URINARY POLYPLOID TUBULAR CELLS REPRESENT A NOVEL PROMISING BIOMARKER OF KIDNEY DAMAGE IN AKI PATIENTS

Acute kidney injury (AKI) is characterized by a rapid and transient decrease in kidney function. AKI is part of an array of conditions collectively defined as acute kidney diseases (AKD). Identification of patients that are more susceptible to AKI/AKD is essential in order to recognize early kidney damage, promote strategies for nephroprotection and ultimately reduce the risk of kidney failure. This is particularly relevant in clinically silent cases, where standard measurements are unreliable. In fact, early kidney damage does not often cause a significant change in urine output or creatinine levels, missing the diagnostic criteria of AKI. Timely detection and quick assessment are necessary to improve patient outcomes, albeit few biomarkers are available for this aim. My research recently identified tubular cell (TC) polyploidy as a novel mechanism of cell adaptation to sustain function during kidney injury. Polyploid TC, which acquire additional genome copies, are shed in the urine in the weeks following AKI, implying they can be used to monitor kidney resilience, as well as disease evolution. Based on this evidence, I hypothesized that the presence and signature of polyploid TC can be leveraged to identify a panel of biomarkers to non-invasively monitor kidney disease.

Single cell RNA-sequencing (scRNA-seq) from mouse and human tissues was employed to define TC polyploidy signature at different days after AKI. In vitro and in vivo transgenic models based on the FUCCI2aR reporter to track polyploid TC were employed to define the relevance of these markers, along with flow cytometry and confocal microscopy. Starting from 2018, urine samples were collected from patients admitted to the Intensive Care Unit (ICU) of AOU Careggi. A subcohort of these patients underwent surgery with various indications, and a fraction of them exhibited post-operative AKI. As controls, we enrolled critically ill patients who underwent surgery without developing AKI. Urine collection was performed 4h, 24h, 48h and 72h post-surgery, RNA was extracted to performed qPCR analysis. Additionally, an ELISA assay was developed for urine testing.

scRNA-seq on mouse kidneys at different days after ischemic AKI revealed the presence of early and late hypertrophy gene expression along the pseudotime of polyploidy acquisition. Starting from polyploid TC signature, we screened the urine of AKI and non-AKI patients admitted to the ICU of AOU Careggi. Out of the 20 genes selected, the level of an early hypertrophy marker (marker X from now on) emerged as associated with the likelihood of developing AKI. Mechanistically, in vivo confocal staining showed that marker X was strongly expressed by polyploid TC early after AKI while it decreased during chronic disease transition. Flow cytometry analysis and in vitro knock-out experiments proved that this marker is required by TC to acquire polyploidy. Biopsy staining showed that marker X is not expressed in healthy kidney while its expression strongly increases in TC acquiring polyploidy early after AKI. Cytospin on urine of healthy and early after AKI patients confirmed the expression of marker X on TC shed in the urine. qPCR quantification in the urine of ICU patients experiencing AKI, showed that marker X concentration is higher in the first hours after surgery and prior to KDIGO-defined AKI events, thus suggesting that this molecule is a putative kidney damage biomarker of early detection. Finally, an ELISA assay was set up to quantify the level of marker X in the urine of AKI vs non-AKI patients.

Our results showed that urine marker X, a protein that controls TC polyploidy acquisition, performed well in early detection of AKI. The implementation of this new biomarker in clinical could improve the ability of physicians to identify the risk of impending AKI, and also prompt future AKI research by permitting a better classification of AKI patients.