**1. Introduction**

Acute tubulointerstitial nephritis (ATIN) is an immunomediated disease affecting the tubulointerstitial area of the kidneys. The tubulointerstitium comprises 80% of the kidney surface and is composed of cellular and extracellular matrix components [1]. The specific and identifying pathological picture consists of a cellular infiltrate composed by leukocytes, primarily lymphocytes, but also including eosinophils, macrophages, or plasma cells. A myriad of etiologies can lead to ATIN, although drug-induced ATIN is the most common, accounting for 3–14% of biopsy-proven acute kidney injury (AKI) [2] and 70–90% of ATIN cases [3]. Also, of note, ATIN is related to autoimmune and inflammatory diseases—systemic lupus erythematosus, IgG-4 related disease, and tubulointerstitial nephritis with uveitis (TINU) syndrome, among others—or infectious diseases, such as cytomegalovirus or adenovirus infections.

Type IV hypersensitivity reactions are the underlying pathomechanisms of drug-induced ATIN. The kidneys are adapted to filter a high rate of blood flow that contains proteins and potential antigens, thus leaving them exposed to drugs and their metabolites [4]. Tubular epithelial cells (TECs) are able to process and present these antigens from tubular lumen, working as non-professional antigen-presenting cells (APCs). TECs present antigens to dendritic cells, which in turn migrate to regional lymph nodes, activate specific T cells, and integrate innate and adaptive immune responses [5]. Those activated T

cells infiltrate the renal parenchyma and amplify inflammation through increased secretion of cytokines and the recruitment of other inflammatory cells [4].

The involvement of necroinflammation pathways has been recently described to collaborate in the pathogenesis of drug induced ATIN. It has been hypothesized that drugs can directly damage TECs and induce necroptosis. This is a recently-described form of cell death, halfway between necrosis and apoptosis, leading to the release of proinflammatory cytokines and the recruitment of innate immune system cells. After necroptosis of TECs, intracellular molecules are dropped to the interstitial space and bind to several receptors that recognize danger signals, such as toll-like receptors (TLRs), expressed by immune cells. Signal–receptor interaction leads to the release of proinflammatory cytokines that, in turn, magnify the immune response, triggering further direct TEC necroptosis [6]. A role for necroinflammation has been confirmed in a murine model of cisplatin-induced AKI, but further research is required to confirm the participation of these pathways in human ATIN [7].

Inflammatory phenomena and cellular infiltration lead to tubular dysfunction and (AKI). It is usually difficult for the clinician to distinguish between ATIN and acute tubular necrosis (ATN) in this setting. ATIN may be accompanied by fever, skin rash, arthralgias, or flank pain, contrarily to ATN, and this picture can help guide the diagnosis, but those are not universal findings. The presence of known previous autoimmune conditions, concomitant infections, or recently-administered drugs can also support the hypothesis of ATIN of a specific etiology. The gold standard in ATIN diagnosis is kidney biopsy. Based on the predominance of an inflammatory component in the kidneys of ATIN, some classical and novel biomarkers, reviewed hereunder, may serve in the diagnosis, prognosis, and follow-up of this disease.

#### **2. ATIN Classical Biomarkers**

Urine cellularity and casts have been used classically to find evidence of localized inflammation in the kidneys. Routine optical microscopy examination of the urine samples requires trained personnel and is time-consuming. In recent decades, automated cytometric urinalysis has replaced provider-performed urine microscopy. The information obtained here may guide the diagnosis but has limitations. Occasionally, urine sediment can be negative despite the existence of inflammatory kidney disease, and the presence of cells and crystals is not always specific to a certain pathology. Also, automated examination is less precise for diagnosis than laboratory-based microscopy examination. Thus, although useful, urine sediment examination should always be accompanied by knowledge of the clinical context [8].

Sterile leukocyturia is a common finding in ATIN patients. Depending on the series, the prevalence of leukocyturia ranges within 50–70% of all-cause-ATIN cases [9,10]. Interestingly, leukocyturia is an almost universal finding in drug-related and especially in antibiotic-related ATIN, while it is found merely in about 50% of ATIN patients related to autoimmune diseases [11].

Due to the Type IV hypersensitivity basis of drug-induced ATIN and the usual presence of eosinophils in kidney biopsy specimens [6]., eosinophiluria was considered a classical biomarker in ATIN. The belief inthe utility of this parameter is based on a small-case series. Although the increased sensitivity was noted using Hansel stain instead of Wright stain [12], eosinophiluria is no longer considered sensitive or specific. In the largest cohort studied, Muriithi et al. found 31% sensitivity and 68% specificity for the diagnosis of ATIN among 566 patients with AKI, and found no utility of eosinophiluria in the distinction between ATIN and ATN [13]. Patients with urinary tract abnormalities, urinary tract infections, acute tubular necrosis, and glomerulonephritis also exhibit eosinophiluria [14].

Other findings from urine microscopic examination are white blood cell (WBC) casts, which are either not sensitive and not specific for ATIN diagnosis. WBC casts are found in urine from patients with inflammatory kidney diseases, not only ATIN. They have also been seen in other conditions such as glomerulonephritis and pyelonephritis. Surprisingly, less than 14% of ATIN patients present WBC in urine microscopy according to the published series. Red blood cell (RBC) casts were once thought to be specific for glomerular disease, however up to one third of ATIN patients exhibit RBC, probably related to disruption of interstitial blood vessels and leakage of erythrocytes to the tubular lumen [15]. Granular casts are the most frequent casts seen in ATIN, up to 95% [16]. depending on the series, but they are also very common in ATN.
