Scopoletin alleviates high glucose-induced toxicity in human renal proximal tubular cells via inhibition of oxidative damage, epithelial-mesenchymal transition, and fibrogenesis.

BACKGROUND: Recent years of evidence suggest the crucial role of renal tubular cells in developing diabetic kidney disease. Scopoletin (SCOP) is a plant-based coumarin with numerous biological activities. This study aimed to determine the effect of SCOP on renal tubular cells in developing diabetic kidney disease and to elucidate mechanisms. METHODS AND RESULTS: In this study, SCOP was evaluated in vitro using renal proximal tubular (HK-2) cells under hyperglycemic conditions to understand its mechanism of action. In HK-2 cells, SCOP alleviated the high glucose-generated reactive oxygen species (ROS), restored the levels of reduced glutathione, and decreased lipid peroxidation. High glucose-induced alteration in the mitochondrial membrane potential was markedly restored in the SCOP-treated cells. Moreover, SCOP significantly reduced the high glucose-induced apoptotic cell population in the Annexin V-FITC flow cytometry study. Furthermore, high glucose markedly elevated the mRNA expression of fibrotic and extracellular matrix (ECM) components, namely, transforming growth factor (TGF)-ß, alfa-smooth muscle actin (α-SMA), collagen I, and collagen III, in HK-2 cells compared to the untreated cells. SCOP treatment reduced these mRNA expressions compared to the high glucose-treated cells. Collagen I and TGF-ß protein levels were also significantly reduced in the SCOP-treated cells. Further findings in HK-2 cells revealed that SCOP interfered with the epithelial-mesenchymal transition (EMT) in the high glucose-treated HK-2 cells by normalizing E-cadherin and downregulating the vimentin and α-SMA proteins. CONCLUSIONS: In conclusion, SCOP modulates the high glucose-generated renal tubular cell oxidative damage and accumulation of ECM components and may be a promising molecule against diabetic nephropathy.

Bioenergetics disruption, oxidative stress, and inflammation as underlying mechanisms of tramadol-induced nephrotoxicity.

Tramadol (TR), a commonly prescribed pain reliever for moderate to severe pain, has been associated with kidney damage. This study investigates TR-induced nephrotoxicity mechanisms, focusing on its effects on renal proximal tubular cells (PTCs). The study findings demonstrate that TR disrupts PTC bioenergetic processes, leading to oxidative stress and inflammation. Significant toxicity to PTCs was observed with estimated effective concentration 50 values of 9.8 and 11.5 µM based on 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, respectively. TR also interferes with the function of PTC transporters, including organic cation uptake transporter 1, organic cation transporter 2, P-glycoprotein, and multidrug resistance protein 2. Furthermore, bioenergetics assays showed that TR reduced the activities of mitochondrial complexes I and III, adenosine triphosphate production, mitochondrial membrane potential, and oxygen consumption rate while increasing lactate release. TR also increased the production of reactive oxygen species, lipid peroxidation thiobarbituric acid reactive substances end products, and the expression of the NRf2 gene while decreasing reduced glutathione (GSH-R) stores and catalase and superoxide dismutase antioxidant activities. Additionally, TR increased the production of inflammatory cytokines (TNF-α and IL-6) and their coding genes expression. Interestingly, the study found that antioxidants like GSH-R, inhibitors of IL-6 and TNF-α, and mitochondrial activating Co-Q10 could protect cells against TR-induced cytotoxicity. The study suggests that TR causes nephrotoxicity by disrupting bioenergetic processes, causing oxidative stress and inflammation, but antioxidants and agents targeting mitochondria may have protective and curative potential.

Redefining the Role of ADAM17 in Renal Proximal Tubular Cells and Its Implications in an Obese Mouse Model of Pre-Diabetes.

Acute and chronic kidney lesions induce an increase in A Disintegrin And Metalloproteinase domain 17 (ADAM17) that cleaves several transmembrane proteins related to inflammatory and fibrotic pathways. Our group has demonstrated that renal ADAM17 is upregulated in diabetic mice and its inhibition decreases renal inflammation and fibrosis. The purpose of the present study was to analyze how Adam17 deletion in proximal tubules affects different renal structures in an obese mice model. Tubular Adam17 knockout male mice and their controls were fed a high-fat diet (HFD) for 22 weeks. Glucose tolerance, urinary albumin-to-creatinine ratio, renal histology, and pro-inflammatory and pro-fibrotic markers were evaluated. Results showed that wild-type mice fed an HFD became obese with glucose intolerance and renal histological alterations mimicking a pre-diabetic condition; consequently, greater glomerular size and mesangial expansion were observed. Adam17 tubular deletion improved glucose tolerance and protected animals against glomerular injury and prevented podocyte loss in HFD mice. In addition, HFD mice showed more glomerular macrophages and collagen accumulation, which was prevented by Adam17 deletion. Galectin-3 expression increased in the proximal tubules and glomeruli of HFD mice and ameliorated with Adam17 deletion. In conclusion, Adam17 in proximal tubules influences glucose tolerance and participates in the kidney injury in an obese pre-diabetic murine model. The role of ADAM17 in the tubule impacts on glomerular inflammation and fibrosis.

Praliciguat inhibits progression of diabetic nephropathy in ZSF1 rats and suppresses inflammation and apoptosis in human renal proximal tubular cells.

Praliciguat, a clinical-stage soluble guanylate cyclase (sGC) stimulator, increases cGMP via the nitric oxide-sGC pathway. Praliciguat has been shown to be renoprotective in rodent models of hypertensive nephropathy and renal fibrosis. In the present study, praliciguat alone and in combination with enalapril attenuated proteinuria in the obese ZSF1 rat model of diabetic nephropathy. Praliciguat monotherapy did not affect hemodynamics. In contrast, enalapril monotherapy lowered blood pressure but did not attenuate proteinuria. Renal expression of genes in pathways involved in inflammation, fibrosis, oxidative stress, and kidney injury was lower in praliciguat-treated obese ZSF1 rats than in obese control rats; fasting glucose and cholesterol were also lower with praliciguat treatment. To gain insight into how tubular mechanisms might contribute to its pharmacological effects on the kidneys, we studied the effects of praliciguat on pathological processes and signaling pathways in cultured human primary renal proximal tubular epithelial cells (RPTCs). Praliciguat inhibited the expression of proinflammatory cytokines and secretion of monocyte chemoattractant protein-1 in tumor necrosis factor-α-challenged RPTCs. Praliciguat treatment also attenuated transforming growth factor-ß-mediated apoptosis, changes to a mesenchyme-like cellular phenotype, and phosphorylation of SMAD3 in RPTCs. In conclusion, praliciguat improved proteinuria in the ZSF1 rat model of diabetic nephropathy, and its actions in human RPTCs suggest that tubular effects may contribute to its renal benefits, building upon strong evidence for the role of cGMP signaling in renal health.

Fatty acids bound to albumin induce prostaglandin E2 production in human renal proximal tubular epithelial cell line HK-2.

Fatty acids bound to albumin have been reported to be involved in various responses in renal proximal tubular cells following albumin overload, leading to progression of tubulointerstitial damage in the kidneys. In addition, it has been reported that prostaglandin E2 (PGE2) plays an important role in nephrotoxicity. The aim of this study was to examine whether albumin-bound fatty acids induce PGE2 production in human renal proximal tubular epithelial cell line HK-2. Fatty acid-bearing human serum albumin increased PGE2 release in the culture medium in concentration-dependent and time-dependent manners, but fatty acid-depleted albumin had no effect on PGE2 production. Next, we investigated the effect of arachidonic acid, a precursor of eicosanoids, on PGE2 production. Arachidonic acid with fatty acid-free albumin significantly enhanced the release of PGE2 into the medium in a concentration-dependent manner. Furthermore, we examined the effect of arachidonic acid on mRNA expression of hypoxia inducible factor-1α (HIF-1α). Arachidonic acid increased HIF-1α mRNA expression in a concentration-dependent manner. These findings suggest that fatty acids, at least in part arachidonic acid, bound to albumin increase PGE2 production and expression of HIF-1α mRNA and protein, possibly resulting in various cell responses induced by albumin overload.

Lack of Cathepsin D in the Renal Proximal Tubular Cells Resulted in Increased Sensitivity against Renal Ischemia/Reperfusion Injury.

Cathepsin D is one of the major lysosomal aspartic proteases that is essential for the normal functioning of the autophagy-lysosomal system. In the kidney, cathepsin D is enriched in renal proximal tubular epithelial cells, and its levels increase during acute kidney injury. To investigate how cathepsin D-deficiency impacts renal proximal tubular cells, we employed a conditional knockout CtsDflox/-; Spink3Cre mouse. Immunohistochemical analyses using anti-cathepsin D antibody revealed that cathepsin D was significantly decreased in tubular epithelial cells of the cortico-medullary region, mainly in renal proximal tubular cells of this mouse. Cathepsin D-deficient renal proximal tubular cells showed an increase of microtubule-associated protein light chain 3 (LC3; a marker for autophagosome/autolysosome)-signals and an accumulation of abnormal autophagic structures. Renal ischemia/reperfusion injury resulted in an increase of early kidney injury marker, Kidney injury molecule 1 (Kim-1), in the cathepsin D-deficient renal tubular epithelial cells of the CtsDflox/-; Spink3Cre mouse. Inflammation marker was also increased in the cortico-medullary region of the CtsDflox/-; Spink3Cre mouse. Our results indicated that lack of cathepsin D in the renal tubular epithelial cells led to an increase of sensitivity against ischemia/reperfusion injury.

Protein kinase Cε targets respiratory chain and mitochondrial membrane potential but not F0 F1 -ATPase in renal cells injured by oxidant.

We have previously shown that protein kinase Cε (PKCε) is involved in mitochondrial dysfunction in renal proximal tubular cells (RPTC). This study examined mitochondrial targets of active PKCε in RPTC injured by the model oxidant tert-butyl hydroperoxide (TBHP). TBHP exposure augmented the levels of phosphorylated (active) PKCε in mitochondria, which suggested translocation of PKCε to mitochondria after oxidant exposure. Oxidant injury decreased state 3 respiration, adenosine triphosphate (ATP) production, ATP content, and complex I activity. Further, TBHP exposure increased ΔΨm and production of reactive oxygen species (ROS), and induced mitochondrial fragmentation and RPTC death. PKCε activation by overexpressing constitutively active PKCε exacerbated decreases in state 3 respiration, complex I activity, ATP content, and augmented RPTC death. In contrast, inhibition of PKCε by overexpressing dnPKCε mutant restored state 3 respiration, respiratory control ratio, complex I activity, ΔΨm , and ATP production and content, but did not prevent decreases in F0 F1 -ATPase activity. Inhibition of PKCε prevented oxidant-induced production of ROS and mitochondrial fragmentation, and reduced RPTC death. We conclude that activation of PKCε mediates: (a) oxidant-induced changes in ΔΨm , decreases in mitochondrial respiration, complex I activity, and ATP content; (b) mitochondrial fragmentation; and (c) RPTC death. In contrast, oxidant-induced inhibition of F0 F1 -ATPase activity is not mediated by PKCε. These results show that, in contrast to the protective effects of PKCε in the heart, PKCε activation is detrimental to mitochondrial function and viability in RPTC and mediates oxidant-induced injury.

Pioglitazone induces hypoxia-inducible factor 1 activation in human renal proximal tubular epithelial cell line HK-2.

We previously reported that fatty acid-bearing albumin but not fatty acid-depleted albumin induces hypoxia-inducible factor-1 (HIF-1) activation in human renal proximal tubular epithelial cell line HK-2. Then, an increase in mRNA expression of peroxisome proliferator-activated receptor gamma (PPARγ) was observed on treatment with fatty acid-bearing albumin but not fatty acid-depleted albumin. The aim of this study was to determine whether a PPARγ agonist, pioglitazone, induces HIF-1 activation or not. Treatment with pioglitazone induced HIF-1α mRNA as well as PPARγ mRNA expression in a concentration dependent manner. In addition, pioglitazone increased HIF-1 target genes such as the mRNAs of glucose transporter 1 (GLUT1) and breast cancer resistance protein (BCRP/ABCG2), in a concentration-dependent manner. Consistent with the increases in GLUT1 and ABCG2 mRNAs, protein expression of GLUT1 and BCRP was increased by pioglitazone. In addition, GLUT inhibitor phloretin-sensitive D-[3H]glucose uptake activity and BCRP inhibitor Ko143-sensitive accumulation of Hoecsht33342, a BCRP substrate, were significantly enhanced by treatment with pioglitazone. These findings suggest that PPARγ activation by pioglitazone leads to HIF-1 protein expression induction followed by changes in HIF-1 target gene expression and protein product activity.

Deletion of protein kinase C-ε attenuates mitochondrial dysfunction and ameliorates ischemic renal injury.

Previously, we documented that activation of protein kinase C-ε (PKC-ε) mediates mitochondrial dysfunction in cultured renal proximal tubule cells (RPTC). This study tested whether deletion of PKC-ε decreases dysfunction of renal cortical mitochondria and improves kidney function after renal ischemia. PKC-ε levels in mitochondria of ischemic kidneys increased 24 h after ischemia. Complex I- and complex II-coupled state 3 respirations were reduced 44 and 27%, respectively, in wild-type (WT) but unchanged and increased in PKC-ε-deficient (KO) mice after ischemia. Respiratory control ratio coupled to glutamate/malate oxidation decreased 50% in WT but not in KO mice. Activities of complexes I, III, and IV were decreased 59, 89, and 61%, respectively, in WT but not in KO ischemic kidneys. Proteomics revealed increases in levels of ATP synthase (α-subunit), complexes I and III, cytochrome oxidase, α-ketoglutarate dehydrogenase, and thioredoxin-dependent peroxide reductase after ischemia in KO but not in WT animals. PKC-ε deletion prevented ischemia-induced increases in oxidant production. Plasma creatinine levels increased 12-fold in WT and 3-fold in KO ischemic mice. PKC-ε deletion reduced tubular necrosis, brush border loss, and distal segment damage in ischemic kidneys. PKC-ε activation in hypoxic RPTC in primary culture exacerbated, whereas PKC-ε inhibition reduced, decreases in: 1) complex I- and complex II-coupled state 3 respirations and 2) activities of complexes I, III, and IV. We conclude that PKC-ε activation mediates 1) dysfunction of complexes I and III of the respiratory chain, 2) oxidant production, 3) morphological damage to the kidney, and 4) decreases in renal functions after ischemia.

Protein kinase C-α interaction with F0F1-ATPase promotes F0F1-ATPase activity and reduces energy deficits in injured renal cells.

We showed previously that active PKC-α maintains F0F1-ATPase activity, whereas inactive PKC-α mutant (dnPKC-α) blocks recovery of F0F1-ATPase activity after injury in renal proximal tubules (RPTC). This study tested whether mitochondrial PKC-α interacts with and phosphorylates F0F1-ATPase. Wild-type PKC-α (wtPKC-α) and dnPKC-α were overexpressed in RPTC to increase their mitochondrial levels, and RPTC were exposed to oxidant or hypoxia. Mitochondrial levels of the γ-subunit, but not the α- and ß-subunits, were decreased by injury, an event associated with 54% inhibition of F0F1-ATPase activity. Overexpressing wtPKC-α blocked decreases in γ-subunit levels, maintained F0F1-ATPase activity, and improved ATP levels after injury. Deletion of PKC-α decreased levels of α-, ß-, and γ-subunits, decreased F0F1-ATPase activity, and hindered the recovery of ATP content after RPTC injury. Mitochondrial PKC-α co-immunoprecipitated with α-, ß-, and γ-subunits of F0F1-ATPase. The association of PKC-α with these subunits decreased in injured RPTC overexpressing dnPKC-α. Immunocapture of F0F1-ATPase and immunoblotting with phospho(Ser) PKC substrate antibody identified phosphorylation of serine in the PKC consensus site on the α- or ß- and γ-subunits. Overexpressing wtPKC-α increased phosphorylation and protein levels, whereas deletion of PKC-α decreased protein levels of α-, ß-, and γ-subunits of F0F1-ATPase in RPTC. Phosphoproteomics revealed phosphorylation of Ser(146) on the γ subunit in response to wtPKC-α overexpression. We concluded that active PKC-α 1) prevents injury-induced decreases in levels of γ subunit of F0F1-ATPase, 2) interacts with α-, ß-, and γ-subunits leading to increases in their phosphorylation, and 3) promotes the recovery of F0F1-ATPase activity and ATP content after injury in RPTC.

Cinnamaldehyde and nitric oxide attenuate advanced glycation end products-induced the Jak/STAT signaling in human renal tubular cells.

Cinnamaldehyde is a major and a bioactive compound isolated from the leaves of Cinnamomum osmophloeum kaneh. It possesses anti-diabetic properties in vitro and in vivo and has anti-inflammatory and anti-cancer effects. To explore whether cinnamaldehyde was linked to altered advanced glycation end products (AGE)-mediated diabetic nephropathy, the molecular mechanisms of cinnamaldehyde responsible for inhibition of AGE-reduced nitric oxide (NO) bioactivity in human renal proximal tubular cells were examined. We found that raising the ambient AGE concentration causes a dose-dependent decrease in NO generation. Cinnamaldehyde significantly reverses AGE-inhibited NO generation and induces high levels of cGMP synthesis and PKG activation. Treatments with cinnamaldehyde, the NO donor S-nitroso-N-acetylpenicillamine, and the JAK2 inhibitor AG490 markedly attenuated AGE-inhibited NOS protein levels and NO generation. Moreover, AGE-induced the JAK2-STAT1/STAT3 activation, RAGE/p27(Kip1) /collagen IV protein levels, and cellular hypertrophy were reversed by cinnamaldehyde. The ability of cinnamaldehyde to suppress STAT activation was also verified by the observation that it significantly increased SCOS-3 protein level. These findings indicate for the first time that in the presence of cinnamaldehyde, the suppression of AGE-induced biological responses is probably mediated by inactivating the JAK2-STAT1/STAT3 cascade or activating the NO pathway.

Fatty acid-bearing albumin but not fatty acid-depleted albumin induces HIF-1 activation in human renal proximal tubular epithelial cell line HK-2.

Recently, we found that albumin overload induces expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) protein and several HIF-1 target genes in human renal proximal tubular epithelial cell line HK-2. In this study, the role of albumin-bound fatty acids in the albumin-induced HIF-1 activation was studied. The enhancing effect of fatty acid-bearing human serum albumin [FA(+)HSA] treatment on HIF-1α protein expression was much greater than that of fatty acid-depleted human serum albumin [FA(-)HSA] treatment. The FA(+)HSA treatment induced HIF-1 target gene mRNAs such as those of glucose transporter 1 (GLUT1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and breast cancer resistance protein (BCRP) in concentration-dependent manners, while FA(-)HSA caused no significant increases in these mRNAs. Consistent with increased GLUT1 mRNA, GLUT1 protein expression and GLUT inhibitor cytochalasin B-sensitive d-[(3)H]glucose uptake activity were significantly enhanced by treatment with FA(+)HSA, but not with FA(-)HSA. These findings indicate that fatty acids bound to albumin play a crucial role in albumin-induced HIF-1 activation followed by changes in HIF-1 target gene expression and protein product activity.

Renin angiotensin system modulates mTOR pathway through AT2R in HIVAN.

Mammalian target of rapamycin (mTOR) has been reported to contribute to the development of HIV-associated nephropathy (HIVAN). We hypothesized that HIV may be activating renal tissue mTOR pathway through renin angiotensin system (RAS) via Angiotensin Receptor Type II receptor (AT2R). Renal tissues of Vpr transgenic and Tg26 (HIVAN) mice displayed enhanced phosphorylation of mTOR and p70S6K. Aliskiren, a renin inhibitor attenuated phosphorylation of both mTOR and p70S6K in renal tissues of HIVAN mice. Interestingly, Angiotensin Receptor Type I (AT1R) blockade did not modulate renal tissue phosphorylation of mTOR in HIVAN mice; on the other hand, AT2R blockade attenuated renal tissue phosphorylation of mTOR in HIVAN mice. In vitro studies, both renin and Ang II displayed enhanced mouse tubular cell (MTC) phosphorylation of p70S6K in a dose dependent manner. HIV/MTC also displayed enhanced phosphorylation of both mTOR and p70S6K; interestingly this effect of HIV was further enhanced by losartan (an AT1R blocker). On the other hand, AT2R blockade attenuated HIV-induced tubular cell phosphorylation of mTOR and p70S6K, whereas, AT2R agonist enhanced phosphorylation of mTOR and p70S6K. These findings indicate that HIV stimulates mTOR pathway in HIVAN through the activation of renin angiotensin system via AT2R.

Blockage of Akt activation suppresses cadmium-induced renal tubular cellular damages through aggrephagy in HK-2 cells.

We have reported that an environmental pollutant, cadmium, promotes cell death in the human renal tubular cells (RTCs) through hyperactivation of a serine/threonine kinase Akt. However, the molecular mechanisms downstream of Akt in this process have not been elucidated. Cadmium has a potential to accumulate misfolded proteins, and proteotoxicity is involved in cadmium toxicity. To clear the roles of Akt in cadmium exposure-induced RTCs death, we investigated the possibility that Akt could regulate proteotoxicity through autophagy in cadmium chloride (CdCl2)-exposed HK-2 human renal proximal tubular cells. CdCl2 exposure promoted the accumulation of misfolded or damaged proteins, the formation of aggresomes (pericentriolar cytoplasmic inclusions), and aggrephagy (selective autophagy to degrade aggresome). Pharmacological inhibition of Akt using MK2206 or Akti-1/2 enhanced aggrephagy by promoting dephosphorylation and nuclear translocation of transcription factor EB (TFEB)/transcription factor E3 (TFE3), lysosomal transcription factors. TFEB or TFE3 knockdown by siRNAs attenuated the protective effects of MK2206 against cadmium toxicity. These results suggested that aberrant activation of Akt attenuates aggrephagy via TFEB or TFE3 to facilitate CdCl2-induced cell death. Furthermore, these roles of Akt/TFEB/TFE3 were conserved in CdCl2-exposed primary human RTCs. The present study shows the molecular mechanisms underlying Akt activation that promotes cadmium-induced RTCs death.

Mitotherapy inhibits against tenofovir induced nephrotoxicity on rat renal proximal tubular cells.

Tenofovir, as nucleotide reverse transcriptase inhibitors (NRTIs), is used to prevent and cure HIV/AIDS. Ample evidence confirmed that the nephrotoxicity of tenofovir has been linked to mitochondrial dysfunction. It seems that transplantation with healthy mitochondria instead of damaged mitochondria may be a beneficial approach to therapy. Therefore, it decided to investigate the impact of mitotherapy on tenofovir against renal proximal tubular cells (RPTCs) toxicity by measurement of oxidative stress and cytotoxicity biomarkers and restoring of mitochondrial function on isolated mitochondria. EC50 of tenofovir was achieved at 40 µM following 2 h incubation in Earle's solution (pH = 7.4; 37 °C). Freshly isolated mitochondria (80 µg/ml) were added to damage RPTCs affected by tenofovir in treated groups. One Way ANOVA analysis showed that healthy mitochondrial transplantation decreased oxidative stress biomarkers following tenofovir toxicity in RPTCs. Our data revealed that mitotherapy makes cell survival possible in RPTCs affected by tenofovir. In addition, it supposed that a novel and ideal strategy for the treatment of chemicals-induced nephrotoxicity.

Mitochondrial Transplantation Alleviates Doxorubicin-Induced Toxicity in Rat Renal Cells.

Background: Doxorubicin (DOX) is used in the treatment of various cancers and has good effectiveness. However, its therapeutic use is limited due to its effects on various organs and healthy cells. Doxorubicin can affect the kidneys and cause toxicity. Evidence shows that DOX induces nephrotoxicity through oxidative stress. Objectives: In this research, we examined the effect of mitochondrial transplantation on improving mitochondrial and cellular toxicity caused by DOX on renal proximal tubular cells (RPTCs). Methods: The research measured 7 toxicity parameters, including cell lysis, reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) decline, GSH and GSSG content, lipid peroxidation (LPO), adenosine triphosphate (ATP) content, and Caspase-3 activity (the final mediator of apoptosis). Active fresh mitochondria were prepared from Wistar rat kidney. Results: The findings indicated that DOX caused cytotoxicity in RPTCs. Additionally, DOX induced oxidative stress by increasing the level of reactive oxygen species, reducing glutathione content, and elevating lipid peroxidation. Moreover, it led to damage to the mitochondrial membrane, increased caspase-3 activity, and decreased ATP content. Mitochondrial transplantation, as a new therapeutic approach, reduced oxidative stress, mitochondrial membrane damage, and apoptosis caused by DOX in RPTCs. Furthermore, this therapeutic approach increased the ATP content in RPTCs. Conclusions: Our study suggests that this therapeutic approach could be helpful in the treatment of drug-induced nephrotoxicity.

Isoliquiritin Ameliorates Cisplatin-Induced Renal Proximal Tubular Cell Injury by Antagonizing Apoptosis, Oxidative Stress and Inflammation.

Acute kidney injury (AKI) is a clinical syndrome characterized by morbidity, mortality, and cost. Cis-diamminedichloroplatinum (cisplatin) is a chemotherapeutic agent used to treat solid tumors and hematological malignancies, but its side effects, especially nephrotoxicity, limit its clinical application. Isoliquiritin (ISL), one of the major flavonoid glycoside compounds in licorice, has been reported to have anti-apoptotic, antioxidant, and anti-inflammatory activities. However, the effect and mechanism of ISL on cisplatin-induced renal proximal tubular cell injury remain unknown. In this study, mouse proximal tubular cells (mPTCs) and human proximal tubule epithelial cells (HK2) were administered increasing concentrations of ISL from 7.8125 to 250 µM. Moreover, mPTC and HK2 cells were pretreated with ISL for 6-8 h, followed by stimulation with cisplatin for 24 h. CCK-8 assay was performed to evaluate the cell viability. Apoptosis and reactive oxygen species (ROS) of cells were measured by using flow cytometer and western blotting. Our results showed that ISL had no obvious effect on cell viability. ISL decreased cisplatin-induced cell injury in a dose-dependent manner. ISL also protected against cisplatin-induced cell apoptosis. Meanwhile, the enhanced protein levels of Bax, cleaved caspase-3/caspase-3 ratio, levels of Pp-65/p-65, levels of IL-6, and the production of ROS induced by cisplatin were significantly attenuated by ISL treatment. Moreover, ISL markedly increased the protein levels of Bcl-2 and SOD2, which were reduced by cisplatin stimulation. These results showed that ISL ameliorated cisplatin-induced renal proximal tubular cell injury by antagonizing apoptosis, oxidative stress and inflammation.

Nephroprotective potential of Panduratin A against colistin-induced renal injury via attenuating mitochondrial dysfunction and cell apoptosis.

Colistin is a last-resort polypeptide antibiotic widely used to treat against multidrug-resistant Gram-negative bacterial infections. However, this treatment is associated with nephrotoxicity. The aim of this study was to examine the potential protective effect of panduratin A, a bioactive compound of Boesenbergia rotunda, on colistin-induced nephrotoxicity in both in vivo and in vitro models. Intraperitoneal injection of 15 mg/kg colistin for 7 days markedly promoted renal tubular degeneration, increased blood urea nitrogen (BUN) levels, and upregulated the expression of renal injury biomarker and apoptosis proteins. In addition, treatment with colistin increased oxidative stress and apoptosis in mice kidney tissues. Interestingly, these defects were attenuated when co-administered of colistin with panduratin A (2.5 or 25 mg/kg). The underlying mechanisms of panduratin A attenuating colistin toxicity was investigated in human renal proximal tubular cells (RPTEC/TERT1). The mechanisms by which colistin-triggered cytotoxicity was determined by analysis of cell death, reactive oxygen species (ROS) levels, mitochondria function as well as the expression of proteins related to apoptosis pathway. Colistin treatment (200 µg/ml) significantly increased cell apoptosis, elevated ROS production, reduced mitochondrial membrane potential, and decreased anti-apoptotic protein (Bcl-2) expression. These effects were notably suppressed by co-treatment with panduratin A (5 µM). Collectively, panduratin A exerts as a novel nephroprotective agent to protect against colistin-induced renal injury by attenuating mitochondrial damage and renal cell apoptosis.

Recent advances in nanotechnology-based drug delivery systems for the kidney.

The application of nanotechnology in medicine has the potential to make a great impact on human health, ranging from prevention to diagnosis and treatment of disease. The kidneys are the main organ of the human urinary system, responsible for filtering the blood, and concentrating metabolic waste into urine by means of the renal glomerulus. The glomerular filtration apparatus presents a barrier against therapeutic agents based on charge and/or molecular size. Therefore, drug delivery to the kidneys faces significant difficulties resulting in treatment failure in several renal disorders. Accordingly, different strategies have recently being explored for enhancing the delivery of therapeutic agents across the filtration barrier of the glomerulus. Nanosystems with different physicochemical properties, including size, shape, surface, charge, and possessing biological features such as high cellular internalization, low cytotoxicity, controllable pharmacokinetics and biodistribution, have shown promising results for renal therapy. Different types of nanoparticles (NPs) have been used to deliver drugs to the kidney. In this review, we discuss nanotechnology-based drug delivery approaches for acute kidney injury, chronic kidney disease, renal fibrosis, renovascular hypertension and kidney cancer.

A Cycle of Altered Proteasome and Reactive Oxygen Species Production in Renal Proximal Tubular Cells.

AIMS: An intricate relationship exists between the mitochondrial function and proteasome activity. Our recent report showed in a rat model of renal transplantation that mitochondrial dysfunction precedes compromised proteasome function and this results in a vicious cycle of mitochondrial injury and proteasome dysfunction. In this study, we studied whether reactive oxygen species (ROS) has a role in proteasome alteration in renal cells and vice versa. METHODS: We used the genomic and pharmacologic approach on rat normal kidney proximal tubular (NRK) cell lines. First, we knocked down ß5 or Rpt6 subunit of the proteasome using small interfering RNA (siRNA) in NRK cells. We also treated NRK cells with Bortezomib, a proteasome inhibitor, and peroxynitrite (a potent ROS). RESULTS: Studies with RNA interference showed increased mitochondrial ROS following knockdown of ß5 or Rpt6 subunit in NRK cells. Similarly, pharmacological inhibition of the proteasome in NRK cells using Bortezomib also showed an increase of mitochondrial ROS in a dose-dependent manner. Next, exposing NRK cells to different concentrations of peroxynitrite provided evidence that the higher levels of peroxynitrite exposure decreased the key subunits (ß5 and α3) of the proteasome in NRK cells. CONCLUSION: Our results suggest that proteasome inhibition/downregulation increases ROS, which then impairs proteasome subunits in renal proximal tubular cells.