Expression of bcl-2 and bax in regenerating rat renal tubules following ischemic injury

1997 ◽  
Vol 272 (5) ◽  
pp. F640-F647 ◽  
Author(s):  
D. P. Basile ◽  
H. Liapis ◽  
M. R. Hammerman
Keyword(s):  
Proximal Tubule ◽  
Ischemic Injury ◽  
Fold Increase ◽  
Renal Tubules ◽  
Proximal Tubule Cells ◽  
Protein Levels ◽  
Bax Protein ◽  
Adult Kidney ◽  
Predictable Pattern

To define potential roles for bcl-2 and bax in adult kidney as regulators of regeneration, their expressions were characterized postischemic injury. A 2.1-fold increase in levels of renal bcl-2 mRNA occurred within 24 h of injury relative to levels in kidney of sham-operated control rats. The levels of bcl-2 mRNA remained elevated for 3 days but returned to baseline by day 5 postischemia. In situ hybridization of kidneys from sham-operated rats demonstrated faint expression of bcl-2 mRNA localized diffusely throughout the nephron. After renal injury, the expression of bcl-2 mRNA was markedly enhanced in regenerating proximal tubule cells relining the basement membrane. Immunohistochemistry showed a similar localization for bcl-2 protein. Levels of bax mRNA in kidney were elevated beginning at 24 h postischemia and remained elevated for 7 days postinjury. Bax mRNA and bax protein were colocalized to regenerating proximal tubules postischemia and were prominently expressed in papillary proliferations. We conclude that the expressions of bcl-2 and bax in kidney are enhanced in a predictable pattern following acute ischemic injury. Our findings suggest that these regulators of apoptosis play key roles in the process of repair of the damaged proximal tubule postischemia.

Abstract 564: Differential Expression and Regulation of Angiotensinogen in Renal Proximal Tubule Cells From S1, S2 and S3 Segments

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Ryousuke Satou ◽  
Kathleen S Hering-Smith ◽  
L G Navar
Keyword(s):  
Proximal Tubule ◽  
Kidney Injury ◽  
Specific Cell ◽  
S2 Cells ◽  
Ang Ii ◽  
Proximal Tubule Cells ◽  
Protein Levels ◽  
Pathogenic Factors ◽  
Renal Proximal Tubule Cells

In angiotensin II (Ang II)-dependent hypertension, intrarenal angiotensinogen (AGT) augmentation induced by Ang II and associated pathogenic factors including interleukin 6 (IL-6) cause further elevation of intratubular Ang II production, leading to the progression of hypertension and kidney injury. Recent studies have suggested that renal proximal straight tubules (S3 segment) are the main source of intrarenal AGT and that S1 and S2 segments do not express AGT mRNA under normal conditions. However, AGT expression and its regulation by Ang II and/or IL-6 in each proximal tubule segment have not been demonstrated an in vitro setting. The availability of specific cell lines derived from mouse S1, S2 and S3 segments provided an opportunity to decisively determine each segments’ capability to express AGT and respond to stimuli. Thus, this study was performed to determine AGT expression and its response to stimulation with Ang II and IL-6 in S1, S2 and S3 cell line. Basal AGT mRNA and protein levels were detected by RT-PCR and western blot analysis. Basal levels of Ang II type 1 receptor (AT1R) and STAT3, which is a transcription factor in IL-6 signaling pathway, were also measured. In addition, the cells were incubated with 100 nM Ang II and/or 400 nM IL-6 for 24 h. Basal AGT levels in S1 and S3 cells were lower than in mouse whole kidney (0.09-fold and 0.33-fold compared with mouse whole kidney). S2 cells exhibited the highest basal AGT levels (4.15-fold) among these cells. In S1 cells, AGT expression was stimulated by IL-6 (1.89 ± 0.32, ratio to control) and co-stimulation with Ang II and IL-6 (1.85 ± 0.28) although Ang II alone did not alter AGT levels. In S2 cells, only the co-stimulation increased AGT expression (1.35 ± 0.01). No changes were observed by similar treatments in S3 cells. Basal AT1R levels were lower in S3 than in S1 and S2 cells (0.97 ± 0.09 in S2, 0.32 ± 0.07 in S3, ratio to S1). S1 cells showed the highest basal levels of STAT3. Basal STAT3 levels in S3 cells were lower than that in S1 and S2 cells. These results indicate that S2 cells are main source of intrarenal AGT which can be augmented by Ang II and IL-6 during the development of Ang II-dependent hypertension. Furthermore, low basal levels of AT1R and STAT3 in S3 cells explain why these cells do not respond to Ang II and IL-6.

scholarly journals Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney

2010 ◽  
Vol 299 (6) ◽  
pp. F1496-F1506 ◽  
Author(s):  
Alan C. Pao ◽  
Aditi Bhargava ◽  
Francesca Di Sole ◽  
Raymond Quigley ◽  
Xinli Shao ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Proximal Tubule ◽  
Cell Surface Expression ◽  
Thick Ascending Limb ◽  
Proximal Tubule Cells ◽  
Mammalian Kidney ◽  
Tubule Cells ◽  
Exchange Activity

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na+) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na+/H+ exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na+/H+ exchange activity by Na+-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na+/H+ exchange activity by >30%. Moreover, the sgk2-mediated increase in Na+/H+ exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na+ transport through NHE3 in the proximal tubule.

scholarly journals Antenatal betamethasone attenuates the angiotensin-(1–7)-Mas receptor-nitric oxide axis in isolated proximal tubule cells

2017 ◽  
Vol 312 (6) ◽  
pp. F1056-F1062 ◽  
Author(s):  
Yixin Su ◽  
Jianli Bi ◽  
Victor M. Pulgar ◽  
Mark C. Chappell ◽  
James C. Rose
Keyword(s):  
Nitric Oxide ◽  
Proximal Tubule ◽  
Cell Model ◽  
Primary Cultures ◽  
Specific Effect ◽  
Renal Tubules ◽  
Proximal Tubule Cells ◽  
Mas Receptor ◽  
Tubule Cells

We previously reported a sex-specific effect of antenatal treatment with betamethasone (Beta) on sodium (Na+) excretion in adult sheep whereby treated males but not females had an attenuated natriuretic response to angiotensin-(1–7) [Ang-(1–7)]. The present study determined the Na+ uptake and nitric oxide (NO) response to low-dose Ang-(1–7) (1 pM) in renal proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to Beta or vehicle. Data were expressed as percentage of basal uptake or area under the curve for Na+ or percentage of control for NO. Male Beta RPTC exhibited greater Na+ uptake than male vehicle cells (433 ± 28 vs. 330 ± 26%; P < 0.05); however, Beta exposure had no effect on Na+ uptake in the female cells (255 ± 16 vs. 255 ± 14%; P > 0.05). Ang-(1–7) significantly inhibited Na+ uptake in RPTC from vehicle male (214 ± 11%) and from both vehicle (190 ± 14%) and Beta (209 ± 11%) females but failed to attenuate Na+ uptake in Beta male cells. Beta exposure also abolished stimulation of NO by Ang-(1–7) in male but not female RPTC. Both the Na+ and NO responses to Ang-(1–7) were blocked by Mas receptor antagonist d-Ala7-Ang-(1–7). We conclude that the tubular Ang-(1–7)-Mas-NO pathway is attenuated in males and not females by antenatal Beta exposure. Moreover, since primary cultures of RPTC retain both the sex and Beta-induced phenotype of the adult kidney in vivo they appear to be an appropriate cell model to examine the effects of fetal programming on Na+ handling by the renal tubules.

Endotoxin infusion in rats induces apoptotic and survival pathways in hearts

2000 ◽  
Vol 279 (5) ◽  
pp. H2053-H2061 ◽  
Author(s):  
Treena E. McDonald ◽  
Michelle N. Grinman ◽  
Chris M. Carthy ◽  
Keith R. Walley
Keyword(s):  
Fold Increase ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Mrna Levels ◽  
Apoptotic Pathway ◽  
Protein Levels ◽  
Bax Protein ◽  
Survival Pathways ◽  
Baseline Values

Inflammatory mediators of sepsis induce apoptosis in many cell lines. We tested the hypothesis that lipopolysaccharide (LPS) injection in vivo results in induction of early apoptotic and survival pathways as well as evidence of late-stage apoptosis in the heart. Hearts were collected from control rats and at 6, 12, and 24 h after LPS injection (4 mg/kg). Activation of an apoptotic pathway was identified by a 1,000-fold increase in caspase-3 activity at 24 h ( P < 0.05). Confirmation of these results occurred when terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining identified myocardial cells undergoing DNA fragmentation with significant levels at 24 h post-LPS injection. LPS also caused early proapoptotic mRNA (Bax) to increase (16% at 24 h, P < 0.05), whereas the Bax protein initially decreased (35% at 6 h, P < 0.05) and then returned to baseline values by 24 h. Six hours after LPS injection, Bcl-2 (early prosurvival) mRNA levels increased, whereas its protein levels decreased (70%, P < 0.05) and then returned to baseline levels by 24 h. Mitochondrial cytochrome c levels decreased, suggestive of mitochondrial involvement. Thus involvement of proapoptotic and prosurvival pathways in the heart occurs during a septic inflammatory response.

Gentamicin suppresses endotoxin-driven TNF-α production in human and mouse proximal tubule cells

2007 ◽  
Vol 293 (4) ◽  
pp. F1373-F1380 ◽  
Author(s):  
Richard A. Zager ◽  
Ali C. M. Johnson ◽  
Adam Geballe
Keyword(s):  
Protein Synthesis ◽  
Proximal Tubule ◽  
Renal Tubules ◽  
Mrna Accumulation ◽  
Chemokine Production ◽  
Proximal Tubule Cells ◽  
Gram Negative ◽  
Essentially Normal ◽  
Tnf Α ◽  
Tubule Cells

Gentamicin is a mainstay in treating gram-negative sepsis. However, it also may potentiate endotoxin (LPS)-driven plasma TNF-α increases. Because gentamicin accumulates in renal tubules, this study addressed whether gentamicin directly alters LPS-driven tubular cell TNF-α production. HK-2 proximal tubular cells were incubated for 18 h with gentamicin (10–2,000 μg/ml). Subsequent LPS-mediated TNF-α increases (at 3 or 24 h; protein/mRNA) were determined. Gentamicin effects on overall protein synthesis ([35S]methionine incorporation), monocyte chemoattractant protein-1 (MCP-1) levels, and LPS-stimulated TNF-α generation by isolated mouse proximal tubules also were assessed. Finally, because gentamicin undergoes partial biliary excretion, its potential influence on gut TNF-α/MCP-1 mRNAs was probed. Gentamicin caused striking, dose-dependent inhibition of LPS-driven TNF-α production (up to 80% in HK-2 cells/isolated tubules). Surprisingly, this occurred despite increased TNF-α mRNA accumulation. Comparable changes in MCP-1 were observed. These changes were observed at clinically relevant gentamicin concentrations and despite essentially normal overall protein synthetic rates. Streptomycin also suppressed LPS-driven TNF-α increases, suggesting an aminoglycoside drug class effect. Gentamicin doubled basal TNF-α mRNA in cecum and in small intestine after LPS. Gentamicin can suppress LPS-driven TNF-α production in proximal tubule cells, likely by inhibiting its translation. Overall preservation of protein synthesis and comparable MCP-1 suppression suggest a semiselective blockade within the LPS inflammatory mediator cascade. These results, coupled with increases in gut TNF-α/MCP-1 mRNAs, imply that gentamicin may exert protean, countervailing actions on systemic cytokine/chemokine production during gram-negative sepsis.

scholarly journals Steroidogenic acute regulatory-related lipid transfer domain protein 5 localization and regulation in renal tubules

2009 ◽  
Vol 297 (2) ◽  
pp. F380-F388 ◽  
Author(s):  
Yu-Chyu Chen ◽  
Renate K. Meier ◽  
Shirong Zheng ◽  
Syed J. Khundmiri ◽  
Michael T. Tseng ◽  
...  
Keyword(s):  
Er Stress ◽  
Proximal Tubule ◽  
Mouse Kidney ◽  
Cell Periphery ◽  
Type I ◽  
Renal Tubules ◽  
Lipid Transfer ◽  
Proximal Tubule Cells ◽  
Cholesterol Levels ◽  
Tubule Cells

STARD5 is a cytosolic sterol transport protein that is predominantly expressed in liver and kidney. This study provides the first report on STARD5 protein expression and distribution in mouse kidney. Immunohistochemical analysis of C57BL/6J mouse kidney sections revealed that STARD5 is expressed in tubular cells within the renal cortex and medullar regions with no detectable staining within the glomeruli. Within the epithelial cells of proximal renal tubules, STARD5 is present in the cytoplasm with high staining intensity along the apical brush-border membrane. Transmission electronmicroscopy of a renal proximal tubule revealed STARD5 is abundant at the basal domain of the microvilli and localizes mainly in the rough endoplasmic reticulum (ER) with undetectable staining in the Golgi apparatus and mitochondria. Confocal microscopy of STARD5 distribution in HK-2 human proximal tubule cells showed a diffuse punctuate pattern that is distinct from the early endosome marker EEA1 but similar to the ER membrane marker GRP78. Treatment of HK-2 cells with inducers of ER stress increased STARD5 mRNA expression and resulted in redistribution of STARD5 protein to the perinuclear and cell periphery regions. Since recent reports show elevated ER stress response gene expression and increased lipid levels in kidneys from diabetic rodent models, we tested STARD5 and cholesterol levels in kidneys from the OVE26 type I diabetic mouse model. Stard5 mRNA and protein levels are increased 2.8- and 1.5-fold, respectively, in OVE26 diabetic kidneys relative to FVB control kidneys. Renal free cholesterol levels are 44% elevated in the OVE26 mice. Together, our data support STARD5 functioning in kidney, specifically within proximal tubule cells, and suggest a role in ER-associated cholesterol transport.

The characterisation and uptake of paraquat in cultured baboon kidney proximal tubule cells (bPTC)

2001 ◽  
Vol 20 (2) ◽  
pp. 90-99 ◽  
Author(s):  
R Machaalani ◽  
V Lazzaro ◽  
G G Duggin
Keyword(s):  
Proximal Tubule ◽  
Mdck Cells ◽  
Distal Tubule ◽  
Genetic Mutation ◽  
Immunoperoxidase Staining ◽  
Renal Tubules ◽  
Trypan Blue Exclusion ◽  
Proximal Tubule Cells ◽  
Morphological Studies ◽  
Tubule Cells

A primary culture of baboon proximal tubule cells (bPTC) was prepared and characterised using LLC-PK1 cells of proximal tubule origin and MDCK cells of distal tubule origin, as positive and negative references, respectively. The proximal tubular origin of the bPTC was determined by morphological studies, immunoperoxidase staining and the expression of proximal tubule markers alkaline phosphatase and gammaglutamyltransferase. The hypothesis that paraquat (PQ) is transported by the bPTC was investigated. The cytotoxic threshold for PQ in these cells was determined and compared to the LLC-PK1 and MDCK cells. Furthermore, this study investigated the transport of the monovalent cation tetraethyl ammonium (TEA) and the polyvalent cation cimetidine in the bPTC and demonstrated their effect on the cellular uptake of PQ. The cytotoxic threshold of PQ in the bPTC, determined by cellular viability studies using the method of Trypan blue exclusion, is 0.05 mM at 2 h incubation. The LC50 after 24 his 76, 61 and 455 pM for the bPTC, LLC-PK1 and MDCK cells, respectively. This indicates that proximal tubule cells are more susceptible to PQ toxicity compared to distal tubule cells, which is consistent with clinical PQ toxicity where renal damage is found predominantly in the proximal renal tubules. The cations PQ and cimetidine were actively transported by the bPTC. The uptake of PQ (0.05 mM) commenced after 15 min whereas cimetidine (0.5 mM) uptake was evident after 2 min. Furthermore, cimetidine was shown to compete with PQ for uptake in the bPTC. Coincubating PQ (0.05 mM) and cimetidine (0.5 mM) for 60 min resulted in an approximate 50% decrease in PQ uptake. The cation TEA was not transported by the bPTC suggesting either a genetic mutation or complete absence of the transporter for TEA in the cells. The results suggest that PQ may be transported by the same cation transporter as cimetidine and not TEA, indicating PQ uptake in the bPTC to be via a polyvalent organic cation transporter.

scholarly journals Heat Shock Protein 70 and CHIP Promote Nox4 Ubiquitination and Degradation within the Losartan Antioxidative Effect in Proximal Tubule Cells

2015 ◽  
Vol 36 (6) ◽  
pp. 2183-2197 ◽  
Author(s):  
Andrea F. Gil Lorenzo ◽  
Valeria V. Costantino ◽  
Martin López Appiolaza ◽  
Valeria Cacciamani ◽  
Maria E. Benardon ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Proximal Tubule ◽  
Ros Generation ◽  
Antioxidative Effect ◽  
Proximal Tubule Cells ◽  
Caveolin 1 ◽  
Protein Levels ◽  
Hsp70 Protein

Background: Angiotensin II/Angiotensin II type 1 receptor (AT1R) effects are dependent on ROS production stimulated by NADPH oxidase activation. Hsp70 regulates a diverse set of signaling pathways through their interactions with proteins. CHIP is a E3 ubiquitin ligase that targets proteins for polyubiquitination and degradation. Aim: We study whether Hsp70/CHIP contribute to the negative regulation of Nox4 after AT1R blockage. Methods/Results: Primary culture of proximal tubule epithelial cells (PTCs) from SHR and WKY were stimulated with Angiotensin II (AII) or treated with Losartan (L) or Losartan plus Angiotensin II (L+AII). Losartan decreased AT1R and Nox4 while enhancing caveolin-1 and Hsp70 protein expression in SHR PTCs. Immunoprecipitation and immunofluorescence proved interaction and colocalization of increased Hsp70/CHIP with decreased Nox4 in SHR PTCs (L) vs (All). Hsp72 knockdown resulted in enhanced Nox4 protein levels, NADPH oxidase activity and ROS generation in (L+AII) revealing that Losartan was unable to abrogate AII effects on Nox4 expression and oxidative activity. Moreover, MG132 exposed PTCs (L) demostrated blocked ubiquitinated Nox4 degradation and increased colocalization of Nox4/Ubiquitin by inmunofluorescence. Conversely, Hsp72 depletion reduced Nox4/Ubiquitin colocalization causing Nox4 upregulation due to proteosomal degradation inhibition, although Losartan treatment. Conclusion: Our study demonstrates that Hsp70 and CHIP mediates the ubiquitination and proteasomal degradation of Nox4 as part of the antioxidative effect of Losartan in SHR.

Transepithelial fluxes of adenosine and 2′-deoxyadenosine across human renal proximal tubule cells: roles of nucleoside transporters hENT1, hENT2, and hCNT3

2009 ◽  
Vol 296 (6) ◽  
pp. F1439-F1451 ◽  
Author(s):  
Adam N. Elwi ◽  
Vijaya L. Damaraju ◽  
Michelle L. Kuzma ◽  
Delores A. Mowles ◽  
Stephen A. Baldwin ◽  
...  
Keyword(s):  
Cell Surface ◽  
Proximal Tubule ◽  
Apical Cell ◽  
Nucleoside Transporters ◽  
Proximal Tubule Cells ◽  
Protein Levels ◽  
Anti Cancer ◽  
Proximal Tubular ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

This study examined the roles of human nucleoside transporters (hNTs) in mediating transepithelial fluxes of adenosine, 2′-deoxyadenosine, and three purine nucleoside anti-cancer drugs across polarized monolayers of human renal proximal tubule cells (hRPTCs), which were shown in previous studies to have human equilibrative NT 1 (hENT1) and 2 (hENT2) and human concentrative NT 3 (hCNT3) activities ( 11 ). Early passage hRPTCs were cultured on transwell inserts under conditions that induced formation of polarized monolayers with experimentally accessible apical and basolateral domains. Polarized hRPTC cultures were monitored for inhibitor sensitivities and sodium-dependence of the following: 1) transepithelial fluxes of radiolabeled adenosine, 2′-deoxyadenosine, fludarabine (9-β-d-arabinosyl-2-fluoroadenine), cladribine (2-chloro-2′-deoxyadenosine), and clofarabine (2-chloro-2′-fluoro-deoxy-9-β-d-arabinofuranosyladenine); 2) mediated uptake of radiolabeled adenosine, 2′-deoxyadenosine, fludarabine, cladribine, and clofarabine from either apical or basolateral surfaces; and 3) relative apical cell surface hCNT3 protein levels. Transepithelial fluxes of adenosine were mediated from apical-to-basolateral sides by apical hCNT3 and basolateral hENT2, whereas transepithelial fluxes of 2′-deoxyadenosine were mediated from basolateral-to-apical sides by apical hENT1 and basolateral human organic anion transporters (hOATs). The transepithelial fluxes of adenosine, hCNT3-mediated cellular uptake of adenosine, and relative apical cell surface hCNT3 protein levels correlated positively in polarized hRPTCs. The purine nucleoside anti-cancer drugs fludarabine, cladribine, and clofarabine, like adenosine exhibited apical-to-basolateral fluxes. Collectively, this evidence suggested that apical hCNT3 and basolateral hENT2 are involved in proximal tubular reabsorption of adenosine and some nucleoside drugs and that apical hENT1 and basolateral hOATs are involved in proximal tubular secretion of 2′-deoxyadenosine.

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