Potential Therapeutic Targets for Cisplatin-Induced Kidney Injury: Lessons from Other Models of AKI and Fibrosis

Abstract

The effectiveness of cisplatin, a mainstay in the treatment of many solid organ cancers, is hindered by dose-limiting nephrotoxicity. Cisplatin causes AKI in 30% of patients. Patients who do not develop AKI by clinical standards during treatment are still at risk for long-term decline in kidney function and the development of CKD. The connection between AKI and CKD has become increasingly studied, with renal fibrosis a hallmark of CKD development. To prevent both the short- and long-term effects of cisplatin, researchers must use models that reflect both types of pathology. Although a lot is known about cisplatin-induced AKI, very little is known about the mechanisms by which repeated low levels of cisplatin lead to fibrosis development. In this review, strategies used in various rodent models to prevent kidney injury, its progression to fibrosis, or both, are examined to gain mechanistic insights and identify potential therapeutic targets for cisplatin-induced kidney pathologies. Reviewing the results from these models highlights the diverse and highly complex role of cell death, cell senescence, endoplasmic reticulum stress, autophagy, and immune cell activation in acute and chronic kidney injuries. The use of several models of kidney injury is needed for development of agents that will prevent all aspects of cisplatin-induced kidney injury.

Cisplatin is an inorganic platinum-based drug that was FDA approved as an anticancer agent in 1978.¹ It is a standard therapy used to treat many solid organ cancers.² The major mechanism of action of cisplatin involves binding DNA and forming adducts, leading to apoptosis or cell cycle arrest. Cisplatin effectiveness is reduced by its dose-limiting nephrotoxicity. Approximately 30% of patients administered cisplatin develop AKI.² There are no approved agents to treat AKI, and thus its development requires suspending cisplatin treatment.

After AKI, the kidney initiates wound-healing processes to recover. Although the repair processes after AKI can restore function, in some patients, the normal repair processes go awry, causing progressive renal damage and leading to development of CKD.³ The primary features of such maladaptive repair include cell cycle arrest, profibrotic cytokine production, myofibroblast accumulation, chronic immune cell activation, and chronic vascular impairment.³

Cisplatin-induced AKI is commonly modeled in rodents via a single high dose (20–30 mg/kg) of cisplatin. After treatment, renal function sharply declines, and the animals must be euthanized within 3–4 days. This model mirrors the development of AKI in the clinic; however, it does not allow for long-term studies of the effects of cisplatin-induced kidney injury and represents only patients with severe AKI.^1,4

Approximately 70% of patients treated with cisplatin do not develop AKI by clinical criteria, but may still be at risk for long-term renal impairment. Skinner et al. followed pediatric patients treated with cisplatin who did not develop clinical AKI; however, 10 years after treatment, 11% had reduced GFR and 15% displayed symptoms of nephrotoxicity.⁵ To model this, our laboratory^6–9 and others^10–15 developed an animal model to study cisplatin-induced kidney injury that involves repeated weekly low doses of cisplatin (7–9 mg/kg) for 4 weeks. A repeated low-dose cisplatin (RLDC) regimen allows mice to survive <6 months after treatment without showing clinical signs of AKI. Mice develop renal fibrosis during the dosing window and have progressive loss of kidney function after cisplatin treatment.⁸

Together, these models facilitate study of renal pathologies on either end of the spectrum of cisplatin-induced kidney injury. Models are needed that allow for AKI development and recovery, using intermediate doses of cisplatin (10–15 mg/kg), but optimization is required so that animal survival is not an obstacle.^10,15 The RLDC and high-dose models of cisplatin-induced kidney injury are well characterized and depict how different biologic processes are triggered after acute or chronic cisplatin treatment. Common therapeutic targets of both cisplatin-induced AKI and fibrosis are needed, and potential drug candidates need testing in both models.

In this review, we compare how targeting cell death, senescence, endoplasmic reticulum (ER) stress, autophagy, and immune cell activation has different effects on renal outcomes in the high-dose cisplatin model and models of renal fibrosis (Supplemental Table 1). Because the RLDC model is fairly new, we examine the results from studies of fibrosis following the ischemia-reperfusion (IR) injury and unilateral ureteral obstruction (UUO) models. It is important to note these models of fibrosis may differ from the RLDC model of fibrosis. A discussion of their key similarities and differences may be found elsewhere.¹⁶

Cell Death, Cell Cycle Arrest, and Senescence

In the high-dose cisplatin model, AKI development depends on cell death.^2,17,18 Cisplatin induces necrosis and intrinsic, extrinsic, and ER stress–associated pathways of apoptosis.^17,18 Intrinsic apoptosis is a key driver of AKI in the high-dose cisplatin model (Figure 1). Global knockout of the proapoptotic Bcl-2–associated X protein conferred resistance to cisplatin-induced AKI in mice by decreasing the number of apoptotic cells.¹⁹ Additionally, inhibition and genetic depletion of tumor protein 53 (p53)–attenuated cisplatin-induced AKI in mice, demonstrating that DNA damage is a major contributor to intrinsic apoptosis in acute cisplatin nephrotoxicity.²⁰

Figure 1.

Mechanisms of cisplatin-induced AKI after a single high dose (20–30 mg/kg) of cisplatin. Pointed arrow heads denote promotion of process, solid block lines indicate inhibition of process. Supporting references: 19, 20, 31, 37, 38, 45, 46, and 58-60.

The RLDC model does not induce robust apoptotic or necrotic cell death,⁷ but whether other forms of cell death are involved has not been examined. A variety of cell death pathways (for example, apoptosis, necrosis, necroptosis, ferroptosis, NETosis, and mitophagy^21–24) have been documented in other models of renal fibrosis and CKD, but the extent to which they drive fibrotic development is unclear. Proximal tubule–specific knockout of proapoptotic Bcl-2 proteins, Bcl-2–associated X protein and Bcl-2 homologous antagonist/killer, protected from apoptosis and renal fibrosis in the UUO model.²⁵ Proximal tubule–specific p53 deletion reduced apoptosis and interstitial fibrosis after IR injury.²⁶ However, in the UUO model, global deletion of p53 attenuated apoptosis but did not confer protection from fibrosis 20 days after injury.²⁷ Attenuation of fibrosis with p53 inhibition has been observed at earlier points in time after UUO, but protection was attributed to reduction of cell cycle arrest at the G2/M phase rather than apoptosis.²⁸ These results suggest apoptosis plays complex and cell type–specific roles in renal fibrosis. As with apoptosis, in-depth studies of nonapoptotic forms of cell death in fibrosis development are greatly needed.

A single high dose of cisplatin induces widespread cell death, whereas RLDC causes very little cell death, with surviving cells undergoing sublethal changes, such as cell cycle arrest (Figure 2). This may be due to repeated insults to tubule epithelial cells. Administration of diphtheria toxin to transgenic mice expressing proximal tubule–specific simian diphtheria toxin receptors directly injures proximal tubules.²⁹ A single dose of diphtheria toxin allowed for replacement of lost tubule cells and recovery of kidney function. Repeated injury to tubules via multiple doses of diphtheria toxin induced either senescence or a prolonged dedifferentiated state of the proximal tubules, inducing maladaptive repair, fibrosis, and glomerulosclerosis.²⁹ This study suggests fibrosis is driven by proximal tubule cells that survive injury rather than the loss of proximal tubule cells itself.

Figure 2.

Mechanisms of cisplatin-induced fibrosis after repeated low dose (7–9 mg/kg) cisplatin treatment. Solid line arrows indicate relationships directly examined in the literature. Dotted line arrows represent relationships studied in other models of renal fibrosis that we propose may also be occurring in the RLDC model. Supporting references: 25-30, 39-42, 47-49, and 61-67.

Cell cycle arrest at the G2/M phase contributes to fibrotic outcomes in renal models of injury. It is associated with high levels of JNK signaling and secretion of profibrotic cytokines, including TGFβ. Pharmacologic inhibition of JNK reduces fibrosis after IR injury.³⁰ Data suggest that RLDC causes cell cycle arrest and senescence mediated by JNK signaling.⁷ Cisplatin is known to induce JNK phosphorylation and activation.¹⁷ In the high-dose model of cisplatin-induced kidney injury, JNK activation promotes apoptosis and inflammation, whereas inhibition of JNK reduces renal damage.³¹ JNK activation also promotes G2/M cell cycle arrest when activated by TGFβ. This alternative activation identifies JNK signaling as a balancing point between apoptosis and cell cycle arrest, with environmental stimuli determining the outcome.³² In RLDC, p-JNK and TGFβ are increased, accompanying elevated expression of cyclin-dependent kinase inhibitor 2a, a known marker of cellular senescence.^7,8 Therefore, RLDC-induced fibrosis may be driven by tubule G2/M cell cycle arrest, senescence, or both, as repair of renal damage is attempted. This needs more in-depth investigation.

ER Stress and Autophagy

ER stress can lead to outcomes ranging from apoptosis to adaptation. Responses to ER stress depend on the extent of cellular damage and the duration of the insult. With acute insults, cells need only to withstand stress for a short period of time. Recovery depends on how quickly the cell can mount the unfolded protein response and clear accumulated unfolded proteins. With chronic stress, the cell must undergo a functional change that is more permanent, with a prolonged unfolded protein response allowing cells to adapt and escape cell death.³³ The level and duration of ER stress determines if cells die or adapt and survive. One method of ER stress adaptation is autophagy³⁴ (discussed below).

In models of AKI, ER stress is induced, and the development of fibrosis and its pathologic effects are attributed to apoptotic and autophagic induction.³⁵ The role of ER stress is studied in vivo using many strategies, including pharmacologic intervention and C/EBP homologous protein (CHOP)–deficient mice. Although CHOP classically is studied as an inducer of proapoptotic genes, it also regulates prosurvival genes.³⁶

In the high-dose cisplatin model, the α₂ adrenergic agonist dexmedetomidine attenuated ER stress via decreased CHOP expression,³⁷ preventing apoptosis and AKI in rats. Similarly, the GPR120 activator TUG891 inhibited ER stress induction, blocked apoptosis, and protected from cisplatin-induced AKI.³⁸ Although these pharmacologic agents might act through alternative mechanisms, both studies attributed the protective effects to ameliorating ER stress, decreasing CHOP expression, and reducing apoptosis.

In models of renal fibrosis, the role of ER stress may be more complex. In the IR model of injury and fibrosis, both CHOP knockout mice and pharmacologic inhibition of ER stress were protective, accompanied by reductions in apoptosis, inflammation, and tubule autophagy.^39,40 Compared with wild-type mice, CHOP knockout mice have reduced expression of autophagy-associated proteins and mitigated fibrosis after UUO.^41,42 Noh et al. suggest that excessive autophagy can contribute to kidney injury via induction of apoptosis.⁴¹ These studies in models of renal fibrosis demonstrate how the ER stress response can lead to both autophagy and apoptosis in kidney disease, processes that may function in opposition or coordination, depending on the length and strength of stimuli. In the high-dose model of cisplatin, high levels of ER stress activation accompany apoptosis (Figure 1). In contrast, the RLDC model may result in sustained, low levels of ER stress, leading to long-term upregulation of autophagy that may be maladaptive (Figure 2).

Autophagy is a form of cellular “self-eating” that can be both protective and detrimental.^43,44 Studies have shown that cisplatin-induced AKI is associated with induction of autophagy, and data suggest it is protective.¹⁸ Autophagy can help reduce levels of cisplatin-induced apoptosis by ameliorating the effects of cellular damage from oxidative stress and mitochondrial dysfunction.⁴³ In the high-dose cisplatin model, autophagy inhibition exacerbated kidney injury by increasing DNA damage, p53 activation, protein aggregation, and apoptosis.^45,46

In models of fibrosis, the role of autophagy is still unclear. It has been argued that long-term upregulation of autophagy might contribute to renal damage.⁴⁴ In a model of IR injury, mice with specific deletion of autophagy related 5 in proximal tubule cells of the S3 segment had reduced interstitial fibrosis and cellular senescence 30 days after reperfusion. Interestingly, at 2 hours postreperfusion, autophagy-deficient mice had increased levels of cell death. This led to the hypothesis that fibrosis is being driven by proximal tubule cells that survive initial injury via increased autophagy and become senescent later.⁴⁷

Mice with distal tubule–specific conditional knockout of autophagy-related 7 had increased levels of TGFβ and renal fibrosis after UUO.⁴⁸ In contrast to the findings of this study, mice with proximal tubule–specific conditional knockout of autophagy-related 7 had decreased levels of renal fibrosis after UUO.⁴⁹ Pharmacologic inhibitors of autophagy (chloroquine and 3-methyladenine) also prevented UUO-induced fibrosis.⁴⁹ Interestingly, TGFβ expression was not altered when autophagy was inhibited.⁴⁹ These studies indicate the autophagy in renal fibrosis may play roles that depend on context and cell type.⁴³ The majority of evidence suggests elevated autophagy can decrease cell death, providing protection from AKI (Figure 1),^45,46 but under chronic stress, the cells that escape death become sublethally injured and undergo senescence, contributing to fibrosis development (Figure 2).^47,50,51

Immune Response

Renal immune cell infiltration and activation may differ in high-dose cisplatin and RLDC models (Figure 3). Inflammation is a major mediator of AKI in the high-dose cisplatin model. Inflammation and renal fibrosis are also known to be closely linked.⁵² Cisplatin treatment induces upregulation of many different proinflammatory cytokines and chemokines, including TNFα.^9,17,18

Figure 3.

Immune cell involvement in cisplatin-induced AKI and renal fibrosis, ointed arrow heads denote promotion of process, solid block lines indicate inhibition of process, lines with no arrow ending denote no effect on process. Supporting references: 59-67. CDDP, cisplatin.

Inhibition of TNFα signaling attenuated cisplatin-induced kidney injury in the high-dose model and decreased production of other inflammatory mediators, indicating it plays a central role in activation of the cytokine response.^53,54 In contrast, TNFα-deficient mice had worse fibrosis compared with wild-type mice 4 weeks after UUO.⁵⁵ However, neutralization of TNFα reduced renal fibrosis and improved function up to 1 week after UUO.⁵⁶ These conflicting results could be due to the different points in time observed, indicating TNFα may be pathogenic in early processes but important for longer-term recovery processes. These studies demonstrate the diverse functions the immune response can have at different stages of renal injury and progression to fibrosis, highlighting the importance of more in-depth studies of immune responses in chronic injury models.

We have observed increased expression of TNFα and other inflammatory cytokines in the mouse kidney after RLDC treatment.^8,9 This cytokine response attracts immune cells to the kidney. Neutrophils, macrophages, T cells, and dendritic cells are the major responders to high-dose cisplatin-induced injury.^2,18 Our laboratory has shown RLDC leads to a significant increase in immune cells in the kidney after four doses⁸; analysis of myeloid cells revealed a significant increase in M2 macrophages.⁹ Although further study is needed to evaluate the role of these inflammatory responders in RLDC, studies in other models of both AKI and renal fibrosis can provide some insight into which targets should be examined.

T cell–deficient (nu/nu) mice were protected from injury after a single high dose of cisplatin.⁵⁷ Furthermore, CD4 T cell–deficient mice showed greater protection compared with CD8 T cell–deficient mice.⁵⁷ In contrast, depletion of CD11c dendritic cells using the diphtheria toxin receptor model–sensitized mice to cisplatin-induced AKI, indicating dendritic cells may play a nephroprotective role.⁵⁸ Lastly, although macrophages were shown to infiltrate the kidney after cisplatin treatment, depletion of macrophages with liposome-encapsulated clodronate had no effect on development of AKI.⁵⁹

The role of immune cells in renal fibrosis may be more complex. RAG-1 mice deficient in T and B cells had less fibrosis development after UUO compared with wild-type mice. Furthermore, reconstitution of CD4 T cells reversed this protection, whereas CD8 T cell reconstitution did not.⁶⁰ Additionally, CD8 T cell–knockout mice were reported to have increased renal fibrosis after UUO, whereas pharmacologic depletion of CD4 T cells decreased renal fibrosis.⁶¹ These results suggest a pathogenic role for CD4 T cells in renal fibrosis. However, a study by Ravichandran et al. found that depletion of CD4 T cells did not protect mice from RLDC-induced fibrosis, and it hindered the efficacy of cisplatin, accelerating subcutaneous tumor growth.⁶² This study indicates CD4 T cells would not be a viable target in cisplatin-induced kidney injury. It also demonstrates that immune cells may play different roles in the different models.

Studies of IR injury showed that global depletion of macrophages via liposome-encapsulated clodronate attenuated development of renal fibrosis. Adoptive transfer of M2 macrophages after global depletion reversed this beneficial effect, whereas M1 transfer did not, suggesting a role for M2 macrophages in the development of fibrosis post-IR injury.⁶³ Global depletion of macrophages with liposome-encapsulated clodronate after development of IR-induced AKI prevented recovery from injury, indicating the timing and coordination of M1 and M2 responses play a role in injury and recovery.⁶⁴ Depletion of macrophages and dendritic cells with liposome-encapsulated clodronate before UUO-attenuated renal fibrosis.^65,66 Altogether, these studies indicate the diverse and complex role of immune cells in kidney disease. The role of the immune system in renal fibrosis is made more complex by the transitions that occur from proinflammatory to prorepair phenotypes. Using only the high-dose model of cisplatin-induced kidney injury does not allow for the study of immune cell clearance and tissue repair. Further studies are needed to elucidate the role of specific immune cell subtypes in the different phases of RLDC-induced fibrosis.

Nephroprotective agents can have differential success in the high-dose cisplatin model and the RLDC model. This is likely due to the inherent biologic differences in these two models with respect to the induction of cell death/senescence, autophagy, ER stress induction and response, and immune-cell involvement. The high-dose cisplatin-induced kidney injury model displays high levels of cell death and ER stress, whereas the RLDC model of injury causes lower levels of ER stress and cell death, leading to more sublethal injury and cellular senescence. The RLDC model is also complicated by chronic immune-cell activity in the kidney during recovery. Evaluating the success of different therapeutic strategies in these models may help shed light on the different nephrotoxic mechanisms underlying high-dose cisplatin and RLDC treatment.

Modeling cisplatin-induced kidney injury in rodents is challenging. One challenge is overcoming the differences in pharmacokinetics between rodents and humans. Mice are known to have a higher peak plasma concentration and shorter half-life of platinum compared with humans.⁶⁷ With the differences in cisplatin handling, the best way to model human nephrotoxicity is to match pathologies induced by different dosing regimens. Because cisplatin is administered with different dosing regimens, depending on the cancer type, it follows that many models of nephrotoxicity should be used in rodent studies.

Another challenge in modeling cisplatin-induced kidney injury is comparing manifestations of kidney injury in mice and humans. Although kidney histopathology can be readily evaluated in rodents, this is not the case in humans, because patients who develop AKI after cisplatin treatment rarely undergo kidney biopsy. Blood urea nitrogen and serum creatinine are relied on as markers of kidney function for both rodents and humans. Electrolyte disturbances such as hypokalemia and hypomagnesemia are also closely monitored in human patients,⁶⁸ but are largely ignored in rodents, although it is possible to perform these analyses.⁶⁹ Finally, patients who receive cisplatin have cancer, and the effect of cancer on cisplatin-induced kidney pathologies has not been well studied in rodents. In sum, rodent studies largely ignore some key aspects of human cisplatin-induced AKI, which may be essential for understanding this disease.

Despite these challenges, notable similarities in rodent and human AKI development after cisplatin treatment have been observed. Sex differences are noted in both mice and humans, with females more sensitive than males to cisplatin-induced AKI.^70,71 Studies have also been undertaken to identify genetic predictors of cisplatin nephrotoxicity in humans,^72,73 but direct comparisons to rodent models have not been investigated in depth. Although controversy exists over methods of analysis, some studies have identified a polymorphism (rs316019) in the organic cation transporter 2 gene SLC22A2 as a predictor of cisplatin nephrotoxicity.^74,75 Similarly, organic cation transporter 2 deletion or inhibition in rodents confers resistance to cisplatin-induced AKI.^74–76 This suggests similar cisplatin handling in rodents and humans.

It is important to consider both acute and chronic renal injury when developing nephroprotective agents. Patients exhibit a wide range of responses to cisplatin in the clinic. Some develop AKI after a single low dose, whereas others may receive multiple doses without displaying signs of clinical AKI. Understanding the biologic processes occurring in both the high-dose cisplatin and RLDC models will elucidate the pathologic processes occurring in all categories of patients, leading to better treatment of AKI and prevention of CKD.

Disclosures

All authors have nothing to disclose.

Funding

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases grants R01DK124112 (to L.J. Siskind) and F31DK126400 (to S.M. Sears).

Supplemental Material

This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2020101455/-/DCSupplemental.

Supplemental Table 1. Strategies to prevent cisplatin-induced AKI and fibrosis.