Tubular epithelial progenitors are excreted in urine during recovery from severe acute kidney injury and are able to expand and differentiate in vitro

Abstract

Background Acute kidney injury (AKI) is a serious condition associated with chronic kidney disease, dialysis requirement and a high risk of death. However, there are specialized repair mechanisms for the nephron, and migrated committed progenitor cells are the key players. Previous work has described a positive association between renal recovery and the excretion of tubular progenitor cells in the urine of kidney transplant recipients. The aim of this work was to describe such structures in non-transplanted AKI patients and to focus on their differentiation. Methods Morning urine was obtained from four patients with AKI stage 3 and need for RRT on a consecutive basis. Urine sediment gene expression was performed to assess which part of the tubular or glomerular segment was affected by injury, along with measurement of neprilysin. Urine output and sediment morphology were monitored, viable hyperplastic tubular epithelial clusters were isolated and characterized by antibody or cultured in vitro. These cells were monitored by phase contrast microscopy, gene, and protein expression over 9 days by qPCR and confocal immunofluorescence. Furthermore, UMOD secretion into the supernatant was quantitatively measured. Results Urinary neprilysin decreased rapidly with increasing urinary volume in ischemic, toxic, nephritic, and infection-associated AKI, whereas the decrease in sCr required at least 2 weeks. While urine output increased, dead cells were present in the sediment along with debris followed by hyperplastic agglomerates. Monitoring of urine sediment for tubular cell-specific gene transcript levels NPHS2 (podocyte), AQP1 and AQP6 (proximal tubule), and SLC12A1 (distal tubule) by qPCR revealed different components depending on the cause of AKI. Confocal immunofluorescence staining confirmed the presence of intact nephron-specific epithelial cells, some of which appeared in clusters expressing AQP1 and PAX8 and were 53% positive for the stem cell marker PROM1. Isolated tubule epithelial progenitor cells were grown in vitro, expanded, and reached confluence within 5–7 days, while the expression of AQP1 and UMOD increased, whereas PROM1 and Ki67 decreased. This was accompanied by a change in cell morphology from a disproportionately high nuclear/cytoplasmic ratio at day 2–7 with mitotic figures. In contrast, an apoptotic morphology of approximately 30% was found at day 9 with the appearance of multinucleated cells that were associable with different regions of the nephron tubule by marker proteins. At the same time, UMOD was detected in the culture supernatant. Conclusion During renal recovery, a high replicatory potential of tubular epithelial progenitor cells is found in urine. In vitro expansion and gene expression show differentiation into tubular cells with marker proteins specific for different nephron regions.

Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells

“…Previous authors have used dual immunocytochemistry to identify the origin of decoy cells and whether they originate from the ureterorenal region or urinary bladder [14]. We used AQP1 as a marker for proximal tubular cells [39,40].…”

Renal progenitors derived from urine for personalized diagnosis of kidney diseases

“…So far, few studies have explicitly explored the utility of RPC in human urine for the early diagnosis and risk stratification of CKD [4]. Recent studies have shown that excretion rate of RPC in urine of patients with kidney transplanted AKI and with stage 3 non-transplanted AKI indicated kidney function recovery [68][69][70]. The increased number of RPC in urine might indicate an attempt of tissue regeneration, which may turn out to be an unsuccessful process resulting in further CKD progression [68][69][70].…”

“…Recent studies have shown that excretion rate of RPC in urine of patients with kidney transplanted AKI and with stage 3 non-transplanted AKI indicated kidney function recovery [68][69][70]. The increased number of RPC in urine might indicate an attempt of tissue regeneration, which may turn out to be an unsuccessful process resulting in further CKD progression [68][69][70]. Overall, RPC in urine might provide insight into the severity of kidney damage and regenerative processes after AKI, opening novel opportunities for predicting kidney function recovery and the risk of progression to CKD (shown in Fig.…”