Pioglitazone protects against cisplatin induced nephrotoxicty in rats and potentiates its anticancer activity against human renal adrenocarncinoma cell linesDIONISIO, DEKYDOMINGUEZ, ANGELICAGABRITO, CHRISTIAN JOHNGIRONELLA, ROCHELLEGISON, CHRISTINE BEATRICE

TERMS BADGE - Bisphenol A diglycidyl ether DMSO - Dimethyl sulfoxide GSH Glutathione MDA Malondialdehyde NF-B Nuclear factor kappa B PPAR- Peroxisome proliferator activated receptor gamma

I. APPRAISING DIRECTNESSPatientsRats with cisplatin induced nephrotoxicityHuman renal adenocarcinoma cell lines

ExposureGroupI: Normal control rats injected IP with0.1 mL diluted DMSOII: Cisplatin injected rats, 7mg/kg IP with Normal Saline SolutionIII: Pioglitazone 3mg/kg orally one hour before Cisplatin injectionIV: BADGE 10 mg/kg IP one hour before CisplatinV: 10 mg/kg BADGE half hour before oral administration of Pioglitazone 3mg/kg then one hour later, cisplatin was also injected by the same previous dose

Outcome Reduction of cisplatin-induced nephrotoxicityPotentiation of anticancer effect of cisplatin

II. APPRAISING VALIDITY1. Were patients randomly assigned to treatment groups?The animals were randomly assigned to five groups (n=6). The rats received the same food, housing and environmental conditions.

2. Was allocation concealed?The allocation is not concealed in the experiment.

3. Were baseline characteristics similar at the start of the trial?The baseline characteristics are similar at the start of the trial. All are adult male Wistar rats weighing 275+10g and were of similar age 61-66 days.

4. Were patients blinded to treatment assignment?Technically, the patients/ subjects in this study which are adult male Wistar rats were blinded since they do not have the capacity to know what was being introduced to their system.

5. Were caregivers blinded to treatment assignment?The caregivers/ researchers in the study were not blinded and they fully know the treatment regimen for each of the study groups which they need to administer.

6. Were outcome assessors blinded to treatment assignment?Likewise, the outcome assessors/ researchers are the ones evaluating the treatment outcome and they have full knowledge of the drug being evaluated in this study.

7. Were all patients analyzed in the groups to which they were originally randomized?All patients/ animal subjects were analyzed prior to randomization with the following parameters Adult male Wistar rats weighing 275 + 10 g was obtained from Faculty of Veterinary Medicine, Zagazig University, Egypt. All rats were similar age of about 61-66 days.

8. Was follow up rate adequate?There was no follow up in the study.

III. APPRAISING RESULTS1. How large was the effect of the treatment?Cisplatin induced a significant increase in serum creatinine level, increased lipid peroxidation and reduced GSH content in renal tissue, increased TNF- and NF-kB genes and reduced PPAR- gene expression in comparison to normal control at p

Pioglitazone significantly reduced serum creatinine level compared to cisplatin alone at p

Combination of pioglitazone and cisplatin resulted in a shift down in cell viability of ACHN cells.

A small probability of p

2. How precise was the estimate of the treatment effect?The estimation of the treatment effect was precise because it was based on significant statistical data.

IV. ASSESSING APPLICABILITY1. Are there biologic issues that may affect applicability of treatment? (Consider the influence of sex, co-morbidity, race, age, and pathology)The treatment was done only in adult male rats. There is no evidence for effect of treatment for females.

2. Are the socio-economic issues affecting applicability of treatment?There were no socio-economic issues affecting applicability of treatment.

V. INDIZIDUALIZING THE RESULTSWhat is the likely effect of the treatment in your individual patient?Pioglitazone may represent a novel therapeutic approach for the prevention of cisplatin-induced nephrotoxicity and potentiating its anticancer effect.

Pioglitazone increased the expression of PPAR- in the renal tissue. This mediated the nephroprotective action of pioglitazone, through the inhibition of NF-B signaling pathway, TNF- expression, and macrophage infiltration and antioxidant effects in cisplatin treated rats without interfering with the anticancer effect of cisplatin.

ADDITIONAL QUESTIONSIf you have patients with nephrotoxicity caused by cisplatin, can you give pioglitazone as a treatment? Pioglitazone can be given as a PROTECTIVE AGENT to reduce nephrotoxicity caused by Cisplatin.

The study focused on the prevention of cisplatin-induced nephrotoxicity. In the said study, pioglitazone was given orally one hour before the administration of Cisplatin. Afterwards, different parameters were used to measure nephrotoxicity of the experimental animals. The study suggests that pioglitazone protected the kidney against cisplatin-induced nephrotoxicity through its interaction with PPAR- receptors and anti-oxidant properties.

If you give pioglitazone, will you expect less toxicity? Less renal problem?Yes. The results of the study suggest that pioglitazone had nephroprotective effect on cisplatin-induced nephrotoxicity.

The study revealed that pioglitazone decreased serum creatinine and ameliorated tubular necrosis without changing serum urea levels in cisplatin treated rats. In addition, the study also suggests that pioglitazone had anti-inflammatory effect in cisplatin treated kidneys. Histopathological examination on the kidneys of the rats treated with pioglitazone showed less tubular necrosis and less glomerular necrosis which indicate that the rats had less renal damage.

Does pioglitazone potentiate anticancer activity of cisplatin?Yes. Pioglitazone alone had cytotoxic effect against adenocarcinoma cell lines (ACHN).

In the study, pioglitazone potentiated the cytotoxic effect of cisplatin against ACHN cell lines. However, the cytotoxic effect was abolished by prior administration of BADGE. The cytotoxic effect was evaluated through in-vitro study using human renal adenocarcinoma cell lines. The results obtained were supported by different studies.

What are the parameters used to measure nephrotoxicity? Blood samplesSerum Creatinine used to evaluate renal dysfunction; increased serum creatinine is an indication of impaired renal functionSerum urea used to assess kidney function.

BUN - measures the amount of nitrogen contained in the urea. High BUN levels can indicate kidney dysfunction.Malondialdehyde (MDA) - is a major lipid peroxidation product that is mutagenic and tumorigenic. It is used to determine lipid peroxidation.Glutathione (GSH) used to measure antioxidant status.

Histopathological Examination of the KidneyTubular necrosisCongestion of glomeruli, capillary and venulesTubular cast and dilatationMononuclear infiltration in interstitiumThickened basement membrane of glomerulusGlomerular necrosis

Is the methodology accurate?Yes. The parameters used in the study were accurate to determine the renal function of the experimental animals. Although some studies suggest that test for uric acid levels should be included. Test for uric acid also serve as marker of nephrotoxicity. Some studies show that uric acid levels of rats treated with cisplatin were elevated.

Will you accept the conclusion of the study? Yes. The data obtained from the in-vivo study of rats were sufficient to evaluate the nephroprotective effect of pioglitazone on cisplatin-induced nephrotoxicity.

While the in-vitro study on the human renal adenocarcinoma cells was sufficient to assess the cytotoxic property of the drugs and their effects when used in combination. The results presented were also supported by different studies. All these supporting studies confirmed current observation that Pioglitazone has nephroprotective effect and it potentiates cisplatin cytotoxic effect on human adenocarcinoma cell lines. .