Effect of creatine and pioglitazone on Hk-2 cell line cisplatin nephrotoxicity

Abstract

Cisplatin is a chemotherapeutic agent, which is used in the treatment of various solid organ cancers, and its main dose limiting side effect of cisplatin is nephrotoxicity. The aim of this study is to investigate the role of pioglitazone and creatine on cisplatin nephrotoxicity in vitro. Real-time cell analyzer system (RTCA) was used for real-time and time-dependent analysis of the cellular response of HK-2 cells following incubation with cisplatin and combination with creatine or pioglitazone hydrochloride. First, half-maximal inhibitory concentrations (IC50) of cisplatin, creatine and pioglitazone were calculated by RTCA system. Afterwards creatine and pioglitazone was administered with serial dilutions under RTCA system. IC50 dose for cisplatin was 7.69 M × 10⁻⁵ at 24th hour and 3.93 M × 10⁻⁶ at 48th hour. IC50 dose for pioglitazone was 1.61 M × 10⁻³ at 24th hour and 2.85 M × 10⁻⁴ at 48th hour. Although cells were treated the dose of 40,225 mM creatine, IC50 dose could not been reached. Neither pioglitazone nor creatine had additional protective effect in any dose. Consequently, beneficial effect of creatine and pioglitazone on cisplatin-induced cell death could not be found. Further studies and clinical trials are needed to evaluate the effect of different doses of these drugs in cisplatin-induced nephrotoxicity.

• Drug toxicity
• cisplatin
• creatine
• pioglitazone

Introduction

Cisplatin is a chemotherapeutic agent which is used in the treatment of a variety of solid organ cancers such as lung, testis, neck, ovary and breast.1 Nephrotoxicity is a serious dose limiting adverse effect of cisplatin treatment.2,3 Several possible mechanisms such as DNA damage, mitochondrial dysfunction, oxidative stress, activation of caspase formation and apoptosis have been proposed to explain mechanism of nephrotoxicity.4–7 Nevertheless, cisplatin-induced nephrotoxicity has not been clearly illuminated and prevention strategies remain unclear. Therefore, more effective preventive ways are still being investigated.

Creatine is a popular supplement to enhance physical performance. The dietary intake and endogenous production of creatine matches the spontaneous degradation of phosphocreatine and creatine to creatinine.8 Recently, several articles have indicated the various effects of creatine including cytoprotective features through antioxidant-like effect, mitochondrial genome stabilization and decrease in oxygen consumption besides its ergogenic role.9

Pioglitazone is an anti-diabetic drug that belongs to thiazolidinediones and acts as an agonist of peroxisome proliferator activated receptor gamma (PPARg). Along with its anti-inflammatory and antioxidant effects, pioglitazone has been reported to have protective effects on cerebral, myocardial and renal ischemia.10–15

Real-time cell analyzer (RTCA) system is a label-free and non-invasive method which works with the basis of measurement of the electrical impedance on integrated microelectrodes in microwell plates. After the interaction of cells with electrodes, ionic environments alter and modifying impedance is determined in each well by RTCA system. Hence, cell number monitoring can be done and viability can be measured in real time. Results are displayed as cell index value. RTCA is a useful method since the system has several advantages like less interfering with normal cell metabolism and dynamic real-time cell viability measurement.16,17

The aim of this study is to investigate the role of pioglitazone and creatine on cisplatin nephrotoxicity in vitro. This is the first study to search for the possible effects of pioglitazone and creatine in the HK-2 cells exposed to cisplatin.

Material and methods

Cell culture

Experimental studies were carried out using HK-2 cells (CRL-2190-American Type Culture Collection), which are human proximal tubular cells immortalized by transduction with human papilloma virus 16 E6/E7 genes. Cells were cultured in keratinocyte serum-free (KSF) medium (Gibco/BRL 17005-042) supplemented with 5 ng/mL recombinant epidermal growth factor and 0.05 mg/mL bovine pituitary extract at 37 °C in 5% CO2 in a humidified incubator.

Cell treatment and viability

Real-time cell viability analysis The xCELLigence RTCA system (Roche Applied Science, Mannheim, Germany) was used for real-time and time-dependent analysis of the cellular response of HK-2 cells following incubation with cisplatin (ORNA, Istanbul, Turkey) and combination of cisplatin with creatine (General Nutrition Corporation, Pittsburgh, PA) or pioglitazone hydrochloride (CHEMOS GmbH, Regenstauf, Germany).

Cells were seeded at a density of 5 × 10³ cells/well in a 96-well plate and allowed to attach for 24 hour. The medium was then replaced with the medium containing cisplatin and creatine or pioglitazone was combined with serial dilutions. The medium and drug combinations renewed at 48th hour. First, half-maximal inhibitory concentrations (IC50) of cisplatin and pioglitazone were calculated by RTCA system to determine the drug potency and the concentration of the drug that is required for 50% inhibition of cell viability. Although we treated creatine with the dose of 40,225 mM, IC50 dose could not been reached.

Creatine and cisplatin were dissolved in medium, while PI was dissolved in DMSO. Cisplatin was used as IC50 drug concentration of 75 µM. Creatine concentrations were 10 mM, 2.5 mM, 0.625 mM, 0.1562 mM and pioglitazone concentrations were 100 µM, 25 µM, 6.25 µM, 1.5625 µM, 0.3906 µM. After treatment, the cells were allowed to grow for 72 hours with monitoring every 30 minutes. Control experiments were carried out using the only growth culture medium without any additive. Cell proliferation was then monitored by RTCA system (xCELLigence and RTCASoftwareV1.2.1). Each experiment was carried out in triplicate. Obtained cell index measurements were normalized according to time of drug administration and referred as normalized cell index in figures.

Results

IC50 dose for cisplatin was 7.69 M × 10⁻⁵ (R²: 0.97) at 24th hour and 3.93 M × 10⁻⁶ (R²: 0.95) at 48th hour. IC50 dose for pioglitazone was 1.61 M × 10⁻³ (R²: 0.96) at 24th hour and 2.85 M × 10⁻⁴ (R²: 0.91) at 48th hour. After addition of creatine to cisplatin treated cells with serial dilutions at 24th hour, all cell series proliferated at 33rd hour and from this point cell viability progressively decreased to end of 72nd hours (Figure 1). In cell line treated with cisplatin and 6.25 µM pioglitazone cell proliferation retained until 34th hour and cell viability began to decrease at the end of 72nd hours. In cell line treated with cisplatin and 100 µM pioglitazone cell proliferation retained until 31st hour, beginning from 34th hour cell viability decreased to end of 72nd hours. Other pioglitazone dose combinations were parallel to free cisplatin administration (Figure 2). These results indicate that neither pioglitazone nor creatine addition had protective effect in any dose.

Figure 1. After cisplatin and creatine addition to HK-2 cells at 24th hour, cell viability graphics in the course of 72 hours.

Figure 2. After cisplatin and pioglitazone addition to HK-2 cells at 24th hour, cell viability graphics in the course of 72 hours.

Discussion

Cisplatin is accumulated in both proximal and distal nephrons. The S3 segment of the proximal tubules has the highest concentration of cisplatin, followed by the distal collecting tubule and the S1 segment in the proximal tubule.18,19 Cisplatin-induced cytotoxicity results from its interaction with DNA and resultant DNA damage.20 Cisplatin especially tends to accumulate in mitochondria and cells which have densely mitochondria are much more sensitive to toxicity. Since renal proximal tubule cells have highest densities of mitochondria in the kidney, this segment is more susceptible to cisplatin-induced cell damage.21 Therefore, we used renal proximal tubular cell line in this toxicity model. In addition to DNA damage, cisplatin inhibits fatty acid oxidation that is the main source of energy for the proximal tubule through reduction in PPAR-α mediated expression of genes involved in cellular fatty acid utilization.21–23 Cisplatin also inhibits the mitochondrial complexes I to IV of the respiratory chain and induces decrease in intracellular ATP levels.24

Creatine can prevent cell damage with two possible mechanisms: stabilization of cellular membranes and maintenance of ATP.18 In addition to these Nomura et al. 25 reported that creatine has an anti-inflammatory effect on endothelial cells. After ischemic insults, ATP production through oxidative pathways is reduced resulting in cell damage. Sharov et al.26 showed that exogenous phosphocreatine administration attenuated ischemic damage and reduced necrotic zone with maintenance of membrane integrity. Berneburg et al.27 reported that there was a link between mitochondrial DNA mutations and cellular energy metabolism. In addition, authors showed that mitochondrial mutagenesis could be normalized by creatine supplementation. On the contrary of this in a limited number of patients (n = 15), Kornblum et al.28 reported that high dose creatine treatment has no beneficial effect on skeletal muscle energy metabolism in patients with genetically proven single mitochondrial DNA deletion.

Besides, activation of intrinsic and extrinsic apoptotic cascades, production of reactive oxygen species are other known pathways of cisplatin-induced renal cell death.21 In this sense, several substances were used to ameliorate cisplatin-induced cell damage such as free radical scavengers, iron chelators and other antioxidants.29–33 Oxidative damage to mtDNA may lead to loss of membrane potential, reduced ATP synthesis and cell death.34 Several reports have been published arguing that creatine supplementation enhances oxidative phosphorylation.35–37 Guidi et al.38 reported that creatine has a protective effect on oxidatively-injured mitochondrial and nuclear DNAs. Sestili et al.39 reported that there is a cytoprotective effect of creatine on oxidatively injured cultured mammalian cells through direct antioxidant activity via a scavenging mechanism. Due to all these mechanism and cytoprotective effects of creatine, we used creatine to prevent cisplatin-induced cell death but we have not seen any significant effect on cell viability in RTCA system. Since there was no cytotoxic effect of creatine, further studies with higher doses of creatine might be carried out in vitro or in vivo.

Apoptosis is induced by several pathways in cisplatin-induced cell damage.7 Wei et al.40 reported that cisplatin-induced apoptosis was attenuated in the Bax protein-deficient mice and authors showed Bax protein and cisplatin-induced apoptosis association. In a renal ischemia-reperfusion injury animal model, Hu et al.15 reported that pioglitazone pretreatment provide up-regulation of expression of Bcl-2 proteins and down-regulation of expression of Bax protein in kidney. Through this way mitochondrial activity and cell integrity are maintained. Pioglitazone has been shown to have beneficial effect on septic shock via suppression of inflammatory responses.12 Cisplatin treatment activates mitogen-activated protein kinase (MAPK) pathways and inhibition of these ways reduces apoptosis, caspase activation, inflammation and renal injury.7 Pioglitazone has ability of suppression of MAPK, nuclear factor-kappa B signaling pathways and inhibition of tumor necrosis factor alpha.10,41 In addition to this, pioglitazone can suppress oxidative stress and damage through improving antioxidant capacity.42,43 Recent studies showed that there is a relationship between cisplatin and sirtuin genes.44 One of the sirtuin genes, SIRT1 is a part of cellular response to cisplatin and overexpression of SIRT1 ameliorated cisplatin-induced cytotoxicity.45 Recent literature supported that pioglitazone treatment increased SIRT1 expression.46,47 For all these reason, in our study, we used pioglitazone as another possible protective drug for cisplatin-induced cell death. However, administration of pioglitazone does not seem to have a beneficial effect on cell viability compromised by cisplatin (Figure 2).

In conclusion, although it is observed that cisplatin nephrotoxicity is attributed to several factors including accumulation, oxidative stress, apoptosis and inflammation, we could not find any beneficial effect of creatine and pioglitazone on cisplatin-induced cell death. Further studies and clinical trials are needed to evaluate the effect of different doses of these drugs and/or other possible drugs, which may affect the ways mentioned above in cisplatin-induced nephrotoxicity.

Declaration of interest

The authors report no declarations of interest. This study was supported by grants from the Ondokuz Mayis University Research Fund with the number PYO.TIP.1901.10.014