Lansoprazole promotes cisplatin‐induced acute kidney injury via enhancing tubular necroptosis

Abstract Acute kidney injury (AKI) is the main obstacle that limits the use of cisplatin in cancer treatment. Proton pump inhibitors (PPIs), the most commonly used class of medications for gastrointestinal complications in cancer patients, have been reported to cause adverse renal events. However, the effect of PPIs on cisplatin‐induced AKI remains unclear. Herein, the effect and mechanism of lansoprazole (LPZ), one of the most frequently prescribed PPIs, on cisplatin‐induced AKI were investigated in vivo and in vitro. C57BL/6 mice received a single intraperitoneal (i.p.) injection of cisplatin (18 mg/kg) to induce AKI, and LPZ (12.5 or 25 mg/kg) was administered 2 hours prior to cisplatin administration and then once daily for another 2 days via i.p. injection. The results showed that LPZ significantly aggravated the tubular damage and further increased the elevated levels of serum creatinine and blood urea nitrogen induced by cisplatin. However, LPZ did not enhance cisplatin‐induced tubular apoptosis, as evidenced by a lack of significant change in mRNA and protein expression of Bax/Bcl‐2 ratio and TUNEL staining. Notably, LPZ increased the number of necrotic renal tubular cells compared to that by cisplatin treatment alone, which was further confirmed by the elevated necroptosis‐associated protein expression of RIPK1, p‐RIPK3 and p‐MLKL. Furthermore, LPZ deteriorated cisplatin‐induced inflammation, as revealed by the increased mRNA expression of pro‐inflammatory factors including, NLRP3, IL‐1β, TNF‐α and caspase 1, as well as neutrophil infiltration. Consistently, in in vitro study, LPZ increased HK‐2 cell death and enhanced inflammation, compared with cisplatin treatment alone. Collectively, our results demonstrate that LPZ aggravates cisplatin‐induced AKI, and necroptosis may be involved in the exacerbation of kidney damage.


| INTRODUC TI ON
Cisplatin is a potent first-line chemotherapy agent for many malignancies, such as lung and ovarian cancers. However, the utility of cisplatin in the clinic is limited mainly because of its nephrotoxicity.
It has been reported that 20%-40% of cancer patients exposed to cisplatin experience acute kidney injury (AKI), which is associated with a significantly increased risk of mortality. 1 Patients that survive AKI are predisposed to develop chronic kidney disease (CKD). 2 Since there is a lack of an effective therapeutic approach for cisplatininduced AKI prevention and treatment, 3,4 risk factors that induce or promote AKI should be addressed during cisplatin treatment as carefully as possible.
In clinical practice, cisplatin, a high emetogenic chemotherapy drug, is often used in combination with proton pump inhibitors (PPIs). PPIs are among the most commonly used medications to treat a variety of indications, including gastroesophageal reflux disease and gastroduodenal disease. 5 Evidence shows that approximately 65% of cancer patients receiving acid-reducing agents receive PPI treatment for gastroesophageal reflux. 6 Although PPIs are considered to be highly safe and well-tolerated, they cause some adverse renal events. Recent large clinical population-based cohort studies have shown that PPIs are associated with an increased risk of AKI, incident CKD, and progression to end-stage renal disease in different patient populations compared with that in histamine-2 (H2) receptor antagonist users or PPI non-users. [7][8][9][10] Additionally, PPIs are often overused without reasonable indications or medical prescription, leaving us to question the number of different populations at risk of adverse renal events. 11 In this regard, we need to evaluate the effects of PPI use under coadministration with cisplatin.
A retrospective cohort study showed that the combination of PPIs alleviated the nephrotoxicity induced by the anti-tumour drugs cisplatin and fluorouracil, as evidenced by the decrease in blood urea nitrogen (BUN) (not serum creatinine [Scr]) and delayed onset of renal function failure, compared to that in non-users. 12 However, given the small size and the observational nature of this data, further investigation of co-treatment of PPIs and cisplatin-associated renal outcomes is needed.
A recent in vitro study focused on the mechanisms of PPIinduced nephrotoxicity, pointing out that tubular cell death may be a contributing factor. 13  Pathological score of tubular injury in the kidneys. (D and E) Blood samples were collected to measure the concentration of serum creatinine (Scr) and blood urea nitrogen (BUN). Data are presented as mean ± standard error (SE). All data of each group are analyzed using oneway analysis of variance (ANOVA). ns: not significant; *P < .05; **P < .01; and ***P < .001 are major factors that determine the outcome of cisplatin-induced AKI. 14 Morphology analyses of cisplatin-induced AKI have shown that tubular cells mainly undergo necrosis and apoptosis. 14 We were curious to learn whether PPIs affect cisplatin-induced renal damage via cell death and inflammation. To test this hypothesis, we assessed apoptosis, necroptosis and inflammation using lansoprazole, a commonly used PPI, in a cisplatin-induced AKI model.

| Measurement of kidney function
Blood samples were collected for kidney function measurement.

| Histological evaluation of kidney damage
Kidney tissue was fixed with Carnoy's solution for 24 hours and embedded in paraffin as previously described. 15 Renal morphology changes were examined on renal tissue slices (3 μm) by periodic acid-Schiff (PAS) staining and haematoxylin and eosin (HE) staining. Tubular dilatation, tubular cell necrosis and cast formation were scored from 0 to 4 as a percentage of the whole cortical area of the kidney slices. 16 0, normal; 1, <10%; 2, 10%-25%; 3, 26%-75%; 4, >75%. The morphometric measurements were performed in a blinded manner.

| Quantitative real-time PCR (RT-qPCR)
Total RNA was extracted from kidney tissues using RNAiso Plus (Takara Bio Inc). PrimeScript RT Master Mix (Takara Bio Inc) was used to reverse transcribe the total RNA into cDNA according to the manufacturer's instructions. RT-qPCR was performed using SYBR Premix Ex Taq II (Takara Bio Inc) with a Light Cycler 480 system (Roche). Sequences of the primers used were as follows:

| Western blotting analysis
Western blotting was performed as previously described. 17  Image J software (NIH, Bethesda, MD) was used for densitometric analysis.

| Apoptosis detection
Apoptosis was determined by TUNEL staining (ApopTag Peroxidase Kit, EMD Millipore) in paraffin-embedded renal tissues (3 μm), according to the manufacturer's instructions. The TUNEL-positive cells in 10 high-power fields at 400× magnification were counted. nuclear staining. Images were acquired using a fluorescence microscope (Olympus IX81, Olympus), and 10 high-power fields at 400× magnification were randomly chosen for neutrophil number counting.

| Electron microscopy
Ultrastructure analysis of renal proximal tubule cells was performed by transmission electron microscopy. Mouse renal tissue samples were fixed, dehydrated, embedded and stained as previously described. 17 Ultrathin sections were examined using a JEM-1400 transmission electron microscope (Jeol Ltd.).

| Statistical analysis
Results are presented as the mean ± standard error. All statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, Inc). One-way analysis of variance with Tukey's or Bonferroni's post-hoc test was used to compare parameters among groups. Statistical significance was defined as P < .05.   Figure 1A. According to PAS staining, there were no significant differences in morphology between the LPZ and CON groups ( Figure 1B

| Exacerbation of cisplatin-induced AKI by lansoprazole is independent of tubular apoptosis
A recent study revealed that PPIs nephrotoxicity is associated with tubular cell death. 13 Since tubular apoptosis is one of the major cellular process changes in cisplatin-induced nephrotoxicity, 20 we questioned whether lansoprazole exacerbates cisplatin-induced renal damage via apoptosis. The TUNEL assay showed that cisplatin treatment led to significant tubular cell apoptosis, while no further increase in apoptosis was observed after co-treatment with different doses of lansoprazole (Figure 2A,B). Moreover, the critical apoptosis regulators Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) were tested at both the mRNA and protein levels. The Bax/Bcl-2 ratio has been hypothesized to increase apoptosis. 21 A remarkable increase in the Bax/Bcl-2 ratio at both the mRNA and protein levels indicated apoptosis in renal tissue after cisplatin treatment. No further increase in the Bax/Bcl-2 ratio was detected following addition of different doses of lansoprazole ( Figure 2C-E). Taken together, these data suggest that apoptosis may not explain why lansoprazole aggravates cisplatin-induced AKI.

| Lansoprazole increases cisplatin-induced tubular necroptosis
Next, we estimated the occurrence of necroptosis, another common form of tubular cell death in cisplatin-induced renal injury. 19 Necroptosis, a type of programmed necrosis, can be triggered by tolllike receptors, interferons, death receptors and other mediators. 22

RIPK3 and its substrate MLKL are considered crucial players of this
pathway. 23 Here, we investigated the role of necroptosis in the exacerbation of cisplatin-induced kidney injury by lansoprazole. Tubular necrosis was estimated in HE-stained renal tissues ( Figure 3A).  Figure 3C). Subsequently, we examined necroptotic biomarkers, including RIPK1, p-RIPK3 and p-MLKL. We detected necroptosis in the renal tissue after cisplatin treatment, as evidenced by the significant increase in RIPK1, p-RIPK3 and p-MLKL expression ( Figure 4). Both p-MLKL and p-RIPK3 expression showed a further significant increase in the CIS + LPZ groups compared to that in the CIS group. RIPK1 expression also showed a slight increase in the CIS + LPZ 12.5 group but showed a significant increase in the CIS + LPZ 25 group compared to that in the CIS group.
In in vitro study, cisplatin were used to stimulate HK-2 cells for 24 hours at 80 μmol/L. 19 Different doses of LPZ, 2.5, 5, 10 and 50 μmol/L, were co-treated with cisplatin, among which the concentration of 5 μmol/L is within the therapeutic range in clinic treatment. 24 Consistently, increased cell death was observed in CIS + LPZ groups, compared with CIS group, revealed by propidium iodide (PI) stain with flow cytometry ( Figure S1). PARP-1, RIPK1, p-RIPK3 and p-MLKL expression were also tested by western blot. The core necroptosis execution component, p-MLKL, was significantly increased after co-treated with LPZ, compared with cisplatin administration alone ( Figure S2). Altogether, these results demonstrate that LPZ aggravates cisplatin-induced tubular necroptosis.

| Lansoprazole enhances cisplatin-induced renal inflammation
In vivo data have shown that necroptosis plays a critical role in triggering inflammation. 25 Next, we were keen to determine whether lansoprazole enhances cisplatin-induced renal inflammation. We cisplatin treatment alone ( Figure 5A-C). NLRP3, caspase 1 and IL-1β were also tested at the protein level, and it showed a slight increase in the CIS + LPZ 12.5 group and a significant increase in the CIS + LPZ 25 group compared to that in the CIS group ( Figure 5D,E and Figure S3). Besides, we also test IL-6 and IL-18 by western blot in in vitro study. The result shows that both IL-6 and IL-18 increased in CIS + LPZ groups, compared with CIS group ( Figure S4). In addition, we tested neutrophil infiltration in kidney tissues, and the result was consistent with the expression of pro-inflammatory factors ( Figure 6A,B). Collectively, these data suggest that lansoprazole increases cisplatin-induced renal inflammation.

| D ISCUSS I ON
In the present study, we examined the effect of lansoprazole, a commonly used PPI, on kidney injury induced by cisplatin treatment. Our data demonstrate that lansoprazole aggravates cisplatin-induced AKI. Necroptosis and inflammation may contribute to this aggravated renal injury, but this exacerbation is independent of apoptosis ( Figure 7).
In this study, a dose of 12.5 mg/kg or 25 mg/kg lansoprazole was used according to the conversion of the clinical dosage (lansoprazole, 1 or 2 mg/kg/d). The pattern of lansoprazole treatment was also based on clinical practice in cisplatin-treated patients. Our findings showed that lansoprazole aggravated cisplatin-induced AKI.
However, in another similar study, rats were pretreated with PPIs (omeprazole, 1.8 mg/kg and 3.6 mg/kg) before cisplatin treatment, in which the doses of omeprazole were below the range of clinical treatment and showed a renoprotective effect. 26 The inconsistencies in these outcomes may be owing to the different doses and administration pattern of PPIs.
Recently, an in vitro study revealed that tubular cell death may contribute to omeprazole nephrotoxicity. 13 Therefore, we investigated the role of renal tubular cell death in exacerbated renal function after cisplatin and lansoprazole co-treatment. Renal tubular cell death (apoptosis and necrosis) has been hypothesized as a major factor in cisplatin-induced nephrotoxicity. 27 Over the past two decades, apoptosis in tubular epithelial cells has been well studied and has been considered to be a promising therapeutic target. 28 Furthermore, additional efforts should be made to elucidate the precise mechanism by which PPIs enhance necroptosis in the setting of cisplatin-induced renal injury. Although, some efforts that we have made give us some hints that this may be associated with increased production of mitochondrial ROS ( Figure S5). Our findings also need to be proven in human studies.
Taken together, our results suggest that lansoprazole aggravates AKI induced by cisplatin and that necroptosis may be involved in the exacerbation of this kidney damage.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science Writing-review & editing (supporting).

DATA AVA I L A B I L I T Y S TAT E M E N T
All data included in this study are available upon request by contact with the corresponding author.