalized by endocytosis.

scholarly journals Presence of an extensive clathrin coat on the apical plasmalemma of the rat kidney proximal tubule cell.

1984 ◽  
Vol 98 (5) ◽  
pp. 1630-1636 ◽  
Author(s):  
J S Rodman ◽  
D Kerjaschki ◽  
E Merisko ◽  
M G Farquhar
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Specific Binding ◽  
Rat Kidney ◽  
Light Chains ◽  
Kidney Cortex ◽  
Immunoperoxidase Staining ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Kidney Proximal Tubule

The nature of the cytoplasmic coat present on the apical invaginations of the kidney proximal tubule cell was investigated by immuneoverlay and immunocytochemistry of renal brush borders with anticlathrin antibodies. When kidney cortex was prepared for electron microscopy using methods that enhance visualization of clathrin coats, the apical invaginations at the base of the brush border microvilli were seen to be backed by a nearly continuous coating which resembles but is more extensive than the lattice-like clathrin coats found around brain coated vesicles. When isolated brush border fractions were prepared under conditions that preserve the coats, separated by SDS PAGE, and transferred to nitrocellulose, the presence of clathrin heavy and light chains was detected by immuneoverlay using two different affinity-purified anticlathrin IgGs--one that we prepared, which detects only the clathrin light chains, and the other, prepared by Louvard et al. ( Louvard , D., C. Morris, G. Warren, K. Stanley, F. Winkler , and H. Reggio , 1983, EMBO [Eur. Mol. Biol. Organ.] J., 2:1655-1664), which detects both the heavy and light chains. As viewed by light microscopy (immunofluorescence or immunoperoxidase), staining with both anticlathrins was concentrated at the base of the proximal tubule microvilli. Immunoelectron microscopic localizations carried out on brush border fractions (using peroxidase and gold conjugates) demonstrated specific binding of anticlathrin IgGs to the lattice-like cytoplasmic coat. When brush border fractions were reacted with monoclonal antibodies prepared against gp330 and maltase, proteins that serve as markers for the membrane of the apical invaginations and microvilli, respectively ( Kerjaschki , D., L. Noronha - Blob , B. Sacktor , and M. G. Farquhar , 1984, J. Cell Biol., 98:1505-1513), the two proteins retained their restrictive distribution in the brush border. The findings demonstrate (a) that the cytoplasmic coat of the proximal tubule intermicrovillar apical invaginations is composed of clathrin heavy and light chains, and (b) that the differential distribution of proteins in these two brush border microdomains is maintained in appropriately prepared brush border fractions.

scholarly journals Studies on the orientation of brush-border membrane vesicles

1978 ◽  
Vol 172 (1) ◽  
pp. 57-62 ◽  
Author(s):  
W Haase ◽  
A Schäfer ◽  
H Murer ◽  
R Kinne
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Rat Kidney ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Immunological Methods ◽  
Kidney Cortex ◽  
Asymmetric Distribution ◽  
Rat Kidney Cortex

Orientation of rat renal and intestinal brush-border membrane vesicles was studied with two independent methods: electron-microscopic freeze-fracture technique and immunological methods. With the freeze-fracture technique a distinct asymmetric distribution of particles on the two membrane fracture faces was demonstrated; this was used as a criterion for orientation of the isolated membrane vesicles. For the immunological approach the accessibility or inaccessibility of aminopeptidase M localized on the outer surface of the cell membrane to antibodies was used. With both methods we showed that the brush-border membrane vesicles isolated from rat kidney cortex and from rat small intestine for transport studies are predominantly orientated right-side out.

Localization of PEPT1 and PEPT2 proton-coupled oligopeptide transporter mRNA and protein in rat kidney

1999 ◽  
Vol 276 (5) ◽  
pp. F658-F665 ◽  
Author(s):  
Hong Shen ◽  
David E. Smith ◽  
Tianxin Yang ◽  
Yuning G. Huang ◽  
Jürgen B. Schnermann ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Rat Kidney ◽  
Broad Band ◽  
Membrane Vesicles ◽  
Mrna Transcript ◽  
Intestinal Brush Border Membrane ◽  
Outer Stripe ◽  
Polyclonal Antisera ◽  
Pars Convoluta

To determine the renal localization of oligopeptide transporters, Northern blot analyses were performed and polyclonal antisera were generated against PEPT1 and PEPT2, the two cloned rat H+/peptide transporters. Under high-stringency conditions, a 3.0-kb mRNA transcript of rat PEPT1 was expressed primarily in superficial cortex, whereas a 3.5-kb mRNA transcript of PEPT2 was expressed primarily in deep cortex/outer stripe of outer medulla. PEPT1 antisera detected a specific band on immunoblots of renal and intestinal brush-border membrane vesicles (BBMV) with an apparent mobility of ∼90 kDa. PEPT2 antisera detected a specific broad band of ∼85 kDa in renal but not in intestinal BBMV. PEPT1 immunolocalization experiments showed detection of a brush border antigen in S1 segments of the proximal tubule and in the brush border of villi from all segments of the small intestine. In contrast, PEPT2 immunolocalization was primarily confined to the brush border of S3 segments of the proximal tubule. All other nephron segments in rat were negative for PEPT1 and PEPT2 staining. Overall, our results conclusively demonstrate that although PEPT1 is expressed in early regions of the proximal tubule (pars convoluta), PEPT2 is specific for the latter regions of proximal tubule (pars recta).

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