Cisplatin-induced Acute Kidney Injury Research Articles

SOD mineralized zeolitic imidazole framework-8 for the treatment of chemotherapy-related acute kidney injury

Acute kidney injury (AKI), a prevalent and fatal adverse event, seriously affects cancer patients undergoing chemotherapy. The most important pathological mechanism of AKI is oxidative stress from reactive oxygen species (ROS). Currently, ROS scavenging is a promising strategy to manage the risk of chemotherapy-induced AKI. Herein, we successfully synthesized SOD@ZIF-8 nanoparticles by biomimetic mineralization, which were taken up by cells and could improve cell viability by limiting oxidative stress damage, as found in in vitro studies. Moreover, SOD@ZIF-8 nanoparticles exhibit broad-spectrum antioxidant properties in addition to significant renal accumulation in AKI mice, preventing clinically related cisplatin-induced AKI in murine models. AKI alleviation in the model was validated by measuring blood serum, staining kidney tissue, and related biomarkers. SOD@ZIF-8 nanoparticle therapeutic efficiency exceeds NAC, a small molecular antioxidant functioning through free radical scavenging. The results suggest SOD@ZIF-8 nanoparticles as a potential therapeutic option for AKI and other ROS-related disorders.

TRAF1 improves cisplatin-induced acute kidney injury via inhibition of inflammation and metabolic disorders

BackgroundCisplatin-induced acute kidney injury (AKI) is a severe clinical complication with no satisfactory therapies in the clinic. Tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) plays a vital role in both inflammation and metabolism. However, the TRAF1 effect in cisplatin induced AKI needs to be evaluated. MethodsWe observed the role of TRAF1 in eight-week-old male mice and mouse proximal tubular cells both treated with cisplatin by examining the indicators associated with kidney injury, apoptosis, inflammation, and metabolism. ResultsTRAF1 expression was decreased in cisplatin-treated mice and mouse proximal tubular cells (mPTCs), suggesting a potential role of TRAF1 in cisplatin-associated kidney injury. TRAF1 overexpression significantly alleviated cisplatin-triggered AKI and renal tubular injury, as demonstrated by reduced serum creatinine (Scr) and urea nitrogen (BUN) levels, as well as the ameliorated histological damage and inhibited upregulation of NGAL and KIM-1. Moreover, the NF-κB activation and inflammatory cytokine production enhanced by cisplatin were significantly blunted by TRAF1. Meanwhile, the increased number of apoptotic cells and enhanced expression of BAX and cleaved Caspase-3 were markedly decreased by TRAF1 overexpression both in vivo and vitro. Additionally, a significant correction of the metabolic disturbance, including perturbations in energy generation and lipid and amino acid metabolism, was observed in the cisplatin-treated mice kidneys. ConclusionTRAF1 overexpression obviously attenuated cisplatin-induced nephrotoxicity, possibly by correcting the impaired metabolism, inhibiting inflammation, and blocking apoptosis in renal tubular cells. General significanceThese observations emphasize the novel mechanisms associated to metabolism and inflammation of TRAF1 in cisplatin-induced kidney injury.

Genetic and pharmacological inhibition of GRPR protects against acute kidney injury via attenuating renal inflammation and necroptosis

Gastrin-releasing peptide (GRP) binds to its receptor (GRP receptor [GRPR]) to regulate multiple biological processes, but the function of GRP/GRPR axis in acute kidney injury (AKI) remains unknown. In the present study, GRPR is highly expressed by tubular epithelial cells (TECs) in patients or mice with AKI, while histone deacetylase 8 may lead to the transcriptional activation of GRPR. Functionally, we uncovered that GRPR was pathogenic in AKI, as genetic deletion of GRPR was able to protect mice from cisplatin- and ischemia-induced AKI. This was further confirmed by specifically deleting the GRPR gene from TECs in GRPRFlox/Flox//KspCre mice. Mechanistically, we uncovered that GRPR was able to interact with Toll-like receptor 4 to activate STAT1 that bound the promoter of MLKL and CCL2 to induce TEC necroptosis, necroinflammation, and macrophages recruitment. This was further confirmed by overexpressing STAT1 to restore renal injury in GRPRFlox/Flox/KspCre mice. Concurrently, STAT1 induced GRP synthesis to enforce the GRP/GRPR/STAT1 positive feedback loop. Importantly, targeting GRPR by lentivirus-packaged small hairpin RNA or by treatment with a novel GRPR antagonist RH-1402 was able to inhibit cisplatin-induced AKI. In conclusion, GRPR is pathogenic in AKI and mediates AKI via the STAT1-dependent mechanism. Thus, targeting GRPR may be a novel therapeutic strategy for AKI.

Inhibition of TMEM16A improves cisplatin-induced acute kidney injury via preventing DRP1-mediated mitochondrial fission.

Acute kidney injury (AKI) is associated with high morbidity and mortality. Our previous study has demonstrated that TMEM16A, a Ca2+-activated chloride channel, contributes to renal fibrosis progression in chronic kidney disease. However, whether TMEM16A isinvolved in AKI is still unknown. In this study, we established cisplatin AKI mice model and found that TMEM16A expression was upregulated in the injured kidney. In vivo knockdown of TMEM16A effectively prevented cisplatin-induced tubular cell apoptosis, inflammation and kidney function loss. Western blot and transmission electron microscopy (TEM) revealed that TMEM16A knockdown inhibited Drp1 translocation from the cytoplasm to mitochondria and prevented mitochondrial fission in tubular cells. Consistently, in cultured HK2 cells, knockdown or inhibition of TMEM16A by shRNA or its specific inhibitor suppressed cisplatin-induced mitochondrial fission and its associated energy dysfunction, ROS accumulation, and cell apoptosis via inhibiting Drp1 activation. Further investigation showed that genetic knockdown or pharmacological inhibition of TMEM16A inhibited cisplatin-induced Drp1 Ser-616 site phosphorylation through ERK1/2 signaling pathway, whereas overexpression of TMEM16A promoted this effect. Treatment with Drp1 or ERK1/2 inhibitor could efficiently prevent cisplatin-induced mitochondrial fission. Collectively, our data suggest that TMEM16A inhibition alleviated cisplatin-induced AKI by preventing tubular cell mitochondrial fission through the ERK1/2 / Drp1 pathway. Inhibition of TMEM16A may be a novel therapeutic strategy for AKI.

Silk fibroin peptide self-assembled nanofibers delivered naringenin to alleviate cisplatin-induced acute kidney injury by inhibiting mtDNA-cGAS-STING pathway.

Silk fibroin (SF) has excellent biocompatibility and biodegradability as a biomaterial. The purity and molecular weight distribution of silk fibroin peptide (SFP) make it more suitable for medical application. In this study, SFP nanofibers (molecular weight ∼30kD) were prepared through CaCl2/H2O/C2H5OH solution decomposition and dialysis, and adsorbed naringenin (NGN) to obtain SFP/NGN NFs. In vitro results showed that SFP/NGN NFs increased the antioxidant activity of NGN and protected HK-2cells from cisplatin-induced damage. In vivo results also showed that SFP/NGN NFs protected mice from cisplatin-induced acute kidney injury (AKI). The mechanism results showed that cisplatin induced mitochondrial damage, as well as increased mitophagy and mtDNA release, which activated the cGAS-STING pathway and induced the expression of inflammatory factors such as IL-6 and TNF-α. Interestingly, SFP/NGN NFs further activated mitophagy and inhibited mtDNA release and cGAS-STING pathway. Demonstrated that mitophagy-mtDNA-cGAS-STING signal axis was involved in the kidney protection mechanism of SFP/NGN NFs. In conclusion, our study confirmed that SFP/NGN NFs are candidates for protection of cisplatin-induced AKI, which is worthy of further study.

Tim-3 protects against cisplatin nephrotoxicity by inhibiting NF-κB-mediated inflammation

The impact of Tim-3 (T cell immunoglobulin and mucin domain-containing protein 3) on cisplatin-induced acute kidney injury was investigated in this study. Cisplatin-induced Tim-3 expression in mice kidney tissues and proximal tubule-derived BUMPT cells in a time-dependent manner. Compared with wild-type mice, Tim-3 knockout mice have higher levels of serum creatinine and urea nitrogen, enhanced TUNEL staining signals, more severe 8-OHdG (8-hydroxy-2’ -deoxyguanosine) accumulation, and increased cleavage of caspase 3. The purified soluble Tim-3 (sTim-3) protein was used to intervene in cisplatin-stimulated BUMPT cells by competitively binding to the Tim-3 ligand. sTim-3 obviously increased the cisplatin-induced cell apoptosis. Under cisplatin treatment conditions, Tim-3 knockout or sTim-3 promoted the expression of TNF-α (tumor necrosis factor-alpha) and IL-1β (Interleukin-1 beta) and inhibited the expression of IL-10 (interleukin-10). NF-κB (nuclear factor kappa light chain enhancer of activated B cells) P65 inhibitor PDTC or TPCA1 lowed the increased levels of creatinine and BUN (blood urea nitrogen) in cisplatin-treated Tim-3 knockout mice serum and the increased cleavage of caspase 3 in sTim-3 and cisplatin-treated BUMPT cells. Moreover, sTim-3 enhanced mitochondrial oxidative stress in cisplatin-induced BUMPT cells, which can be mitigated by PDTC. These data indicate that Tim-3 may protect against renal injury by inhibiting NF-κB-mediated inflammation and oxidative stress.

A chemical derivatization-based pseudotargeted LC-MS/MS method for high coverage determination of dipeptides

Dipeptides (DPs) have attracted more and more attention in many research fields due to their important biological functions and promising roles as disease biomarkers. However, the determination of DPs in biological samples is very challenging owing to the limited availability of commercial standards, high structure diversity, distinct physical and chemical characteristics, wide concentration range, and the extensive existence of isomers. In this study, a pseudotargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled with chemical derivatization for the simultaneous analysis of 400 DPs and their constructing amino acids (AAs) in biospecimens is established. Dansyl chloride (Dns-Cl) chemical derivatization was introduced to provide characteristic MS fragments for annotation and improve the chromatographic separation of DP isomers. A retention time (RT) prediction model was constructed using 83 standards (63 DPs and 20 AAs) based on their quantitative structural retention relationship (QSRR) after the Dns-Cl labeling, which largely facilitated the annotation of the DPs without standards. Finally, we applied this method to investigate the profile change of DPs in a cisplatin-induced acute kidney injury (AKI) rat model. The established workflow provides a platform to profile DPs and expand our understanding of these little-studied metabolites.

Preparation, characterization and protective effect of chitosan - Tripolyphosphate encapsulated dihydromyricetin nanoparticles on acute kidney injury caused by cisplatin

Dihydromyricetin (DMY) is a natural dihydroflavonol compound known for its diverse pharmacological benefits. However, its limited stability and bioavailability posed significant challenges for further applications. To address these issues, in this study, an ion crosslinking method was utilized to prepare chitosan nanoparticles that were loaded with DMY. The synthesized chitosan nanoparticles (CS-DMY-NPs) were spherical in shape with particle size and ζ potential of 198.7 nm and 45.05 mV, respectively. Furthermore, in vitro release experiments demonstrated that CS-DMY-NPs had sustained release and protective effects in simulated gastric and intestinal fluids. CS-DMY-NPs exhibited better antioxidant activity by ABTS and DPPH radical scavenging activity than free DMY. In vivo study showed that CS-DMY-NPs alleviated cisplatin-induced kidney damage by inhibiting oxidative stress and proinflammatory cytokines, and had better activity compared to DMY (free). Immunofluorescence data showed that CS-DMY-NPs activated the Nrf2 signaling pathways in a dose-dependent manner to combat cisplatin-induced kidney damage. Our results demonstrate that CS-TPP has good compatibility with DMY, and CS-DMY-NPs exhibited better protective effects against cisplatin-induced acute kidney injury (AKI) than free DMY.

Mitofusin2 Ameliorated Endoplasmic Reticulum Stress and Mitochondrial Reactive Oxygen Species Through Maintaining Mitochondria-Associated Endoplasmic Reticulum Membrane Integrity in Cisplatin-Induced Acute Kidney Injury.

Aims: This study investigated the regulatory effect of Mitofusin2 (Mfn2) on mitochondria-associated endoplasmic reticulum membrane (MAM) integrity and cellular injury in cisplatin-induced acute kidney injury (CP-AKI). Results: CP-AKI mice exhibited decreased expression of Mfn2, increased expression of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), abnormal mitochondrial morphology, and reduced MAMs integrity, accompanied by the activation of mitochondrial reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress (inositol-requiring enzyme 1 [IRE1] and PERK pathways). In in vitro studies, CP-induced mitochondrial ROS, ER-stress activation, and increased apoptosis were accompanied by the downregulation of Mfn2 and MAMs integrity reduction in Boston University mouse proximal tubular cells (BUMPT) and human proximal tubular epithelial cells (HK-2). Pretreatment of BUMPT cells with the Mfn2 plasmid partially restored the integrity of MAMs, negatively controlled IRE1 and PERK pathways, and inhibited cell apoptosis. In contrast, ER-stress and MAMs integrity violations were increased after Mfn2 small-interfering RNA (siRNA) treatment in HK-2 cells under CP treatment. Coimmunoprecipitation analysis demonstrated that Mfn2 interacted with PERK and IRE1. Furthermore, the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), acadesine (AICAR), had a similar effect to Mfn2 plasmid in the regulation of ER stress and MAMs. Conversely, the ER-stress inhibitor, 4-phenylbutyric acid (4-PBA), had no effect on the expression of Mfn2 and MAMs integrity. Innovation and Conclusion: This is the first study to explore the association between MAMs, ER stress, and Mfn2 in CP-AKI. Downregulation of Mfn2 expression abolished the MAMs integrity, and induced ER stress, mitochondrial ROS, and tubular cell apoptosis. This suggests that the Mfn2-MAMs pathway is a potential therapeutic target in CP-AKI. Antioxid. Redox Signal. 40, 16-39. The Ethical Registration number of animal experiment in this study was CSU-2022-01-0095.

Fucoidan-proanthocyanidins nanoparticles protect against cisplatin-induced acute kidney injury by activating mitophagy and inhibiting mtDNA-cGAS/STING signaling pathway

Fucoidan (FU) is a natural polymer from marine organisms, which has been widely studied and applied in drug delivery. In this study, FU nanoparticles loaded with proanthocyanidins (PCs) (FU/PCs NPs) were prepared and their effect and mechanism in protecting cisplatin-induced acute kidney injury (AKI) were studied. The in vitro studies confirmed that FU/PCs NPs increased the antioxidant activity of free PCs and protected the death of human kidney proximal tubule (HK-2) cells induced by cisplatin. Further mechanism studies showed that FU/PCs NPs protected the mitochondrial damage induced by cisplatin, activated mitophagy, inhibited the release of mitochondrial DNA (mtDNA), and inhibited the cGAS/STING signal pathway. The in vivo results also indicated that FU/PCs NPs protected cisplatin-induced AKI, including inhibiting the increase of blood urea nitrogen (BUN) and serum creatinine (SCr) levels induced by cisplatin. The mechanism studies confirmed that cisplatin induced an increase in the expression of mitophagy-related protein Pink/Pakrin, mitochondrial mtDNA release and cGAS/STING expression in mice kidney tissues. Pre-administration of FU/PCs NPs further activated mitophagy, as well as inhibiting mtDNA release and cGAS/STING expression. In conclusion, our research proved the role of mitophagy-mtDNA-cGAS/STING signal was involved in cisplatin-induced AKI.

7-hydroxycoumarin-β-D-glucuronide protects against cisplatin-induced acute kidney injury via inhibiting p38 MAPK-mediated apoptosis in mice

AimsCisplatin is a widely-used drug in the clinical treatment of tumors, but kidney nephrotoxicity is one of the reasons that limits its widespread use. We previously found that 7-hydroxycoumarin-β-D-glucuronide (7-HCG) was one of metabolites of skimmin and highly enriched in the kidneys and maintained a high blood concentration in skimmin-treated rats. Therefore, we investigated whether 7-HCG has a protective effect on cisplatin-induced acute kidney injury. Materials and methodsMale C57BL/6 mice were continuously administered 7-HCG for five days, and on the third day, an intraperitoneal injection of cisplatin was given to induce acute kidney injury. After 72 h, the mice were sacrificed for analysis. Serum and renal tissue were collected for renal function evaluation. RNA sequencing was used to explore mechanism, and further validated by western blot and immunohistochemistry. In addition, pharmacokinetic study of oral 7-HCG administration was performed to examine how much 7-hydroxycoumarin (7-HC) was metabolized and 7-HC possible effect on renal protection. Key findings7-HCG significantly reduced serum BUN and SCR levels, and alleviated pathological damage in renal tissue, and reduced the renal index. RNA sequencing revealed that 7-HCG could reverse p38 MAPK regulation and apoptosis. By western blotting, it was found that 7-HCG could reduce renal injury by reducing p-p38, p-ERK, p-JNK, cleaved-caspase3 and Bax. The immunohistochemical results of cleaved-caspase3 were consistent with western blotting. 7-HCG also significantly reduced the production of ROS in kidney tissue. Pharmacokinetic experiments have shown that 7-HCG in the blood increased rapidly and was eliminated slowly, with an average t1/2β of 18.3 h. And the concentration of 7-HCG in the target organ kidney was about 4 times higher than that in blood. SignificanceOur findings indicate that 7-HCG could exert its protective effect against cisplatin-induced acute kidney injury by inhibiting apoptosis via p38 MAPK regulation and elucidates its pharmacokinetics.

#3768 CDYL IS A THERAPEUTIC TARGET FOR ACUTE KIDNEY INJURY BY REGULATION PYROPTOSIS

Abstract Background and Aims Pyroptosis as a necrosis type related to inflammation, is involved in various diseases, including acute kidney injury (AKI). Here, we report that Chromodomain Y-like (CDYL) is critical for pyroptosis regulation in AKI. Method Cisplatin-induced mouse model and cisplatin-induced TCMK-1 cell model of AKI were established, serum creatinine (Cr) and blood urea nitrogen (BUN) were measured to evaluate kidney function, and renal tissue lesions were observed by HE staining. The expression of gene/protein in relation to inflammation and pyroptosis were measured by qPCR and western blot. The CDYL was overexpression in mice by rapid injection of a volume of naked plasmid DNA solution (30 ug) through the tail vein 2 hours before intraperitoneal injection of cisplatin. Results The results indicate that CDYL was increased in cisplatin-induced and sepsis-induced AKI, and overexpression of CDYL in mice would significantly increase the expressions of NGAL, NLRP3, Caspase-1, GSDMD, IL-1β, MCP-1, TNFα and IL-6 (Fig. 1). RNA-seq shown that the genes were mainly enriched in fatty acid metabolic process when CDYL overexpression, and FABP4 was finally identified as the key downstream factor (Fig. 2). Overexpression of CDYL in cisplatin-induced AKI further aggravated renal injury compared with the model mice, as indicated by the increased inflammation and pyroptosis protein expression, and worse tissue architecture, while the knockout of FABP4 will attenuate the injury (Fig. 3). In addition, treatment with CDYL inhibitors could attenuate inflammation and pyroptosis in cisplatin-induced renal injury (Fig. 4) and cisplatin-stimulated TCMK-1 cells (Fig. 5). Conclusion CDYL played a role in inflammation and pyroptosis regulation in AKI. Inhibition of CDYL could suppress renal inflammatory response and pyroptosis, and improve kidney function via modulation of FABP4 expression. The results highlighted that CDYL might represent a potential therapeutic target against AKI.

#4967 GASDERMIN D IS REQUIRED FOR CONTROL OF NECROPTOTIC CELL DEATH IN AKI

Abstract Background and Aims Contradicting previous reasoning, it is now established that necrotic rather than apoptotic cell death pathophysiologically drives acute tubular necrosis during AKI. Whereas involvement of necroptosis and ferroptosis has been demonstrated by various groups, the role of pyroptosis, a highly immunogenic form of cell death requiring proteolytic activation of gasdermin protein family members, remains uncertain upon AKI. Thus, we aimed to investigate the role of pyroptosis and its mechanism of action in AKI. Method We set to detect gasdermin D (GSDMD) expression in murine renal samples after ischemia/reperfusion injury (AKI) by immunohistochemistry. Furthermore, we investigated GSDMD-deficient mice in IRI and cisplatin-induced AKI. To conclude on mechanisms, we isolated fresh murine renal tubules and measured LDH release over time. We generated MLKL/GSDMDdko mice to investigate the interplay of these caspase-dependent forms of regulated necrosis in AKI. Results After IRI, we detected a specific GSDMD signal in areas surrounding necrotic tubules, whereas no such signal could be detected within necrotic areas. Whereas interference with other forms of regulated necrosis leads to reduced injury, GSDMD-deficient mice demonstrated higher levels of serum creatinine and urea as well as more severe tubular injury compared to wildtypes. In line with the findings of immunohistochemistry, we only found GSDMD protein expression in whole kidney lysates but not isolated renal tubules; and no difference in spontaneous cell death propagation was detectable. Co-deletion of MLKL (effector protein of necroptosis) reversed the sensitization to AKI by GSDMD-deficiency. These results could be reproduced in cisplatin-induced AKI. Conclusion Here, we found an unexpected protective role for GSDMD, the effector protein of pyroptosis, in two different models of AKI. Mechanistically, our studies indicate the effect of GSDMD to function outside the tubular compartment, specifically surrounding areas of tubular necrosis. These infiltrating innate immune cells appear to interfere with tubular necroptosis in a non-cell autonomous manner. Alongside with these mechanistic insights, our data urge caution when inhibition of pyroptosis is therapeutically considered.

Dihydromyricetin attenuates cisplatin-induced acute kidney injury by reducing oxidative stress, inflammation and ferroptosis

BackgroundCisplatin is effective against various types of cancers. However, its clinical application is limited owing to its adverse effects, especially acute kidney injury (AKI). Dihydromyricetin (DHM), a flavonoid derived from Ampelopsis grossedentata, has varied pharmacological activities. This research aimed to determine the molecular mechanism for cisplatin-induced AKI. MethodsA murine model of cisplatin-induced AKI (22 mg/kg, I.P.) and a HK-2 cell model of cisplatin-induced damage (30 μM) were established to evaluate the protective function of DHM. Renal dysfunction markers, renal morphology and potential signaling pathways were investigated. ResultsDHM decreased the levels of renal function biomarkers (blood urea nitrogen and serum creatinine), mitigated renal morphological damage, and downregulated the protein levels of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. It upregulated the expression levels of antioxidant enzymes (superoxide dismutase and catalase expression), nuclear factor-erythroid-2-related factor 2 (Nrf2) and its downstream proteins, including heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic (GCLC) and modulatory (GCLM) subunits, thus eventually reducing cisplatin-induced reactive oxygen species (ROS) production. Moreover, DHM partially inhibited the phosphorylation of the active fragments of caspase-8 and -3 and mitogen-activated protein kinase and restored glutathione peroxidase 4 expression, which attenuated renal apoptosis and ferroptosis in cisplatin-treated animals. DHM also mitigated the activation of NLRP3 inflammasome and nuclear factor (NF)-κB, attenuating the inflammatory response. In addition, it reduced cisplatin-induced HK-2 cell apoptosis and ROS production, both of which were blocked by the Nrf2 inhibitor ML385. ConclusionsDHM suppressed cisplatin-induced oxidative stress, inflammation and ferroptosis probably through regulating of Nrf2/HO-1, MAPK and NF-κB signaling pathways.

#5658 TUBULE-SPECIFIC CYCLIN-DEPENDENT KINASE 12 KNOCKDOWN POTENTIATES KIDNEY INJURY THROUGH TRANSCRIPTIONAL ELONGATION DEFECTS

Abstract Background and Aims Direct tubular injury caused by several medications, especially chemotherapeutic drugs, is a common cause of AKI. Inhibition or loss of cyclin-dependent kinase 12 (CDK12) triggers a transcriptional elongation defect that results in deficiencies in DNA damage repair, producing genomic instability in a variety of cancers. Notably, 10-25% of individuals developed AKI after treatment with a CDK12 inhibitor, and the potential mechanism is not well understood. Method Here, we established a cisplatin-induced AKI mouse model with CDK12 knockdown mice and created stable in vitro models with CDK12 knockdown tubular epithelial cells. Cisplatin nephrotoxicity was induced by intraperitoneal injection of cisplatin (20 mg/kg). After 2 days following cisplatin treatment, blood and kidney tissues were harvested. Renal function and histology were evaluated. DDR, tubular apoptosis, cell proliferation were evaluated by immunofluorescence assays, real-time PCR, flow cytometer and western blot analysis. Notably, we performed single-molecule real-time sequencing (SMRT), a long-read sequencing platform, to identify the formation of novel transcripts involved in the above events. Results We found that CDK12 was downregulated in the renal tubular epithelial cells in both patients with AKI and murine AKI models. Moreover, tubular cell-specific knockdown of CDK12 in mice enhanced cisplatin-induced AKI through promotion of genome instability, apoptosis, and proliferative inhibition, whereas CDK12 overexpression protected against AKI. Using the single molecule real-time (SMRT) platform on the kidneys of CDK12RTEC+/− mice, we found that CDK12 knockdown targeted Fgf1 and Cast through transcriptional elongation defects, thereby enhancing genome instability and apoptosis. Conclusion These data demonstrated that CDK12 knockdown could potentiate the development of AKI by altering the transcriptional elongation defect of the Fgf1 and Cast genes, and more attention should be given to patients treated with CDK12 inhibitors to prevent AKI.

#4248 COMBINED EFFECT OF DEHYDRATION, HYPERTENSION, AND AGE ON RENAL FRAILTY

Abstract Background and Aims Renal frailty (RF) is a premorbid and, at least partly, modifiable condition arising from diminished renal functional reserve and defective adaptive response capacity predisposing to acute kidney injury (AKI). RF ensues from subclinical wear or distortion of the renal haemodynamic and tubular homeostatic responses that defend the renal excretory function from supervening circumstances (i.e., stressors such as drugs). In this work, we aimed to study the impact of RF generated by the combined effect of the hydration state, hypertension and ageing on the nephrotoxicity developed by a low dose of cisplatin in the rat. Method Young Wistar rats, young spontaneously hypertensive rats (SHR), and aged Wistar rats were used. Rats in each group were randomly subject to four experimental conditions: - Control: rats with ad libitum water intake, receiving vehicle (0.9% NaCl, i.p). - Cisplatin: rats with ad libitum water intake, treated with 2.5 mg/kg, i.p. cisplatin. - Water deprivation: rats deprived of water for 48 h, receiving vehicle (0.9% NaCl, i.p.). - Water deprivation + cisplatin: rats deprived of water for the 48 h prior to 2.5 mg/kg i.p. cisplatin administration. Tail vein blood and 24-hour urine samples were collected at basal time (B), after water deprivation (D0), and 4 days after cisplatin/vehicle administration (D4, day of maximum kidney damage). Haematocrit was measured and plasma osmolality, plasma creatinine (Jaffe reaction) and urea (Jung method) concentrations were determined. Urine samples were analysed for volume (urinary flow), osmolality, and proteinuria (Bradford method). Body fluid composition was measured by bioimpedance spectroscopy with an ImpediVETTM VetBIS1 device. Results All rats showed clear signs of dehydration after 48 hours of water deprivation: weight loss, increases in plasma osmolality and haematocrit (more pronouncedly in young Wistar and SHR than in aged animals), and reduced flow of a highly concentrated urine (i.e., with elevated osmolality). Bioimpedance analysis revealed a parallel loss of intracellular and extracellular water and net loss of total body water (explaining most of weight loss), with no changes in the liquid distribution between the intracellular and extracellular compartments. Dehydration was reverted after 4 days of rehydration in vehicle-treated rats, and no alteration of renal function was observed due to water deprivation. Administration of cisplatin in young, normohydrated Wistar rats showed minimal loss of renal function that was not worsened by water deprivation. Hypertensive rats showed completely normal renal function when cisplatin was administered under normohydration. Interestingly, however, dehydrated SHR did suffer a cisplatin-induced AKI (significant increases in plasma creatinine, plasma urea and proteinuria). In aged Wistar rats, cisplatin compromised renal function (as supported by increases in plasma creatinine and urea concentration), and this effect was significantly amplified by water deprivation. Conclusion This study suggests that dehydration in fully competent young rats is not in itself a significant risk factor for AKI, while the combination of dehydration and hypertension in young rats significantly increases the risk of AKI. Furthermore, ageing poses animals in a state of frailty that renders their kidneys unable to respond to stressors (such as a low dose of cisplatin). Frailty is boldly worsened in dehydrated aged animals. In perspective, models reproducing conditions of increased vulnerability to AKI provide useful tools to search for biomarkers pre-emptively predicting undesired health outcomes before exposure to stressors, and for developing preventing strategies. This study was supported by Project PI21/01226, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, and a grant from the Consejería de Educación, Junta de Castilla y León (IES160P20), Spain, co-funded by FEDER funds. Noelia Diaz-Morales is recipient of a Juan de la Cierva-Formación postdoctoral contract (FJC2020-043205-I) funded by MCIN/AEI/10.13039/501100011033 and European Union “NextGenerationEU/PRTR”.

#2920 MAGNESIUM DEFICIENCY ABROGATES THE RENOPROTECTIVE EFFECT OF DPP-4 INHIBITOR ON CISPLATIN-INDUCED KIDNEY INJURY

Abstract Background and Aims Cisplatin is an effective chemotherapeutic agent, but often induces acute kidney injury (AKI). Magnesium (Mg) deficiency is often found in cancer patients, and the nephrotoxicity of cisplatin is exacerbated under Mg deficiency [1]. We previously reported the potential of Dipeptidyl peptidase-4 (DPP-4) inhibitor to attenuate cisplatin nephrotoxicity in rats and diabetic cancer patients [2, 3]. However, it remains to be investigated whether DPP-4 inhibitor can preserve its renoprotective effect under Mg deficiency. The purpose of this study was to examine whether DPP-4 inhibitor can prevent cisplatin-induced AKI even under Mg deficiency. Method Sprague Dawley rats received Mg-deficient diet for 7days to induce Mg deficiency. AKI was induced in rats by injecting cisplatin intravenously. Oral administration of a DPP-4 inhibitor, once a day, was started 1day before injecting cisplatin. Mg sulfate was once injected intraperitoneally just before injecting cisplatin to correct Mg deficiency (Fig. 1A). By using our previous cohort data [3], we divided diabetic cancer patients treated with high-dose cisplatin (>50 mg/m2)-containing regimens into 4 groups according to the use/unused of Mg sulfate or DPP-4 inhibitor. The change of estimated glomerular filtration rate (eGFR) within 2 weeks after cisplatin treatment was compared between the groups. Results At the peak of AKI (day 5), Mg sulfate supplementation significantly attenuated the increase of blood urea nitrogen (BUN), and combination of Mg sulfate and DPP-4 inhibitor further suppressed the increase of BUN as compared to rats received only cisplatin. However, DPP-4 inhibitor alone did not attenuate the increase of BUN under Mg deficiency (Fig. 1B). The result of serum creatinine (sCr) was consistent with that of BUN (Fig. 1C). This effect was associated with a reduced renal cell death as evaluated with the terminal uridine nick-end labeling (TUNEL)+ cells (Fig. 1D).The change of eGFR was significantly less in the patients treated with both of DPP-4 inhibitors/Mg supplementation, compared to those without DPP-4 inhibitors/Mg supplementation. The effect was not found in patients with either DPP-4 inhibitor alone or Mg supplementation alone as compared to those without DPP-4 inhibitor/Mg supplementation (Fig. 2). Conclusion Our results suggested that Mg deficiency must be corrected to exert the renoprotective effect of DPP-4 inhibitor on cisplatin nephrotoxicity. The molecular mechanisms underlying this phenomenon remain to be defined.

A risk prediction model for cisplatin-induced acute kidney injury in patients with head and neck cancer who receive chemoradiotherapy: A supplement analysis of a phase II/III trial of JCOG1008.

12104 Background: Cisplatin-induced acute kidney injury (C-AKI) is a common reason for treatment interruption in head and neck cancer patients who receive chemoradiotherapy (CRT). We developed a risk prediction model for C-AKI in patients from a randomized phase II/III trial of JCOG1008, which demonstrated that CRT with weekly cisplatin at 40 mg/m2 (weekly arm) was noninferior to 3-weekly cisplatin at 100 mg/m2 (3-weekly arm) in terms of overall survival for postoperative high-risk head and neck cancer ( J Clin Oncol 2022; 40: 1980-90). Methods: Two hundred fifty-one patients received RT with a total of 66 Gy/33 fr concurrently with 3-weekly or weekly cisplatin. AKI was defined using the AKI Network classification/staging system as an increase in serum creatinine of ≥0.3 mg/dL or a 1.5-fold or greater increase from baseline (≥ stage I) by 30 days after the completion of CRT. AKI risk prediction models were developed by combining explanatory variables which were expected to contribute, including age, sex, ECOG performance status, primary site, pathological stage, diabetes mellites, hypertension, main nutritional method, treatment arm, serum albumin, serum magnesium, and creatinine clearance (CCr) at registration. A logistic regression model with the Akaike information criterion was used to explore the optimal model. Results: Ninety-four of 251 (37.5%) patients developed C-AKI in this study. The optimal C-AKI risk prediction model was composed of four factors, including a primary site (hypopharynx/larynx or oral cavity/oropharynx, odds ratio [OR] 1.92 [95% CI, 1.10 to 3.30]), treatment arm (3-weekly arm or weekly arm, OR 1.75 [95% CI, 1.02 to 3.00]), serum albumin (≤ 3.5 g/dL or > 3.5 g/dL, OR 2.06 [95% CI, 1.11 to 3.87]) and CCr ( < 90 mL/min or ≥ 90 mL/min, OR 1.85 [95% CI, 1.05 to 3.26]). The incidence of C-AKI increased in accordance with a total count of the four factors (Table): with a total count of 0-1, 2 and 3-4, the incidence of clinically relevant stage II/III C-AKI was 2.9%, 11.5%, and 22.6%, respectively. When total count was ≤1, the positive and negative predictive values for stage II/III C-AKI development were 16.1% and 97.0%, respectively. Conclusions: We developed a risk prediction model for C-AKI in head and neck cancer patients who receive CRT using primary site, cisplatin administration method, serum albumin, and CCr. The high negative predictive value of this prediction model may help rule out the development of moderate to severe C-AKI. Clinical trial information: jRCTs031180135 . [Table: see text]

Paeoniflorin protects against cisplatin-induced acute kidney injury through targeting Hsp90AA1-Akt protein-protein interaction.

Paeonia lactiflora Pall has been used in Chinese Medicine for thousands of years, especially having anti-inflammatory, sedative, analgesic and other ethnic pharmacological effects. Moreover, Paeoniflorin is the main active ingredient of the Paeonia lactiflora Pall, and most are used in the treatment of inflammation-related autoimmune diseases. In recent years, studies have found that Paeoniflorin has a therapeutic effect on a variety of kidney diseases. Cisplatin (CIS) is limited in clinical use due to its serious side effects, such as renal toxicity, and there is no effective method for prevention. Paeoniflorin (Pae) is a natural polyphenol which has a protective effect against many kidney diseases. Therefore, our study is to explore the effect of Pae on CIS-induced AKI and the specific mechanism. Firstly, CIS induced acute renal injury model was constructed in vivo and in vitro, and Pae was continuously injected intraperitoneally three days in advance, and then Cr, BUN and renal tissue PAS staining were detected to comprehensively evaluate the protective effect of Pae on CIS-induced AKI. We then combined Network Pharmacology with RNA-seq to investigate potential targets and signaling pathways. Finally, affinity between Pae and core targets was detected by molecular docking, CESTA and SPR, and related indicators were detected in vitro and in vivo. In this study, we first found that Pae significantly alleviated CIS-AKI in vivo and in vitro. Through network pharmacological analysis, molecular docking, CESTA and SPR experiments, we found that the target of Pae was Heat Shock Protein 90 Alpha Family Class A Member 1 (Hsp90AA1) which performs a crucial function in the stability of many client proteins including Akt. RNA-seq found that the KEGG enriched pathway was PI3K-Akt pathway with the most associated with the protective effect of Pae which is consistent with Network Pharmacology. GO analysis showed that the main biological processes of Pae against CIS-AKI include cellular regulation of inflammation and apoptosis. Immunoprecipitation further showed that pretreatment with Pae promoted the Hsp90AA1-Akt protein-protein Interactions (PPIs). Thereby, Pae accelerates the Hsp90AA1-Akt complex formation and leads to a significant activate in Akt, which in turn reduces apoptosis and inflammation. In addition, when Hsp90AA1 was knocked down, the protective effect of Pae did not continue. In summary, our study suggests that Pae attenuates cell apoptosis and inflammation in CIS-AKI by promoting Hsp90AA1-Akt PPIs. These data provide a scientific basis for the clinical search for drugs to prevent CIS-AKI.

One-year reductions in cisplatin-related chronic kidney disease (CKD) in patients with head and neck (HNC) cancer treated with avasopasem manganese: A prespecified analysis from the phase 3 ROMAN trial.

6066 Background: Cisplatin-induced acute kidney injury (AKI), acute kidney disease (AKD), and chronic kidney disease (CKD) are associated with increased production of reactive oxygen species, notably superoxide, in conjunction with alterations in mitochondrial electron transport chain complex activities in tubular epithelial cells (Mapuskar et al. Antioxidants 2021;10:1329). These pathologies can be targeted and thereby inhibited by increasing superoxide dismutase activity, which converts superoxide to hydrogen peroxide. The investigational new drug avasopasem manganese (AVA), a selective dismutase mimetic, ameliorated cisplatin-induced kidney injury in mice (Mapuskar et al. Redox Biol 2019;20:98). Preclinical results also demonstrate that AVA does not interfere with cisplatin anticancer activity (Mohanty et al. Cancer Res 2018;78[13_suppl]:Abstract 2929). Consistent with this, a retrospective analysis of 1- and 2-year kidney function of a limited subset of patients treated in a randomized, placebo (PBO)-controlled phase 2b trial of AVA to reduce severe oral mucositis (SOM) from radiotherapy and cisplatin (CRT) suggested that AVA prevented or reduced cisplatin-related CKD compared to PBO (Steinbach et al. J Clin Oncol 2020;38[15_suppl]:Abstract 12071). Methods: The ROMAN phase 3 trial (NCT03689712; ITT n = 407) of CRT in combination with AVA or PBO demonstrated improved incidence, duration, and onset of SOM by AVA (n = 241) vs PBO (n = 166) (Anderson et al. J Clin Oncol 2022;40[16_suppl]:Abstract 6005). In ROMAN, prospectively defined follow-up of serum creatinine (sCr) and estimated glomerular filtration rates (eGFR) were assessed every 3 months for 1 year following 7 weeks of CRT. Results: At 1-year follow-up, AVA was associated with significant improvements in preservation of eGFR ( P= 0.0008) and sCr ( P= 0.006) levels vs PBO. Grade 3+ CKD (eGFR < 60 mL/min) results through 12 months show 10% AVA vs 20% PBO (relative risk 0.55, P= 0.0043). Regarding cisplatin schedule, patients receiving Q3W therapy show grade 3+ CKD of 12% AVA vs 22% PBO; patients receiving QW therapy show AVA 9% vs PBO 19%. During treatment, nominal reduction of the incidence of AKI adverse events (0.8% AVA vs 3.6% PBO) was also observed. Conclusions: At 1-year follow-up, AVA appears to reduce cisplatin-related CKD in the study population and may also reduce cisplatin-related AKI during treatment. These results carry significance beyond CRT for HNC, potentially impacting platinum-containing regimens in other cancers. Clinical trial information: NCT03689712 .