**3. Cisplatin associated acute kidney injury**

## **3.1 Background**

Due to their role in the metabolism and excretion of xenobiotics, kidneys are particularly vulnerable to drug-induced toxicities [1]. The renal tubular epithelial cells have significant capacity for uptake of drugs, and this can result in high intracellular concentrations, which can lead to toxicities and development of acute kidney injury. Cisplatin is a widely used chemotherapy drug that accumulates in renal tubular cells causing acute kidney injury [5]. Cisplatin is used as part of chemotherapy regimens for the treatment of a wide spectrum of malignancies such as testicular, head and neck, ovarian, lung, cervical, and bladder cancers. Cisplatin accumulates in the tubular epithelial cells through organic cation [20] and copper transporters [21], which in turn activates a plethora of signaling pathways that culminate in epithelial cell death, inflammation, and kidney injury [5]. Tubulointerstitial injury is the predominant lesion observed during cisplatin nephrotoxicity, wherein both proximal and distal tubules are affected and display significant necrosis. It is observed that renal function improves in most patients, however a subset of patients can develop chronic renal impairment [22].

Cisplatin nephrotoxicity can be mimicked in murine models through single [16, 23] or multiple [24] injections. The single injection induced AKI is the most widely used model of cisplatin nephrotoxicity. In this model, a single intraperitoneal injection (10–30 mg/kg) results in development of AKI within 2–3 days.

### **3.2 Methods**


afternoon (2 PM) leads to more consistent injury and this might be related to the circadian regulation of organic cation transporters that are involved in renal uptake of cisplatin [25].

3.On day three post-cisplatin injection, sacrifice the mice by carbon dioxide euthanasia followed by serum and renal tissue collection for further analysis. Kidney injury can then be evaluated by serum analysis [blood urea nitrogen (BUN) and creatinine], histological analysis [hematoxylin–eosin (H&E) staining], and examination of renal expression of injury biomarkers [kidney injury molecule-1 (KIM1) and neutrophil gelatinase-associated lipocalin (NGAL)] [26, 27]. Blood urine nitrogen and enzymatic assay-based creatinine measurements can be performed in serum or plasma samples using commercially available kits. For histological analysis, mouse kidneys are harvested and embedded in paraffin and tissue sections (4–5μm) are stained with H&E using standard methods. Histopathological scoring can be conducted by examining 10 consecutive ×100fields per section from at least 3 mice/group. Tubular damage is then scored by calculating the percentage of tubules that showed dilation, epithelium flattening, cast formation, loss of brush border and nuclei, and denudation of the basement membrane using a previously [28] described injury scale: 0, no damage; 1, <25%; 2, 25–50%; 3, 50–75%; and 4, >75%.
