Development and nationwide validation of kidney graft injury markers using urinary exosomes and microvesicles (complete English translation of the Japanese version)

Kidney transplantation is the standard renal replacement therapy. Despite the requirement of immunosuppressants to suppress any immunological reaction against alloimmunity, kidney transplantation improves the life expectancy and quality of life of patients with end-stage kidney disease as compared to dialysis therapies. Although the outcome of kidney transplantation has been improving following the development of immunosuppressants and the increased understanding of proper management for graft rejection, kidney grafts tend to lose their function due to allograft rejections, as well as problems such as recurrence of the original disease, toxicity of immunosuppressants, development of metabolic disorders, and glomerular overload [2]. Improving survival of kidney grafts is thus a challenge and an unsolved issue.

The diagnosis of graft injury (including graft rejection) relies on clinical manifestations such as a decrease in urine volume and fever, urine and blood analyses, blood chemical analysis, and radiographic evaluation such as ultrasonography, computed tomography, or radioisotope imaging. However, the definitive diagnosis is still made by graft pathology [3, 4]. A graft biopsy can generally be performed safely; however, there are risks for patients in the early period after kidney transplantation and for those taking antithrombotic agents because of slight invasiveness. Moreover, the final diagnosis takes several days. Thus, there is a need for a non-invasive biomarker assay that can yield a correct diagnosis of graft injuries with a comparable performance to histology. An assay of chemical substances such as neutrophil gelatinase-associated lipocalin [6, 18] and liver-type fatty acid-binding protein [19], which have been proposed to be enhanced in tubular injury; however, a diagnostic modality that can detect several types of graft injury is ideal. We focused on exosomes in patient urine in this study. Exosomes are microvesicles discharged from cells and include cell membrane components, protein, DNA, mRNA, and miRNA. Additionally, exosomes have been focused on as an information source since the late 1990s [20]. The intercellular signal from renal injury and lymphocytes is included. Strictly, the sizes of exosomes and microvesicles are 50–100 nm and 100–1000 nm, respectively, but they are often collectively referred to as EVs [20]. EVs are located in blood or fluids such as bile or ascites [13] and can be recovered from any part of the body. They are an ideal biomarker source for investigating kidney or urinary tract disease [5, 21, 22] because EVs from urine can be recovered non-invasively. The efficacy of EV evaluation by RT-PCR in nephritis [11] diabetic kidney disease [12], and bladder cancer [10] has been previously established. Furthermore, EVs are generally retrieved by an ultracentrifugation method; however, this procedure is complicated, and yields limited measurable samples [23]. As an alternative, we explored the seamless assay system for recovery of EV, extraction of mRNA, and generation of cDNA, establishing a protocol for the rapid management of multiple samples [9]. A critical step during the mRNA assay is preventing damage by RNase among urine samples contaminated in recovery or storage. Thus far, we have recovered urine samples by way of ordinal sample handling for urinalysis and consecutive freezing preservation within a few hours, yielding RNA that was successfully measured. This may be because EVs are covered with cellular lipid membranes; RNA is thus protected from temperature changes and RNase, preventing its degradation. Consequently, EVs are an ideal source of information [20].

In this study, we evaluated KGI using the measurement of mRNA obtained from urine EVs after having previously introduced the usefulness of a single gene, ANXA1, in the detection of graft injury in a single center analysis of kidney injury model [14]. Subsequently, a nationwide survey including the search of candidate genes by NGS was developed to verify this result.

Here, 39 candidate genes selected based on our preparation study were analyzed using qPCR from 127 patients. CXCL9, CXCL10, SPDEF, SPNS2, and UMOD showed statistical differences between some graft rejection types. Among these, CXCL9/CXCL10 and UMOD were shown to be significant biomarkers of TCMR, as their expression showed robust enhancements in samples from patients with TCMR; in contrast, there was no increase in the expression of these genes in samples from patients with antibody-mediated rejection. Previous literature has stated that the chemokines CXCL9 and CXCL10 are significant biomarkers for detecting allograft rejection in animal models and a clinical multiple-institute study. Our present study clearly supports these results [7, 8, 15, 16]. In this study, the detection of TCMR by single genes other than CXCL9 or CXCL10 was difficult; however, a combination of multiple candidate mRNA generated reliable diagnostic formula and became the promising biomarker instead of graft biopsy and pathology in the diagnosis of KGI. For example, we also determined that UMOD can be an alternative biomarker for TCMR detection. UMOD is a gene-encoding uromodulin, also called Tamm-Horsfall protein. Uromodulin, a kidney-specific protein located in the medullary thick ascending limb of the loop of Henle, is reportedly a predictor of tissue injury in patients with anti-neutrophil cytoplasmic antibody-related nephritis [24]. Moreover, UMOD expression in urine EVs is a predictive biomarker of the development of diabetic kidney disease in patients with type 2 diabetes [12].

B4GALT1 expression was increased in cABMR but decreased in cCNIT. Both KGIs induce gradual arteriole stenosis and consecutive tissue injury as a result of chronic ischemic changes. B4GALT4 is a promising gene biomarker for distinguishing between these two events and has critical significance given the contrary management of immunosuppressant dosing for these conditions. B4GALT1 is a gene encoding glycosyltransferase and influences B cell activation [25] and has been used as a predictive marker for disease progression and prognosis in malignancy [26]. The relationship between B4GALT4 and kidney injuries has not been well studied. In the present study, SPNS2 was also nominated as a biomarker gene and has similar expression patterns to B4GALT1. SPNS2 plays a role in anti-fibrotic and anti-inflammatory processes in human kidney gene tissue [27].

SLC12A1 was identified as a candidate marker for reflecting the severity of IFTA by qPCR analysis. NKCC exists on the cell surface and has two variants, NKCC1 and NKCC2; NKCC2 is expressed only in kidney tissue and encoded by SLC12A1 [28]. However, the role of NKCC in graft fibrosis and tubular atrophy is not understood.

Finally, POTEM and HAVCR1 were nominated as the candidate biomarkers for the detection of BChS, which is supposed to correlate with graft loss. HAVCR1, also called TIM-1, is a known biomarker of kidney injury and has been proven to be a candidate marker for chronic KGI. While POTEM was also identified as a candidate biomarker, further study is needed regarding its mechanism of involvement in the progression of chronic graft damage.

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