4.1.2. miRNAs

miRNAs, which we already analyzed as candidate biomarkers in the setting of DGF and AR, are also opening new perspectives in this setting. Recent studies have proposed sets of urinary and renal biopsy miRNAs as prognostic biomarkers of IFTA and CAD [163].

Aberrant urinary mi-R21 and miR200b expression was associated with IFTA and CAD [157].

Plasma circulating levels of miR-150, miR-192, miR-200b, and miR-423-3p were significantly different between patients with IFTA and those with stable renal Tx and accurately identified IFTA (AUC = 0.87; sensitivity = 78%; specificity = 91%) [158].

In another study, plasma expression of miR-21, miR-142-3p, and miR-155 were upregulated in IFTA and mi-R 21 levels were positively correlated with eGFR [159].

On the contrary, miR-145-5p expression in blood cells was significantly downregulated in IFTA and could discriminate it from many other active lesions, such as TCMR, ABMR, borderline-rejection, and from a condition of stable graft function [160].

Another area of active research is that of epigenetic modifications of immunity genes on progression to IFTA: epigenetic mechanisms such as hypomethylation could directly enhance their expression and also indirectly modulate it by regulating miRNAs [164].

#### 4.1.3. Biomarkers of EMT and EndMT

IF is determined by massive deposition of EM, which is mainly produced by activated myofibroblasts probably derived from several cell types, especially renal tubular cells, through EMT.

This process, promoted by several factors such as oxidative stress and mitochondrial dysfunction due to IRI, deeply alters epithelial cell properties, determining loss of polarity and cell–cell adhesion and assumption of a mesenchymal phenotype, characterized by markedly increased production of EM [165].

More recently, activated myofibroblasts have been shown to arise also from renal endothelial cells through a similar process, EndMT, already mentioned in the section on DGF [166].

Both EMT and EndMT lead to abnormal production of EM and consequently play a key role in the pathogenesis of allograft IFTA [161]. Several histological and urinary EMT biomarkers have been proposed (Table 8), whereas more recent, initial evidence on potential EndMT biomarkers in KTx is available. Biomarkers of both processes will be analyzed.


**Table 8.** Potential biomarkers for epithelial-to-mesenchymal transition (EMT).

#### (a) Biomarkers of EMT

#### *Histological biomarkers*

In a recent study, renal expression of CD45, vimentin (VIM), and periostin (POSTN) correlated with iIFTA and POSTN was the strongest predictor of graft loss. Of interest, its expression was inversely correlated with 25(OH)VitD levels, suggesting that these might influence graft fibrosis [167].

Smad ubiquitination regulatory factor 1 (Smurf1) is part of Smurf1/Akt/mTOR/P70S6K signaling pathway, activated by TNF-α and involved in EMT. Of interest, Bortezomib blunted progression of EMT and IF by inhibiting TNF-α production and consequently expression of Smurf1, suggesting that this could be an EMT biomarker with diagnostic and therapeutic value [168].

Tubular expression of VIM and β-catenin, biomarkers of EMT, in protocol biopsy performed 3 months after KTx, was an independent risk factor for IFTA and eGFR decline up to 4 years post-transplant in CsA-treated recipients [169].

Finally, an interesting area of research is that of cellular senescence. This is associated with an inflammatory, "senescence-associated secretory phenotype" (SASP) which is tightly connected to EMT and CAD. Senescence markers (e.g., p16INK4a) could therefore be considered as potential surrogate biomarkers of EMT [170].

#### *Urinary biomarkers*

An interesting non-invasive biomarker of EMT is the ratio between VIM and CD45 relative to uroplakin 1a (UPK) urinary mRNA, which has been shown to correlate with intensity of VIM renal expression measured with immunostaining in per-protocol renal biopsies [171].

Other studies adopting a whole transcriptomic analysis approach identified specific urinary transcriptomic patterns associated with pEMT. Unbiased pathway analysis revealed that these patterns expressed increased inflammation and reduced metabolic functions, suggesting that they may be effective to detect subclinical immune response leading to EMT and graft fibrosis [172].

#### (b) Biomarkers of EndMT

Three biomarkers of EndMT, fascin1, vimentin, and heat shock protein 47, were strongly expressed in endothelial cells of peritubular capillaries in ABMR as compared to stable patients and predicted late graft dysfunction (up to 4 years since ABMR diagnosis) better than histological lesions. These results suggest that they may be reliable in identifying persistent endothelial activation and evolution towards cABMR [173].

In vitro and in vivo experimental studies demonstrated that EndMT may promote IF by targeting the TGF-β/Smad and Akt/mTOR/p70S6K signaling pathways, indicating that components of these pathways may be a potential source of EndMT biomarkers [174].

Finally, E Glover et al. analyzed evidence of miRNAs regulation of EndMT from experimental studies and their potential impact on kidney and other solid organ allograft dysfunction in a recent review. However, clinical studies in humans are needed to confirm their role as EndMT biomarkers [175].
