Mitochondrial aquaporin-8 in renal proximal tubule cells: evidence for a role in the response to metabolic acidosis

2012 ◽  
Vol 303 (3) ◽  
pp. F458-F466 ◽  
Author(s):  
Sara M. Molinas ◽  
Laura Trumper ◽  
Raúl A. Marinelli
Keyword(s):  
Metabolic Acidosis ◽  
Protein Expression ◽  
Proximal Tubule ◽  
Ammonia Excretion ◽  
Renal Proximal Tubule ◽  
In Vivo Studies ◽  
Proximal Tubule Cell ◽  
Inner Mitochondrial Membrane ◽  
Ammonia Transport ◽  
In Cells

Mitochondrial ammonia synthesis in proximal tubules and its urinary excretion are key components of the renal response to maintain acid-base balance during metabolic acidosis. Since aquaporin-8 (AQP8) facilitates transport of ammonia and is localized in inner mitochondrial membrane (IMM) of renal proximal cells, we hypothesized that AQP8-facilitated mitochondrial ammonia transport in these cells plays a role in the response to acidosis. We evaluated whether mitochondrial AQP8 (mtAQP8) knockdown by RNA interference is able to impair ammonia excretion in the human renal proximal tubule cell line, HK-2. By RT-PCR and immunoblotting, we found that AQP8 is expressed in these cells and is localized in IMM. HK-2 cells were transfected with short-interfering RNA targeting human AQP8. After 48 h, the levels of mtAQP8 protein decreased by 53% ( P < 0.05). mtAQP8 knockdown decreased the rate of ammonia released into culture medium in cells grown at pH 7.4 (−31%, P < 0.05) as well as in cells exposed to acid (−90%, P < 0.05). We also evaluated mtAQP8 protein expression in HK-2 cells exposed to acidic medium. After 48 h, upregulation of mtAQP8 (+74%, P < 0.05) was observed, together with higher ammonia excretion rate (+73%, P < 0.05). In vivo studies in NH4Cl-loaded rats showed that mtAQP8 protein expression was also upregulated after 7 days of acidosis in renal cortex (+51%, P < 0.05). These data suggest that mtAQP8 plays an important role in the adaptive response of proximal tubule to acidosis possibly facilitating mitochondrial ammonia transport.

scholarly journals Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism

2016 ◽  
Vol 310 (11) ◽  
pp. F1229-F1242 ◽  
Author(s):  
Hyun-Wook Lee ◽  
Gunars Osis ◽  
Mary E. Handlogten ◽  
Wouter H. Lamers ◽  
Farrukh A. Chaudhry ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Glutamine Synthetase ◽  
Proximal Tubule ◽  
Ammonia Excretion ◽  
Renal Proximal Tubule ◽  
Acid Excretion ◽  
Ammonia Metabolism ◽  
Adaptive Changes ◽  
Multiple Components

Glutamine synthetase (GS) catalyzes the recycling of NH4+ with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phospho enolpyruvate carboxykinase, and Na+-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression.

scholarly journals Angiotensin II type 1 receptor-associated protein deficiency attenuates sirtuin1 expression in an immortalised human renal proximal tubule cell line

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Takahiro Yamaji ◽  
Akio Yamashita ◽  
Hiromichi Wakui ◽  
Kengo Azushima ◽  
Kazushi Uneda ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Protein Expression ◽  
Proximal Tubule ◽  
Cell Line ◽  
Renal Proximal Tubule ◽  
Sirtuin 1 ◽  
Epithelial Cell Line ◽  
Proximal Tubule Cell ◽  
Deficient Mice

Abstract The proximal tubule is a particularly important site for ageing-related kidney damage. Sirtuin 1 (SIRT1), an NAD+ (nicotinamide adenine dinucleotide)-dependent deacetylase in the proximal tubule, may be involved in renal injury associated with ageing. However, the mechanisms of SIRT1 regulation remain to be elucidated. We recently reported that angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP)-deficient mice displayed age-associated renal function decline and tubulointerstitial fibrosis. Our data showed that SIRT1 protein expression was reduced in ATRAP-deficient mice, although the relationship between ATRAP deficiency and age-associated renal fibrosis is still not fully understood. It is, therefore, necessary to investigate how ATRAP affects SIRT1 protein expression to resolve ageing-associated kidney dysfunction. Here, since ageing studies are inherently lengthy, we used an ex vivo model of the proximal tubule to determine the role of ATRAP in SIRT1 protein expression. We first generated a clonal immortalised human renal proximal tubule epithelial cell line (ciRPTEC) expressing AT1R and ATRAP. Using this cell line, we demonstrated that ATRAP knockdown reduced SIRT1 protein expression in the ciRPTEC but did not alter SIRT1 mRNA expression. Thus, ATRAP likely mediates SIRT1 protein abundance in ciRPTEC.

PKC-α-mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells

2005 ◽  
Vol 288 (6) ◽  
pp. F1227-F1235 ◽  
Author(s):  
Deanne H. Hryciw ◽  
Carol A. Pollock ◽  
Philip Poronnik
Keyword(s):  
Proximal Tubule ◽  
Fluorescent Protein ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Apical Surface ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Albumin Endocytosis ◽  
In Cells

One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular filtrate by constitutive receptor-mediated endocytosis. In the opossum kidney (OK) renal proximal tubule cell line, inhibition of protein kinase C (PKC) reduces albumin uptake, although the isoforms involved and mechanisms by which this occurs have not been identified. We used pharmacological and molecular approaches to investigate the role of PKC-α in albumin endocytosis. We found that albumin uptake in OK cells was inhibited by the pan-PKC blocker bisindolylmaleimide-1 and the isoform-specific PKC blockers Gö-6976 and 2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimethanol dimethyl ether, indicating a role for PKC-α. Overexpression of a kinase deficient PKC-α(K368R) but not wild-type PKC-α significantly reduced albumin endocytosis. Western blot analysis of fractionated cells showed an increased association of PKC-α-green fluorescent protein with the membrane fraction within 10–20 min of exposure to albumin. We used phalloidin to demonstrate that albumin induces the formation of clusters of actin at the apical surface of OK cells and that these clusters correspond to the location of albumin uptake. These clusters were not present in cells grown in the absence of albumin. In cells treated either with PKC inhibitors or overexpressing kinase-deficient PKC-α(K368R) this actin cluster formation was significantly reduced. This study identifies a role for PKC-α in constitutive albumin uptake in OK cells by mediating assembly of actin microfilaments at the apical membrane.

scholarly journals Renal Proximal Tubule Cell Cannabinoid-1 Receptor Regulates Bone Remodeling and Mass via a Kidney-to-Bone Axis

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 414
Author(s):  
Saja Baraghithy ◽  
Yael Soae ◽  
Dekel Assaf ◽  
Liad Hinden ◽  
Shiran Udi ◽  
...  
Keyword(s):  
Bone Mass ◽  
Proximal Tubule ◽  
Bone Remodeling ◽  
Kidney Function ◽  
Bone Cells ◽  
Critical Role ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Protective Effects ◽  
Cannabinoid 1 Receptor

The renal proximal tubule cells (RPTCs), well-known for maintaining glucose and mineral homeostasis, play a critical role in the regulation of kidney function and bone remodeling. Deterioration in RPTC function may therefore lead to the development of diabetic kidney disease (DKD) and osteoporosis. Previously, we have shown that the cannabinoid-1 receptor (CB1R) modulates both kidney function as well as bone remodeling and mass via its direct role in RPTCs and bone cells, respectively. Here we employed genetic and pharmacological approaches that target CB1R, and found that its specific nullification in RPTCs preserves bone mass and remodeling both under normo- and hyper-glycemic conditions, and that its chronic blockade prevents the development of diabetes-induced bone loss. These protective effects of negatively targeting CB1R specifically in RPTCs were associated with its ability to modulate erythropoietin (EPO) synthesis, a hormone known to affect bone mass and remodeling. Our findings highlight a novel molecular mechanism by which CB1R in RPTCs remotely regulates skeletal homeostasis via a kidney-to-bone axis that involves EPO.

Influence of Na+ intake on dopamine-induced inhibition of renal cortical Na(+)-K(+)-ATPase

1990 ◽  
Vol 258 (1) ◽  
pp. F52-F60 ◽  
Author(s):  
I. Seri ◽  
B. C. Kone ◽  
S. R. Gullans ◽  
A. Aperia ◽  
B. M. Brenner ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Sodium Intake ◽  
Dietary Sodium ◽  
Acid Decarboxylase ◽  
Proximal Tubule Cell ◽  
Maximal Velocity ◽  
Tubule Cell ◽  
Amino Acid Decarboxylase ◽  
Cell Cytosol ◽  
In Cells

The enzyme L-amino acid decarboxylase (L-AADC), found in abundance in rat proximal tubule cell cytosol, converts L-dopa to dopamine. Dopamine, in turn, suppresses proximal tubule sodium transport by inhibiting Na(+)-K(+)-ATPase activity. We sought to determine whether changes in dietary sodium intake in rats lead to adaptation of dopamine formation and dopamine-induced Na(+)-K(+)-ATPase inhibition. In rats on a high-salt (HS) diet, the maximal velocity (Vmax) of renal cortical L-AADC was 78 +/- 19% higher than that in rats on a low-salt (LS) diet. The Michaelis constant (Km) of the enzyme remained unchanged. In renal cortical tubule cell suspensions the L-dopa-induced inhibition of ouabain-sensitive oxygen consumption (QO2) was significantly greater in rats on HS diet than in rats on LS diet. Furthermore, L-dopa completely inhibited the nystatin-induced rise in QO2 in the HS but not in the LS group. Carbidopa, an inhibitor of L-AADC, abolished the L-dopa-induced inhibition of nystatin-stimulated QO2 in cells from HS rats and was without significant effect in cells from LS rats. L-Dopa-stimulated K+ efflux was greater in cells from HS rats at 28 +/- 1 nmol.min-1.mg protein-1, compared with 7 +/- 6 nmol.min-1.ng protein-1 in cells from LS rats. By contrast, ouabain-stimulated K+ efflux did not differ between the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of xenobiotic transporters in the human renal proximal tubule cell line RPTEC/TERT1

2015 ◽  
Vol 30 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Lydia Aschauer ◽  
Giada Carta ◽  
Nadine Vogelsang ◽  
Eberhard Schlatter ◽  
Paul Jennings
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Tubule Cell

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death

2012 ◽  
Vol 303 (2) ◽  
pp. F266-F278 ◽  
Author(s):  
Šárka Lhoták ◽  
Sudesh Sood ◽  
Elise Brimble ◽  
Rachel E. Carlisle ◽  
Stephen M. Colgan ◽  
...  
Keyword(s):  
Er Stress ◽  
Proximal Tubule ◽  
Lipid Accumulation ◽  
Cell Injury ◽  
Apoptotic Cell ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Proximal Tubule Cells ◽  
Tubule Cells

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca2+-independent phospholipase A2 (iPLA2β), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.

Interleukin-6 stimulates α-MG uptake in renal proximal tubule cells: involvement of STAT3, PI3K/Akt, MAPKs, and NF-κB

2007 ◽  
Vol 293 (4) ◽  
pp. F1036-F1046 ◽  
Author(s):  
Yu Jin Lee ◽  
Jung Sun Heo ◽  
Han Na Suh ◽  
Min Young Lee ◽  
Ho Jae Han
Keyword(s):  
Protein Expression ◽  
Proximal Tubule ◽  
Signaling Pathways ◽  
Interleukin 6 ◽  
Renal Proximal Tubule ◽  
Expression Level ◽  
Protein Expression Level ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Recent studies have shown that interleukin 6 (IL-6) acts on the cellular proliferation-activating transduction signals during cellular regeneration. Therefore, this study examined the effect of IL-6 on the activation of Na+/glucose cotransporters (SGLTs) and its related signaling pathways in primary cultured renal proximal tubule cells (PTCs). IL-6 increased the level of α-methyl-d-[14C]glucopyranoside (α-MG) uptake in time- and dose-dependent manners. IL-6 also increased SGLT1 plus SGLT2 mRNA and protein expression level. The IL-6 receptors (IL-6Rα and gp130) were expressed in PTCs. In addition, genistein and herbimycin A completely blocked the IL-6-induced increases in α-MG uptake and the protein expression level of SGLTs. On the other hand, IL-6 increased the level of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate-sensitive cellular reactive oxygen species (ROS), and IL-6-induced increases in α-MG uptake and the protein expression level of SGLTs were blocked by ascorbic acid or taurine (antioxidants). IL-6 also increased the phosphorylation of signal transducer and activator of transcription-3 (STAT3), phosphoinositide-3 kinase (PI3K)/Akt, and mitogen-activated protein kinases (MAPKs) in a time-dependent manner. A pretreatment with STAT3 inhibitor LY 294002, an Akt inhibitor, or MAPK inhibitors significantly blocked the IL-6-induced increase in α-MG uptake. In addition, IL-6 increased the level of nuclear factor-κB (NF-κB) phosphorylation. A pretreatment with SN50 or BAY 11-7082 also blocked the IL-6-induced increase in α-MG uptake. In conclusion, IL-6 increases the SGLT activity through ROS, and its action in renal PTCs is associated with the STAT3, PI3K/Akt, MAPKs, and NF-κB signaling pathways.

Formate: A Critical Intermediate for Chloride Transport in the Proximal Tubule

Physiology ◽  
1987 ◽  
Vol 2 (5) ◽  
pp. 160-164
Author(s):  
LP Karniski ◽  
PS Aronson
Keyword(s):  
Formic Acid ◽  
Proximal Tubule ◽  
Cell Membranes ◽  
Chloride Transport ◽  
Critical Role ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Active Uptake

Recent experiments unexpectedly suggest that formate plays a critical role in chloride transport across cell membranes. In particular, active uptake of chloride in the renal proximal tubule cell occurs by chloride-formate exchange. Formate recycles from lumen to cell via nonionic diffusion of uncharged formic acid. In this manner, small amounts of formate can facilitate resorption of large quantities of chloride.

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