Dorottya K. de Vries

Autologous Bone Marrow-Derived Mesenchymal Stromal Cells for the Treatment of Allograft Rejection After Renal Transplantation: Results of a Phase I Study

Despite excellent short‐term results, long‐term survival of transplanted kidneys has not improved accordingly. Although alloimmune responses and calcineurin inhibitor‐related nephrotoxicity have been identified as main drivers of fibrosis, no effective treatment options have emerged. In this perspective, mesenchymal stromal cells (MSCs) are an interesting candidate because of their immunosuppressive and regenerative properties. Of importance, no other clinical studies have investigated their effects in allograft rejection and fibrosis. We performed a safety and feasibility study in kidney allograft recipients to whom two intravenous infusions (1 million cells per kilogram) of autologous bone marrow (BM) MSCs were given, when a protocol renal biopsy at 4 weeks or 6 months showed signs of rejection and/or an increase in interstitial fibrosis/tubular atrophy (IF/TA). Six patients received MSC infusions. Clinical and immune monitoring was performed up to 24 weeks after MSC infusions. MSCs fulfilled the release criteria, infusions were well‐tolerated, and no treatment‐related serious adverse events were reported. In two recipients with allograft rejection, we had a clinical indication to perform surveillance biopsies and are able to report on the potential effects of MSCs in rejection. Although maintenance immunosuppression remained unaltered, there was a resolution of tubulitis without IF/TA in both patients. Additionally, three patients developed an opportunistic viral infection, and five of the six patients displayed a donor‐specific downregulation of the peripheral blood mononuclear cell proliferation assay, not reported in patients without MSC treatment. Autologous BM MSC treatment in transplant recipients with subclinical rejection and IF/TA is clinically feasible and safe, and the findings are suggestive of systemic immunosuppression.

Oxidative damage in clinical ischemia/reperfusion injury: a reappraisal.

AIMS Ischemia/reperfusion (I/R) injury is a common clinical problem. Although the pathophysiological mechanisms underlying I/R injury are unclear, oxidative damage is considered a key factor in the initiation of I/R injury. Findings from preclinical studies consistently show that quenching reactive oxygen and nitrogen species (RONS), thus limiting oxidative damage, alleviates I/R injury. Results from clinical intervention studies on the other hand are largely inconclusive. In this study, we systematically evaluated the release of established biomarkers of oxidative and nitrosative damage during planned I/R of the kidney and heart in a wide range of clinical conditions. RESULTS Sequential arteriovenous concentration differences allowed specific measurements over the reperfused organ in time. None of the biomarkers of oxidative and nitrosative damage (i.e., malondialdehyde, 15(S)-8-iso-prostaglandin F2α, nitrite, nitrate, and nitrotyrosine) were released upon reperfusion. Cumulative urinary measurements confirmed plasma findings. As of these negative findings, we tested for oxidative stress during I/R and found activation of the nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of oxidative stress signaling. INNOVATION This comprehensive, clinical study evaluates the role of RONS in I/R injury in two different human organs (kidney and heart). Results show oxidative stress, but do not provide evidence for oxidative damage during early reperfusion, thereby challenging the prevailing paradigm on RONS-mediated I/R injury. CONCLUSION Findings from this study suggest that the contribution of oxidative damage to human I/R may be less than commonly thought and propose a re-evaluation of the mechanism of I/R.

Renal ischemia-reperfusion induces a dysbalance of angiopoietins, accompanied by proliferation of pericytes and fibrosis

Endothelial cells (ECs) are highly susceptible to hypoxia and easily affected upon ischemia-reperfusion (I/R) during renal transplantation. Pericytes and angiopoeitins play important role in modulating EC function. In the present study, we investigate the effect of renal I/R on the dynamics of angiopoietin expression and its association with pericytes and fibrosis development. Male Lewis rats were subjected to unilateral renal ischemia for 45 min followed by removal of the contralateral kidney. Rats were killed at different time points after reperfusion. Endothelial integrity (RECA-1), pericytes [platelet-derived growth factor receptor-β (PDGFR-β)], angiopoietin-2 (Ang-2)/angiopoietin-1 (Ang-1) expression, and interstitial collagen deposition (Sirius red and α-smooth muscle actin) were assessed using immunohistochemistry and RT-PCR. Our study shows an increase in protein expression of Ang-2 starting at 5 h and remaining elevated up to 72 h, with a consequently higher Ang-2/Ang-1 ratio after renal I/R (P < 0.05 at 48 h). This was accompanied by an increase in protein expression of the pericytic marker PDGFR-β and a loss of ECs (both at 72 h after I/R, P < 0.05). Nine weeks after I/R, when renal function was restored, we observed normalization of the Ang-2/Ang-1 ratio and PDGFR-β expression and increase in cortical ECs, which was accompanied by fibrosis. Renal I/R induces a dysbalance of Ang-2/Ang-1 accompanied by proliferation of pericytes, EC loss, and development of fibrosis. The Ang-2/Ang-1 balance was reversed to baseline at 9 wk after renal I/R, which coincided with restoration of cortical ECs and pericytes. Our findings suggest that angiopoietins and pericytes play an important role in renal microvascular remodeling and development of fibrosis.

The L-arginine/NO pathway in end-stage liver disease and during orthotopic liver and kidney transplantation : Biological and analytical ramifications

The L-arginine/nitric oxide (L-Arg/NO) pathway is altered in liver and kidney diseases. However, the status of the L-Arg/NO pathway during and after orthotopic transplantation is insufficiently investigated and findings are uncertain because of analytical shortcomings. Also, most human studies have focused on individual members of the L-Arg/NO pathway such as nitrate or asymmetric dimethylarginine (ADMA). In the present article we report on a pilot study investigating extensively the status of the L-Arg/NO pathway before and during orthotopic liver transplantation (OLT). By using fully validated, highly sensitive and specific GC-MS and GC-MS/MS methods nitrite, nitrate, ADMA and its hydrolysis product dimethylamine (DMA), L-arginine and L-ornithine were measured in blood and urine. Our study gives strong evidence of the exceptional importance of hepatic dimethylarginine dimethylaminohydrolase (DDAH) activity for the elimination of systemic ADMA. In end-stage liver disease the synthesis of NO and ADMA as well as the DDAH activity are elevated. However, increase in DDAH activity is insufficient to efficiently eliminate overproduced ADMA. The transplanted liver graft is capable of clearing ADMA in a rapid and sufficient manner. In contrast to studies from other groups, our study shows that in OLT as well as in living donor kidney transplantation, the second study reported here, reperfusion of the graft does not cause drastic alterations to the L-Arg/NO pathway with regard to NO synthesis. In the OLT study the concentration of circulating L-arginine fell temporally dramatically, while L-ornithine levels increased diametrically, most likely due to elevation of arginase activity. However, the relatively long-lasting decrease in plasmatic L-arginine in OLT seems not to have affected NO synthesis after reperfusion. Our OLT study suggests that liver reperfusion is associated with greatly elevated activity of proteolytic and hydrolytic enzymes including DDAH and arginase. Suppression of proteolytic and hydrolytic activity in transplantation could be a useful measure to improve outcome and remains to be investigated in further studies on larger patient collectives. The importance of analytical chemistry in this area of research is also discussed in this article.

Mesenchymal stromal cells in renal ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury is an inevitable consequence of organ transplantation and a major determinant of patient and graft survival in kidney transplantation. Renal I/R injury can lead to fibrosis and graft failure. Although the exact sequence of events in the pathophysiology of I/R injury remains unknown, the role of inflammation has become increasingly clear. In this perspective, mesenchymal stromal cells (MSCs) are under extensive investigation as potential therapy for I/R injury, since MSCs are able to exert immune regulatory and reparative effects. Various preclinical studies indicate the beneficial effects of MSCs in ameliorating renal injury and accelerating tissue repair. These versatile cells have been shown to migrate to sites of injury and to enhance repair by paracrine mechanisms instead of by differentiating and replacing the injured cells. The first phase I studies of MSCs in human renal I/R injury and kidney transplantation have been started, and results are awaited soon. In this review, preliminary results and opportunities of MSCs in human renal I/R injury are summarized. We might be heading towards a cell-based paradigm shift in the treatment of renal I/R injury.

Silencing of MiRNA-126 in Kidney Ischemia Reperfusion is Associated with Elevated SDF-1 Levels and Mobilization of Sca-1+/Lin- Progenitor Cells

Integrity of the capillary network in the kidney is essential in the recovery from ischemia/ reperfusion injury (IRI), a phenomenon central to kidney transplantation and acute kidney injury. MicroRNA- 126 (miR-126) is known to be important in maintaining vascular homeostasis by facilitating vascular regeneration and modulating the mobilization of vascular progenitor cells. Stromal cell-derived factor 1 (SDF-1), important in the mobilization of vascular progenitor cells, is a direct target of miR-126 and modulation of miR-126 was previously shown to affect the number of circulating Sca-1(+)/Lin(-) vascular progenitor cells in a mouse model for hind limb ischemia. Here, we assessed the in vivo contribution of miR-126 to progenitor cell mobilization and kidney function following IRI in mice. A three day follow up of blood urea levels following kidney IRI demonstrated that systemic antagomir silencing of miR-126 did not impact the loss or subsequent restoration of kidney function. However, whole kidney lysates displayed elevated gene expression levels of Sdf-1, Vegf-A and eNOS after IRI as a result of systemic silencing of miR-126. Furthermore, FACS-analysis on whole blood three days after surgery revealed a marked up regulation of the number of circulating Sca-1(+)/Lin(-) progenitor cells in the antagomir-126 treated mice, in an ischemia dependent manner. Our data indicate that silencing of miR-126 can enhance renal expression of Sdf-1 after IRI, leading to the mobilization of vascular progenitor cells into the circulation.

Donor pre-treatment in clinical kidney transplantation: a critical appraisal.

Kidney transplantation represents one of the medical achievements of the 20th century. However, its continued success is limited by the increasing shortage of donor grafts. As a result, more kidney grafts from marginal donors are being considered for transplantation, with concomitantly more initial graft injury and limited organ and patient survival. This has led to an increased need for interventions aiming to optimize and preserve graft quality. Interventions within the donor may protect against ischemia/reperfusion injury, and therefore, donor pre‐treatment is a promising strategy to increase graft function and survival. During the last decade, diverse donor pre‐treatment interventions have been explored in animal studies. Moreover, the first human trials concerning donor pre‐treatment in kidney transplantation have provided encouraging results. Unfortunately, it remains difficult to determine how and where to intervene in the multifactorial and complex processes that affect the donor kidney. Moreover, ethical matters play a critical role in donor interventions, and pre‐treatment should principally not have any potentially unfavorable effects on other organs to be transplanted or on the living donor. This review provides an overview of promising therapeutical strategies for donor pre‐treatment in kidney transplantation and discusses the clinical trials that have been conducted thus far.

Pitfalls in urinary complement measurements

Local activation of the complement system has been associated with ischemia/reperfusion injury following kidney transplantation and tubular injury under proteinuric conditions. The soluble terminal complement complex sC5b-9 is a stable end-product of the complement cascade, and as such a promising urinary biomarker. In the early post-transplant period we found high urinary levels of sC5b-9, significantly correlating with the degree of proteinuria, suggesting activation of filtered complement components at the tubular epithelial surface of the kidney. However, when mimicking proteinuria in vitro by exposing serum (or blood) to urine (both negative for sC5b-9), we found extensive generation of sC5b-9 in urine. This process was inhibited by EDTA, confirming activation of the complement system. In conclusion, although sC5b-9 is an attractive urinary biomarker, one should be aware of the risk of extra-renal complement activation independent of a renal contribution. This may be of special interest when measuring urinary sC5b-9 following kidney transplantation in which procedure-related (microscopic) hematuria and proteinuria are common.

The hypoxanthine-xanthine oxidase axis is not involved in the initial phase of clinical transplantation-related ischemia-reperfusion injury

The hypoxanthine-xanthine oxidase (XO) axis is considered to be a key driver of transplantation-related ischemia-reperfusion (I/R) injury. Whereas interference with this axis effectively quenches I/R injury in preclinical models, there is limited efficacy of XO inhibitors in clinical trials. In this context, we considered clinical evaluation of a role for the hypoxanthine-XO axis in human I/R to be relevant. Patients undergoing renal allograft transplantation were included (n = 40) and classified based on duration of ischemia (short, intermediate, and prolonged). Purine metabolites excreted by the reperfused kidney (arteriovenous differences) were analyzed by the ultra performance liquid chromatography-tandem mass spectrometer (UPLCMS/MS) method and tissue XO activity was assessed by in situ enzymography. We confirmed progressive hypoxanthine accumulation (P < 0.006) during ischemia, using kidney transplantation as a clinical model of I/R. Yet, arteriovenous concentration differences of uric acid and in situ enzymography of XO did not indicate significant XO activity in ischemic and reperfused kidney grafts. Furthermore, we tested a putative association between hypoxanthine accumulation and renal oxidative stress by assessing renal malondialdehyde and isoprostane levels and allantoin formation during the reperfusion period. Absent release of these markers is not consistent with an association between ischemic hypoxanthine accumulation and postreperfusion oxidative stress. On basis of these data for the human kidney we hypothesize that the role for the hypoxanthine-XO axis in clinical I/R injury is less than commonly thought, and as such the data provide an explanation for the apparent limited clinical efficacy of XO inhibitors.

Equivalent Long-term Transplantation Outcomes for Kidneys Donated After Brain Death and Cardiac Death: Conclusions From a Nationwide Evaluation

Background Despite growing waiting lists for renal transplants, hesitations persist with regard to the use of deceased after cardiac death (DCD) renal grafts. We evaluated the outcomes of DCD donations in The Netherlands, the country with the highest proportion of DCD procedures (42.9%) to test whether these hesitations are justified. Methods This study included all procedures with grafts donated after brain death (DBD) (n = 3611) and cardiac death (n = 2711) performed between 2000 and 2017. Transplant outcomes were compared by Kaplan Meier and Cox regression analysis, and factors associated with short (within 90 days of transplantation) and long-term graft loss evaluated in multi-variable analyses. Findings Despite higher incidences of early graft loss (+ 50%) and delayed graft function (+ 250%) in DCD grafts, 10-year graft and recipient survival were similar for the two graft types (Combined 10-year graft survival: 73.9% (95% CI: 72.5–75.2), combined recipient survival: 64.5% (95 CI: 63.0–66.0%)). Long-term outcome equivalence was explained by a reduced impact of delayed graft function on DCD graft survival (RR: 0.69 (95% CI: 0.55–0.87), p < 0.001). Mid and long-term graft function (eGFR), and the impact of incident delayed graft function on eGFR were similar for DBD and DCD grafts. Interpretation Mid and long term outcomes for DCD grafts are equivalent to DBD kidneys. Poorer short term outcomes are offset by a lesser impact of delayed graft function on DCD graft survival. This nation-wide evaluation does not justify the reluctance to use of DCD renal grafts. A strong focus on short-term outcome neglects the superior recovery potential of DCD grafts.