A tubule cell model for ifosfamide nephrotoxicity

Mechanisms leading to ifosfamide (IF)-induced renal damage have not been fully elucidated. Recent work suggests that localized renal tubular metabolism of IF and the production of the nephrotoxic chloroacetaldehyde may lead to nephrotoxicity. Presently no pharmacological method to reduce IF nephrotoxicity has been identified. The objectives of this study were to establish a tubule cell model for IF nephrotoxicity, to verify whether renal proximal tubular cells have the necessary cytochrome P450 (CYP) enzymes to oxidize IF, and whether they can metabolize IF to chloroacetaldehyde. CYP3A, and 2B mRNA and protein were identified in LLCPK-1 cells. The cells metabolized the R- and S-IF enantiomers to their respective 2- and 3-dechloroethylifosfamide metabolites, by-products of chloroacetal dehyde formation. Metabolite production was both time and concentration-dependent. IF did not affect cell viability. In contrast, glutathione-depleted cells showed time and dose-dependent damage. The presence of the relevant CYP enzymes in renal tubular cells along with their ability to metabolize IF to its 2- and 3-dechloroethylifosfamide metabolites suggests that nephrotoxic damage may result from the localized production of chloroacetaldehyde. Glutathione is a major defence mechanism against IF toxicity, thus pharmacological methods for replenishing intracellular glutathione may be effective in modulating IF-induced nephrotoxicity. Key words: LLCPK-1, metabolism, ifosfamide, renal, CYP3A, CYP2B.

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Abstract 210: Stem Cell Conditioned Culture Media Attenuated Albumin-induced Epithelial-mesenchymal Transition in Renal Tubular Cells

Hypertension ◽

10.1161/hyp.62.suppl_1.a210 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Junping Hu ◽

Weiqing Han ◽

Qing Zhu ◽

Pin-Lan Li ◽

Ningjun Li

Keyword(s):

Epithelial Mesenchymal Transition ◽

Culture Media ◽

Mesenchymal Transition ◽

Tubular Cells ◽

Protein Levels ◽

Renal Tubular Cells ◽

Proximal Tubular ◽

Renal Proximal Tubular Cells ◽

Renal Proximal Tubular ◽

Renal Tubular

Mesenchymal stem cells (MSCs) have been shown to be a promising therapy for many different diseases. Stem cell conditioned culture media (SCM) exhibit similar beneficial effects as MSCs. Albuminuria-induced epithelial-mesenchymal transition (EMT) plays an important role in progressive renal tubulointerstitial fibrosis in chronic renal disease. The present study tested the hypothesis that SCM inhibit albumin-induced EMT in cultured renal tubular cells. SCM were obtained by culturing rat adult MSCs for 3 days. Cultured renal proximal tubular cells were incubated with rat albumin (20μg/ml) and treated with SCM or control culture media. Our results showed that 48 h albumin incubation stimulated EMT in renal proximal tubular cells as shown by significant decrease in the protein levels of epithelial marker E-cadherin from 2.30 ± 0.27 to 0.87 ± 0.11 ( P < 0.05) and increase in the protein levels of mesenchymal marker fibroblast-specific protein 1 (FSP-1) (2.18±0.33 folds, P < 0.05). SCM treatment significantly inhibited these albumin-induced changes in E-cadherin and FSP-1 by 2.33±0.17 and 1.95±0.23 folds ( P < 0.05), respectively. Meanwhile, albumin increased the mRNA levels of pro-inflammatory factor monocyte chemoattractant protein-1 (MCP)-1 by nearly 30 folds compared with control. SCM almost abolished the increase of MCP-1 induced by albumin. Furthermore, Western blot results displayed that albumin rapidly decreased the cytosolic levels and increased the nuclear levels of NF-κB, indicating a translocation of NF-κB; immunofluorescence microscopy also demonstrated that albumin induced NF-κB translocation from the cytosol into nucleus. SCM blocked the translocation of NF-κB into nucleus. These results suggest that SCM attenuated albumin-induced EMT in renal tubular cells via inhibiting NF-κB activation and inflammation, which may serve as a new therapeutic approach for chronic kidney diseases. (Supported by NIH grant HL89563 and HL106042)

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Autophagy Deficiency in Renal Proximal Tubular Cells Leads to an Increase in Cellular Injury and Apoptosis under Normal Fed Conditions

International Journal of Molecular Sciences ◽

10.3390/ijms21010155 ◽

2019 ◽

Vol 21 (1) ◽

pp. 155 ◽

Cited By ~ 3

Author(s):

Chigure Suzuki ◽

Isei Tanida ◽

Juan Alejandro Oliva Trejo ◽

Soichiro Kakuta ◽

Yasuo Uchiyama

Keyword(s):

Renal Injury ◽

Kidney Injury ◽

Renal Tissue ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Renal Tubular Cells ◽

Proximal Tubular ◽

Renal Proximal Tubular Cells ◽

Renal Proximal Tubular ◽

Renal Tubular

Renal proximal tubular epithelial cells are significantly damaged during acute kidney injury. Renal proximal tubular cell-specific autophagy-deficient mice show increased sensitivity against renal injury, while showing few pathological defects under normal fed conditions. Considering that autophagy protects the proximal tubular cells from acute renal injury, it is reasonable to assume that autophagy contributes to the maintenance of renal tubular cells under normal fed conditions. To clarify this possibility, we generated a knock out mouse model which lacks Atg7, a key autophagosome forming enzyme, in renal proximal tubular cells (Atg7flox/flox;KAP-Cre+). Analysis of renal tissue from two months old Atg7flox/flox;KAP-Cre+ mouse revealed an accumulation of LC3, binding protein p62/sequestosome 1 (a selective substrate for autophagy), and more interestingly, Kim-1, a biomarker for early kidney injury, in the renal proximal tubular cells under normal fed conditions. TUNEL (TdT-mediated dUTP Nick End Labeling)-positive cells were also detected in the autophagy-deficient renal tubular cells. Analysis of renal tissue from Atg7flox/flox;KAP-Cre+ mice at different age points showed that tubular cells positive for p62 and Kim-1 continually increase in number in an age-dependent manner. Ultrastructural analysis of tubular cells from Atg7flox/flox;KAP-Cre+ revealed the presence of intracellular inclusions and abnormal structures. These results indicated that autophagy-deficiency in the renal proximal epithelial tubular cells leads to an increase in injured cells in the kidney even under normal fed conditions.

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Empagliflozin Ameliorates Free Fatty Acid Induced-Lipotoxicity in Renal Proximal Tubular Cells via the PPARγ/CD36 Pathway in Obese Mice

International Journal of Molecular Sciences ◽

10.3390/ijms222212408 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12408

Author(s):

Chiang-Chi Huang ◽

Chia-An Chou ◽

Wei-Yu Chen ◽

Jenq-Lin Yang ◽

Wen-Chin Lee ◽

...

Keyword(s):

Body Weight Gain ◽

Specific Inhibitor ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Renal Tubular Cells ◽

Cd36 Expression ◽

Proximal Tubular ◽

Renal Proximal Tubular Cells ◽

Renal Proximal Tubular ◽

Renal Tubular

High serum levels of free fatty acids (FFAs) could contribute to obesity-induced nephropathy. CD36, a class B scavenger receptor, is a major receptor mediating FFA uptake in renal proximal tubular cells. Empagliflozin, a new anti-diabetic agent, is a specific inhibitor of sodium-glucose co-transporter 2 channels presented on renal proximal tubular cells and inhibits glucose reabsorption. In addition, empagliflozin has shown renoprotective effects. However, the mechanism through which empagliflozin regulates CD36 expression and attenuates FFA-induced lipotoxicity remains unclear. Herein, we aimed to elucidate the crosstalk between empagliflozin and CD36 in FFA-induced renal injury. C57BL/6 mice fed a high-fat diet (HFD) and palmitic acid-treated HK-2 renal tubular cells were used for in vivo and in vitro assessments. Empagliflozin attenuated HFD-induced body weight gain, insulin resistance, and inflammation in mice. In HFD-fed mice, CD36 was upregulated in the tubular area of the kidney, whereas empagliflozin attenuated CD36 expression. Furthermore, empagliflozin downregulated the expression of peroxisome proliferator-activated receptor (PPAR)-γ. Treatment with a PPARγ inhibitor (GW9662) did not further decrease PPARγ expression, whereas a PPARγ antagonist reversed this effect; this suggested that empagliflozin may, at least partly, decrease CD36 by modulating PPARγ. In conclusion, empagliflozin can ameliorate FFA-induced renal tubular injury via the PPARγ/CD36 pathway.

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Prolactin and dopamine 1-like receptor interaction in renal proximal tubular cells

AJP Renal Physiology ◽

10.1152/ajprenal.00582.2009 ◽

2010 ◽

Vol 299 (1) ◽

pp. F49-F54 ◽

Cited By ~ 13

Author(s):

Susanne Crambert ◽

Agneta Sjöberg ◽

Ann-Christine Eklöf ◽

Fernando Ibarra ◽

Ulla Holtbäck

Keyword(s):

Protein Kinase ◽

Atpase Activity ◽

Proximal Tubular Cells ◽

Dopamine System ◽

Tubular Cells ◽

Proximal Tubular ◽

Renal Proximal Tubular Cells ◽

Renal Proximal Tubular ◽

Renal Tubular ◽

Renal Dopamine

Prolactin is a natriuretic hormone and acts by inhibiting the activity of renal tubular Na+-K+-ATPase activity. These effects require an intact renal dopamine system. Here, we have studied by which mechanism prolactin and dopamine interact in Sprague-Dawley rat renal tissue. Na+-K+-ATPase activity was measured as ouabain-sensitive ATP hydrolysis in microdissected renal proximal tubular segments. Intracellular signaling pathways were studied by a variety of different techniques, including Western blotting using phosphospecific antibodies, immunoprecipitation, and biotinylation assays. We found that dopamine and prolactin regulated Na+-K+-ATPase activity via similar signaling pathways, including protein kinase A, protein kinase C, and phosphoinositide 3-kinase activation. The cross talk between prolactin and dopamine 1-like receptors was explained by a heterologous recruitment of dopamine 1-like receptors to the plasma membrane in renal proximal tubular cells. Prolactin had no effect on Na+-K+-ATPase activity in spontaneously hypertensive rats, a rat strain with a blunted response to dopamine. These results further emphasize the central role of the renal dopamine system in the interactive regulation of renal tubular salt balance.

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mTOR contributes to ER stress and associated apoptosis in renal tubular cells

AJP Renal Physiology ◽

10.1152/ajprenal.00629.2014 ◽

2015 ◽

Vol 308 (3) ◽

pp. F267-F274 ◽

Cited By ~ 18

Author(s):

Guie Dong ◽

Yu Liu ◽

Lei Zhang ◽

Shuang Huang ◽

Han-Fei Ding ◽

...

Keyword(s):

Er Stress ◽

Kidney Diseases ◽

Renal Diseases ◽

Therapeutic Effects ◽

Tubular Cells ◽

Tunicamycin Treatment ◽

Renal Tubular Cells ◽

Renal Proximal Tubular Cells ◽

Renal Proximal Tubular ◽

Induced Apoptosis

ER stress has been implicated in the pathogenesis of both acute and chronic kidney diseases. However, the molecular regulation of ER stress in kidney cells and tissues remains poorly understood. In this study, we examined tunicamycin-induced ER stress in renal proximal tubular cells (RPTC). Tunicamycin induced the phosphorylation and activation of PERK and eIF2α within 2 h in RPTC, which was followed by the induction of GRP78 and CHOP. Consistently, tunicamycin also induced apoptosis in RPTC. Interestingly, mTOR was activated rapidly during tunicamycin treatment, as indicated by phosphorylation of both mTOR and p70S6K. Inhibition of mTOR with rapamycin partially suppressed the phosphorylation of PERK and eIF2a and the induction of CHOP and GRP78 induction during tunicamycin treatment. Rapamycin also inhibited apoptosis during tunicamycin treatment and increased cell survival. Collectively, the results suggest that mTOR plays a regulatory role in ER stress, and inhibition of mTOR may have potential therapeutic effects in ER stress-related renal diseases.

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Sodium-dependent phosphate cotransporter type 1 sequence polymorphisms in male patients with gout

Annals of the Rheumatic Diseases ◽

10.1136/ard.2008.106856 ◽

2009 ◽

Vol 69 (6) ◽

pp. 1232-1234 ◽

Cited By ~ 44

Author(s):

Wako Urano ◽

Atsuo Taniguchi ◽

Naohiko Anzai ◽

Eisuke Inoue ◽

Yoshikatsu Kanai ◽

...

Keyword(s):

Nucleotide Polymorphisms ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Renal Tubular Cells ◽

Sodium Dependent ◽

Proximal Tubular ◽

Male Patients ◽

Renal Proximal Tubular Cells ◽

Renal Proximal Tubular

ObjectivesMolecular biological approaches have recently identified urate transporters in renal proximal tubular cells. Human sodium-dependent phosphate cotransporter type 1 encoded by SLC17A1 is a urate transporter localised to the renal proximal tubular cells and candidate molecule to secret urate from renal tubular cells to urine. This study investigated the roles of SLC17A1 in the development of gout.Patients and MethodsSingle nucleotide polymorphisms in the human SLC17A1 gene (rs1165176, rs1165151, rs1165153, rs1165196, rs1165209, rs1165215, rs1179086, rs3799344 and rs3757131) were selected, and an association study was conducted using male patients with gout (n=175) and male controls (n=595).ResultsThere were significant differences between gout and control groups in the distribution of genotypes at rs1165196 (T806C; Ile269Thr, odds ratio (OR) 0.55, p=0.0035), rs1179086 (OR 0.57, p=0.0018) and rs3757131 (OR 0.54, p=0.0026). In controls, T806C alone had no effect on serum uric acid (sUA) levels. However, T806C showed significant interaction with a reduction of sUA in obese individuals (body mass index ≥25) using multiple regression analysis.ConclusionsOur data suggest that SLC17A1 polymorphisms are associated with the development of gout.

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Response gene to complement 32 interacts with Smad3 to promote epithelial-mesenchymal transition of human renal tubular cells

AJP Cell Physiology ◽

10.1152/ajpcell.00204.2010 ◽

2011 ◽

Vol 300 (6) ◽

pp. C1415-C1421 ◽

Cited By ~ 25

Author(s):

Xia Guo ◽

Pedro A. Jose ◽

Shi-You Chen

Keyword(s):

Epithelial Mesenchymal Transition ◽

Extracellular Matrix Protein ◽

Proximal Tubular Cells ◽

Response Gene ◽

Mesenchymal Transition ◽

Tubular Cells ◽

Renal Tubular Cells ◽

Proximal Tubular ◽

Renal Proximal Tubular Cells ◽

Renal Proximal Tubular

Previous studies demonstrate that response gene to complement 32 (RGC-32) mediates transforming growth factor-β1-induced epithelial-mesenchymal transition (EMT) of human renal proximal tubular cells. However, the mechanisms underlying RGC-32 function remain largely unknown. In the present study, we found that RGC-32 function in EMT is associated with Smad3. Coexpression of RGC-32 and Smad3, but not Smad2, induces a higher mesenchymal marker α-smooth muscle actin (α-SMA) protein expression as compared with RGC-32 or Smad3 alone, while knockdown of Smad3 using short hairpin interfering RNA blocks RGC-32-induced α-SMA expression. These data suggest that RGC-32 interacts with Smad3, but not Smad2, in the regulation of EMT. In addition to α-SMA, RGC-32 and Smad3 also synergistically activate the expression of extracellular matrix protein fibronectin and downregulate the epithelial marker E-cadherin. RGC-32 colocalizes with Smad3 in the nuclei of renal proximal tubular cells. Coimmunoprecipitation assays showed that Smad3, but not Smad2, physically interacts with RGC-32 in renal proximal tubular cells. Mechanistically, RGC-32 and Smad3 coordinate the induction of EMT by regulating the EMT regulators Slug and Snail. Taken together, our data demonstrate for the first time that RGC-32 interacts with Smad3 to mediate the EMT of human renal proximal tubular cells.

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