scholarly journals Divalent metal transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes: implications for renal handling of protein-metal complexes

2006 ◽  
Vol 290 (6) ◽  
pp. F1525-F1533 ◽  
Author(s):  
Marouan Abouhamed ◽  
Jakub Gburek ◽  
Wei Liu ◽  
Blazej Torchalski ◽  
Andreas Wilhelm ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Rt Pcr ◽  
Divalent Metal Transporter 1 ◽  
Rat Kidney Cortex ◽  
Divalent Metal Transporter ◽  
Alexa Fluor ◽  
Metal Transporter ◽  
Late Endosomes

The H+-coupled polyligand transport protein divalent metal transporter 1 (DMT1) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associated with iron acquisition and storage. Interestingly, it is also highly expressed in the kidney, yet its function in this tissue is unknown. The aim of this study was to determine the cellular location of DMT1 in proximal tubule cells as a first step to determining the role of this protein in the kidney. To do this we performed RT-PCR and immunostaining experiments using rat kidney and the S1 proximal tubule-derived WKPT-0293 Cl.2 cell line. RT-PCR revealed that mRNAs encoding all four DMT1 splice variants were present in RNA extracted from rat kidney cortex or WKPT-0293 Cl.2 cells. Immunostaining of rat kidney cortex or WKPT-0293 Cl.2 cells showed that DMT1 protein was expressed intracellularly and was not present in the plasma membrane. Expression of DMT1 partially colocalized with the late endosomal/lysosomal proteins LAMP1 and cathepsin-L. Using immunogold labeling, DMT1 was shown to be expressed in the membranes of late endosomes/lysosomes. Uptake of Alexa Fluor 546-transferrin was only observed following application to the apical membrane of WKPT-0293 Cl.2 cells. Within these cells, Alexa Fluor 546-transferrin colocalized with DMT1. In conclusion, renal proximal tubular cells express DMT1 in the membranes of organelles, including late endosomes/lysosomes, associated with processing of apically sequestered transferrin. These findings have implications for renal iron handling and possibly for the handling of nephrotoxic metals that are also DMT1 ligands, including Cd2+.

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 THE POLARITY OF THE PROXIMAL TUBULE CELL IN RAT KIDNEY

1972 ◽  
Vol 54 (2) ◽  
pp. 232-245 ◽  
Author(s):  
Hans-G Heidrich ◽  
Rolf Kinne ◽  
Eva Kinne-Saffran ◽  
Kurt Hannig
Keyword(s):  
Proximal Tubule ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Rat Kidney ◽  
High Specific Activity ◽  
Kidney Cortex ◽  
Proximal Tubule Cell ◽  
Surface Charges ◽  
Tubule Cell ◽  
Rat Kidney Cortex

Two different membrane fractions were obtained from a brush-border fraction of rat kidney cortex by using their different electrical surface charges in preparative free-flow electrophoresis. One membrane fraction contained only morphologically intact microvilli and was characterized by a high specific activity of alkaline phosphatase. The other fraction morphologically resembled classical plasma membranes by possessing junctional complexes and a high Na-K-ATPase activity The contamination of the isolated membrane fractions by other cell organelles was extremely low These two fractions represent the apical (luminal) and the basal (interstitial) area of the renal proximal tubule cell membrane and clearly demonstrate the polarity of this cell.

scholarly journals Immunoelectron Microscopic Localization of the Electrogenic Na/HCO3 Cotransporter in Rat and Ambystoma Kidney

2000 ◽  
Vol 11 (12) ◽  
pp. 2179-2189
Author(s):  
ARVID B. MAUNSBACH ◽  
HENRIK VORUM ◽  
TAE-HWAN KWON ◽  
SØREN NIELSEN ◽  
BRIAN SIMONSEN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Rat Kidney Cortex ◽  
C Terminus ◽  
Basolateral Plasma Membrane ◽  
Intracellular Vesicles

Abstract. Immunofluorescence analysis has revealed that electrogenic Na+/HCO3- (NBC1) is expressed in the proximal tubule of rat kidney and in the proximal and distal tubules of the salamander Ambystoma tigrinum kidney. The present study was undertaken to define the detailed subcellular localization of the NBC1 in rat and Ambystoma kidney using high-resolution immunoelectron microscopy. For this purpose, two rabbit polyclonal antibodies raised against amino acids 928 to 1035 and amino acids 1021 to 1035 of the C-terminus of rat kidney (rkNBC1) were developed. The affinity-purified antibodies revealed a strong band of approximately 140 kD in immunoblots of membranes from rat kidney cortex but no signal in membranes isolated from outer and inner medulla. Deglycosylation reduced the apparent molecular weight to approximately 120 kD, corresponding to the predicted molecular weight. A similar but weaker band was also present in membranes isolated from the lateral part of Ambystoma kidney. In rat kidney, immunohistochemistry confirmed the presence of rkNBC1 in convoluted segments of the proximal tubules. In ultrathin cryosections or Lowicryl HM20 sections from rat kidney cortex, distinct immunogold labeling was associated with the basolateral plasma membrane of segments S1 and S2 of proximal tubules, whereas in S3 no labeling was observed. The labeling density was similar at the basal and lateral plasma membrane and was specifically associated with the inner surface of the membrane consistent with the internal position of the C-terminus of the transporter. In contrast, rkNBC1 was absent from the apical plasma membrane and not observed in intracellular vesicles, including those closely associated with basolateral plasma membrane. In Ambystoma kidney, a weak labeling was present in the basolateral membrane of the proximal tubule and stronger labeling was observed in the late distal segment. The results demonstrate that rkNBC1 is expressed only in segment S1 and segment S2 of rat proximal tubule as well as Ambystoma proximal and late distal tubule and that rkNBC1 is present in both basal and lateral plasma membranes and absent in intracellular vesicles of the apical plasma membrane.

scholarly journals Impaired renal function and development in Belgrade rats

2014 ◽  
Vol 306 (3) ◽  
pp. F333-F343 ◽  
Author(s):  
Tania Veuthey ◽  
Dana Hoffmann ◽  
Vishal S. Vaidya ◽  
Marianne Wessling-Resnick
Keyword(s):  
Iron Status ◽  
Rat Kidney ◽  
Kidney Injury ◽  
Renal Physiology ◽  
Renal Metabolism ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Metal Transporter ◽  
Morphological Studies ◽  
Kidney Injury Molecule 1

Belgrade rats carry a disabling mutation in the iron transporter divalent metal transporter 1 (DMT1). Although DMT1 plays a major role in intestinal iron absorption, the transporter is also highly expressed in the kidney, where its function remains unknown. The goal of this study was to characterize renal physiology of Belgrade rats. Male Belgrade rats died prematurely with ∼50% survival at 20 wk of age. Necropsy results indicated marked glomerular nephritis and chronic end-stage renal disease. By 15 wk of age, Belgrade rats displayed altered renal morphology associated with sclerosis and fibrosis. Creatinine clearance was significantly lower compared with heterozygote littermates. Urinary biomarkers of kidney injury, including albumin, fibrinogen, and kidney injury molecule-1, were significantly elevated. Pilot morphological studies suggest that nephrogenesis is delayed in Belgrade rat pups due to their low iron status and fetal growth restriction. Such defects in renal development most likely underlie the compromised renal metabolism observed in adult b/b rats. Belgrade rat kidney nonheme iron levels were not different from controls but urinary iron and transferrin levels were higher. These results further implicate an important role for the transporter in kidney function not only in iron reabsorption but also in glomerular filtration of the serum protein.

Epidermal growth factor receptor regulation in rat kidney: two models of renal growth

1989 ◽  
Vol 257 (6) ◽  
pp. F1059-F1064 ◽  
Author(s):  
M. T. Behrens ◽  
A. L. Corbin ◽  
M. K. Hise
Keyword(s):  
Folic Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Rat Kidney Cortex ◽  
Epidermal Growth

The binding of 125I-labeled epidermal growth factor (EGF) to plasma membranes prepared from rat kidney cortex was studied following unilateral nephrectomy, a model of proximal tubule cell hypertrophy, and following the administration of folic acid, a model of proximal tubule cell hyperplasia. Binding of 125I-EGF was a linear function of basolateral membrane protein content and time of incubation. Specific binding to luminal brush-border membranes was not evident in these studies. Neither insulin nor insulin-like growth factor I could displace EGF binding, indicating that binding was specific. Scatchard analysis revealed a single binding site. The KD in sham-operated animals 48 h after surgery was 11.2 +/- 1.4 nM, whereas Bmax averaged 95.2 +/- 4.1 fmol/mg protein (n = 3). Similar values were obtained in nephrectomized animals. The Bmax of folic acid-untreated animals averaged 212.5 +/- 6.9 fmol/mg 48 h after administration, whereas that of vehicle-injected controls averaged 85.4 +/- 9.2 (n = 3, P less than 0.001). Differences in binding were not related to changes in affinity, ligand degradation by the preparations, or receptor binding of endogenous EGF. These data indicate that regeneration following folic acid administration is associated with an upregulation of proximal nephron EGF receptors that may play an important role in the mitogenic response.

Expression of Na-P(i) cotransport in rat kidney: localization by RT-PCR and immunohistochemistry

1994 ◽  
Vol 266 (5) ◽  
pp. F767-F774 ◽  
Author(s):  
M. Custer ◽  
M. Lotscher ◽  
J. Biber ◽  
H. Murer ◽  
B. Kaissling
Keyword(s):  
Transport System ◽  
Polyclonal Antibodies ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Expression Cloning ◽  
Kidney Cortex ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Rat Kidney Cortex ◽  
Proximal Tubular

We have recently identified a rat kidney cortex Na-dependent transport system for phosphate (P(i)) by expression cloning (NaP(i)-2) (S. Magagnin, A. Werner, D. Markovich, V. Sorribas, G. Stange, J. Biber, and H. Murer. Proc. Natl. Acad. Sci. USA 90: 5979, 1993). In this study we have used reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry to establish the sites of expression of the NaP(i)-2-related mRNA and protein. RT-PCR was performed with single microdissected nephron segments. From these experiments we conclude that NaP(i)-2 mRNA is predominantly expressed in the proximal tubules of superficial and deep nephrons. No NaP(i)-2 mRNA was detected in the thick ascending limb of Henle's loop; however, faint NaP(i)-2 related PCR products were also observed in collecting ducts. Expression of the NaP(i)-2 protein was examined with the use of polyclonal antibodies raised against synthetic NaP(i)-2-derived peptides. Strong specific anti-NaP(i)-2 antiserum-mediated immunofluorescence was found in the convoluted part of proximal tubules and gradually decreased along the straight part. Immunofluorescence indicated that the NaP(i)-2 protein is present in the brush border of proximal tubular cells. In addition, NaP(i)-2-specific immunofluorescence was also observed in subapical vesicles. The described distribution of the NaP(i)-2 protein is in agreement with previously described nephron sites of P(i) reabsorption in the rat kidney and therefore suggests that the NaP(i)-2 transport system represents an Na-P(i) cotransporter involved in proximal tubular apical transport of phosphate.

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 Divalent Metal Transporter 1 Knock-Down Modulates IL-1β Mediated Pancreatic Beta-Cell Pro-Apoptotic Signaling Pathways through the Autophagic Machinery

2021 ◽  
Vol 22 (15) ◽  
pp. 8013
Author(s):  
Taewook Kang ◽  
Honggang Huang ◽  
Thomas Mandrup-Poulsen ◽  
Martin R. Larsen
Keyword(s):  
Cell Death ◽  
Divalent Metal ◽  
Β Cells ◽  
Β Cell ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Knock Down ◽  
Metal Transporter ◽  
Cell Functions ◽  
Protective Proteins

Pro-inflammatory cytokines promote cellular iron-import through enhanced divalent metal transporter-1 (DMT1) expression in pancreatic β-cells, consequently cell death. Inhibition of β-cell iron-import by DMT1 silencing protects against apoptosis in animal models of diabetes. However, how alterations of signaling networks contribute to the protective action of DMT1 knock-down is unknown. Here, we performed phosphoproteomics using our sequential enrichment strategy of mRNA, protein, and phosphopeptides, which enabled us to explore the concurrent molecular events in the same set of wildtype and DMT1-silenced β-cells during IL-1β exposure. Our findings reveal new phosphosites in the IL-1β-induced proteins that are clearly reverted by DMT1 silencing towards their steady-state levels. We validated the levels of five novel phosphosites of the potential protective proteins using parallel reaction monitoring. We also confirmed the inactivation of autophagic flux that may be relevant for cell survival induced by DMT1 silencing during IL-1β exposure. Additionally, the potential protective proteins induced by DMT1 silencing were related to insulin secretion that may lead to improving β-cell functions upon exposure to IL-1β. This global profiling has shed light on the signal transduction pathways driving the protection against inflammation-induced cell death in β-cells after DMT1 silencing.

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