Protective Effect Of Panduratin A On Cisplatin-Induced Apoptosis Of Human Renal Proximal Tubular Cells And Acute Kidney Injury in Mice
Mar 18, 2022
Background: Cisplatin is effective chemotherapy but its main side effect, acute kidney injury, limits its use. Pandurate A, a bioactive compound extracted from Boesenbergia rotunda, shows several biological activities such as anti-oxidative effects. The present study investigated the nephroprotective effect of pandurate A on the cisplatin-induced renal injury. Methods: We investigated the effect of pandurate A on the toxicity of cisplatin in both mice and human renal cell cultures using RPTEC/TERT1 cells. Results: The results demonstrated that pandurate A ameliorates cisplatin-induced renal toxicity in both mice and RPTEC/ TERT1 cells by reducing apoptosis. Mice treated with a single intraperitoneal (i.p.) injection of cisplatin (20 mg/kg body weight (BW)) exhibited renal tubule injury and impaired kidney function as shown by histological examination and increased serum creatinine. Co-administration of pandurate A (50 mg/kg BW) orally improved kidney function and ameliorated renal tubule injury of cisplatin by inhibiting activation of extracellular signal-regulated kinase (ERK)1/2 and caspase 3. In human renal proximal tubular cells, cisplatin-induced cell apoptosis by activating pro-apoptotic proteins (ERK1/2 and caspase 3), and reducing the anti-apoptotic protein (Bcl-2). These effects were significantly ameliorated by co-treatment with pandurate A. Interestingly, pandurate A did not alter the intracellular accumulation of cisplatin. It did not alter the anti-cancer efficacy of cisplatin in either human colon or non-small cell lung cancer cell lines. Conclusions: The present study highlights pandurate A has a potential protective effect on cisplatin's nephrotoxicity.
Keywords: chemotherapy; acute kidney injury; panduratin A; human renal proximal tubule; anti-apoptosis; renal; kidney
CISTANCHE WILL IMPROVE KIDNEY/RENAL DIALYSIS
INTRODUCTION
Nephrotoxicity induced by drugs is a major problem in the clinical setting because the use of nephrotoxic drugs is often unavoidable. Nephrotoxicity can be defined by renal injuries including glomerular damage and tubular injury leading to impairment of renal function.1) Common drugs associated with kidney injury are anti-inflflammatories, antibiotics, and chemotherapeutic agents, such as cisplatin.2–4) Cisplatin is a broad-spectrum, high potency chemotherapeutic drug.5) However, approximately one out of three patients receiving cisplatin treatment suffers from nephrotoxicity6,7) and electrolyte imbalance.8,9) Cisplatin-induced nephrotoxicity is characterized by proximal tubule injury, vascular injury, and inflammation.4) Accumulation of cisplatin in renal cells induces multiple pathways that promote cell death. Reactive oxygen species (ROS) has been identified as a key mediator in cisplatin nephrotoxicity.10,11) Additionally, ROS induces nephrotoxicity via activation of mitogen-activated protein kinases (MAPKs).10,12,13) Several studies have reported that cisplatin itself can directly stimulate extracellular signal-regulated kinase (ERK)1/2 (a member of MAPKs) both in vitro and in vivo. 14,15)
Certain phytochemicals ameliorate cisplatin-induced nephrotoxicity by inhibiting ROS accumulation and apoptosis.16–18) Although nephroprotective effects of these phytochemicals have been reported in preclinical studies, there are still no approved drugs from phytochemicals for cisplatin's nephrotoxicity (see detail in review).19) Current clinical practice only provides supportive treatments to recover renal function.20) Therefore, it is still important to search for effective agents to prevent or treat cisplatin-induced nephrotoxicity. Pandurate A is an interesting phytochemical. It is a cyclohexanol chalcone isolated from Boesenbergia rotunda, a plant used in traditional medicine and food.21) Panduratin A has been shown to prevent or treat oxidative stress, inflammation, and metabolic disease.22,23) Injury of renal proximal tubules caused by cisplatin is mediated, at least in part, via increased oxidative stress and inflammation.11) Therefore, we investigated the effect of pandurate A on the toxicity of cisplatin in both animals and cell cultures using RPTEC/TERT1 cells. We also tested whether co-treatment with pandurate A affects cisplatin’s anticancer activity in human cancer cell lines.
MATERIALS AND METHODS
Chemicals Thiazolyl blue tetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), cisplatin, and compound C (AMP-activated protein kinase (AMPK) inhibitor) were purchased from Sigma-Aldrich (MO, U.S.A.). 3 H-1-Methyl-4-phenylpyridinium (3 H-MPP+) was purchased from PerkinElmer, Inc. (Bangkok, Thailand). Annexin V- fluorescein isothiocyanate (FITC) apoptosis detection kit was purchased from BD Biosciences (CA, U.S.A.). Primary antibodies for p-ERK1/2 (Cat. No. 9102S), ERK1/2 (Cat. No. 9101S), Bcl-2 (Cat. No. 2872T), caspase-3 (Cat. No. 9662S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat. No. 2118S), p-AMPKα (Cat. No.2531S), AMPKα (Cat. No. 5832S), Bax (Cat. No. 5023) and β-actin (Cat. No. 4970T) antibodies were obtained from Cell Signaling (MA, U.S.A.) and anti-neutrophil gelatinase-associated lipocalin (NGAL) antibody (Cat. No. STCSC-515876) was purchased from Santa Cruz Biotechnology (CA, U.S.A.). Pandurate A >98% purity determined by HPLC was isolated from Boesenbergia rotunda as previously described by our group.24) The rhizomes of Boesenbergia rotunda were collected from Kanchanaburi, Thailand. The plant was identified by Tuanta Sematong. The voucher specimen (No. BKF 68909) has been deposited at the Forest Herbarium, Royal Forestry Department, Bangkok.
CISTANCHE WILL IMPROVE KIDNEY/RENAL FUNCTION
Animals Male C57BL/6 mice (8-weeks old) were purchased from Nomura Siam International Co., Ltd. (Bangkok, Thailand). Animal Care and Use Protocol No. MUSC61-063-464 has been approved by the Institutional Animal Care and Use Committee, MUSC–IACUC. The mice were allowed to freely access food and water. After one week of acclimatization, mice were divided randomly and administered treatments as follows: normal saline by a single intraperitoneal (i.p.) injection on day 4 (control group); pandurate A (50 mg/kg body weight (BW)) by oral gavage for 7 d (pandurate A group); cisplatin (20 mg/kg BW) by a single i.p. injection on day 4 (cisplatin group); pandurate A (50 mg/kg BW/d) by oral gavage for 7 d and cisplatin (20 mg/kg BW) by a single i.p. injection on day 4 (co-treatment group). On day 7, mice were deeply anesthetized by thiopental sodium. Blood was drawn and centrifuged for 10 min at 3000 pm. The collected supernatants were kept at −80 °C until the renal function was measured. Kidneys were collected for the measurement of protein expressions and histopathological studies.
Determination of Renal Function and Histological Examination Renal function was determined by measuring serum creatinine using Stanbio Creatinine Liquicolor (NY, U.S.A.) and blood chemistry analyzer, Licenza (Rome, Italy). To examine cisplatin-induced renal tubular damage, mouse kidneys were fixed in 4% paraformaldehyde. The kidney slices were stained with hematoxylin and eosin (H&E) photographed by a light microscope. The percentage of tubular injury was used to evaluate the renal tubular injury: 0 = no tubular injury 1=<10%; 2 = 10–25%; 3 = 26–50%; 4 = 51–75%; 5=>75%. Slides were blindly scored by a pathologist. The mean score for each group of animals was calculated by counting 10 different fields of a slide.
Cell Lines RPTEC/TERT1 cells, colon cancer cells (HCT116) and non-small cell lung cancer cells (A549) were purchased from American Type Culture Collection (VA, U.S.A.). Briefly, RPTEC/TERT1 cells were cultured in complete Dulbecco’s modified Eagle’s medium/F-12 medium (DMEM/F12) as previously described.25) HCT116 and A549 cells were cultured in DMEM/F12 and RPMI1640 medium, respectively, supplemented with penicillin-streptomycin antibiotics and 10% fetal bovine serum (FBS). Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% O2.
Cell Viability Assay and Cell Apoptosis Analysis Cell viability were determined by exposing the RPTEC/TERT1 cells to 0.5mg/mL MTT reagent for 1h at 37℃C. The reagents were removed, the formazan salt formed was dissolved in dimethyl sulfoxide (DMSO)and detected by EnVision microplate reader at 570nm absorbance. Cell viability was reported as a percentage of control. Annexin V and propidium iodide (PIstaining analysis were used to determine cell apoptosis. Briefly, RPTEC/TERT1 cells were detached by 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). The cell suspensions were incubated with Annexin V-FITC and PI in the dark at 4°C for 15min followed by twice washing with binding buffer. Apoptotic cells were counted by flow cytometer and expressed as a percent of total cells.
Assessment of Intracellular ROS Accumulation ROS levels were determined by DCFH-DA assay. RPTEC/TERT1 cells were incubated with 10uM DCFH-DA and incubated for 30min at 37°C. Next, DCFH-DA dye was removed and cells were washed with phosphate-buffered saline(PBS). Intracellular ROS level was measured at wavelengths of 485 and 530nm for excitation and emission, respectively. Intracellular ROS accumulation is reported as a percentage of fluorescent intensity relative to control cells.
CISTANCHE WILL IMPROVE KIDNEY/RENAL DISEASE
Platinum Accumulation Analysis Accumulated platinum was measured in both digested RPTEC/TERT1 cells and mouse kidneys by UNICAM 989 QZ AA spectrometer (Geleen, Netherlands). Ten microliters of 1% Triton X-100 and 400uL of 1% nitric acid were added to each 100μL sample. Samples were then incubated for at least 1h. Prior to platinum detection, each sample was diluted 10:1 with 1% nitric acid. The platinum concentration was calculated using linear regression of a platinum standard curve prepared using 2.5mg/mL of cisplatin-diluted in 1% nitric acid. The platinum was calculated as ng/cell number or ng/kidney weight.
3H-MMP* Uptake Assay Uptake 3H-MPP+ into RPTEC/TERT1 cells was determined using our previous method.25)Briefly, cells were washed with warm transport buffer and further incubated for 20min. The cell monolayers were incubated with H-MPP+(10nM) followed by washing with ice-cold transport buffer. The samples were collected and radioactive activity of H-MPP+ was measured using a liquid scintillation counter.
Western Blotting Analysis Protein expressions were processed according to our previous study.26)Extracted proteins from mouse kidney and RPTEC/TERT1 cells were centrifuged at 12000rpm for 20min at 4°C. Equal amounts of proteins isolated from cells and tissue were denatured and separated by 10-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were transferred onto nitrocellulose membranes followed by 1h non-specific protein blocking using nonfat dry milk(5%). The membranes were incubated with primary antibody for 24h and were then incubated with secondary antibody for 1 h. The intensity of protein expression was detected using chemiluminescent HRP substrate and quantified by ImageJ software.
Statistical Analysis Data are expressed as mean ± standard deviation (S.D.). Data were analyzed by one-way ANOVA using Tukey’s posthoc tests (GraphPad Prism 8.0). A significance is considered when the p-value is less than 0.05.
RESULTS Panduratin A Ameliorates Cisplatin-Induced Acute Kidney Injury We investigated the effect of pandurate A on kidney injury caused by cisplatin in mice. We first observed the general toxicity by measuring body weight change in mice following treatment with normal saline, pandurate A (50 mg/kg BW), cisplatin (20 mg/kg BW), and both cisplatin (20 mg/kg BW) and pandurate A (50 mg/kg BW). A signifies- cant decreased body weight in the cisplatin-treated mice was found. The body weight loss was mitigated by co-treatment with pandurate A (Fig. 1A). Compared with the control, kidney function which was measured by serum creatinine level showed that mice-treated pandurate A alone did not alter serum creatinine level; whereas the cisplatin-treated mice had significantly increased serum creatinine levels, indicating impaired kidney function. The serum creatinine level in co-administration of pandurate A was significantly decreased compared with cisplatin-treated mice (Fig. 1B). In addition, the accumulation of platinum in kidney tissue was not signified- cantly different in mice treated with cisplatin alone compared with mice co-treated with cisplatin and pandurate A (Fig. 1C). These data indicate that pandurate A did not affect cisplatin content in renal tissue. The kidneys obtained from mice treated with vehicle or pandurate A alone showed no marked pathological changes. However, cisplatin-treated mice showed more tubular damage compared with vehicle-treated mice as shown by the increase in degeneration and desquamation, formation of luminal casts, mononuclear cell infiltration, karyomegaly, inter-tubular haemorrhagia, and dilation. These pathological changes were significantly attenuated by co-administration of pandurate A (Fig. 2A, Table 1). The expression levels of NGAL (a nephrotoxicity biomarker), and ERK1/2 and cleaved caspase 3 (pro-apoptotic proteins) were determined. Our study showed that mice treated with cisplatin significantly increased expression of NGAL, p-ERK1/2, and cleaved caspase 3 compared with vehicle-treated mice. Co-treatment with pandurate A significantly reduced these proteins induced by cisplatin treatment (Fig. 2B).
Pandurate A Prevents Cisplatin-Induced Cytotoxicity in Human Renal Proximal Tubular Cells by Inhibiting Apoptotic Signaling Pathways Cisplatin (50 µM) signifies- cantly decreased viability of RPTEC/TERT1 cells following 72 h incubation. Interestingly, co-treatment with pandurate A (1 and 5 µM) significantly increased cell viability (Fig.
3A). Panduratin A’s protective effect against the cytotoxicity of cisplatin was confirmed by the measurement of cell apoptosis. As expected, exposure of the cells to 50 µM cisplatin for 48 h significantly increased apoptosis of RPTEC/TERT1 cells. This effect was attenuated by pandurate A (Fig. 3B). We examined whether the effect of pandurate A on cisplatin’s toxicity required activation of AMPK. As shown in Fig. 3C, protein expression of p-AMPK, an active form of AMPK, was increased by pandurate A whereas it was decreased by cisplatin. In addition, the protective effect of pandurate A under AMPK inhibition was determined. Interestingly, incubation of the cells with 10 µM compound C, an inhibitor of AMPK, did not attenuate the protective effect of pandurate A on cell viability (Fig. 3D). These results indicate that the protective effect of pandurate A might be mediated by AMPK-independent mechanisms. We next examined the effect of pandurate A on certain proteins involved in cell apoptosis induced by cisplatin. As shown in Fig. 4, cisplatin significantly increased pro-apoptotic proteins p-ERK1/2 and cleaved caspase 3. Cisplatin decreased the expression of anti-apoptotic protein Bcl-2. Co-treatment with pandurate A reversed the expression of these proteins. Since ROS is a major factor involved in renal cell apoptosis induced by cisplatin,11,27) we determined the effect of panduratin A on ROS accumulation due to cisplatin. Cisplatin significantly increased intracellular levels of ROS. Interestingly, panduratin A significantly reduced cisplatin-induced ROS accumulation.
Panduratin A Does Not Affect Cellular Accumulation of Cisplatin OCT2 mediates cisplatin transport into the human renal proximal tubular cells.289 To examine the possibility of panduratin A reducing cisplatin accumulation, we measured the transport function of OCT2. The results showed that incubation of cells with 5uM panduratin A for 10min and 24h had no effect on the cellular accumulation of H-1-methyl-4-phe-nylpyridinium (H-MPP+), a substrate of OCT2. Next, the effect of panduratin A on cisplatin cellular accumulation was confirmed. Panduratin A had no significant effect on the platinum accumulation in the human renal cells(Fig. 5).
The Protective Effect of Panduratin A Does Not Affect the Anti-cancer Activity of Cisplatin Panduratin A's effect on cisplatin's toxicity in cancer cell lines was determined. Vi-ability of HCT116 and A549 cells which are colon and lung cancer cell lines, respectively, was significantly reduced 72h after treatment with cisplatin （50μuM）. Co-treatment with panduratin A（5μM） did not attenuate the cytotoxicity of cisplatin. Interestingly, panduratin A by itself also reduced the
DISCUSSION
The present study reveals that panduratin A reduces cis-platin nephrotoxicity without impairing cisplatin's anticancer activity in colon and non-small cell lung cancer cell lines. We showed that co-treatment with panduratin A attenuates acute kidney injury in mice caused by cisplatin. Although the present study shows the protective effect on cisplatin-induced kidney injury, the primary target of panduratin A is not identified. Panduratin A could activate AMPK in renal proximal tubular cells(Fig. 3) and other cell types.30-2)It seems unlikely that the protective effect of panduratin A requires activation of AMPK as evidence showed that inhibition of AMPK using chemical inhibitors did not alter the protective effect of Pandu-rating A. Panduratin A improved renal function as shown by reduced serum creatinine. Cisplatin-impaired renal function is correlated with renal tubule injuries such as degeneration, luminal cast formation, haemorrhagia, and dilation. The renal tubule injury results correlated well with our data showing the induction of NGAL expression in kidney tissue of mice treated with cisplatin. In addition, this evidence is supported by the previous study showing NGAL is markedly expressed in renal tubules following acute kidney injury induced by cisplatin.3)Interestingly, co-treatment of the mice with panduratin A attenuated cisplatin-induced renal tubule injury. The histological analysis showed that cisplatin increased mononuclear cell infiltration and this effect was abolished by panduratin A. These results imply that panduratin A, which is previously shown an anti-inflammation,24.3435)might attenuate the inflammatory effect of cisplatin in renal tissue. MAPK activation could induce cell death.2415)Our results showed that cisplatin stimulated activation of ERK1/2 and caspase 3. Expressions of these proteins were attenuated by panduratin A.
Multiple pathways contributing to cisplatin-induced nephrotoxicity(including apoptosis, oxidative stress, and inflammation) have been reported.4We used the RPTEC/TERT1 cell line to identify mechanisms of panduratin A ameliorating toxicity caused by cisplatin in human renal proximal tubular cells. RPTEC/TERT1 cells have been suggested as a renal cell model for xenobiotic-induced toxicities.36Panduratin A exhibits a protective effect in respect of cisplatin cytotoxicity as shown by a decrease in RPTEC/TERT1 cell apoptosis. The mechanisms of cisplatin's toxicity in renal proximal tubular cells are complex. ERK1/2 and caspase 3 activation and Bcl-2 depletion have been implicated in renal cell apoptosis due to cisplatin.373)Consistent with previous studies,the present study showed that cisplatin increases expression of p-ERK1/2 and cleaved caspase 3 and decreases Bcl-2 expression. Our results indicate that panduratin A suppresses cisplatin-induced apoptotic signaling proteins, p-ERK1/2 and cleaved caspase 3, and maintains anti-apoptotic protein. Bcl-2. In addition to the activation of ERK1/2 and caspase 3, increased intracellular ROS accumulation is a key factor in renal cell apoptosis due to cisplatin.139A previous study reported that panduratin A protects against oxidative damage in hepatocytes by reducing intracellular ROS formation caused by reactive intermediates.4The decrease in ROS accumulation following treatment with panduratin A could be mediated by either inhibition of ROS production or increase in ROS scavenging reaction. We reported that panduratin A increased glutathione level, an ROS scavenging molecule, which might reduce ROS accumulation in renal tissue (see supplementary materials). However, we do not rule out other mechanisms are involved in ROS reduction. Our results revealed that panduratin A reduces intracellular ROS accumulation, which contributes to panduratin A's protective effect. The intracellular level of cisplatin is the primary factor affecting the severity of cisplatin-induced toxicity. Renal transporters such as OCT2, copper transporters and multidrug and toxin extrusion transporter-1 (MATE1) have been reported to contribute to cisplatin transport.4)However, transport of cisplatin into the renal proximal tubular cells is primarily mediated by OCT2. Inhibition of OCTs has been shown to attenuate cisplatin-induced renal toxicity both in cultured renal cells and animals.发9，42）However， it seems likely that panduratin A does not affect OCT2 transport function or accumulation of cisplatin suggesting panduratin A's protective effect against cisplatin's toxicity is not mediated by reducing transport or accumulation of cisplatin.
CISTANCHE WILL IMPROVE KIDNEY/RENAL PAIN
Panduratin A exhibits renoprotective effects via the inhibition of renal cell apoptosis. If panduratin A also inhibited cancer cell apoptosis, it would reduce the chemotherapy efficacy. We, therefore, tested whether panduratin A reduces cisplatin's anticancer activity. Significantly, co-treatment with Pandu-rating A does not alter the anti-cancer activity of cisplatin in either colon or non-small lung cell cancer cells. Moreover, panduratin A itself is toxic to both cancer cell lines. The results indicate that panduratin A's effect is selective, affecting normal renal cells and cancer cells differently. Although pan-duration A might be a good agent for preventing of cisplatin-induced nephrotoxicity, the information concerning the pharmacological effects of panduratin A under clinically relevant in xenograft model should be investigated in the future.
CONCLUSION
Our present study demonstrated that panduratin A provides a marked protective effect on cisplatin’s nephrotoxicity by reducing oxidative stress and inhibiting ERK1/2 and caspase 3 activations. The protective effect of panduratin A did not alter the anti-cancer activity of cisplatin in cancer cells. Even though blocking some injurious events may only have partial renoprotective effects, panduratin A might be a good candidate agent to alleviate cisplatin’s nephrotoxicity because it reduces the toxicity of cisplatin via multiple mechanisms.
