Cisplatin Injury Research Articles

Kidney double positive T cells have distinct characteristics in normal and diseased kidneys

Multiple types of T cells have been described and assigned pathophysiologic functions in the kidneys. However, the existence and functions of TCR+CD4+CD8+ (double positive; DP) T cells are understudied in normal and diseased murine and human kidneys. We studied kidney DPT cells in mice at baseline and after ischemia reperfusion (IR) and cisplatin injury. Additionally, effects of viral infection and gut microbiota were studied. Human kidneys from patients with renal cell carcinoma were evaluated. Our results demonstrate that DPT cells expressing CD4 and CD8 co-receptors constitute a minor T cell population in mouse kidneys. DPT cells had significant Ki67 and PD1 expression, effector/central memory phenotype, proinflammatory cytokine (IFNγ, TNFα and IL-17) and metabolic marker (GLUT1, HKII, CPT1a and pS6) expression at baseline. IR, cisplatin and viral infection elevated DPT cell proportions, and induced distinct functional and metabolic changes. scRNA-seq analysis showed increased expression of Klf2 and Ccr7 and enrichment of TNFα and oxidative phosphorylation related genes in DPT cells. DPT cells constituted a minor population in both normal and cancer portion of human kidneys. In conclusion, DPT cells constitute a small population of mouse and human kidney T cells with distinct inflammatory and metabolic profile at baseline and following kidney injury.

Adipose Stromal Cell-Derived Secretome Attenuates Cisplatin-Induced Injury In Vitro Surpassing the Intricate Interplay between Proximal Tubular Epithelial Cells and Macrophages.

(1) Background: The chemotherapeutic drug cisplatin exerts toxic side effects causing acute kidney injury. Mesenchymal stromal cells can ameliorate cisplatin-induced kidney injury. We hypothesize that the MSC secretome orchestrates the vicious cycle of injury and inflammation by acting on proximal tubule epithelial cells (PTECs) and macrophages individually, but further by counteracting their cellular crosstalk. (2) Methods: Conditioned medium (CM) from adipose stromal cells was used, first assessing its effect on cisplatin injury in PTECs. Second, the effects of cisplatin and the CM on macrophages were measured. Lastly, in an indirect co-culture system, the interplay between the two cell types was assessed. (3) Results: First, the CM rescued PTECs from cisplatin-induced apoptosis by reducing oxidative stress and expression of nephrotoxicity genes. Second, while cisplatin exerted only minor effects on macrophages, the CM skewed macrophage phenotypes to the anti-inflammatory M2-like phenotype and increased phagocytosis. Finally, in the co-culture system, the CM suppressed PTEC death by inhibiting apoptosis and nuclei fragmentation. The CM lowered TNF-α release, while cisplatin inhibited macrophage phagocytosis, PTECs, and the CM to a greater extent, thus enhancing it. The CM strongly dampened the inflammatory macrophage cytokine secretion triggered by PTECs. (4) Conclusions: ASC-CM surpasses the PTEC-macrophage crosstalk in cisplatin injury. The positive effects on reducing cisplatin cytotoxicity, on polarizing macrophages, and on fine-tuning cytokine secretion underscore MSCs' CM benefit to prevent kidney injury progression.

Inhibition of the HMGB1/RAGE axis protects against cisplatin-induced ototoxicity via suppression of inflammation and oxidative stress.

As an anti-tumor drug widely used in the clinic, cisplatin is limited by its ototoxic side effects associated with various factors, including inflammatory responses. Receptor for Advanced Glycation Endproducts (RAGE) recognizes damage-associated molecular patterns (DAMPs) and promotes stress and inflammation. This study intended to determine the potential behavior of the HMGB1/RAGE axis after cisplatin injury and whether it has a protective effect after inhibiting this pathway. We used FPS-ZM1, a RAGE inhibitor, to modulate the axis of HMGB1/RAGE in neonatal mouse cochlear explants and C57BL/6 mice in vivo. Apoptosis was identified by Annexin V-FITC/PI assay, Cleaved Caspase-3, and TUNEL staining. Reactive oxygen species (ROS) level was assessed by MitoSOX Red and CellROX Green assay. The expression of proteins associated with the HMGB1/RAGE axis and apoptosis was observed by western blotting. The expression of inflammatory cytokines was evaluated by qPCR. The protective effect of HMGB1/RAGE knockdown was also assessed on cisplatin-induced ototoxicity. These results demonstrated that cisplatin could activate the HMGB1/RAGE pathway in cochlear hair cells and release inflammatory factors. Pretreatment with FPS-ZM1 alleviated cisplatin-induced ototoxicity in vivo and in vitro. Knocking down HMGB1 and RAGE achieved specific protective effects. Altogether, inhibiting HMGB1/RAGE axis can reverse the increase of ROS accumulation, the activation of apoptosis, and the production of inflammatory reactions after cisplatin injury. FPS-ZM1 could resist the ototoxicity of cisplatin by suppressing the HMGB1/RAGE signal pathway, and it may be considered the new otoprotective potential strategy for hearing loss.

Sulfide:quinone oxidoreductase alleviates ferroptosis in acute kidney injury via ameliorating mitochondrial dysfunction of renal tubular epithelial cells

Ferroptosis is iron-dependent and regulates necrosis caused by lipid peroxidation and mitochondrial damage. Recent evidence has revealed an emerging role for ferroptosis in the pathophysiology of acute kidney injury (AKI). Sulfide:quinone oxidoreductase (SQOR) is a mitochondrial inner membrane protein highly expressed in the renal cortex. However, the effects of SQOR on ferroptosis and AKI have not been elucidated. In this study, we evaluated the effects of SQOR in several AKI models. We observed a rapid decrease in SQOR expression after cisplatin stimulation in both in vivo and in vitro models. SQOR-deletion mice exhibit exacerbated kidney impairment and ferroptosis in renal tubular epithelial cells following cisplatin injury. Additionally, our results showed that the overexpression of SQOR or ADT-OH (the slow-releasing H2S donor) preserved renal function in the three AKI mouse models. These effects were evidenced by lower levels of serum creatinine (SCr), blood urea nitrogen (BUN), renal neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule 1 (KIM-1). Importantly, SQOR knockout significantly aggravates cisplatin-induced ferroptosis by promoting mitochondrial dysfunction in renal tubular epithelial cells (RTECs). Moreover, online database analysis combined with our study revealed that SYVN1, an upregulated E3 ubiquitin ligase, may mediate the ubiquitin-mediated degradation of SQOR in AKI. Consequently, our results suggest that SYVN1-mediated ubiquitination degradation of SQOR may induce mitochondrial dysfunction in RTECs, exacerbating ferroptosis and thereby promoting the occurrence and development of AKI. Hence, targeting the SYVN1-SQOR axis could be a potential therapeutic strategy for AKI treatment.

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) protects hair cells from cisplatin-induced ototoxicity in vitro: possible relation to the activities of p38 MAPK signaling pathway.

The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) gene encodes rate-limiting enzyme in cholesterol biosynthesis, which is related to cell proliferation and mitochondrial function. The present study was designed to explore the expression of HMGCR in murine cochlear hair cells and HEI-OC1 cells and the possible mechanisms underpinning the actions of HMGCR in cisplatin-induced ototoxicity, with special attention given to p38 mitogen-activated protein kinase (MAPK) activities in vitro. The expressions of HMGCR, p-p38, cleaved caspase-3 and LC3B was measured by immunofluorescence and western blot. JC-1 staining and MitoSOX Red were used to detect mitochondria membrane potential (MMP) and reactive oxygen species (ROS) levels respectively. The apoptosis of auditory cells was assessed by TUNEL staining and flow cytometry. Protein levels of bcl2/bax and beclin1 were examined by western blot. We found that HMGCR was widely expressed in the auditory cells, of both neonatal mice and 2-month-old mice, in cytoplasm, nucleus and stereocilia. Moreover, 30μM cisplatin elicited the formation of ROS, which, in turn, led to HMGCR reduction, activating p38 kinase-related apoptosis and autophagy in auditory cells. Meanwhile, co-treatment with ROS scavenger at a concentration of 2mM, N-acetyl-L-cysteine (NAC), could alleviate the aforementioned changes. In addition, HMGCR silencing resulted in higher p38 MAPK-mediated apoptosis and autophagy under cisplatin injury. Taken together, we demonstrate that, for the first time, that HMGCR is expressed in the cochlear. Furthermore, HMGCR exerts protective benefit on auditory cells against cisplatin-mediated injury stimulated by ROS, culminating in regulation of p38 MAPK-dependent apoptosis and autophagy.

TIGIT mediates experimental ischemia reperfusion and nephrotoxic acute kidney injury in mice

Abstract Background: We unexpectedly found upregulated expression of T cell immunoreceptor with Ig and ITIM domains (TIGIT) on kidney CD4 T cells after ischemia reperfusion (IR) using RNA-Seq. However, TIGIT role in kidney T cells and during acute kidney injury (AKI) is poorly understood. Methods: Effect of TIGIT on murine kidney T cells was assessed at baseline and after IR injury by flow cytometry and single cell RNA sequencing (scRNA-Seq). B6 wild type (WT) and TIGIT knockout (KO) mice were subjected to IR and cisplatin-induced AKI. TIGIT expression was assessed in AKI patients using the Kidney Precision Medicine Project (KPMP) scRNA-Seq dataset. Results: We found increased kidney CD4+TIGIT+ central memory (10.0±2.3 vs 0.9±0.1%, p=0.001) cells compared to CD4+TIGIT− cells in WT mice. TIGIT KO mice had reduced serum creatinine (SCr) after IR (24h Scr, 1.1±0.2 vs 2.7±0.1 mg/dL; p≤0.001) and cisplatin (72h Scr, 0.8±0.1 vs 1.4±0.1 mg/dL; p=0.0002) injury compared to WT mice. TIGIT KO kidneys had reduced necrotic tubules in outer medulla after IR (48.8±4.7% vs 73.0±1.5%; p=0.001) and cisplatin (11.5±3.0% vs 31.3±4.0%; p=0.008) injury than WT kidneys. Gene set enrichment analysis of scRNA-Seq data showed enrichment of inflammatory genes in Th17 cells from WT kidney and oxidative phosphorylation and mTORC1 signaling genes in TIGIT KO kidney. Human KPMP data demonstrated significant TIGITexpression in kidney T/NK cells from AKI patients compared to controls (p≤0.0001). Conclusions: TIGIT mediates both IR and nephrotoxic AKI in mice and its expression increases in kidney T cells from AKI patients. TIGIT is a promising novel therapeutic target for AKI, and also relevant due to increasing efforts to develop TIGIT blockade to treat cancers. American Society of Nephrology, Johns Hopkins University Ed Kraus Award, National Kidney Foundation of Maryland and Delaware

Rapamycin Perfluorocarbon Nanoparticle Mitigates Cisplatin-Induced Acute Kidney Injury.

For nearly five decades, cisplatin has played an important role as a standard chemotherapeutic agent and been prescribed to 10-20% of all cancer patients. Although nephrotoxicity associated with platinum-based agents is well recognized, treatment of cisplatin-induced acute kidney injury is mainly supportive and no specific mechanism-based prophylactic approach is available to date. Here, we postulated that systemically delivered rapamycin perfluorocarbon nanoparticles (PFC NP) could reach the injured kidneys at sufficient and sustained concentrations to mitigate cisplatin-induced acute kidney injury and preserve renal function. Using fluorescence microscopic imaging and fluorine magnetic resonance imaging/spectroscopy, we illustrated that rapamycin-loaded PFC NP permeated and were retained in injured kidneys. Histologic evaluation and blood urea nitrogen (BUN) confirmed that renal structure and function were preserved 48 h after cisplatin injury. Similarly, weight loss was slowed down. Using western blotting and immunofluorescence staining, mechanistic studies revealed that rapamycin PFC NP significantly enhanced autophagy in the kidney, reduced the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as decreased the expression of the apoptotic protein Bax, all of which contributed to the suppression of apoptosis that was confirmed with TUNEL staining. In summary, the delivery of an approved agent such as rapamycin in a PFC NP format enhances local delivery and offers a novel mechanism-based prophylactic therapy for cisplatin-induced acute kidney injury.

SIRT3/GLUT4 signaling activation by metformin protect against cisplatin-induced ototoxicity in vitro.

Cisplatin is highly effective for killing tumor cells. However, as one of its side effects, ototoxicity limits the clinical application of cisplatin. The mechanisms of cisplatin-induced ototoxicity have not been fully clarified yet. SIRT3 is a deacetylated protein mainly located in mitochondria, which regulates a variety of physiological processes in cells. The role of SIRT3 in cisplatin-induced hair cell injury has not been founded. In this study, primary cultured cochlear explants exposed to 5μM cisplatin, as well as OC-1 cells exposed to 10μM cisplatin, were used to establish models of cisplatin-induced ototoxicity in vitro. We found that when combined with cisplatin, metformin (75μM) significantly up-regulated the expression of SIRT3 and alleviated cisplatin-induced apoptosis of hair cells. We regulated the expression of SIRT3 to explore the role of SIRT3 in cisplatin-induced auditory hair cell injury. Overexpression of SIRT3 promoted the survival of auditory hair cells and alleviated the apoptosis of auditory hair cells. In contrast, knockdown of SIRT3 impaired the protective effect of metformin and exacerbated cisplatin injury. In addition, we found that the protective effect of SIRT3 may be achieved by regulating GLUT4 translocation and rescuing impaired glucose uptake caused by cisplatin. Our study confirmed that upregulation of SIRT3 may antagonize cisplatin-induced ototoxicity, and provided a new perspective for the study of cisplatin-induced ototoxicity.

C-phycocyanin alleviated cisplatin-induced oxidative stress and inflammation via gut microbiota-metabolites axis in mice.

C-phycocyanin is a natural protein extracted from Spirulina platensis. We aim to investigate the preventive effect of C-phycocyanin on cisplatin chemotherapy-induced oxidative damage and inflammation. The result showed that C-phycocyanin treatment reduced cisplatin-induced mortality and inflammation including decreased levels of serum IL6, kidney MCP1, and liver IL1β. Furthermore, C-phycocyanin also exerted antioxidant effects on mice, including increased GSH-Px, GGT, and GSH levels in the liver and increased CAT and SOD levels in the kidney. HepG2 cells experiments showed that C-phycocyanin exhibited none of the prevention effects on cisplatin injury. Faecalibaculum showed the greatest reduction among genera after cisplatin treatment, which was related to the enrichment of Romboutsia and Lactobacillus genera. C-phycocyanin treatment reduced the populations of harmful bacteria of Enterococcus faecalis, which was positively correlated with inflammation induced by cisplatin. C-phycocyanin increased the contents of 23-nordeoxycholic acid and β-muricholic acid. Moreover, C-phycocyanin increased amino acid-related metabolites, Nα-acetyl-arginine and trimethyl-lysine contents, and decreased fatty acid esters of hydroxy fatty acids (FAHFAs) contents. In conclusion, C-phycocyanin inhibited inflammation via the 23-nordeoxycholic acid-Enterococcus faecalis-inflammation axis, and enhanced the antioxidant capacity of kidney via Lactobacillus-NRF2 pathway. C-phycocyanin alleviated cisplatin injury via the modulation of gut microbiota, especially Lactobacillus and Enterococcus, as well as regulation of metabolites, especially bile acid and FAHFAs, which highlight the effect of C-phycocyanin and provide a new strategy to prevent cisplatin injury.

P53/sirtuin 1/NF-κB Signaling Axis in Chronic Inflammation and Maladaptive Kidney Repair After Cisplatin Nephrotoxicity.

Chronic inflammation contributes to maladaptive kidney repair, but its regulation is unclear. Here, we report that sirtuin 1 (SIRT1) is downregulated after repeated low-dose cisplatin (RLDC) injury, and this downregulation leads to p65 acetylation and consequent NF-κB activation resulting in a persistent inflammatory response. RLDC induced the down-regulation of SIRT1 and activation of NF-κB, which were accompanied by chronic tubular damage, tubulointerstitial inflammation, and fibrosis in mice. Inhibition of NF-κB suppressed the production of pro-inflammatory cytokines and fibrotic phenotypes in RLDC-treated renal tubular cells. SIRT1 activation by its agonists markedly reduced the acetylation of p65 (a key component of NF-κB), resulting in the attenuation of the inflammatory and fibrotic responses. Conversely, knockdown of SIRT1 exacerbated these cellular changes. At the upstream, p53 was activated after RLDC treatment to repress SIRT1, resulting in p65 acetylation, NF-κB activation and transcription of inflammatory cytokines. In mice, SIRT1 agonists attenuated RLDC-induced chronic inflammation, tissue damage, and renal fibrosis. Together, these results unveil the p53/SIRT1/NF-κB signaling axis in maladaptive kidney repair following RLDC treatment, where p53 represses SIRT1 to increase p65 acetylation for NF-κB activation, leading to chronic renal inflammation.

Cisplatin exposure acutely disrupts mitochondrial bioenergetics in the zebrafish lateral-line organ

Cisplatin is a commonly used chemotherapeutic agent that causes debilitating high-frequency hearing loss. No targeted therapies currently exist to treat cisplatin ototoxicity, partly because the underlying mechanisms of cisplatin-induced hair cell damage are not completely defined. Zebrafish may offer key insights to cisplatin ototoxicity because their lateral-line organ contains hair cells that are remarkably similar to those within the cochlea but are optically accessible, permitting observation of cisplatin injury in live intact hair cells. In this study, we used a combination of genetically encoded biosensors in zebrafish larvae and fluorescent indicators to characterize changes in mitochondrial bioenergetics in response to cisplatin. Following exposure to cisplatin, confocal imaging of live intact neuromasts demonstrated increased mitochondrial activity. Staining with fixable fluorescent dyes that accumulate in active mitochondria similarly showed hyperpolarized mitochondrial membrane potential. Zebrafish expressing a calcium indicator within their hair cells revealed elevated levels of mitochondrial calcium immediately following completion of cisplatin treatment. A fluorescent ROS indicator demonstrated that these changes in mitochondrial function were associated with increased oxidative stress. After a period of recovery, cisplatin-exposed zebrafish demonstrated caspase-3-mediated apoptosis. Altogether, these findings suggest that cisplatin acutely disrupts mitochondrial bioenergetics and may play a key role in initiating cisplatin ototoxicity.

C-Myb protects cochlear hair cells from cisplatin-induced damage via the PI3K/Akt signaling pathway

The transcription factor c-Myb is vital for cell survival, proliferation, differentiation, and apoptosis. We have previously reported that c-Myb knockdown exacerbates neomycin-induced damage to cochlea cells. However, the function and regulation of c-Myb in the mammalian inner ear remains unclear. Here, we first found that the expression of c-Myb in cochlear HCs was downregulated after cisplatin damage in vivo. Next, to investigate the role of c-Myb in HCs treated with cisplatin, the recombinant virus AAV-ie-CAG-Myb-HA (AAV-c-Myb) that overexpresses c-Myb was constructed and transfected into HCs. The protein expression of c-Myb was effectively up-regulated in cultured cochlear HCs after the virus transfection, which increased cochlear HC viability, decreased HC apoptosis and reduced intracellular reactive oxygen species (ROS) levels after cisplatin injury in vitro. The overexpression of c-Myb in HCs after AAV-c-Myb transfection in vivo also promoted HC survival, improved the hearing function of mice and reduced HC apoptosis after cisplatin injury. Furthermore, c-Myb-HC conditional knockout mice (Prestin; c-Myb-cKO) in which c-Myb expression is downregulated only in cochlear OHCs were generated and the cisplatin-induced HCs loss, apoptosis and hearing deficit were all exacerbated in Prestin; c-Myb-cKO mice treated with cisplatin in vivo. Finally, mechanistic studies showed that upregulation of the PI3K/Akt signaling pathway by c-Myb contributed to the increased HC survival after cisplatin exposure in vitro. The findings from this work suggest that c-Myb might serve as a new target for the prevention of cisplatin-induced HC damage and hearing loss.

Preferential siRNA delivery to injured kidneys for combination treatment of acute kidney injury

Acute kidney injury (AKI) is characterized by a sudden loss of renal function and is associated with high morbidity and mortality. Tumor suppressor p53 and chemokine receptor CXCR4 were both implicated in the AKI pathology. Here, we report on the development and evaluation of polymeric CXCR4 antagonist (PCX) siRNA carrier for selective delivery to injured kidneys in AKI. Our results show that PCX/siRNA nanoparticles (polyplexes) provide protection against cisplatin injury to tubule cells in vitro when both CXCR4 and p53 are inhibited. The polyplexes selectively accumulate and are retained in the injured kidneys in cisplatin and bilateral ischemia reperfusion injury models of AKI. Treating AKI with the combined CXCR4 inhibition and p53 gene silencing with the PCX/sip53 polyplexes improves kidney function and decreases renal damage. Overall, our results suggest that the PCX/sip53 polyplexes have a significant potential to enhance renal accumulation in AKI and deliver therapeutic siRNA.

DNA damage is overcome by TRIP13 overexpression during cisplatin nephrotoxicity.

Cisplatin is a commonly used chemotherapeutic agent to treat a wide array of cancers that is frequently associated with toxic injury to the kidney due to oxidative DNA damage and perturbations in cell cycle progression leading to cell death. In this study, we investigated whether thyroid receptor interacting protein 13 (TRIP13) plays a central role in the protection of the tubular epithelia following cisplatin treatment by circumventing DNA damage. Following cisplatin treatment, double-stranded DNA repair pathways were inhibited using selective blockers to proteins involved in either homologous recombination or non-homologous end joining. This led to increased blood markers of acute kidney injury (AKI) (creatinine and neutrophil gelatinase–associated lipocalin), tubular damage, activation of DNA damage marker (γ-H2AX), elevated appearance of G2/M blockade (phosphorylated histone H3 Ser10 and cyclin B1), and apoptosis (cleaved caspase-3). Conditional proximal tubule–expressing Trip13 mice were observed to be virtually protected from the cisplatin nephrotoxicity by restoring most of the pathological phenotypes back toward normal conditions. Our findings suggest that TRIP13 could circumvent DNA damage in the proximal tubules during cisplatin injury and that TRIP13 may constitute a new therapeutic target in protecting the kidney from nephrotoxicants and reduce outcomes leading to AKI.

Knockdown of sorcin increases HEI-OC1 cell damage induced by cisplatin in vitro

Hearing loss caused by ototoxic drugs is a kind of acquired hearing loss. Cisplatin is one of the most commonly used drugs and its main action sites are hair cells (HCs). Sorcin is a drug-resistant calcium-binding protein belonging to the small penta-EF-hand protein family. Sorcin is highly expressed in many tissues, including bone, heart, brain, lung, and skin tissues. Single-cell RNA sequencing showed that sorcin was expressed in the outer HCs of mice, but its role remained unknown. We also found that sorcin was highly expressed in the cytoplasm of cochlear HCs and HEI-OC1 cells. After cisplatin injury, the expression of sorcin in HCs and HEI-OC1 cells decreased significantly. SiRNA transfection technology was used to knock down the expression of sorcin. The results showed that the number of apoptotic cells, the expression of cleaved caspased-3, and the expression of Bax increased while the anti-apoptotic factor Bcl-2 decreased in the siRNA-Sorcin + CIS group. The observed increase in apoptosis was related to the increase of reactive oxygen species (ROS) and the destruction of the mitochondrial membrane potential (MMP). Finally, we found that the downregulated sorcin worked by activating the P-ERK1/2 signaling pathway. Overall, this study showed that sorcin can be used as a new target to prevent the ototoxicity of platinum drugs.

Ginsenoside Rg1 and Resveratrol Alleviate Acute Kidney Injury Induced by Cisplatin via Downregulation of Autophagy in Mice

Background: Cisplatin, a chemotherapeutic agent, is widely used in the treatment of malignant tumors. Nephrotoxicity, especially acute kidney injury (AKI), is the most common and severe adverse reaction of cisplatin. Resveratrol and ginsenoside Rg1, two natural products, have been found to have renal protective effects. However, the effects and the mechanisms in cisplatin-induced AKI need further investigation. Methods: The mouse models of cisplatin-induced AKI and several treatment groups were established. Male C57BL/6 mice were divided into five groups: saline control group, cisplatin injury group, resveratrol treatment group, Rg1 treatment group, resveratrol and Rg1 combined treatment group. Serological analysis of serum urea nitrogen was aimed to reflect the function of kidney, and histological analysis of renal tissue sections was aimed to assess the damage of proximal convoluted tubules. The expression levels of autophagy-related proteins Beclin 1 and LC3 were detected by western blotting and qRT-PCR respectively. Results: The renal function was improved and renal damage was alleviated in Rg1 and resveratrol alone or combined treatment groups compared with the cisplatin injury group. For the mechanism, treatment with Rg1 and resveratrol alone or in combination decreased the expressions of Beclin 1 both at protein and mRNA levels, decreased LC3II/I protein levels, indicating that autophagy was inhibited by treatment with Rg1 and resveratrol alone or in combination. Conclusion: Resveratrol and Rg1 alleviated the kidney damage caused by cisplatin, and reduced autophagy was involved in the renoprotective effects of resveratrol and Rg1 against cisplatin-induced AKI. This study may provide new evidence to alleviate cisplatin-induced AKI.

Vitamin D/VDR attenuate cisplatin-induced AKI by down-regulating NLRP3/Caspase-1/GSDMD pyroptosis pathway

Vitamin D/Vitamin D receptor (VDR) has been shown to inhibit the NF-κB-mediated inflammatory effects. Up-regulation of the NLRP3(Recombinant NLR Family, Pyrin Domain Containing Protein 3)/Caspase-1/GSDMD (Gasdermin D) pathway through NF-κb is one of the key mechanisms leading to pyroptosis. This study aims to explore the effects of vitamin D/VDR on the pyroptosis pathway in cisplatin induced acute kidney injury (AKI) models. Our results showed that in wide type mice, renal function loss, tissue injury and cell death induced by cisplatin were alleviated by pretreatment of high-dose paricalcitol(a VDR agonist) accompanied with up-regulated VDR and decreased expression of NLRP3, GSDMD-N, Cleaved-Caspase-1 and mature Interleukin- 1β (features of pyroptosis). While, in VDR knock out mice, cisplatin induced more severer renal injury and further increased pyroptosis related protein than the wild type mice and the effect of paricalcitol were also eliminated. In tubular cell specific VDR-over expressing mice, those renal injury index as well as pyroptosis phenotype were significantly reduced by low-dose paricalcitol pretreatment with upregulated VDR expression compared with WT mice. In vitro data using gain and lose function experiments in Human tubular epithelial cell (HK-2) were consistent with the observation as in vivo work. Our further experiments in both animal and cell culture work has found that the level of IκBα(Inhibitor of NF-κB) were decreased and the nuclear level of NF-κB p65 of renal tubular cells were increased after cisplatin injury while VDR activation by paricalcitol could reverse up-regulation of nuclear NF-κB p65 with reduced cell pyroptosis. These data suggested that vitamin D/VDR could alleviate cisplatin-induced acute renal injury partly by inhibiting NF-κB-mediated NLRP3/Caspase-1/GSDMD pyroptosis.

Nrf2 activation protects auditory hair cells from cisplatin-induced ototoxicity independent on mitochondrial ROS production

Cisplatin is a well-known and commonly used chemotherapeutic agent. However, cisplatin-induced ototoxicity limits its clinical use. Previous studies have shown an important role of reactive oxygen species (ROS) accumulation in the pathogenesis of cisplatin-induced ototoxicity. In many cell types, the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant response element (ARE) protect against oxidative stress by suppressing ROS. Here our results showed that cisplatin injury reduced Nrf2 expression and inhibited Nrf2 translocation in HEI−OC1 cells and Nrf2 activator tert-butylhydroquinone (TBHQ) rescued hair cells from cisplatin induced apoptosis by suppressing the total cellular ROS accumulation. Moreover, we found that decreased ROS accumulation induced by TBHQ didn’t depend on mitochondrial derived ROS production, indicating that Nrf2 activation alleviated cisplatin induced oxidative stress and apoptosis through mitochondrial-independent ROS production. Therefore, we provide a potential strategy of prevention and treatment for cisplatin-induced ototoxicity by Nrf2 activation. In conclusion, Nrf2 activation protects auditory hair cells from cisplatin-induced ototoxicity through suppressing the total cellular ROS levels which arise from sources other than mitochondria.

FDA‐approved PARP Inhibitors are Cytoprotective against Cisplatin Injury in Renal Proximal Tubular Cells

Cisplatin is a potent chemotherapeutic drug used to treat many solid cancers. However, its dose‐limiting side effect is nephrotoxicity, leading to acute kidney injury and chronic kidney disease. We previously reported that genetic or pharmacologicial inhibition of poly (ADP‐ribose) polymerase 1 (PARP1) protects against cisplatin nephrotoxicity.PARP1 inhibitors including olaparib, niraparib, and rucaparib are currently FDA approved for treatment of ovarian cancer. In this study, we tested the effect of the above FDA‐approved PARP inhibitors in cisplatin nephrotoxicity. Human HK‐2, pig LLC‐PK1, and mouse HUMPT renal proximal tubular cells (PTC) were subjected to one cycle of treatment with high‐dose cisplatin (1 to 1.5 mM) or four cycles of treatment with low‐dose cisplatin (1 to 3 μM). Each cycle consisted of 6 hours of treatment with cisplatin and 18 hours of recovery in cisplatin‐free medium. The FDA‐approved PARP inhibitors (300 pM to 1 μM) or the same volume of dimethylformamide (vehicle) were added immediately before the first treatment cycle and incubated continuously. The 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay performed to determine cell viability, lactate dehydrogenase (LDH) release assay to determine cell membrane disruption, and propidium iodide (PI) staining to determine necrotic cell death.All of the PARP inhibitors improved cell viability after the high‐dose cisplatin treatment in renal PTC. Increased percentages of LDH release and PI‐positive cells observed after one cycle of treatment with high‐dose cisplatin were significantly abrogated by the PARP inhibitors. Moreover, combined treatment with the PARP inhibitors showed an additive effect on cell viability in renal PTC treated with high‐dose cisplatin. The PARP inhibitors also enhanced cell viability after the four cycles of treatment with low‐dose cisplatin in renal PTC.Thus, our findings suggest that the FDA‐approved PARP inhibitors can protect efficaciously against death of PTC during cisplatin nephrotoxicity and could be developed as potential therapeutic agents to improve cisplatin nephrotoxicity.Support or Funding InformationThis study is supported by NIH grants DK‐116987, DK‐120533, AHA grants 15GRNT25080031 (BJP), 15POST25130003 (HSJ), and NRF grant NRF‐2019R1F1A1041410 (JK).

Cyclophilin D Sequesters PPARα in Mitochondria to Impair Fatty Acid Oxidation in Cisplatin‐induced Acute Kidney Injury

ObjectiveKidney is a highly metabolic organ and ATP depletion is critical to acute kidney injury (AKI). Mitochondrial fatty acid oxidation (FAO) is the preferred energy source of the kidney and is inhibited during AKI. A pivotal role for the mitochondrial matrix protein cyclophilin D (CypD) in regulating overall energy metabolism is being unraveled. We hypothesize that mitochondrial interaction between CypD and PPARα in renal proximal tubule (PT) where is highly susceptible to acute stimuli modulates FAO in cisplatin‐induced AKI (cisplatin AKI).MethodsUsing genetic and pharmacological intervention and protein‐protein interaction studies, we investigated whether PT‐CypD modulates FAO in cisplatin AKI through mitochondrial CypD‐PPARα binding and its sequestration.ResultsCisplatin injury resulted in histological and functional damage in the kidney, along with downregulated mitochondrial FAO, increased intrarenal lipid accumulation and declined ATP level. However, PT‐specific deletion of CypD protected the malevolent effects. Immunoprecipitation and BioID methods demonstrated mitochondrial translocation of PPARα and its binding to CypD and sequestration. This led to inhibition of nuclear translocation of PPARα and reduced transcription of FAO genes during cisplatin AKI. Genetic or pharmacological inhibition of CypD suppressed mitochondrial CypD‐PPARα binding in cisplatin AKI, preventing the impairment of FAO and intracellular lipid accumulation.ConclusionsThese results uncover a novel mechanism by which mitochondrial interaction between CypD and PPARα impairs FAO in cisplatin AKI. Targeting their interaction may be a potential therapeutic strategy to prevent energy depletion and cell death in AKI.Support or Funding InformationThis work was supported by NIH grants DK‐116987, DK‐120533 and American Heart Association (AHA) Grant in Aid 15GRNT25080031 (BJP), AHA postdoctoral fellowship Grant 15POST25130003 (HSJ), and grants (NRF‐2016R1C1B2012080 and NRF‐2019R1F1A1041410) from the National Research Foundation of Korea (JK).