Spironolactone but not Eplerenone Exacerbates Cisplatin Nephrotoxicity

Cisplatin is a highly successful chemotherapeutic agent used for the treatment of solid tumors. However, nephrotoxicity is a limiting factor that occurs in 30% of patients under treatment. Many mechanisms are involved in cisplatin-induced nephrotoxicity, such as epithelial and endothelial injury, inflammation, oxidative stress, and renal vasoconstriction. The mineralocorticoid receptor (MR) has an important role in inflammation and vascular function. MR blockage and ablation have been shown to be effective in preventing renal ischemia-reperfusion injury and cyclosporine A-induced nephrotoxicity. We investigated whether MR antagonism with spironolactone or eplerenone could prevent cisplatin-induced nephrotoxicity. Here, we show that spironolactone treatment exacerbates nephrotoxicity in mice treated with acute and long-term cisplatin regimes. Moreover, spironolactone potentiated the toxicity induced by cisplatin treatment in a cell viability assay in human embryonic kidney cells. In contrast, eplerenone neither prevented nor increased cisplatin toxicity in mice or cultured cells. Thus, our studies support recent findings showing that spironolactone potentiates cisplatin-induced cytotoxicity, independently of mineralocorticoid receptor inhibition.


Introduction
Cisplatin (CIS) is an antineoplastic drug used in chemotherapy with a high rate of success to treat solid tumors. However, its use is limited by its side effects, especially nephrotoxicity, which affects 30% of patients treated with CIS (1,2). CIS-induced nephrotoxicity is due to several mechanisms, such as direct epithelial and endothelial injury, renal vasoconstriction, inflammation, oxidative stress, hypoxia, and apoptosis (2)(3)(4). Mineralocorticoid receptor (MR) antagonism is widely used for the treatment of cardiovascular complications of heart failure (5)(6)(7). Our group demonstrated that MR antagonism protects against acute kidney injury (AKI) induced by Cyclosporin A (8) and ischemia-reperfusion, two conditions characterized by sustained vasoconstriction, resulting in epithelial and endothelial injury (9)(10)(11). MR antagonism also attenuates the AKI-to-chronic kidney disease transition in a bilateral renal ischemia-reperfusion model (12)(13)(14). These observations in rodent models have been extended to the large white pig ischemia-reperfusion model (11,13).
Our main objective was to analyze whether pharmacological MR antagonism can protect kidneys against nephrotoxicity induced by CIS.

Experimental protocols
All procedures used and described for animal experiments were carried out in accordance with the National and European guidelines for animal experimental research and were approved by the Ethical Committee for Animal Experiments of the Sorbonne University (#7594), France. The animals were housed in controlled-climate conditions with a 12-hour light-dark cycle and provided free access to food and water.
Eight-week-old male C57Bl/6 mice (Janvier Laboratories, France) were used for pharmacological experiments with spironolactone (Sigma-Aldrich), eplerenone (Inspra, Pfizer), and CIS (Sigma-Aldrich). Spironolactone (20 mg/kg) was administered by oral gavage (vehicle: methylcellulose) in a single dose, 48, 24, and 1h before each CIS injection. Eplerenone (200 mg/kg/day) was administered by oral gavage in two doses of 100 mg/kg, 48, 24, and 1 h before CIS injection. The doses of spironolactone and eplerenone were chosen according to our past experience and based on previously published studies in mice (15)(16)(17)(18)(19)(20)(21)(22)(23). Acute kidney injury was induced with one single dose of CIS (20 mg/kg) i.p and mice were euthanized three days later. Longterm CIS treatment consisted of repeated dosing with one dose of CIS (7 mg/kg) i.p per week for four weeks. Mice were euthanized three days after the last injection.

Renal Parameters analysis
Blood sample was taken via cardiac punction and plasma urea and creatinine concentrations were determined with an automatic analyzer (Konelab 20i, Thermoscientific). Urine was collected in metabolic cages over 24 hours after 3 days period of habituation and protein concentration were determined with Konelab 20i.

RNA extraction and real-time PCR
Total RNA was extracted from the kidneys with TRIZOL reagent (Life Technologies,  Table 1.

Histological analysis
The kidneys were fixed in Bouin's fixative solution and then dehydrated and embedded in paraffin. Sections (4 m) were cut and stained with Hematoxylin Eosin and Sirius Red. At least six subcortical fields were visualized and analyzed for each mouse using a Leica DM4000 microscope at a magnification of 200X. The acute tubular necrosis score was determined based on the percentage of tubules showing luminal casts, cell detachment, or dilation and assigned according to the following scale: 0 = 0 to 5%, 1 = 6 to 25%, 2 = 26 to 50%, 3 = 51 to 75%, and 4 > 75%. Histology analysis was performed blind to experimental groups to assess tubule-interstitial fibrosis based on the Sirius Red-positive area and assigned according to the following scale: 1 ≤ 25%, 2 = 26 to 50%, 3 = 51 to 75%, and 4 > 75%.

Statistics
The results are expressed as the mean +/-SEM. The significance of between-group differences was assessed by one-way analysis of variance (ANOVA) with the Tukey correction for multiple comparisons. A Student t-test was performed to compare two groups. GraphPad (La Jolla, CA) Prism 6 software was used for statistical analyses and graphical representations. p values < 0.05 were considered to be statistically significant.

Spironolactone but not Eplerenone exacerbates renal dysfunction and renal injury after single-dose administration of cisplatin
Plasma creatinine and plasma urea levels were higher in the CIS group than vehicle (VEH) group and further exacerbated in the CIS+SPIRO group ( Figure 1A-B). The mRNA expression of NGAL, a renal injury marker, and TNF-α, an inflammatory marker, was also higher in the CIS group than VEH group and further elevated in the CIS+SPIRO group ( Figure 1C-D). Tubular injury, assessed by HE staining, was higher in the CIS group than VEH group and further exacerbated in the CIS+SPIRO group ( Figure 1E-H). In contrast to spironolactone, eplerenone neither exacerbated nor protected against the deleterious effects of CIS (Figure 2A-H).

Spironolactone exacerbates nephrotoxicity after long-term cisplatin treatment
In clinical practice, patients receive CIS at low doses repeatedly and for long periods.

Spironolactone but not eplerenone decreases HEK-293 cell viability after cisplatin exposure
We next assessed cell viability by the MTT assay. HEK cells were incubated with spironolactone or eplerenone for 24 h and then exposed to CIS for 24 h. CIS decreased cell viability for all doses tested (25/40/60 µM) ( Figure 4A). The diluent of CIS, spironolactone or eplerenone (NaCl, EthOH or DMSO, respectively) has no effect on viability ( Figure 4A-B). The addition of spironolactone decreased cell viability for all tested concentration of CIS ( Figure 4A). Importantly, eplerenone at 100 orr 200 microM did not affect the cell toxicity of CIS, in contrast to spironolactone ( Figure 4B).

Discussion
Approximately one-third of patients treated with CIS develop nephrotoxicity (1,2). CIS enters renal cells by a facilitated mechanism or a passive route and activates pathways that promote cell death (1,2,26). In addition, CIS induces TNF-α production by tubular cells, triggering an inflammatory response which contributes to tubular cell injury and death (27,28). The renal vasculature is also targeted, resulting in ischemic tubular cell death (2). Part of the nephrotoxicity of CIS is due to vascular damage and vasoconstriction, caused mainly by endothelial dysfunction and abnormal vascular autoregulation (29). CIS can also be directly toxic to the vascular endothelium (29).
Endothelial injury results in overexpression of endothelial cell-adhesion molecules and the binding of leukocytes (30). Vascular congestion and leukocyte infiltration result, leading to the injury and swelling of endothelial cells. Moreover, such obstruction decreases renal blood flow, contributing to the local production of vasoconstrictors and affecting the glomerular filtration rate (29, 31).
Our group previously demonstrated that MR antagonism attenuates kidney injury in various experimental models. Pharmacological MR antagonism by all current classes of MR antagonists (steroidal, such as spironolactone or eplerenone, or non-steroidal, such as BR-4628 and finerenone) protects against acute kidney injury induced by ischemia-reperfusion in mice, rats, and large white pigs (10,11,14,32) as well as cyclosporine-induced nephrotoxicity (8,(10)(11)(12)(13). We hypothesized that a similar benefit of MR antagonism might be observed for acute CIS-induced nephrotoxicity, as several common underlying mechanisms have been proposed, including altered hemodynamics, oxidative stress, and an inflammatory response. Indeed, pharmacological MR antagonism during renal ischemia-reperfusion has highlighted the role of MR-mediated oxidative stress in the acute phase of ischemia (10,11), which leads to decreased renal perfusion and increased renal resistance. Similar observations were made in an acute model of cyclosporin-induced nephrotoxicity, in which MR deletion in smooth muscle cells improved renal perfusion, thus attenuating vascular resistance and modulating L-type Ca 2+ channels (8). Importantly, blunting MR activation in the early phase of IRI (by short-term treatment with MRAs) in rodents or large white pigs has long-term benefits, even in the absence of continued MRA administration, preventing the development of chronic kidney disease with impaired renal function, proteinuria, and kidney fibrosis (13). In the present study, we show that these benefits do not extend to CIS-induced nephrotoxicity, since both acute and chronic CIS-induced nephrotoxicity were not blunted by MRAs given just before and at the time of CIS administration, when the acute phase of nephrotoxicity maybe maximal.
Indeed, we observed the opposite effect with spironolactone, as it aggravated CISinduced nephrotoxicity.
The impact of MR antagonism on CIS nephrotoxicity is controversial: spironolactone has been reported to exacerbate CIS-induced nephrotoxicity in rabbits (33), whereas eplerenone treatment had a protective effect in balb/c mice (34). Moreover, daily spironolactone administration for one week reduced CIS-induced nephrotoxicity caused by a single CIS injection in Wistar albino rats (35). Here, we show that spironolactone, but not eplerenone, exacerbates nephrotoxicity in the C57Bl/6 mouse during acute and chronic treatment with CIS. Eplerenone neither worsened or lessened this effect of CIS treatment. The various effects observed in our study versus those previously reported may be related to the timing of MR antagonist administration, as well as differences in species susceptibility but it may also relate to off target effect of spironolactone, independent of MR blockade.
In fact, it is difficult to understand how two members of the same pharmacological class can display such different effects. We show in the present study that spironolactone potentiates the deleterious effect of CIS on the cellular viability of human kidney cells in culture. This suggests that the in vivo potentiation of CIS is not related to a functional deleterious effect of spironolactone, such as dehydration induced by the diuretic effect of the drug, but rather a direct potentiating effect of spironolactone on CIS toxicity in epithelial cells. A major difference between the two MRAs is that spironolactone also acts as an androgen receptor antagonist or progesterone receptor agonist while eplerenone does not since it is much more specific for MR. While we cannot discard this possibility, previous results rather favor an off-target effect of spironolactone that synergizes with the cytotoxic effects of CIS. Indeed a recent study identified spironolactone in an unbiased screen of molecules which sensitized CIS-dependent cellular toxicity (24). It is well known that CIS affects various DNA-associated process, such as replication and transcription, which leads to cell apoptosis. Nucleotide excision repair (NER) is a mechanism that removes DNA lesions caused by UV irradiation and platinum-based drugs, such as CIS and is expected that the inhibition of NER would be a powerful tool to sensitize cancer cells to chemotherapy (24). Surprisingly the unbiased screening for CIS potentiating agents underlined that spironolactone indeed potentiated the effect of CIS on DNA damage through the inhibition of NER, whereas the more specific MR antagonist eplerenone had no effect (24). The authors reported that spironolactone, but not eplerenone, is a nontoxic NER inhibitor that can increase the sensitivity of CIS and UV irradiation, leading to lower viability of cells (24). Similarly, spironolactone potentiated the cytotoxicity of CIS in human pancreatic cancer cells and human colon cancer cells (36). Indeed, we translated this concept to human kidney cells, showing decreased cell viability upon CIS co-treatment.
The results of our study may have a potential clinical impact that needs to be further explored; the deleterious potentiating effect of spironolactone should be considered in cases of combined treatment with CIS and spironolactone (for example to treat heart failure or resistant hypertension), whereas eplerenone or another class of MR antagonists may be used more safely. The treatment regimen used in the chronic setting in mice in the present study was appropriate to test the prevention of repeated acute renal injury induced by each dose of CIS but do not to test the impact of repeated administration of CIS on top of long-term MRA administration as it could occur in patients. This is a limitation of our study and the impact of different treatment regimen should also be explored in a dedicated animal model.  from H&E-stained sections. n = 6 to 7 per group. One-way analysis of variance was performed. Bar = 100 µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. performed. ****P < 0.0001 vs control groups ****P < 0.0001 CIS vs CIS+SPIRO.