Kidney transplant monitoring by urinary flow cytometry: Biomarker combination of T cells, renal tubular epithelial cells, and podocalyxin-positive cells detects rejection

Creatinine and proteinuria are used to monitor kidney transplant patients. However, renal biopsies are needed to diagnose renal graft rejection. Here, we assessed whether the quantification of different urinary cells would allow non-invasive detection of rejection. Urinary cell numbers of CD4+ and CD8+ T cells, monocytes/macrophages, tubular epithelial cells (TEC), and podocalyxin(PDX)-positive cells were determined using flow cytometry and were compared to biopsy results. Urine samples of 63 renal transplant patients were analyzed. Patients with transplant rejection had higher amounts of urinary T cells than controls; however, patients who showed worsening graft function without rejection had similar numbers of T cells. T cells correlated with histological findings (interstitial inflammation p = 0.0005, r = 0.70; tubulitis p = 0.006, r = 0.58). Combining the amount of urinary T cells and TEC, or T cells and PDX+ cells, yielded a significant segregation of patients with rejection from patients without rejection (all p < 0.01, area under the curve 0.89–0.91). Urinary cell populations analyzed by flow cytometry have the potential to introduce new monitoring methods for kidney transplant patients. The combination of urinary T cells, TEC, and PDX-positive cells may allow non-invasive detection of transplant rejection.

Here we hypothesize that cellular signatures of different urinary cells will reflect different elements of the renal transplant pathology. Specifically, assuming that T cells and monocytes/macrophages will reflect intrarenal inflammation; TEC will indicate tubular damage; and podocytes, specifically podocalyxin-positive (PDX-positive) cells, will mirror glomerular pathology, we are interested to know whether the combination of these cells would allow a more precise, non-invasive differentiation of renal transplant rejection from other transplant pathologies, as compared to monitoring only singular cell subsets. In this study, we analyze urinary cell populations of CD4 + and CD8 + T cells, monocytes/macrophages, TEC, and PDX-positive cells to evaluate correlations with respect to allograft rejection vs. non-rejection. The overall goal of this analysis is to establish a non-invasive diagnostic tool to monitor kidney transplant patients.

Urinary tubular epithelial cells and podocalyxin-positive cells can be detected by flow cytometry.
Urinary TEC were detected using a pan-cytokeratin reactive antibody as lineage marker for epithelial cells, CD10 (also called neutral endopeptidase, NEP, CALLA) as a marker for TEC originating in the proximal tubular system 16,17 and epithelial cell adhesion molecule (EPCAM) as a marker for distal TEC 18,19 . Therefore, proximal urinary TEC were defined as cytokeratin and CD10 positive cells, and distal TEC as cytokeratin and EPCAM positive cells. Urinary podocalyxin positive cells were analyzed as a surrogate for urinary podocytes. Specificity of the antibody binding was demonstrated using matching isotype controls (Fig. 1).   TEC. According to the histological diagnosis of the renal biopsy, 14 patients had T cell-mediated rejection, 7 patients had antibody-mediated rejection, and 18 patients had no signs of rejection (representative biopsy pictures are depicted in Fig. 2). Five patients had inconclusive biopsies and were excluded from the analysis. As shown in Fig. 3, overall urinary cell quantity varied between the described groups. Different cell types were compiled for each group in planet plots: The highest total cell number was found in biopsy patients without rejection (No RX). In these patients, urinary cells consisted mainly of monocytes/macrophages and TEC. The largest T cell population was found in patients with T cell mediated rejection (TCMR). Patients with proven antibody-mediated rejection (ABMR) had the lowest total cell number, as well as the lowest cell numbers in all different cell types.

Urinary cell composition varies between
Urinary TEC, PDX-positive cells, and T cells did not correlate with dipstick results for hemoglobin, protein, or leukocytes.
Correlation of different urinary cell populations with histopathological lesions. To assess our hypothesis that different urinary cell populations reflect specific renal pathologies, we examined the correlations of patients' urinary numbers with the respective histopathological findings, using the BANFF classification of 2013 20 , which was the most recent at the time of the described analysis (Fig. 4).
Urinary PDX-positive cells correlated with the histological scores of glomerular injury; however, this correlation was unexpectedly negative. In detail, the quantity of urinary PDX-positive cells negatively correlated with chronic glomerulopathy score (p = 0.0451, r = −0.4) and glomerulitis (p = 0.0322, r = −0.41), and this correlation was mainly driven by high podocyte counts in patients with a score of 0 in the respective BANFF elements.
Peritubular capillaritis did not correlate with T cells, PDX+ cells or TEC.

Total amounts of urinary immune cells, tubular epithelial cells, and podocalyxin-positive cells only moderately separate patients with rejection.
Next, we assessed whether certain subsets of urinary cells would allow non-invasive detection of acute graft rejection. To this end, we also included a further control group of 24 patients with stable renal graft function without renal biopsy.
In patients with TCMR, significantly higher amounts of immune cells were observed for CD3 + CD8 + and CD3 + CD4 + T cells (p < 0.05), compared to patients with ABMR. Although the T cells tended to be higher in the TCMR group compared to No RX group, there was no good separation of these groups and no significant difference. Unexpectedly, the patients with No RX had significantly higher urinary counts of PDX-positive cells, proximal TEC, and distal TEC than patients with TCMR (p < 0.01) or ABMR (p < 0.01 for PDX-positive cells, p < 0.001 for CD10 + TEC, p < 0.01 for EPCAM + TEC) (Fig. 5).

Increased proportions of T cells per tubular epithelial cells and T cells per podocalyxin-positive cells in patients with allograft rejection.
As the different patient groups showed different patterns of cells in their urine, we next assessed whether a combination of certain cell subsets would increase the diagnostic yield (Fig. 6). Calculating the ratios of all T cells per TEC, patients with TCMR or ABMR showed a significantly higher ratio compared to the patients with No RX (p < 0.01). No significant difference was observed between patients with TCMR and ABMR.
Similarly, when normalizing the amount of urinary T cells to the amount of PDX-positive cells, a significantly higher ratio was observed in patients with rejection (TCMR or ABMR) than in patients without rejection (p < 0.01). Again, no significant difference was observed between patients with TCMR and ABMR.
Calculating ROC curves, we observed a good segregation of patients with rejection (T cell-mediated or antibody-mediated) from patients who had graft deterioration without rejection (AUC 0.90 for T cells/TEC total, AUC 0.89 for T cells/CD10 + TEC, AUC 0.91 for T cell/ECPAM + TEC, AUC 0.89 for T cells/PDX-positive cells).

Discussion
Kidney allograft rejection is a constant concern in renal transplant patients. Hence, non-invasive biomarkers that can help in monitoring kidney transplant patients and identify rejection are of great interest. Here, we assessed whether the quantification of urinary immune cells, TEC, and PDX-positive cells would allow non-invasive allograft rejection. While the single subsets of urinary cells showed only modest separation of patients with and without rejection, the combination of urinary T cells, TEC, and PDX-positive cells allowed non-invasive detection of patients with acute rejection. Urinary T cells detected by flow cytometry have been described in various renal diseases and seem to directly reflect renal inflammation, making them a promising biomarker [21][22][23] . Several groups have assessed urinary T cells as a biomarker for renal transplant rejection and have reported elevated T cells in patients with acute rejection 9-12 . However, urinary T cells alone have not consistently delineated patients with and without rejection in all studies. www.nature.com/scientificreports www.nature.com/scientificreports/ Therefore, we analyzed whether combining urinary T cells with urinary monocytes/macrophages, TEC, and PDX-positive cells might increase the diagnostic yield of urinary cells in the transplant setting.
Recent research has shown that renal epithelial cells express cytokeratin as stress responders and potential biomarkers 24,25 . In order to distinguish proximal from distal TEC, we used CD10 and EPCAM as they were described by previous authors [16][17][18][19] . By combining these markers, we were able to establish detection of urinary proximal and distal TEC.  www.nature.com/scientificreports www.nature.com/scientificreports/ Urinary podocyte-associated molecules have been reported as a biomarker for various renal diseases [26][27][28] . Podocalyxin is a surface antigen of podocytes and regulates cell adhesion and morphology 29 . Podocyte damage is supposed to be accompanied by podocyte detachment. Their sheds and fragments are found in the urine as podocalyxin-positive elements 13 . However, podocalyxin is not exclusively expressed by podocytes and therefore is not a specific marker 30 . Furthermore, while staining for TEC and urinary immune cells did allow for identification of circumscribed cell populations (at least in patients with increased amounts), staining for podocalyxin was challenging and did not result in the identification of a clear cell population. Therefore, the cell population referred to as PDX-positive in this paper should not be assumed to be a pure podocyte population and must be interpreted as podocytes with caution.
Assuming that a renal graft rejection induces destruction and detachment of TEC and podocytes, we were surprised to find the largest cell populations in patients without rejection. At least for podocytes, increased shedding after renal transplantation has been reported before, potentially due to podocyte stress 31 . In patients receiving renal biopsy, as expected, the amount of urinary T cells correlated with the histological element, thus reflecting inflammation. However, counter to our prediction, the amount of TEC did not reflect the tubular or glomerular damage observed in the biopsy, whereas PDX-positive cells did negatively correlate with chronic transplant glomerulopathy and glomerulitis. The significance of the high amounts of TEC and PDX-positive cells observed in patients without graft rejection is presently unclear.
In clinical practice, non-invasive detection of graft rejection would enable early diagnosis and would potentially facilitate monitoring and tailoring of the patient's immunosuppression. Considering that patients without rejection showed high numbers of TEC and PDX-positive cells but low numbers of T cells, we identified a combination of biomarkers as a non-invasive tool for kidney transplant evaluation: the ratio of T cells to TEC or to PDX-positive cells seems capable of distinguishing humoral and acute rejection from no rejection. Specifically, the clearest distinction was achieved by considering the ratio of T cells per EPCAM + TEC (AUC = 0.91). Whether these results can be confirmed in an independent study and can be used to assess treatment responses remains to be determined.
In summary, here we report flow cytometry monitoring of urinary cell populations as a potential method for monitoring kidney transplant patients. Increased amounts of T cells in the urine of patients with rejection support our previous assumption that urinary immune cells mirror intrarenal inflammation. Unexpectedly, urinary TEC and PDX-positive cells did not reflect damage in the respective compartments, and the significance of their www.nature.com/scientificreports www.nature.com/scientificreports/ presence in urine is presently unknown. Combining counts of urinary T cells, TEC, and PDX-positive cells, however, may provide a useful biosignature for non-invasive detection of renal graft rejection in clinical routine.

Materials and Methods
Patients and controls. Urine samples from 63 renal transplant patients were collected and analyzed for urinary cell populations utilizing flow cytometry. Detailed patient characteristics are displayed in Tables 1 and  2, individual immunosuppression details are included as supplemetary information. Of these patients, 39 were admitted due to renal allograft deterioration and underwent renal biopsy as a standard procedure for diagnosing allograft rejection (biopsy cohort). Urine was collected within three days before or after the biopsy.
The current study divided these patients into three groups according to their histological diagnosis: patients who had been classified as affected by acute cellular rejection were considered the TCMR group; patients with proven acute or chronic humoral rejection constituted the ABMR group; and samples without rejection were classified as the No RX group.
Patients with uncertain histological diagnosis due to insufficient biopsy material were excluded from the analysis; however, they are displayed here as separate data points within the diagnostic categories suggested by their respective renal pathologists. Meanwhile, a handful of particular diagnoses excluded biopsy patients from this study: membranous glomerulonephritis, urinary tract infection, allergic interstitial nephritis, focal segmental glomerulosclerosis, and recurrent IgA nephropathy.
An additional cohort of 24 transplant patients with stable graft function was analyzed as a control group. Stable graft function was defined by stable serum creatinine (fluctuations less than 25%) or creatinine decrease on regular outpatient clinic visits, and no admittance related to issues regarding kidney transplant function during the 6 months prior to urine analysis. Of these, 6 cases deserve special mention. One control patient showed an elevation in creatinine 1 month prior to analysis, which resolved within 1 week; the creatinine elevation was of unclear significance. Furthermore, in 5 patients with long-stable transplant function (median years after transplant = 8.39), creatinine had not been measured 6 months prior to urine analysis because outpatient visits were scheduled less frequently; nonetheless, these 5 patients met the criteria of stable creatinine and absence of hospital admittance. All 6 of these cases remained in the Ctrl group.
Urine collection started in March 2016 and ended in March 2017. Samples were collected from patients of the Department of Nephrology, Charité University Hospital, Berlin. Informed consent was obtained from all patients for study participation. Furthermore, Patients agreed and gave written consent to participate and to the www.nature.com/scientificreports www.nature.com/scientificreports/ publication of the findings in a scientific journal. Moreover, our method of biomarker staining was tested on kidney tissue harvested postmortem from deceased patients with the consent of their relatives for postmortem donation of a small kidney sample for research purpose. The Charité University review board granted ethical approval (Charité EA1/152/16 and EA 2/045/18), and the study was conducted according to the ethical guidelines both at our institution and of the Helsinki Declaration.

Establishment of a method for detecting tubular epithelial cells and podocalyxin-positive cells via flow cytometry by staining kidneys from deceased patients.
To establish a staining protocol for the urinary detection of proximal TEC, distal TEC, and PDX-positive cells, human kidneys donated by deceased individuals were digested within 24 hours after death and used. Tissue was digested in RPMI medium containing collagenase VIII and DNase for 30-60 minutes at 37 °C. Red blood cell lysis was performed by BD Pharm Lyse (BD, Franklin Lakes, NJ, USA). Following this, cells were stained with viability dye and separated into dead and living cells. Staining protocol and flow cytometry analysis were equal, as described below.
Staining and flow cytometry. Within 8 hours after collection, a urine dipstick test for each sample was performed (Only nitrite-negative samples were used for analysis.), and urine samples were centrifuged (280 × g, 4 °C, 8 minutes). The pellet was resuspended in phosphate-buffered saline (PBS)/bovine serum albumin (BSA). Prior to the staining, peripheral blood monoclonal cells were isolated using Ficoll-Paque (Density 1.073 g/mL,