Graft Pre-conditioning by Peri-Operative Perfusion of Kidney Allografts With Rabbit Anti-human T-lymphocyte Globulin Results in Improved Kidney Graft Function in the Early Post-transplantation Period-a Prospective, Randomized Placebo-Controlled Trial

Abstract

Introduction: Although prone to a higher degree of ischemia reperfusion injury (IRI), the use of extended criteria donor (ECD) organs has become reality in transplantation. We therefore postulated that peri-operative perfusion of renal transplants with anti-human T-lymphocyte globulin (ATLG) ameliorates IRI and results in improved graft function. Methods: We performed a randomized, single-blinded, placebo-controlled trial involving 50 kidneys (KTx). Prior to implantation organs were perfused and incubated with ATLG (AP) (n = 24 kidney). Control organs (CP) were perfused with saline only (n = 26 kidney). Primary endpoint was defined as graft function reflected by serum creatinine at day 7 post transplantation (post-tx). Results: AP-KTx recipients illustrated significantly better graft function at day 7 post-tx as reflected by lower creatinine levels, whereas no treatment effect was observed after 12 months surveillance. During the early hospitalization phase, 16 of the 26 CP-KTx patients required dialysis during the first 7 days post-tx, whereas only 10 of the 24 AP-KTx patients underwent dialysis. No treatment-specific differences were detected for various lymphocytes subsets in the peripheral blood of patients. Additionally, mRNA analysis of 0-h biopsies post incubation with ATLG revealed no changes of intragraft inflammatory expression patterns between AP and CP organs. Conclusion: We here present the first clinical study on peri-operative organ perfusion with ATLG illustrating improved graft function in the early period post kidney transplantation. Clinical Trial Registration: www.ClinicalTrials.gov, NCT03377283.

Keywords: ATLG; RCT; ischemia reperfusion injury; kidney transplantation; organ preservation.

Figure 1

ATLG binding within the kidney. (A) ATLG binding to kidney structures was detected via binding of anti-rabbit polymer and visualization by Opal570 (red). Autofluorescence of the tissue (green) was kept in order to visualize kidney tissue morphology. ATLG shows no binding to structures within the glomerulus. Original magnification ×200, scale bar = 50 μm. (B) ATLG binds to intravascular and extravasated leukocytes within the kidney. Leukocytes within vessels (filled arrowhead) as well as leukocytes migrated into the kidney (blank arrowhead) show binding of ATLG (red). Autofluorescence of the tissue (green) was kept in order to distinguish tissue structures and to visualize erythrocytes (arrow) and tubules (dotted arrow). Original magnification ×400, scale bar = 20 μm. (C) Abundant leukocytes within a peritubular capillary show ATLG binding (red). Autofluorescence (green) helps to distinguish erythrocytes and tubules. Original magnification ×400. (D) ATLG (red) binds to the wall of peritubular capillaries (arrow) and leukocytes (arrowheads) within the kidney. Original magnification ×400, scale bar = 20 μm. (E) No binding of ATLG was detected in saline perfused control kidneys. Picture was taken with the same exposure time as used for treated kidney tissue sections. Original magnification ×200, scale bar = 50 μm. Representative pictures of n = 2 experiments with different organs are shown.

Figure 2

ATLG kidney perfusion consort diagram.

Figure 3

Clinical parameters following KTX. The functional parameters creatinine and urea as well as the calculated eGFR illustrate a comparable course between kidneys peri-operatively perfused with ATLG or saline eGFR until month 12 post-transplantation. However, ATLG recipients demonstrated significantly better graft function regarding creatinine as compared with controls at days 5 and 7 (p = 0.015 and p = 0.045). ATLG: open boxes, control: filled boxes; data are presented as mean values ± SEM; *p < 0.05.

Figure 4

mRNA analysis of candidate genes in kidney biopsies taken 1 h post-reperfusion. Data from available tissue specimens are shown. mRNA measurement of selected candidate genes indicative for inflammation and adhesion did not reveal significant differences between ATLG-perfused kidneys and control kidneys. ATLG: open boxes, control: filled boxes. Data are presented as mean ± SEM.

Figure 5

Frequencies of major lymphocyte subsets in the peripheral blood of kidney recipients. Recipients who received an ATLG-perfused organ and those who received a saline-perfused organ illustrated similar frequencies of CD3⁺, CD4⁺, CD8⁺ T cells, CD4⁻CD8⁻ T cells, CD19⁺ B cells, and CD56⁺CD3⁻ NK cells. Interestingly, whereas T cell frequencies appeared to decrease in the early postoperative days, there was a significant increase between day 7 post-transplantation (POD 7) and month 6 (p = 0.0078, respectively). Moreover, both patient groups show a significant decrease in CD19⁺ B cells as compared with pre-transplant levels and POD7 (p = 0.001, respectively). ATLG: open boxes, control: filled boxes; data are presented as mean ± SEM; *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001; *, difference within AP; #, difference within CP; ^## < 0.01, ^### < 0.001.

Figure 6

Age-related effects of ATLG perfusion. Creatinine, urea levels and the calculated eGFR are displayed for donor organs grouped into either young (≤55 years, n = 14 for rATG perfused) or old organs (>55 years, n = 10, rATG perfused). Accordingly, donor organs were also grouped in young (≤55 years, n = 12) or old (>55 years, n = 14). ATLG: open boxes, control: filled boxes; data are presented as mean values ± SEM; *p < 0.05, **p ≤ 0.01.

Figure 7

Age-related effects of young and old donor organs. Within the rATG and control group, graft outcome was separately analyzed for young (≤55 years) or old (>55 years) donor age. ATLG: open boxes, control: filled boxes; data are presented as mean values ± SEM; *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 8

Patient and graft survival at 1 year post-transplantation. Whereas no differences in patient as well as graft survival were detected between ATLG-perfused kidneys and control kidneys. (patient survival: AP-KTx 100% vs. CP-KTx 96%, p = 0.38; graft survival: AP-KTx 96% vs. CP-KTx 92%, p = 0.60).