**1. Introduction**

Serum creatinine, urea, cystatin C, and estimation of glomerular filtration rate (GFR) via different equations are currently routinely used biomarkers of kidney graft function in clinical transplantation. Although they are characterized by low cost and rapid accessibility of results, these biomarkers are significantly less sensitive and specific than the aggressive and time-consuming gold standard, i.e., the measurement of GFR by an exogenous marker, such as chromium-51-ethylenediaminetetraacetic acid (51CrEDTA). Many new candidate biomarkers in kidney transplantation have been proposed and tested in recent years, which address specific pathologic processes and not merely glomerular, tubular, or overall kidney graft function. Uromodulin (also known as Tamm–Horsfall's protein) is a urinary mucoprotein that is synthesized only in the thick ascending limb of Henle's loop and early distal convoluted tubules of the kidneys [1]. In addition to this classical tubular secretion, to a

minor degree uromodulin also sorts to the basolateral pole of tubular epithelial cells, as shown by its presence in circulation [2]. The reduced number of tubular cells seen in chronic kidney disease (CKD) due to interstitial fibrosis/tubular atrophy (IF/TA) is paralleled by the reduced urinary and serum concentrations of uromodulin [3–5]. The potential utility of serum [4,6,7] and urine [8] uromodulin measurement in kidney transplant recipients (KTRs) has been studied, showing an association of lower serum uromodulin levels with progression to end-stage renal disease and graft failure. Although normative ranges for serum/plasma uromodulin concentration were established over 30 years ago, its characteristics have not yet been sufficiently identified as a priority in certain instances, resulting in a failure to fully implement uromodulin in clinical practice.

MicroRNAs (miRNAs) are short, endogenous non-coding ribonucleic acids (RNAs) involved in the modulation of gene expression, mainly by inhibition of messenger RNA translation [9,10]. Recent studies have indicated an association of miRNAs with pathological processes following kidney transplantation, such as T-cell or antibody-mediated rejection, delayed graft function, and IF/TA [9–11]. The diagnostic accuracy of such molecules as biomarkers is still questionable, since many of them emerge on the vascular side of the glomerular filtration barrier and can therefore reflect glomerular filtration rather than a specific disease process. We have focused on searching for miRNAs that were among the most studied in the context of fibrosis (anti-fibrotic *miR-29c*) [12], endothelial dysfunction (*miR-126*) [13–15], and immune response (*miR-146a*) [16,17], or might even be involved in more than one physiological and/or pathogenetic process, e.g., *miR-150* [18,19], *miR-155* [16,17], and *miR-223* [20–22]. Moreover, our previous pilot research on miRNA association with certain most common kidney graft pathologies, such as kidney graft rejection and the recurrence of primary glomerular disease, offered interesting insights into a possible connection of selected miRNAs with underlying kidney graft pathology. For details, see also Supplementary Table S1 and Figure S1.

In this study, we investigated the association of serum uromodulin concentration (s-Uromodulin) (which emerges on the urinary side of the filtration barrier) and selected miRNAs (which emerge on the vascular side of the glomerular filtration barrier) with standard biomarkers of kidney graft function, including measurement of 51CrEDTA clearance. The study's aim was firstly to investigate whether any of the proposed biomarkers are associated with the glomerular filtration and renal function in KTRs. Based on these results, the proposed biomarkers could or could not be a reliable indicator of kidney graft associated disease processes. The possible association of reliable biomarker(s) with the course of the associated disease process and kidney graft outcome is a long-term aim of this study protocol.
