Molecular Mechanisms And Biomarkers Associated With Chemotherapy-Induced AKI Ⅱ

NEW HERBAL FORMULATION FOR ACUTE KIDNEY INJURY (AKI) 

1. Introduction 

Acute kidney injury (AKI) is a life-threatening condition with increasing incidence worldwide [1]. It is characterized by a rapid and transient decrease in kidney function, measured as an increase in serum creatinine (sCr) and a reduction in the glomerular filtration rate (GFR) and urine output [1]. AKI is part of an assortment of conditions, defined as acute kidney diseases (AKD) [1]. AKD can occur after an AKI event has ended, but where the deterioration in kidney dysfunction and structural damage persist or when kidney dysfunction evolves slowly without a rapid AKI onset [1]. AKD lasting for >3 months is referred to as chronic kidney disease (CKD) [2]. Approximately 20% to 50% of all patients admitted to the intensive care unit (ICU) develop AKI [3]. In the context of cancer treatment, old and new chemotherapy drugs often cause chemotherapy-associated nephrotoxicity [4,5]; accordingly, up to 17.5% of cancer patients develop AKI, which negatively affects patient survival [6]. Additionally, long term, these AKI events are associated with the progression of CKD, cardiovascular complications, and mortality [7]. 

A common pathological feature of AKI is an injury to tubular epithelial cells (TEC), endothelial damage, and accumulation of inflammatory cells [1]. However, the incomplete understanding of pathophysiology and molecular mechanisms associated with chemotherapy-related nephrotoxicity resulting in AKI has hampered the identification of effective biomarkers for patient stratification. As chemotherapy-related AKI may affect the bioavailability of many chemotherapy drugs, potentially leading to suboptimal treatments, the identification of biomarker profiles predictive of AKI severity and outcome is now more urgent than ever. While routine diagnostic markers, such as sCr and urine output, measure the loss of kidney function following an AKI, they do not identify the preceding pathophysiological changes, such as tubular injury. Numerous urinary biomarkers are in use or have been proposed as indicators of kidney injury [8]. Identifying patients at high risk of developing AKI, together with an awareness of potential drug nephrotoxicity, early recognition, and management of incipient AKI, are vital to reducing cases of established AKI. In this review, we aim to (1) provide an overview of the mechanisms involved in chemotherapy-associated AKI; (2) discuss the biomarkers currently available and propose additional biomarkers for early AKI detection, and (3) raise awareness of chemotherapy-induced AKI and promote collaboration between nephrologists, oncologists, and intensive care specialists for early AKI recognition and effective oncologic patient management. 

2. Epidemiology

According to incidence, prevalence, and mortality trends, the epidemiology of cancer worldwide is dramatically changing [9,10]. Despite the impact of cancer overdiagnosis in correctly estimating the actual impact of cancer on patient survival [11], cancer will likely be the leading cause of death in the following decades [9,10]. Nevertheless, the development of new cancer drugs continues to improve cancer survival rates in high-income countries [11]. Chemotherapy nephrotoxicity is associated with significant kidney manifestations, including AKI, progression to CKD, proteinuria, nephrotic syndrome, and electrolyte disorders [9]. Thus, the interplay between cancer treatment and kidney health is complex. While cancer patients are a population at high risk of developing AKI as a result of their cancer treatment regimes, the physical and psychosocial effects associated with cancer survivorship may make a subsequent CKD diagnosis a difficult health issue to address [12]. 

In recent years, many initiatives have provided operative definitions of AKI, all based on the measurement of sCr and urine output (Table 1). AKI can complicate disease courses and is associated with increased mortality in cancer patients [6]. In a population-based study of 163,071 patients undergoing systemic treatment for cancer in Ontario, the overall cumulative incidence of AKI was 9.3% [13]. A similar study conducted in China demonstrated an incidence of 7.5%, with a higher prevalence among hospitalized patients [14]. The most extensive study to date, accounting for 1.2 million patients in Denmark followed from 1999 to 2006, revealed a 1-year AKI risk of 17.5% [15]. Thus, risk factors for AKI, AKD, and progression to CKD should be carefully assessed [6]. 

The number of cancer patients admitted to the ICU has gradually increased over the last number of decades [16,17]. A recent multicenter, observational study showed that 15% of patients admitted to European ICUs have cancer, with solid tumors being more common than hematological cancer (85% vs. 15%, respectively) [18]. AKI is a severe and frequent complication during critical illness ranging from 54% [19] to 70% [20,21], and it is particularly common in patients with hematological cancer or multiple myeloma [18,22]. Notably, in most cases, AKI is already present on admission to the ICU rather than acquired in the ICU [21]. However, reports vary depending on the criteria used to define AKI [23]. Furthermore, diagnosis and staging of AKI in critically ill patients should consider that sCr concentrations may be artificially low due to cachexia and muscle mass loss, although AKI is already present.

3. Risk Factors

Epidemiology studies have highlighted common risk factors for AKI, also traceable in the general population, and specific risk factors that are tumor-related. In advanced cancer, underlying CKD and diabetes are all associated with an increased risk of AKI [13], together with urinary tract obstruction [14]. In addition, volume depletion, due to fluid loss or confinement into the third space, a common condition in elderly patients, can be easily traced as one of the most common risk factors for AKI [29]. Other drugs, when administered concomitantly with certain cancer drugs, such as diuretics, angiotensin-converting enzyme (ACE)-inhibitors, or proton-pump inhibitors, are associated with increased toxicity. Tumor-specific risk factors are generally a hallmark of certain tumors [30,31]. AKI complicating hematologic malignancies may be due to light chain cast nephropathy in multiple myeloma or tumor lysis syndrome after the initiation of chemotherapy in patients with high-grade lymphomas or leukemias [30,31]. Metastases to the kidney from solid tumors are not uncommon; functional impairment of the kidneys generally requires metastases to both kidneys. This condition occurs mainly with rapidly growing hematologic malignancies, such as lymphoma or acute leukemia [32]. Thrombotic microangiopathy may be associated with primary cancer or, more likely, with therapeutic regimens, such as gemcitabine or vascular endothelial growth factor (VEGF) inhibitors (such as bevacizumab) [33]. Urinary tract obstruction should be considered as a cause of AKI in cancer patients, especially those with malignancies of the bladder, prostate, uterus, or cervix [34]. Intratubular obstruction can be caused by crystals composed of uric acid, xanthine, hypoxanthine, or calcium phosphate [35]. Of note, phosphate metabolism itself is dysregulated during AKI, and hyperphosphatemia can be present as a result of reduced kidney excretion together with increased fibroblast growth factor 23 (FGF-23) levels [36,37]. FGF-23 is a crucial modulator of calcium and phosphate metabolism. In vitro, FGF-23 is overexpressed in osteoblast-like cells exposed to chemotherapeutics [38], while it has been established that FGF-23 may be upregulated in some cancers [39]. Together, this data indicates an interplay between FGF-23, AKI, and chemotherapy administration that should be further investigated. Extrarenal obstruction can be caused by a wide range of malignancies and may indicate metastatic disease [40]. A diagnosis is usually established by imaging studies which typically show hydronephrosis [41]. When dealing with oncology patients, tumor-specific risk factors, together with common risk factors, significantly increase the risk of AKI. Indeed, these patients should receive additional attention to reduce the burden of AKI and CKD.

In critically ill patients with cancer, sepsis, metabolic disturbances (e.g., hypercalcemia and hyperuricemia), and the nephrotoxic effects of anticancer and supportive therapies are common triggers of AKI [18,42,43]. Older age (>65 years), female sex, and coexisting disease processes, including CKD, diabetic kidney disease, volume depletion (e.g., due to vomiting or diarrhea), or renal hypoperfusion (e.g., due to cardiomyopathy, cirrhosis, or the nephrotic syndrome), predispose these patients to potential AKI development [44]. The association between AKI and anticancer therapies seems particularly relevant in the critical care setting [45]. Hyperthermic chemotherapy is a valuable strategy for patients with carcinomatosis [46]. Most surgical patients treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy are admitted to the ICU and frequently develop severe AKI [46,47]. Immune checkpoint inhibitors (ICPi), which are one of the most frequently prescribed anticancer treatments nowadays [48], are associated with a unique spectrum of immune-related adverse events affecting several organs, including the kidneys [49]. Direct renal toxicity of these drugs can have severe consequences and lead to ICU admission. Collectively, a better understanding of the mechanisms linked to chemotherapy-related AKI could potentially help the identification of more specific and sensitive biomarkers.

4. Mechanisms and Clinical Manifestations 

AKI can affect the different portions of the nephron, namely the tubules and the glomeruli, as well as the interstitium and the vasculature [50]. Acute tubular necrosis (ATN) results from direct injury to the tubules and is one of the common manifestations of nephrotoxic AKI [50]. ATN is a dynamic process involving different forms of regulated necrosis, resulting in the synchronization of tubular cell death along the entire tubule [51,52]. Necrotic TEC releases pro-inflammatory molecules that activate resident immune cells in the interstitium, which, in turn, further promote tubular necrosis in a vicious circle [53]. Following AKI, functional recovery occurs via two main mechanisms: (1) clonal expansion of a TEC subset (termed progenitor cells) endowed with regenerative ability to replace lost TEC [54] and (2) polyploidization of differentiated TEC [54]. From an evolutionary point of view, polyploidization appears to be most likely developed to sustain a temporary functional recovery of the kidney that is not accompanied by a structural recovery (which should be sustained by progenitor cells). When structural damage is prolonged, AKI may progress to AKD [1].

As a comprehensive analysis of all the AKI mechanisms associated with cancer is beyond the scope of this review, we will focus specifically on drugs that directly affect the tubules. Cytotoxic chemotherapy, targeted agents, as well as ICPi account for several cases of AKI in patients receiving those treatments. Nephrotoxicity is more frequently observed with cytotoxic agents, likely due to their nonspecific mechanisms of action [44]. Many of the drug-related mechanisms of nephrotoxicity are not well-defined, making it difficult to develop targeted strategies for preventing or minimizing their occurrence. In addition, there is often a lack of standardization for dose adjustment in patients with pre-existing kidney impairment [5]. The mechanisms of nephrotoxicity are summarized in Figures 1 and 2, and Table 2 and described in the following sections. Cytotoxic agents. There are many different classes of nephrotoxic agents employed for cancer treatment, comprising but not limited to alkylating agents, antimetabolites, anytimemicrotubule agents, antibiotics, proteasome inhibitors, and platinum agents. Among these, the most widely used (being prescribed in nearly 50% of all tumor chemotherapies [55]) is cisplatin, a platinum drug. Cisplatin nephrotoxicity may be associated with a protean clinical manifestation [56]. Appropriately timed renal functional assessment may help diagnose cisplatin-associated AKI, as exposure typically exerts a slow rise in sCr five to seven days after administration [56]. Severe AKI requiring kidney replacement therapy (KRT) is uncommon. Hypomagnesemia, a typical feature of cisplatin toxicity, is caused by urinary magnesium wasting, and it is dose-related [57,58].

Figure 1. Chemotherapy-induced nephrotoxicity. (A) Hematoxylin and Eosin staining shows tubular damage in a kidney biopsy of a patient following treatment with a cocktail of cisplatin, carboplatin, etoposide, cyclophosphamide, and vincristine. (B–D) Higher magnification of biopsy is shown in (A). Black circles indicate distal tubular casts. White circles indicate luminal cellular debris.*indicates proximal tubule injury.◦indicates regenerative nuclear atypia. Arrows indicate karyomegaly. Bars100µm. 

Cisplatin-induced nephrotoxicity is associated with oxidative stress and inflammation [59-61]; however, the precise mechanisms of action of the drug remain somewhat unclear [59,62. However, its major reported cytotoxic effect is mediated by its interaction with DNA, which leads to DNA damage and apoptosis induction [63]. Terminally different treated cells, such as TEC, must cope with the accumulation of damage throughout a lifespan [64]. Importantly, DNA damage triggered by cisplatin and the associated DNA damage response (DDR) is an important pathogenic mechanism of AKI following cisplatin treatment [65]. DDR activation may lead to cell cycle arrest [66–68] or, in the presence of severe injury, cell death. However, not only cell cycle arrest but also polyploidy has been recently shown to protect against DNA damage-induced cell death [64]. Cisplatin treatment in humans and rodents is reported to cause karyomegaly in renal tubules [69–71], which could indicate the presence of polyploid TEC. Indeed, renal tubule karyomegaly does not develop immediately, instead requiring successive rounds of nuclear division to increase the ploidy content to a recognizable size [72,73] likely explaining why this feature is frequently missed.

Another commonly used nephrotoxic agent is ifosfamide, an alkylating agent. Its nephrotoxicity is particularly relevant considering that it is mostly observed in pediatric patients [74,75]. Thirty percent of the children treated with ifosfamide will consequently develop CKD [76]. Nevertheless, the reported prevalence of nephrotoxicity ranges from 15% to 60% [77,78]. Clinically, AKI associated with ifosfamide is characterized by tubular dysfunction [75]. Ifosfamide mainly affects the S3 segment of the proximal tubule and/or the distal nephron resulting in Fanconi syndrome. This syndrome is characterized by inadequate reabsorption in the proximal renal tubules, with traceable glucosuria, aminoaciduria, tubular proteinuria, decreased phosphate reabsorption, and type 1 (distal) or type 2 (proximal) renal tubular acidosis, or even nephrogenic diabetes insipidus [75]. A specific risk factor for ifosfamide nephrotoxicity is cumulative drug dose [79,80]. Two drugs with antioxidant properties-mesna and N-acetylcysteine (NAC)-are currently used to limit their toxic effects, although their efficacy has not been tested in clinical trials [74,77,79,81]. Considering the long life expectancy of children and young adults surviving cancer, drug-related nephrotoxicity and its lasting consequences represent a crucial unmet problem in medicine. Among the numerous side effects associated with its metabolites [82–84], ifosfamide reacts with DNA molecules to form intra-and interstrand cross-links, causing the DNA strand to break [85]. Interestingly, ifosfamide has also been associated with karyomegalic nephropathy, further suggesting an interesting association between DNA damage, AKI, and increased ploidy [86–88]. Anti-infective drugs, such as vancomycin, gentamicin, and amphotericin B, are also leading causes of drug-induced nephrotoxicity [89–91]. Their mechanisms of action are not well understood, but their primary target is, in all cases, the proximal tubular cells where they cause oxidative stress [89–91], a well-recognized trigger of DNA damage [92]. Finally, one nephrotoxic manifestation of many cytotoxic agents is rhabdomyolysis [93–95] which is known to cause nephrotoxic AKI [96]. Importantly, we have recently shown that rhabdomyolysis triggers TEC polyploidy in response to damage [54]. 

Collectively, the presence of tubular karyomegaly and polyploidy in response to AKI may play an important role in the pathogenesis of nephrotoxicity, at least for some anti-cancer drugs.

Immune checkpoint inhibitors (ICPi). Immune checkpoint inhibitors (ICPi) are a major class of cancer drugs able to improve prognosis in several cancers. These humanized monoclonal antibodies target inhibitory receptors (CTLA-4, PD-1, LAG-3, TIM-3) and ligands (PD-L1) expressed on T lymphocytes, antigen-presenting cells, and tumor cells, eliciting an anti-tumor response by stimulating the immune system [97–99]. Targeting checkpoints of immune cell activation has been demonstrated to be the most effective approach for activating anti-tumor immune responses. The combination of CTLA-4 and PD-1 blockers increases the response rates in patients, and ipilimumab (anti-CTLA-4) plus nivolumab (anti-PD-1) in combination are particularly effective in different cancer types, such as those affecting the kidney [100]. 

However, patients treated with ICPi are also subject to "immune-related adverse events (IRAEs)", which are common and can affect any organ, including the lung, liver, skin, and kidney [101]. Recent data show differences in the IRAE characteristics caused by different ICPi, and organ-specific effects remain unexplained [102]. ICPi-induced AKI is being observed with increasing frequency in patients. In the largest retrospective study available, AKI occurred at a median time of 16 weeks (IQR 8–32) following ICPi initiation [49]. When a kidney biopsy was performed, the typical lesion associated with AKI was acute tubulointerstitial nephritis (ATIN) [49]. Thus, it is likely that nephrologists will be increasingly charged with diagnosing and managing AKI following ICPi administration [103]. Accordingly, an increasing number of case reports have described kidney complications and AKI associated with the use of ipilimumab and/or nivolumab [103,104]. The first reported cases of AKI linked to nivolumab were described in 2016 [105] and were associated with ATIN. This association raised the possibility that nivolumab therapy may release the suppression of T-cell immunity that normally permits renal tolerance to drugs known to be associated with ATIN [105,106]. In addition, PD-1 knockout mice were shown to spontaneously develop glomerulonephritis [107,108], suggesting that PD-1 inhibitor therapy may drive an autoimmune variant of interstitial nephritis. Though very little has been reported about AKI pathophysiology linked to nivolumab and ipilimumab, AKI appears as a delayed onset following exposure to ICPi [103]. This is in stark contrast with cytotoxic-induced AKI, which is rather immediate. In addition, ICPi-induced AKI presents many features of autoimmune diseases rather than of direct drug-related nephrotoxicity, likely explaining the delayed onset of AKI in these patients. However, the underlying mechanisms of kidney injury are largely unknown (and excellently reviewed here [98]) and warrant further investigation. Nevertheless, a recent paper reported two AKI cases in patients with nivolumab treatment, characterized by the presence of karyomegalic TEC, potentially indicating TEC polyploidy. Of note, most of the enlarged tubular epithelial cells were positive for Ki-67, a cell cycle activation marker [109]. Ki-67 cannot distinguish cells undergoing mitotic or alternative cell cycles, but rather it indicates cell cycle entry [109]. This may indicate that karyomegalic TEC are polyploid cells undergoing multiple rounds of polyploidization [109,110]. 

Targeted agents. Molecular-targeted agents are compounds that target specific molecules involved in the growth and spread of cancer cells [111]. Concerning cytotoxic agents, targeted agents are thought to have fewer side effects and cause less harm to non-cancer cells. Epidermal growth factor receptor inhibitors (EGFR inhibitors) are used extensively to treat various cancers, such as non-small-cell lung cancer, breast, head and neck, and pancreatic cancer [112]. Epidermal growth factor receptor (EGFR) is a transmembrane protein with intrinsic tyrosine kinase activity that can be activated by several ligands, modulating cell differentiation, proliferation, and survival through the EGFR–ERK and EGFR–PI3K–Akt signaling pathways [113,114]. The blockade of EGFR may result in AKI, nephrotic syndrome, and proliferative glomerulonephritis [115,116]. The exact pathogenesis of EGFR inhibitors-associated kidney-related disorders is unclear [112]. However, it should be noted that EGFR is widely expressed in mammalian kidneys [117]. In this respect, functional analysis performed in vivo showed that treatment with an EGFR tyrosine kinase inhibitor (erlotinib, a commonly used anti-cancer agent [118]) delayed renal function recovery after AKI [114]. In contrast, EGFR activation accelerated kidney repair [114]. Indeed, proximal tubular EGFR knock-out (KO) mice showed persistent tubular cell damage in the weeks after AKI compared to wild-type mice [114]. Interestingly, no difference was detected in innate immune system activation and inflammatory cell infiltration [114]. This implies that the delayed recovery rate of EGFR-KO mice is related to a direct effect on TEC rather than a systemic effect. Recently, activation of the EGFR-PI3K-Akt pathway in response to AKI was shown to activate Yes-associated protein (YAP1), promoting kidney repair [119]. YAP1 is the main effector of the highly conserved Hippo pathway [120]. Unlike other signaling pathways, the Hippo pathway does not have dedicated receptors, but it is rather regulated by a network of upstream components [120]. This pathway appears to work as a sensor for tissue integrity, responding and adapting accordingly [120]. Importantly, YAP1 has been reported to control polyploidization in the liver through Akt signaling [121]. In the liver, YAP1 activation turns on Akt signaling, promoting S-Phase Kinase Associated Protein 2 (SPK2) acetylation, resulting in cytoplasmic retention, leading to cell polyploidy [121]. Interestingly, loss of liver kinase B1 (LKB1) in mouse hepatocytes enhances EGFR activation, which leads, in turn, to mitotic slippage and increased cell polyploidization [122]. Collectively, the importance of polyploidization [54,123] and EGFR downstream activation of YAP1 in response to AKI [114] may indicate that blocks of EGFR could profoundly affect the endogenous repair potential of the kidney, especially when administered in combination with cytotoxic agents that are notoriously nephrotoxic, exacerbating their damage. In addition to EGFR inhibitors, targeted agents that have been shown to trigger AKI are BRAF blockers [124,125], B-cell lymphoma-2 inhibitors [126], and BCR-ABL1 and receptor tyrosine kinase inhibitors [127–129]. However, in most cases, the association remains vague and requires thorough investigation.