*General Features and Meaning of a Biomarker*

A biomarker has been defined as "a characteristic that is objectively measured and evaluated as an indicator of a normal biological process, pathogenic process or pharmacological response to a therapeutic intervention" [1,2].

Transplanted kidney is currently monitored through a complex of clinical (e.g., GFR, proteinuria), immunological (e.g., DSA), instrumental (e.g., resistive index at Doppler ultrasound), and histological parameters. Overall these "traditional biomarkers" have many limits related not only to disease, but also to both nephrologists' and pathologists' skills. Even histological examination through renal biopsy, which remains the diagnostic golden standard criterion despite its invasiveness, is hampered by many drawbacks: low sensitivity (e.g., failure to detect subclinical acute rejection), low specificity due to heterogeneity of processes underlying the same lesion (e.g., uncertain interpretation of interstitial fibrosis-tubular atrophy, IFTA), lack of standardization (poor reproducibility, elevated inter-observer variability due to expertise-dependence) and of quantitative thresholds, sampling errors (e.g., failure to detect focal disorders such as Polyomavirus associated nephropathy, PVAN) [3].

New biomarkers have been the focus of intense research over the last decade to overcome these limits and improve allograft monitoring. Most of them are derived from "OMICs" revolution [4] and can be considered the cornerstone of precision medicine, which is based on a proactive approach and aims at predicting and preventing pathological processes by providing earlier and more extensive information than traditional ones [5].

In general, biomarkers can be classified into seven categories with different meaning and aims [5], outlined in Table 1.


**Table 1.** Biomarkers categories and their meaning in renal transplant.

A plethora of new, non-invasive biomarkers measured in either urine or peripheral blood have been studied over the last years, mainly with a diagnostic and prognostic meaning, with different degrees of preclinical and clinical success. Some of them have been validated in independent cohorts and may be already employed in clinical decision-making when kidney biopsy is contraindicated or inconclusive. Other biomarkers, mainly represented by gene expression signatures, have been assessed in kidney tissue and appear to significantly expand information provided by traditional histology [6].

Different pathological processes can cause early and late KTx dysfunction and are outlined in Figure 1. We herein reviewed the current literature on potential biomarkers in three main settings of KTx: ischemia reperfusion injury (IRI) and DGF, AR, and CAD. The latter includes biomarkers for chronic rejection, chronic Calcineurin-Inhibitor (CNI) nephrotoxicity, and PVAN.

**Figure 1.** Timeline of early and late causes of graft dysfunction.

## **2. IRI and DGF**

DGF is a common complication of KTx, which affects short and long-term outcomes, including risk of acute rejection and graft survival. It is often caused by IRI due to long cold ischemia time, especially in kidney from "extended-criteria" donors (ECD) and donation after cardiac death (DCD). The most commonly employed definition for DGF relies on the need for dialysis in the first week after KTx.

Biomarkers measured in the immediate post-Tx would be extremely useful to identify patients at risk of DGF and prevent this common complication, for example delaying start of CNI [7].

Ideally, biomarkers predicting DGF should be available either before KTx, in the donor, or immediately after it, in the recipient. The first option is especially interesting in the current era of increasingly higher risk ECDs [8], as accurate tools to assess kidney quality are needed to help allocate them to the most adequate recipient, or even discard them when considered unsuitable [9].

A lot of potential biomarkers of DGF have been studied and some of them have already been validated in independent cohorts (Tables 2 and 3). Some biomarkers have been analyzed in donor's biological fluids or in the graft (e.g., preservation fluid) before KTx, whereas most of them were studied in the recipient after KTx.

#### *2.1. Donor-Related Biomarkers*

Donor-related biomarkers can be measured in donor biological fluids, in graft preservation fluid, or in the perfusate of machine-perfused kidneys.


#### **Table 2.** Potential biomarkers for DGF.
