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

Patient enrollment and sample collection

Among 700 patients admitted to the Department of Nephrology presenting with stage III AKI, four patients with excretion of nephrospheres in urine were enrolled on a continual basis after giving oral and written informed consent. The study was approved by the ethics committee of the Medical University of Vienna under the number 1043/2016. Catheter urine was obtained every morning from the designated port and was immediately processed.

Cell isolation and in vitro culture

Morning urine was centrifuged in 50 ml conical tubes at 1,500 RPM in order to pellet cells. The resultant cell pellet was suspended in tissue culture medium (RPMI 1640 supplemented with 10% fetal calf serum) which was layered onto Ficoll-Paque Plus (GE Healthcare, Chicago, IL, USA) and centrifuged at 2,000 RPM for 20 min. The interface was taken off and washed using tissue culture medium. The cell pellet was then resuspended and plated into 12-well plates. The second day the culture medium was replaced with tubular cell culture medium. This type of medium was prepared immediately before cell feeding by combining the tissue culture medium with (30%) ProxUp RPTEC Growth Medium (Evercyte, Vienna Austria). Cell feeding was carried out every second day, thereby dead cells were washed off and taken from the well together with old tissue culture medium.

Preparation of cytoslides

Epithelial cells monolayer was washed twice with PBS and then incubated with trypsin/EDTA over 3 min. Trypsin was then inactivated by tissue culture medium and suspended cells were washed with tissue culture medium. A total of 100 µl of cell suspension was applied into the cytocentrifuge funnel which was spun at 1,200 RPM for 3 min. The resultant cytopreparation was air-dried and either immediately stained using a routine H/E staining for visualization of cell density or frozen at −20 °C wrapped in aluminum foil.

Immunofluorescence staining

Cytopreparations of ex vivo urinary cells and of cultured epithelial cells were fixed in acetone for 5 min. A water repellent barrier was drawn around the area where cells had been placed by the cytocentrifuge using a PAP Pen (SCI, science service, Munich, Germany). Following wetting of the cell containing area with 20 µl PBS the antibodies were applied. CD133/1 (Prominin 1), AQP1 (aquaporin 1), PAX8 a kidney specific transcription factor (Kaminski et al., 2016), CD10 (neprilysin). The dilution is described below. Incubation was carried out at room temperature for 2–3 h under constant shaking in a moist chamber. Before application of the secondary antibodies, slides were washed in PBS for 10 min. As secondary antibodies, goat anti-rabbit Alexa Fluor 488 (diluted 1:400) and Rhodamine (TRIC)-conjugated AffiniPur F(ab)2 goat anti-mouse (diluted 1:200) were applied and incubated for 1 h under constant shaking at room temperature. As nuclear staining, DAPI/PBS was incubated for 5 min before final washing for 10 min twice. Slides were mounted in Vectashield mounting medium for immunofluorescence (Vecotor Laboratories, Burlingam, CA, USA) and covered with a coverslip. A Zeiss Axiovert confocal microscope was used for picture recording. Pictures were further processed using Photoshop Version 6.

RNA isolation

The pellet of 7–10 ml morning urine was lysed in 1,000 µl of Trizol and kept for 10 min at room temperature and frozen at −20 °C until RNA isolation. Before addition of 200 µl chloroform the Trizol lysate was thawed. The content was then vigorously shaken and spun at 12,000g for 10 min for phase separation. The aqueous phase was pipetted into a separate tube and mixed with 500 µl isopropanol. Following an incubation period of 10 min at room temperature tubes were spun at 12,000g for 10 min at 4 °C. The RNA pellet was washed with 500 µl 75% ethanol, and the pellet was shortly dried and finally dissolved in about 20 µl RNAse free water. The RNA concentration was evaluated at a Nano drop device.

Reverse transcription and quantitative PCR

Eight hundred ng of RNA was combined with dNTPs and random primers and incubated for 5 min at 65 °C followed by rapid chilling on ice-water. First stand buffer, DTT, and RNaseOUT and Superscript III was added before increasing incubation temperature to 42 °C for cDNA synthesis for 50 min. Enzyme activity was stopped by heating the sample at 70 °C for 10 min. The resultant cDNA concentration was diluted up to 80 µl and out of this 2 µl were added to the qPCR setup mix. This consisted of TaqMan mastermix and gene specific probe sets. Each sample was set up in duplicate in a final volume of 10 µl.

UMOD ELISA from tissue culture

The human uromodulin ELISA was purchased from BioVendor (RD191163200R) and performed as described in the supplied test manual. In brief, tissue culture fluid was applied to the test well accompanied with a standard series as supplied in the test kit. Following 60 min of incubation at room temperature under constant shaking the plate was washed three times with wash buffer. Following this, the diluted biotinylated detection antibody was applied to each well and again incubated for 60 min at room temperature. Following the same washing procedure as above, the streptavidine/HRP was applied to each well and reacted for 30 min. After a final washing step, the ELISA was developed with TMB substrate incubating for 10 min under light protection. The reaction was stopped using the stop solution and read with an ELISA reader. Concentrations were calculated according to the standard curve.

Neprilysin ELISA using urine

The human neprilysin ELISA was purchased from RayBiotech and performed according to the description in the test manual. In brief, frozen urine was thawed mixed and centrifuged at 3,000 RPM before loading. A total of 20 µl of assay diluent were dispensed in each well before loading 80 µl of urine. Samples from time points with disease maximum had to be diluted up to 1:5 in provided assay diluent. Following 2 h incubation at room temperature under constant shaking the plate was washed three times with the provided wash buffer. This was followed by incubation with the biotinylated antibody using a dilution of 1:100 in assay diluent. Following an incubation period of 60 min and washing the plate three times on the plate washer, the streptavidine/HRP conjugate was dispensed into each well at the recommended dilution of 1:300. Following an incubation period of 45 min and washing of the plate (three times) on the plate washer the antibody reaction was developed with TMB at room temperature under light protection. Following the application of 50 µl stop solution the reaction intensity was measured on the ELISA reader and neprilysin concentrations in samples were calculated according to the included standard curve.

Epithelial cell cluster excretion started with an increase in urine output

Among 700 screened patients, four non-KTX AKI stage III patients exhibited nephrosphere excretion in urine and were included into this study. The patient with ischemic AKI was a 34-year-old male, who exhibited peak serum creatinine (sCr)-levels of 13.57 mg/dL (Fig. 1A). The patients with toxic AKI was a 19-year old male with peak sCr-levels of 9.87 mg/dL; the patient admitted with nephritic AKI was a 56-year old male with peak sCr-levels of 11.91 mg/dL, and the patient with infection-associated AKI was a 29-year-old male with a peak sCr of 12.39 mg/dL (Figs. 1C, 1E, and 1G). All patients required RRT during recovery from AKI.

The occurrence of epithelial cell clusters in the urine started with increasing urine volume and represented a patient specific variable (day 2 to day 14) (Table 1, Figs. S1 and S2).

The proximal tubular epithelial cell marker protein neprilysin and nephron derived epithelial cell clusters are found in urine of AKI stage three patients during recovery

On the first day that patients resumed urine production after anuria, urine collection was performed with morphological examination of urine sediment and measurement of gene expression. Morphological follow-up pictures of the urinary sediment are shown from a patient with toxic AKI in Fig. S1 and one with infection-associated AKI in Fig. S2. During the initial phase of renal recovery, patients with AKI of different etiologies: ischemic (Fig. 1A), toxic (Fig. 1C), nephritic (Fig. 1E), and infectious AKI (Fig. 1G) experienced a steady increase in urine output accompanied by decreasing urinary excretion of the cell injury marker neprilysin. The decrease in sCr showed a delay of about 5 days compared with urinary neprilysin as marker of tubular cell damage (Bernardi et al., 2021; Knafl et al., 2017; Pajenda, Mechtler & Wagner, 2017). Peak urinary neprilysin levels were significantly higher in infection-associated AKI (Fig. 1G). Renal recovery in terms of sCr levels and time was comparable among all four types of AKI.

In addition, RNA was extracted from 10ml of urine sediment, reverse transcribed and analyzed for expression of renal epithelial genes. The transcriptome of the tubular epithelial- and podocyte-specific genes, AQP1, AQP6, NPHS2, SCL12A1, was examined over a period of 11 days for toxic and ischemic and of up to 24 days for nephritic and infection-associated AKI. Expression of the podocyte marker gene podocin (NPHS2) varied but was highest on the first day of observation in patients with ischemic AKI (Fig. 1B) and increased by the fifth day in those with toxic AKI (Fig. 1D). Of note, no podocin was detected in the sediment of patients with nephritic AKI (Fig. 1F) and infection-associated AKI (Fig. 1H). Expression of the proximal tubule marker AQP1 increased in all four variants of AKI, toxic, ischemic, nephritic, and infection-associated, reflecting increasing numbers of hyperplastic epithelial cells in the urine (Figs. 1B, 1D, 1F and 1H). A similar pattern was found for the distal tubule cell-specific marker gene SLC12A1. Peak detection was found in patients with nephritic AKI and infection-associated AKI (Figs. 1F and 1H) at day 24, whereas in toxic AKI, peak levels were found during the early phase of recovery.

Naive epithelial cell clusters express the kidney-specific transcription factor PAX8 and the stem cell marker PROM1 (CD133) in confocal immunofluorescence staining

In a first attempt of investigating the origin of urinary sediment cells, naive isolated cells from urine were examined. Immunofluorescence and confocal microscopy were performed to confirm the gene expression pattern and to evaluate the morphology of the excreted cells contributing to the expression landscape shown by qPCR (Figs. 1B, 1D, 1F and 1H). Most of the cell clusters examined expressed AQP1, and a subpopulation was positive for the stem cell marker PROM1 (CD133) (Fig. 2). All of cells visualized in cluster formation showed positive staining for PAX8, a kidney-specific transcription factor (Fig. 2). In the patient with ischemic AKI, no podocin-expressing cells could be detected.

Isolated nephron derived epithelial cells from urine of patients recovering from stage 3 AKI proliferate in in vitro cell culture for a minimum of 7 days

Viable epithelial cells consisting of hyperplastic cell clusters (Fig. 3A) and individual cells were isolated by density gradient sedimentation of urine sediment cells and incubated in culture medium as described in the methods section. A rapid expansion from proliferating foci and migration over the entire free tissue culture plate was observed over the initial 5 days (Fig. 3B). As demonstrated in Fig. 3C a minor number of cells already started to show apoptotic morphology with condensed chromatin and apoptotic bodies on day 7. Fifty percent of the cell population continued to exhibit disproportionate nuclear/cytoplasmic ratios, indicating mitotic and DNA synthesis stages of the cell cycle. This scenario changed with day 8–9 when 70% of adherent growing cells started to round up and detach from the culture plate.

In vitro grown epithelial cells express genes affiliated with podocytes, the proximal and distal tubule, and the loop of Henle

In order to further define the in vitro growth and differentiation of tubular epithelial cells, total RNA of in vitro cultured cells was reverse transcribed, and the resultant cDNA was investigated by qPCR using gene specific probe sets indicative for defined nephron segments such as for proximal tubular epithelial cells (AQP1), the loop of Henle (SLC12A1) and distal tubular epithelia (SLC12A3) and podocytes (NPHS2). This revealed remarkable AQP1 expression in addition to SCL12A1 and NPHS2 (Fig. 4). NPHS2 was detected from day 8 of culture.

In vitro grown cells exhibit PAX8, PROM1 and increasing levels of UMOD

As a next step, we thought it would be of interest to establish whether subpopulations of cells would differentiate into region-specific epithelia by translating specific marker genes into proteins. Therefore, immunofluorescence staining of in vitro cultured cells was performed.

In vitro grown cells also showed positive staining for PAX8, and the protein distribution was spread throughout the cells, with more than 50% of cells also positive for PROM1 (Fig. 5). This changed rapidly over the time of in vitro culture. While the nuclear size decreased, the proportion of PROM1 positive cells minorized, the expression levels of AQP1 and of UMOD increased, reaching a maximum on day 9 (Fig. 5). When testing for the proliferation marker Ki67 on day 5, 15% of cells stained positive (Fig. S3), the percentage of nuclear staining decreased and was almost absent on day 9, while tubular protein expression markers AQP1/UMOD increased (Fig. 5, bottom panel). Testing into different regions of the nephron the SLC12A1 expression was found in 28% of the outgrowing cells at day 8 of culture. Interestingly, these cells were also positive for PROM1 in 9%. Among these cells were 8% undergoing endocycling (Fig. 6). Measurement of UMOD in the culture supernatant increased starting from day 5 and reaching a maximum on days 8–9 (Fig. S3).