De novo SIX2 activation in human kidneys treated with neonatal kidney stem/progenitor cells

During development, nephron structures are derived from a SIX2+ stem cell population. After 36 weeks of gestation, these cells are exhausted, and no new nephrons are formed. We have previously described a non‐invasive strategy to isolate and expand the native SIX2+ kidney stem cells from the urine of preterm neonates, named neonatal kidney stem/progenitor cells (nKSPC). Here, we investigated the safety and feasibility of administering nKSPC into human kidneys discarded for transplantation during normothermic machine perfusion (NMP) and evaluated the regenerative and immunomodulatory potential of nKSPC treatment. We found that nKSPC administration during NMP is safe and feasible. Interestingly, nKSPC induced the de novo expression of SIX2 in proximal tubular cells of the donor kidneys and upregulated regenerative markers such as SOX9 and VEGF. This is the first time that SIX2 re‐expression is observed in adult human kidneys. Moreover, nKSPC administration significantly lowered levels of kidney injury biomarkers and reduced inflammatory cytokine levels via the tryptophan‐IDO‐kynurenine pathway. In conclusion, nKSPC is a novel cell type to be applied in kidney‐targeted cell therapy, with the potential to induce an endogenous regenerative process and immunomodulation.


| INTRODUC TI ON
Chronic kidney disease (CKD) is caused by the irreversible loss of nephrons, the structural, and functional units of the kidney. Global mortality from CKD is estimated at above 1 million people per year. 1 Kidney transplantation is the preferred treatment for patients with end-stage kidney disease (ESKD) and it is anticipated that by 2030, 5.4 million people worldwide will necessitate a kidney graft. 1 Due to the shortage of donor organs, only 25% of patients will receive a transplant. To increase the number of kidneys for transplantation, inferiorquality grafts, such as those from donation after circulatory death (DCD) are now considered, 2 which urges for strategies to improve the quality of these kidneys, such as ex vivo normothermic machine perfusion (NMP). 3,4 NMP of kidneys from marginal donors offers the opportunity to deliver therapies directly to the organ excluding systemic effects and yielding an ideal pre-clinical platform for investigating kidneytargeted cell therapy.
We have previously established a non-invasive strategy to isolate and expand neonatal kidney stem/progenitor cells (nKSPC) from the urine of preterm neonates. 5 nKSPC represent a unique kidney stem/progenitor cell type derived from developing kidneys, the only period in which authentic stem cells are present in the human kidney. [6][7][8] nKSPC have endogenous SIX2 expression, a transcription factor active in kidney cap mesenchyme cells, which is responsible for cells self-renewal and survival, and its cessation drives differentiation of cells to form nephrons. 9 This SIX2+ cells population does not persist in the postnatal kidney, which limits the regenerative potential of the organ upon injury. 7 Still, substantial repair of tubular cells in response to acute kidney injury (AKI), elicits an ongoing discussion about the source of regeneration. Continuous and established AKI trigger maladaptive repair responses, putting patients at high risk of kidney failure, while no therapy exists to promote effective endogenous regeneration, making this, a high priority goal in nephrology. 10 In this study, we administered nKSPC to donor human kidneys during NMP. We show for the first time that treatment with nKSPC induces the de novo expression of SIX2 in proliferating proximal tubule epithelial cells (PTEC) of donor kidneys, suggesting initiation of an endogenous regenerative process. This phenomenon is confirmed in in vitro co-cultures of injured PTEC with nKSPC. Moreover, nKSPC treatment modulates the immune response by reducing proinflammatory cytokines. This immunomodulatory effect relies on the mechanism of activation of the tryptophan-IDO-kynurenine pathway, as shown in vitro and confirmed in perfusate samples.

| Ethical statement
The use of nKSPC was approved by the ethical board of KU Leuven (s53345) and an informed consent form was signed by the parents or legal guardian. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of healthy volunteers at the Red Cross of Mechelen, Belgium, under an informed consent form signed by each donor. In the UK ethical approval was granted by the National Research Ethics Committee (NRES) to use the discarded human kidneys for research (15/NE/0408). Consent for research was obtained by Specialist Nurses in Organ Donation (SNODs) from the donor families.

| Normothermic machine perfusion (NMP)
nKSPC 5 were labeled with 3 μg/ml of the red fluorescent dye CellTracker CM-DiI (Invitrogen) the day before injection into the human kidneys ( Figure S1). 1 × 10 7 nKSPC were added to 50 ml Ringer's solution and flushed into the renal artery (5-10 min) before NMP. NMP was carried out by perfusing the kidneys with an oxygenated red cell-based solution at 36°C and a mean arterial pressure of 75 mm Hg using adapted pediatric cardiopulmonary bypass technology as previously described. 4

| Quantitative polymerase chain reaction (qPCR)
mRNA from cells was isolated using RNeasy Mini or Micro Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. RNA from tissue was extracted using TriPure Isolation Reagent (Sigma-Aldrich). RNA was synthesized to cDNA using a mix of Oligo (dT) 12-18 Primer, random primers, dNTP mix (100 mM), and SuperScript™ III Reverse Transcriptase, all from Invitrogen. qPCR was performed on a CFX96™ Real-Time PCR Detection System, using Platinum™ SYBR™ Green qPCR SuperMix-UDG w/ROX (Thermo Fisher), 10 μM of primers, and applied in kidney-targeted cell therapy, with the potential to induce an endogenous regenerative process and immunomodulation.

K E Y W O R D S
basic (laboratory) research/science, immunosuppression/immune modulation, kidney transplantation/nephrology, regenerative medicine, stem cells, organ perfusion and preservation, tissue injury and repair 1 μl of cDNA (5 ng/μl). qPCR data were retrieved and processed using the CFX Manager™ software (Bio-Rad, USA). All primers used are specified in Table S1.

| Cytokines and NGAL measurements
IL-6 (R&D Systems) and IL-10 (R&D Systems) were measured using ELISA following the manufacturer's instructions. IL-10 assay was carried out on undiluted perfusate. IL-6 samples were optimized to fit within the assay standard curve.

| Immunofluorescence staining
5 μm thickness sections were fixed with 4% formaldehyde and quenched with 0.5% Triton X-100 (Sigma-Aldrich). For blocking, 1% BSA (Sigma-Aldrich) and 0.3% Triton X-100 (Sigma-Aldrich) in PBS were used. Specification of antibodies is described in Table S2 and validation of the SIX2 antibodies is presented in Figure S1.

| In vitro co-cultures
Thirty thousand conditionally immortalized proximal tubule epithelial cells (ciPTEC) were injured with 20 ng/ml of tumor necrosis factorα (TNFα) and 100 ng/mL of interferon-gamma (IFNγ) 11 in hypoxia (1% O 2 ) 12 to mimic the ischemic environment of donor kidneys or in control conditions for 24 h. Then, the medium was removed and ciPTEC was incubated with 30 000 CM-DiI labeled nKSPC or bone marrow-derived mesenchymal stem cells (MSC) for 24. After incubation, cells were fixed and stained for SIX2 following the protocol in 2.5.

| In vitro quantitative assays
Human IDO and prostaglandin E2 (PGE2) were measured in the supernatants of MLR co-cultures using the IDO DuoSet® Elisa  Fifteen microliters of the sample were injected into a HILIC column (Poroshell 120 HILIC-Z PEEK, Agilent).
Data collection was performed using the Xcalibur software (Thermo Scientific). The data analyses were performed by integrating the peak areas (El-Maven-Polly-Elucidata). Absolute concentrations were calculated based on the abundances of the labeled and non-labeled compounds.

| Statistics
All data were analyzed in GraphPad Prism 9.

| nKSPC administration into human donor kidneys during NMP is safe and feasible
In order to evaluate the safety and feasibility of the administration of nKSPC into donor kidneys during NMP, we perfused six human kidneys (n = 6), of which 3 controls (only NMP), and 3 experimental (NMP + nKSPC) ( Table 1). These DCD kidneys were subjected to at least 17 h of cold ischemia time (CIT). Donors' ages ranged from 39 to 67 years and the kidneys were declined for transplantation due to damaged vessels or poor perfusion at the time of retrieval.
The administration of nKSPC had no adverse effects on perfusion parameters or renal function during NMP. All kidneys produced urine and appeared evenly perfused. The renal blood flow (RBF) increased during perfusion in both the nKSPC-treated and control groups. There was no statistically significant difference in RBF levels between the groups (mean; nKSPC 111.4 ± 6.0 vs. control 110.2 ± 33.5 ml/min/100 g = .959; Figure 1A). The nKSPCtreated kidneys produced a higher volume of urine, but this did not reach statistical significance (mean total urine output; nKSPC 1448 ± 105 vs. control 824 ± 245 ml; = .219; Figure 1B). Other measures of renal and tubular cell function, such as creatinine clearance and fractional excretion of sodium, were also similar between the two groups (mean creatinine clearance; nKSPC 3.5 ± 1.9 vs. control 3.0 ± 3.7 ml/min/100 g = .848; Figure 1C), (mean fractional excretion of sodium; nKSPC 27.1 ± 1.0 vs. control 26.4 ± 9.4% = .909; Figure 1D).

| nKSPC can be traced within the perfused kidneys
In order to follow the cells' fate during NMP, nKSPC were prelabeled with a red fluorescent dye. All cells injected into the renal artery were fluorescently red ( Figure S1). Cryosections of biopsies were counterstained with DAPI for nuclear localization. We found nKSPC being present in the renal cortex immediately after infusion (flush), and at the later time points (2, 4, and 6 h) during NMP. By the end of perfusion, at 6 h, the majority of nKSPC were observed in the medullary region ( Figure 2). Control kidneys had no red fluorescence ( Figure S3).

| De novo SIX2 expression in human donor kidneys
Because nKSPC express endogenous SIX2, we analyzed SIX2 expression in the kidney biopsies ( Figure 3; Figure S4).
Before infusion of nKSPC (condition: pre), no SIX2 expression was observed. Immediately after infusion, SIX2+ nKSPC could be traced within the tissue. Strikingly, after 2 h of NMP + nKSPC we observed a de novo expression of SIX2 in the tubular cells of the donor kidneys, which increased at 4 h and peaked at 6 h in the cortex ( Figure 3A,B). This phenomenon was unexpected, since to our knowledge SIX2 re-expression in adult kidneys have never been reported, even upon injury. 7 The SIX2+ cells were mainly co-localized with LTL at the proximal tubules of the kidneys ( Figure 3C), while the control did not show SIX2 expression ( Figure S5).

| De novo SIX2 expression in human proximal tubule cells in vitro
To exclude confounder factors arising from the whole organ or the perfusion system, we developed an in vitro assay to model the PTEC-nKSPC interactions. PTEC were exposed for 24 h to TNFα and IFNγ in hypoxia to mimic the ischemic environment of donor kidneys. These PTEC were then co-cultured with nKSPC in a ratio of 1:1. Healthy or injured PTEC did not express SIX2, but incubation with nKSPC induced its expression similarly to the observations after administration of nKSPC in donor kidneys ( Figure 4). To understand if this phenomenon was unique to nKSPC, the same assays were performed using MSC, which do not express SIX2. In fact, no SIX2 expression arose in injured PTEC upon co-culture with MSC ( Figure 4).

| De novo SIX2 activation in human kidneys is accompanied by tubular cell proliferation and upregulation of regenerative markers
Ischemia activates a variety of intrinsic repair processes and the cell cycle to compensate for cell loss. We evaluated the prolif-  It has been suggested that Sox9+ cells regenerate proximal tubule epithelium after renal injury in mice. 14 We analyzed SOX9 expression in our samples and observed a significant increase in the NMP + nKSPC condition in comparison with NMP control kidneys ( Figure 5B). In addition, activation of hypoxia-inducible factors (HIFs) plays an important role in kidney injury and repair by regulating HIF target genes, 15 such as upregulation of vascular endothelial growth factor (VEGF). 16,17 We measured the expression of HIF-1α and VEGF in the perfused kidneys and found that both factors were significantly upregulated after 4 h of NMP + nKSPC ( Figure 5B). These results corroborate with the concept of an ongoing effective regenerative process induced by NMP + nKSPC.

| nKSPC administration has immunomodulatory effects in human kidneys
MSCs are known to induce immunomodulation in solid organ transplantation. [18][19][20] Likewise, we evaluated the immunomodulatory potential of nKSPC in human kidneys during NMP. nKSPC treatment significantly reduced the expression of the pro-inflammatory cytokines IL-1β, TNFα, IL-2, and IL-8 in comparison with the control kidneys ( Figure 6A). There was a numerical reduction of the proinflammatory cytokine IL-6 in NMP + nKSPC but this did not reach statistical significance ( = .092). Importantly, the biomarker of kidney injury KIM-1 was significantly reduced in NMP + nKSPC in comparison with NMP control ( Figure 6A).
One of the most accepted mechanisms of immunomodulation of MSC is the upregulation of IDO. 21 IDO catalyzes the tryptophan degradation along the kynurenine pathway. Tryptophan depletion in the microenvironment and accumulation of kynurenine, inhibits the activation, proliferation, and functional activity of T cells. 22 We found a significant increase in IDO activity in kidneys treated with NMP + nKSPC in comparison with controls, which was demonstrated by the elevated kynurenine/tryptophan ratio ( Figure 6B).
This immunomodulatory mechanism of nKSPC was also confirmed in vitro (Figure 7).

| nKSPC inhibit allogeneic T cell responses by tryptophan degradation via the IDO pathway
The immunomodulatory effect seen in nKSPC-treated kidneys is of particular interest in the context of kidney transplantation.
F I G U R E 2 nKSPC can be traced within human kidneys perfused in normothermic machine perfusion (NMP). 10 million nKSPC were labeled with a red fluorescent dye (white arrows) and flushed into the kidneys before exvivo NMP. Biopsies from the cortical region were collected before NMP (pre), immediately after nKSPC administration (flush), at 2, 4, and 6 h of perfusion, and from the medullary region at 6 h of NMP.
Cryosections were counterstained only with the nuclear marker DAPI. Scale bar: 10 μm.

F I G U R E 3 nKSPC administration induces de novo SIX2 activation in proximal tubules of injured human kidneys. (A)
Representative images of SIX2 staining (green) in human kidney biopsies before (pre), after nKSPC administration (flush), and after 2, 4, and 6 h of normothermic machine perfusion (NMP) with labeled nKSPC (red). The specific nuclear staining of SIX2 was only observed at 2, 4, and 6 h of NMP at the cortical zone of the kidney. No signal was observed before infusion of nKSPC (pre) nor before starting NMP (flush) or at the medullary region (6 h med). Nuclei were counterstained with DAPI. Dashed arrows point at nuclear SIX2 staining and full arrows point at nKSPC. Scale bar: 50 μm. Zoomed images were added for better appreciation of details due to the known high background for SIX2 antibody. Note nKSPC (red) positive for SIX2 (green) in these merged images (yellow).  Table 1. Results are expressed as mean ± SD of qPCR conducted in triplicate. Statistics were performed using multiple unpaired t tests, comparing NMP versus NMP + nKSPC per time point. * p < .05.
As alloreactive lymphocytes are the primary mediators of kidney graft rejection, we assessed whether nKSPC could inhibit allogenic T cell responses when co-cultured in MLR. We detected that nKSPC hinder T cell proliferation in a dose-dependent manner ( Figure 7A). A ratio of at least 5:1 PBMC/nKSPC (1 × 10 5 : 2 × 10 4 ) was necessary to significantly halt T cell proliferation. As the maximum suppressive effect was desired, in the mechanistic studies we used the ratio 2:1 PBMC/nKSPC (1 × 10 5 : 5 × 10 4 ), which decreased T cell proliferation to 14.4% ± 6.0 of the initial response in the absence of nKSPC.
Next, we analyzed the cytokines and chemokines secreted by nKSPC alone as well as in the MLR with and without the addition of nKSPC. We found that nKSPC at baseline secreted high levels of IL-6 and IL-8 (≥5000 pg/ml) ( Figure 7B). In the MLR without nKSPC high concentrations of INFγ, TNFα, IL-1β, IL-6, IL-8, IL-13, and IL-17A were measured. Importantly, co-culture with nKSPC decreased the release of the pro-inflammatory cytokine TNFα, but increased IFNγ and IL-17A, which are known to mediate the immunosuppressive capacity of MSC. 22,23 Therefore, we primed nKSPC with IFNγ and analyzed the differential gene expression. As shown in Figure 7C, the expression of IDO was highly upregulated (qPCR Ct value from 36 to 21) in comparison with other genes. So, we measured the concentration of soluble IDO released in the supernatant of the MLR, in which cytokines were measured. We observed a steep increase of IDO in MLR + nKSPC, reaching saturation on day 4 ( Figure 7D).
As other mediators affected by IFNγ could be involved in the immunomodulatory mechanism, we also measured the concentration of prostaglandin E2 (PGE2). However, almost no PGE2 could be detected in the secretome of MLR + nKSPC ( Figure 7E), which correlated with no significant upregulation of COX-1 and COX-2 24 genes when nKSPC were primed with IFNγ ( Figure 7C). These results strongly suggest that IDO-driven tryptophan degradation is the major mechanism of nKSPC immunomodulation. Hence, we measured the concentration of tryptophan and kynurenine in the MLR supernatants. MLRs without nKSPC had high levels of tryptophan and very low levels of kynurenine throughout the period of 5 days ( Figure 7F-dashed lines). MLR + nKSPC showed a significant reduction of tryptophan starting from day 3 whereas kynurenine, the tryptophan metabolite, significantly increased ( Figure 7F-full lines). Moreover, adding IDO-inhibitor to the MLR + nKSPC showed a restoration of T cell proliferation ( Figure 7G).

| DISCUSS ION
This study was designed to provide proof of principle evidence that nKSPC can be safely administered to human donor kidneys during NMP. We found that nKSPC treatment induced the re-activation of . Nkspc were treated with mitomycin C to hinder proliferation during co-culture and to ensure the known number of cells. The proliferation rate was quantified after 5 days of co-culture. Data are expressed as mean ± SD of 3 independent experiments conducted in triplicate. 100% of proliferation corresponds to PBMC stimulated with RPMI 1788 cells only. Data were analyzed by one-way ANOVA test: ** p < .01, *** p < .001, **** p < .0001 PBMC stimulated with RPMI 1788 without nKSPC versus PBMC stimulated with RPMI 1788 in co-culture with nKSPC. (B) A multiplex human cytokine detection (MSD U-plex assay) was used to measure levels of secretory factors in cell culture supernatants for 5 consecutive days from nKSPC alone and MLR with or without nKSPC. Black dot, value ≤50 pg/ml; yellow dot, 50 pg/ml ≤ value ≤500 pg/ml; red dot, 500 pg/ml ≤ value ≤5000 pg/ml; green dot, value ≥5000 pg/ ml. HIF-1α promotes kidney tissue repair, being critical for proximal tubule cell survival and facilitating cell proliferation. 15,26 VEGF has been implicated as a critical factor for renal recovery after IRI due to its mitogenic, angiogenic, anti-inflammatory, and antiapoptotic effect. 16 Based on the analysis of these tissue regeneration markers (HIF-1α, VEGF, and SOX9) we can assume the regenerative superiority of the treatment with nKSPC compared to NMP only. were perfused in human kidneys for 7 h and were found in high amounts in the medullary region. 20 The attraction of nKSPC to the renal medulla might be due to the fact that tubules of the S3 segment region are the most sensitive regions to IRI. 28 Not by coincidence, in another study, MSC treatment showed a positive effect on ATP synthesis, which was more pronounced in the medullary region, suggesting increased synthetic functions associated with regeneration of damaged tubules. 27 Whilst perfusion of donor kidneys is an excellent pre-clinical model to test novel treatments with translational potential, they do have high biological variability. This study is limited to a small sample size and a heterogenous group of DCD kidneys declined for transplantation due to damage in three cases, poor in-situ perfusion in two, and a diseased artery in one. All donors had a normal renal function and the kidneys were of good quality at the time of organ retrieval but were subject to prolonged cold ischemic injury. All kidneys functioned well during NMP and produced urine.
The level of RBF was similar between groups suggesting that the nKSPC had no adverse effects. Although one donor was younger and CIT was shorter than in other kidneys (NMP + nKSPC #3), the cytokine measurements and effects of nKSPC treatment to induce SIX2 expression were similar to the other kidneys of the same group.
Kidneys treated with nKSPC had a reduction of inflammatory cytokines and biomarkers of kidney injury. There was a trend toward an increase in the volume of urine produced and lower urinary NGAL. These parameters have been used for the functional assessment of kidneys in NMP and, after transplantation, it was shown that they correlated with improved clinical outcomes. 4 Better performance of these key parameters has also been suggested as a sign of less tissue damage, better cellular metabolism, improved tissue perfusion, and re-establishment of homeostasis. 20 Moreover, NMP + nKSPC administration resulted in lower levels of KIM-1, a transmembrane tubular protein, which is undetectable in normal kidneys but is markedly induced after injury and its high excretion in urine predicts long-term renal graft loss. 29 In conclusion, the administration of nKSPC in NMP of injured human kidneys is safe and feasible. This proof of principle study demonstrated that nKSPC can potentiate immunomodulation and trigger endogenous regeneration. Therefore, nKSPC may serve as a potent source of cells for kidney-targeted cell therapy aiming at the improvement of kidney graft quality ultimately attenuating the problem of shortage of organs available for transplantation.