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

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Abstract P238: Regulation Of G Protein-coupled Receptor Kinase 4 Expression By Serum In Breast Cancer And Renal Proximal Tubule Cells

Hypertension ◽

10.1161/hyp.78.suppl_1.p238 ◽

2021 ◽

Vol 78 (Suppl_1) ◽

Author(s):

Wei Yue ◽

Peng Xu ◽

John J Gildea ◽

Robin A Felder

Keyword(s):

Breast Cancer ◽

Protein Synthesis ◽

Proximal Tubule ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Renal Proximal Tubule ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Renal Proximal Tubule Cells ◽

G Protein Coupled

G protein-coupled receptor kinase 4 (GRK4) is a member of the GRK family which play critical role in regulation of the function of G protein-coupled receptors. Our previous studies have shown that GRK4 not only plays a role in regulating sodium excretion in renal proximal tubule cells but also acts as a stimulator on proliferation of breast cancer cells. Uncontrolled proliferation is a characteristics of cancer cells and GRK4 is upregulated in breast cancer cells. We hypothesized that expression of GRK4 may be regulated differently in cancer and non-cancer cells. To test this hypothesis, expression of GRK4 in response to serum was compared in breast cancer cells and renal proximal tubule cells by Western analysis. In three breast cancer cell lines serum withdrawal caused rapid reduction in the levels of GRK4 which occurred as early as 15 min. GRK4 levels correlated with the concentrations of serum added to the culture media. To determine if growth factors were a critical element for maintaining GRK4 levels in the cells, EGF (10-20 ng/ml) was added to serum free medium for 24 h. There was no increase in GRK4 levels in the cells treated with EGF compared with the serum starvation control. Similarly, serum withdrawal (16 h) led to 40-80% decrease of GRK4 levels in renal proximal tubule cells even in the presence of EFG supplement. Serum feeding for 30 min after starvation dramatically increased the levels of GRK4 in both breast cancer cells and RPTC which exceeded the steady state levels. This rapid recovery of GRK4 protein do not need de novo protein synthesis because pretreatment of the cells with protein synthesis inhibitor, cycloheximide (10 μg/ml, 24 h), did not prevent this event. Expression of GRK2, another member of the GRK family, was not affected by serum starvation. Our results have shown that GRK4 is very sensitive to serum concentration in breast cancer cells as well as in RPTC. Preliminary studies suggest that rapid protein degradation rather than shutting down the protein synthesis plays a major role in this kind of GRK4 regulation. The biological significance of serum regulation of GRK4 in cancer and non-cancerous cells needs further investigation.

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6-Shogaol protects against ischemic acute kidney injury by modulating NF-κB and heme oxygenase-1 pathways

AJP Renal Physiology ◽

10.1152/ajprenal.00182.2019 ◽

2019 ◽

Vol 317 (3) ◽

pp. F743-F756 ◽

Cited By ~ 3

Author(s):

Sang Jun Han ◽

Mihwa Kim ◽

Vivette D. D’Agati ◽

H. Thomas Lee

Keyword(s):

Acute Kidney Injury ◽

Proximal Tubule ◽

Heme Oxygenase ◽

Kidney Injury ◽

Heme Oxygenase 1 ◽

Zinc Protoporphyrin ◽

Mrna Synthesis ◽

Proximal Tubule Cells ◽

Tnf Α ◽

Tubule Cells

Acute kidney injury (AKI) due to renal ischemia-reperfusion (I/R) is a major clinical problem without effective therapy. Ginger is one of the most widely consumed spices in the world, and 6-shogaol, a major ginger metabolite, has anti-inflammatory effects in neuronal and epithelial cells. Here, we demonstrate our novel findings that 6-shogaol treatment protected against renal I/R injury with decreased plasma creatinine, blood urea nitrogen, and kidney neutrophil gelatinase-associated lipocalin mRNA synthesis compared with vehicle-treated mice subjected to renal I/R. Additionally, 6-shogaol treatment reduced kidney inflammation (decreased proinflammatory cytokine and chemokine synthesis as well as neutrophil infiltration) and apoptosis (decreased TUNEL-positive renal tubular cells) compared with vehicle-treated mice subjected to renal I/R. In cultured human and mouse kidney proximal tubule cells, 6-shogaol significantly attenuated TNF-α-induced inflammatory cytokine and chemokine mRNA synthesis. Mechanistically, 6-shogaol significantly attenuated TNF-α-induced NF-κB activation in human renal proximal tubule cells by reducing IKKαβ/IκBα phosphorylation. Furthermore, 6-shogaol induced a cytoprotective chaperone heme oxygenase (HO)-1 via p38 MAPK activation in vitro and in vivo. Consistent with these findings, pretreatment with the HO-1 inhibitor zinc protoporphyrin IX completely prevented 6-shogaol-mediated protection against ischemic AKI in mice. Taken together, our study showed that 6-shogaol protects against ischemic AKI by attenuating NF-κB activation and inducing HO-1 expression. 6-Shogaol may provide a potential therapy for ischemic AKI during the perioperative period.

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Anti-inflammatory and antinecrotic effects of the volatile anesthetic sevoflurane in kidney proximal tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.00412.2005 ◽

2006 ◽

Vol 291 (1) ◽

pp. F67-F78 ◽

Cited By ~ 64

Author(s):

H. Thomas Lee ◽

Mihwa Kim ◽

Michael Jan ◽

Charles W. Emala

Keyword(s):

Cell Death ◽

Proximal Tubule ◽

Volatile Anesthetics ◽

Necrotic Cell Death ◽

Necrotic Cell ◽

Proximal Tubule Cells ◽

Kidney Proximal Tubule ◽

Tnf Α ◽

Tubule Cells ◽

Anti Inflammatory

Renal ischemia-reperfusion (IR) injury is a major clinical problem without effective therapy. We recently reported that volatile anesthetics protect against renal IR injury, in part, via their anti-inflammatory properties. In this study, we demonstrate the anti-inflammatory and antinecrotic effects of sevoflurane in cultured kidney proximal tubule cells and probed the mechanisms of sevoflurane-induced renal cellular protection. To mimic inflammation, human kidney proximal tubule (HK-2) cells were treated with tumor necrosis factor-α (TNF-α; 25 ng/ml) in the presence or absence of sevoflurane. In addition, we studied the effects of sevoflurane pretreatment on hydrogen peroxide (H2O2)-induced necrotic cell death in HK-2 or porcine proximal tubule (LLC-PK1) cells. We demonstrate that sevoflurane suppressed proinflammatory effects of TNF-α evidenced by attenuated upregulation of proinflammatory cytokine mRNA (TNF-α, MCP-1) and ICAM-1 protein and reduced nuclear translocation of the proinflammatory transcription factors NF-κB and AP-1. Sevoflurane reduced necrotic cell death induced with H2O2 in HK-2 cells as well as in LLC-PK1 cells. Sevoflurane treatment resulted in phosphorylation of prosurvival kinases, ERK and Akt, and increased de novo HSP-70 protein synthesis without affecting the synthesis of HSP-27 or HSP-32. We conclude that sevoflurane has direct anti-inflammatory and antinecrotic effects in vitro in a renal cell type particularly sensitive to injury following IR injury. These mechanisms may, in part, account for volatile anesthetics' protective effects against renal IR injury.

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

AJP Renal Physiology ◽

10.1152/ajprenal.00593.2016 ◽

2017 ◽

Vol 312 (6) ◽

pp. F1056-F1062 ◽

Cited By ~ 6

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.

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

AJP Renal Physiology ◽

10.1152/ajprenal.90433.2008 ◽

2009 ◽

Vol 297 (2) ◽

pp. F380-F388 ◽

Cited By ~ 16

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.

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

Human & Experimental Toxicology ◽

10.1191/096032701672136818 ◽

2001 ◽

Vol 20 (2) ◽

pp. 90-99 ◽

Cited By ~ 10

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.

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CD40 ligation stimulates MCP-1 and IL-8 production, TRAF6 recruitment, and MAPK activation in proximal tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.00291.2001 ◽

2002 ◽

Vol 282 (6) ◽

pp. F1020-F1033 ◽

Cited By ~ 52

Author(s):

Hongye Li ◽

Edward P. Nord

Keyword(s):

Protein Kinase ◽

Proximal Tubule ◽

Receptor Activation ◽

Cell Fractionation ◽

Cd40 Ligation ◽

Chemokine Production ◽

Proximal Tubule Cells ◽

Tubule Cells ◽

Cytoplasmic Compartment ◽

Human Proximal Tubule

The mechanism of CD40/CD154-induced chemokine production and its potential role in renal inflammatory disease were explored. Human proximal tubule cells maintained in primary culture were used as the experimental model. With the use of immunocytochemistry, confocal microscopy, and a cell fractionation assay, the CD40 receptor was found to be expressed in the cell membrane of the epithelial cell, and, on engagement by CD154, its cognate ligand, translocated to the cytoplasmic compartment. Engagement of CD40 by CD154 stimulated interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) production, which proceeded via receptor activation of the extracellular signal-regulated kinase (ERK)1/2, stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways. CD40 ligation also engaged tumor necrosis factor receptor-activating factor 6 (TRAF6), as evidenced by colocalization of the activated receptor with TRAF6 in the cytoplasmic compartment, translocation of both proteins from the insoluble to the soluble cell fraction, and coimmunoprecipitation of the two proteins only under ligand-stimulated conditions. Furthermore, an antisense oligodeoxyribonucleotide targeted against TRAF6 mRNA blunted p38 and SAPK/JNK but not ERK1/2 MAPK activities, as well as IL-8 and MCP-1 production, arguing that TRAF6 is an upstream activator. The zinc chelator TPEN, but not the calcium chelator BAPTA, obliterated CD154-evoked MAPK activity and chemokine production, providing indirect evidence for protein-protein interactions playing a critical role in CD40 signaling in these cells. We conclude that in human proximal tubule cells, CD40 and TRAF6 reside in separate low-density, detergent-insoluble membrane microdomains, or rafts, and on activation translocate and associate with one another probably via zinc-finger domains in the soluble or cytoplasmic compartment. TRAF6, in turn, activates SAPK/JNK and p38 MAPK phosphorylation, which in turn stimulates IL-8 and MCP-1 production in these cells.

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Regulation of S6 kinase activity in renal proximal tubule

AJP Renal Physiology ◽

10.1152/ajprenal.1992.263.1.f127 ◽

1992 ◽

Vol 263 (1) ◽

pp. F127-F134 ◽

Cited By ~ 3

Author(s):

R. C. Harris

Keyword(s):

Protein Synthesis ◽

Angiotensin Ii ◽

Proximal Tubule ◽

Renal Mass ◽

Kinase Activity ◽

Renal Cortex ◽

S6 Kinase ◽

Proximal Tubule Cells ◽

Hypertrophic Growth ◽

Tubule Cells

The proximal tubule undergoes hypertrophy in response to loss of functioning renal mass and hyperplasia following injury by ischemia or nephrotoxins. Both hypertrophic growth and cell proliferation are characterized by increases in the rate of protein synthesis. To investigate regulation of protein synthesis in mammalian proximal tubule cells, potential peptide mediators of proximal tubule growth, epidermal growth factor (EGF) and angiotensin II, were studied in cultured rabbit proximal tubule cells. Although only EGF stimulated DNA synthesis, both agonists stimulated protein synthesis. One potential regulatory mechanism of eukaryotic protein synthesis involves phosphorylation of ribosomal protein S6 by activation of a specific serine/threonine kinase (S6 kinase). Both EGF and angiotensin II stimulated S6 kinase activity and S6 phosphorylation. Phorbol 12-myristate 13-acetate was also found to activate S6 kinase, and 24 h of pretreatment to deplete protein kinase C inhibited subsequent S6 kinase activation by a high concentration (10(-6) M) of angiotensin II. To determine whether S6 kinase was also activated in the kidney in vivo, S6 kinase activity was examined after ablation of renal mass. Within 1 h after contralateral nephrectomy, S6 kinase activity increased in rat renal cortex. In summary, both EGF and angiotensin II stimulated protein synthesis and S6 kinase activity in cultured proximal tubule cells, and S6 kinase activity also increased in renal cortex after contralateral nephrectomy.

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