Ivy A. Rosales

Biopsy transcriptome expression profiling to identify kidney transplants at risk of chronic injury: a multicentre, prospective study

BACKGROUND Chronic injury in kidney transplants remains a major cause of allograft loss. The aim of this study was to identify a gene set capable of predicting renal allografts at risk of progressive injury due to fibrosis. METHODS This Genomics of Chronic Allograft Rejection (GoCAR) study is a prospective, multicentre study. We prospectively collected biopsies from renal allograft recipients (n=204) with stable renal function 3 months after transplantation. We used microarray analysis to investigate gene expression in 159 of these tissue samples. We aimed to identify genes that correlated with the Chronic Allograft Damage Index (CADI) score at 12 months, but not fibrosis at the time of the biopsy. We applied a penalised regression model in combination with permutation-based approach to derive an optimal gene set to predict allograft fibrosis. The GoCAR study is registered with ClinicalTrials.gov, number NCT00611702. FINDINGS We identified a set of 13 genes that was independently predictive for the development of fibrosis at 1 year (ie, CADI-12 ≥2). The gene set had high predictive capacity (area under the curve [AUC] 0·967), which was superior to that of baseline clinical variables (AUC 0·706) and clinical and pathological variables (AUC 0·806). Furthermore routine pathological variables were unable to identify which histologically normal allografts would progress to fibrosis (AUC 0·754), whereas the predictive gene set accurately discriminated between transplants at high and low risk of progression (AUC 0·916). The 13 genes also accurately predicted early allograft loss (AUC 0·842 at 2 years and 0·844 at 3 years). We validated the predictive value of this gene set in an independent cohort from the GoCAR study (n=45, AUC 0·866) and two independent, publically available expression datasets (n=282, AUC 0·831 and n=24, AUC 0·972). INTERPRETATION Our results suggest that this set of 13 genes could be used to identify kidney transplant recipients at risk of allograft loss before the development of irreversible damage, thus allowing therapy to be modified to prevent progression to fibrosis. FUNDING National Institutes of Health.

Pretransplant IgG Reactivity to Apoptotic Cells Correlates With Late kidney Allograft Loss

Preexisting serum antibodies have long been associated with graft loss in transplant recipients. While most studies have focused on HLA‐specific antibodies, the contribution of non‐HLA‐reactive antibodies has been largely overlooked. We have recently characterized mAbs secreted by B cell clones derived from kidney allograft recipients with rejection that bind to apoptotic cells. Here, we assessed the presence of such antibodies in pretransplant serum from 300 kidney transplant recipients and examined their contribution to the graft outcomes. Kaplan–Meier survival analysis revealed that patients with high pretransplant IgG reactivity to apoptotic cells had a significantly increased rate of late graft loss. The effect was only apparent after approximately 1 year posttransplant. Moreover, the association between pretransplant IgG reactivity to apoptotic cells and graft loss was still significant after excluding patients with high reactivity to HLA. This reactivity was almost exclusively mediated by IgG1 and IgG3 with complement fixing and activating properties. Overall, our findings support the view that IgG reactive to apoptotic cells contribute to presensitization. Taking these antibodies into consideration alongside anti‐HLA antibodies during candidate evaluation would likely improve the transplant risk assessment.

Intronic locus determines SHROOM3 expression and potentiates renal allograft fibrosis

Fibrosis underlies the loss of renal function in patients with chronic kidney disease (CKD) and in kidney transplant recipients with chronic allograft nephropathy (CAN). Here, we studied the effect of an intronic SNP in SHROOM3, which has previously been linked to CKD, on the development of CAN in a prospective cohort of renal allograft recipients. The presence of the rs17319721 allele at the SHROOM3 locus in the donor correlated with increased SHROOM3 expression in the allograft. In vitro, we determined that the sequence containing the risk allele at rs17319721 is a transcription factor 7-like 2-dependent (TCF7L2-dependent) enhancer element that functions to increase SHROOM3 transcription. In renal tubular cells, TGF-β1 administration upregulated SHROOM3 expression in a β-catenin/TCF7L2-mediated manner, while SHROOM3 in turn facilitated canonical TGF-β1 signaling and increased α1 collagen (COL1A1) expression. Inducible and tubular cell-specific knockdown of Shroom3 markedly abrogated interstitial fibrosis in mice with unilateral ureteric obstruction. Moreover, SHROOM3 expression in allografts at 3 months after transplant and the presence of the SHROOM3 risk allele in the donor correlated with increased allograft fibrosis and with reduced estimated glomerular filtration rate at 12 months after transplant. Our findings suggest that rs17319721 functions as a cis-acting expression quantitative trait locus of SHROOM3 that facilitates TGF-β1 signaling and contributes to allograft injury.

Prolonged Survival of Pig Skin on Baboons After Administration of Pig Cells Expressing Human CD47.

BackgroundSuccessful xenotransplantation will likely depend, in part, on the induction of immunological tolerance, because the high levels of immunosuppression otherwise required would likely have unacceptable side effects. Rapid clearance of administered porcine hematopoietic stem cells by primate macrophages has hampered previous attempts to induce tolerance through mixed hematopoietic chimerism across a pig-to-primate barrier. Phagocytosis is normally inhibited by binding of cell surface protein CD47 to macrophage signal regulatory protein &agr; receptors. However, pig CD47 has previously been shown to be ineffective in transducing signals through primate signal regulatory protein &agr;. MethodsMobilized peripheral blood hematopoietic cells from transgenic swine expressing high or low levels of human CD47 were infused into conditioned baboons at 3 time points over a 9-week period. Xenogeneic peripheral blood chimerism was assessed after each infusion. Split thickness skin grafts from the hematopoietic cell donor swine were placed on recipients 5 weeks after the last cell infusion and 7 weeks after the discontinuation of all immunosuppression to test immune response. ResultsThe level and duration of transient chimerism were substantially greater in baboons receiving hematopoietic cells from a pig expressing high levels of human CD47. Skin graft survival on high CD47 recipients was prolonged as well, in 1 case showing no signs of rejection at least 53 days after placement. ConclusionsProlongation of transient porcine chimerism via transgenic expression of human CD47 in a primate model is associated with an immune modulating effect, leading to markedly prolonged survival of donor swine skin xenografts that may be applicable to clinical solid organ xenotransplantation.

A Bridge to Somewhere: 25-day Survival After Pig-to-Baboon Liver Xenotransplantation.

To the Editor: C linical application of porcine xenogeneic livers remains an attractive option, either as a bridge to allotransplantation or, eventually, to address the current shortage of donor organs. However, until now, progress has been limited. Early work demonstrated reasonable xenogeneic production of essential proteins, but with survival limited to a week or less due to profound thrombocytopenia, bleeding, and notable cholestasis, thought to be due to interspecies incompatibilities. Although previous work in our laboratory did extend survival up to 9 days after liver xenotransplantation (LXT), recipient baboons required intensive platelet transfusions, antifibrinolytic agents, and on average 70 to 80 mLs of packed red blood cell (PRBC) transfusions daily. Based upon our observation of marginal coagulation factor production in baboon recipients of porcine livers, we recently developed a novel approach utilizing the continuous administration of exogenous human coagulation factors after LXT. This demonstrated the ability to control coagulopathy, maintain circulating platelets, and prevent thrombotic microangiopathy (TMA) in our pig-to-baboon model of orthotopic LXT. On the basis of these findings, we have now further extended our study of continuous coagulation factor administration by adding measures to minimize infectious risks, and utilize costimulation blockade. With these modifications, we now are able to report 25-day survival after pig-tobaboon LXT, which represents a major advance towards clinical applicability. The recipient baboon (female, 9.6 kg) underwent orthotopic LXT in a standard bicaval fashion as previously described. The alpha-1,3-galactosyltransferase knock out miniature swine donor (male, 9.4 kg) was porcine cytomegalovirus (CMV) (pCMV)-negative and underwent hepatectomy in a standard fashion with in situ cold perfusion using UW solution (Barr Pharmaceuticals, Pomona, NY). Six hours post-LXT continuous administration of human prothrombin concentrate complex

Haptoglobin or Hemopexin Therapy Prevents Acute Adverse Effects of Resuscitation After Prolonged Storage of Red Cells

Background: Extracellular hemoglobin and cell-free heme are toxic breakdown products of hemolyzed erythrocytes. Mammals synthesize the scavenger proteins haptoglobin and hemopexin, which bind extracellular hemoglobin and heme, respectively. Transfusion of packed red blood cells is a lifesaving therapy for patients with hemorrhagic shock. Because erythrocytes undergo progressive deleterious morphological and biochemical changes during storage, transfusion of packed red blood cells that have been stored for prolonged intervals (SRBCs; stored for 35–40 days in humans or 14 days in mice) increases plasma levels of cell-free hemoglobin and heme. Therefore, in patients with hemorrhagic shock, perfusion-sensitive organs such as the kidneys are challenged not only by hypoperfusion but also by the high concentrations of plasma hemoglobin and heme that are associated with the transfusion of SRBCs. Methods: To test whether treatment with exogenous human haptoglobin or hemopexin can ameliorate adverse effects of resuscitation with SRBCs after 2 hours of hemorrhagic shock, mice that received SRBCs were given a coinfusion of haptoglobin, hemopexin, or albumin. Results: Treatment with haptoglobin or hemopexin but not albumin improved the survival rate and attenuated SRBC-induced inflammation. Treatment with haptoglobin retained free hemoglobin in the plasma and prevented SRBC-induced hemoglobinuria and kidney injury. In mice resuscitated with fresh packed red blood cells, treatment with haptoglobin, hemopexin, or albumin did not cause harmful effects. Conclusions: In mice, the adverse effects of transfusion with SRBCs after hemorrhagic shock are ameliorated by treatment with either haptoglobin or hemopexin. Haptoglobin infusion prevents kidney injury associated with high plasma hemoglobin concentrations after resuscitation with SRBCs. Treatment with the naturally occurring human plasma proteins haptoglobin or hemopexin may have beneficial effects in conditions of severe hemolysis after prolonged hypotension.

Prolonged Survival Following Pig-to-Primate Liver Xenotransplantation Utilizing Exogenous Coagulation Factors and Costimulation Blockade

Since the first attempt of pig‐to‐primate liver xenotransplantation (LXT) in 1968, survival has been limited. We evaluated a model utilizing α‐1,3‐galactosyltransferase knockout donors, continuous posttransplant infusion of human prothrombin concentrate complex, and immunosuppression including anti–thymocyte globulin, FK‐506, methylprednisone, and costimulation blockade (belatacept, n = 3 or anti‐CD40 mAb, n = 1) to extend survival. Baboon 1 remained well until postoperative day (POD) 25, when euthanasia was required because of cholestasis and plantar ulcers. Baboon 2 was euthanized following a seizure on POD 5, despite normal liver function tests (LFTs) and no apparent pathology. Baboon 3 demonstrated initial stable liver function but was euthanized on POD 8 because of worsening LFTs. Pathology revealed C4d positivity, extensive hemorrhagic necrosis, and a focal cytomegalovirus inclusion. Baboon 4 was clinically well with stable LFTs until POD29, when euthanasia was again necessitated by plantar ulcerations and rising LFTs. Final pathology was C4d negative and without evidence of rejection, inflammation, or thrombotic microangiopathy. Thus, nearly 1‐mo rejection‐free survival has been achieved following LXT in two of four consecutive recipients, demonstrating that the porcine liver can support life in primates for several weeks and has encouraging potential for clinical application as a bridge to allotransplantation for patients with acute‐on‐chronic or fulminant hepatic failure.

The Effects of Exogenous Administration of Human Coagulation Factors Following Pig-to-Baboon Liver Xenotransplantation

We sought to determine the effects of exogenous administration of human coagulation factors following pig‐to‐baboon liver xenotransplantation (LXT) using GalT‐KO swine donors. After LXT, baboons received no coagulation factors (historical control, n = 1), bolus administration of a human prothrombin concentrate complex (hPCC; 2.5 mL/kg, n = 2), continuous infusion of hPCC (1.0 mL/h, n = 1) or continuous infusion of human recombinant factor VIIa (1 µg/kg per hour, n = 3). The historical control recipient demonstrated persistent thrombocytopenia despite platelet administration after transplant, along with widespread thrombotic microangiopathy (TMA). In contrast, platelet levels were maintained in bolus hPCC recipients; however, these animals quickly developed large‐vessel thrombosis and TMA, leading to graft failure with shortened survival. Recipients of continuous coagulation factor administration experienced either stabilization or an increase in their circulating platelets with escalating doses. Furthermore, transfusion requirements were decreased, and hepatic TMA was noticeably absent in recipients of continuous coagulation factor infusions compared with the historical control and bolus hPCC recipients. This effect was most profound with a continuous, escalating dose of factor VIIa. Further studies are warranted because this regimen may allow for prolonged survival following LXT.

Magnesium protects against cisplatin-induced acute kidney injury without compromising cisplatin-mediated killing of an ovarian tumor xenograft in mice

Cisplatin, a commonly used chemotherapeutic for ovarian and other cancers, leads to hypomagnesemia in most patients and causes acute kidney injury (AKI) in 25-30% of patients. Previously, we showed that magnesium deficiency worsens cisplatin-induced AKI and magnesium replacement during cisplatin treatment protects against cisplatin-mediated AKI in non-tumor-bearing mice (Solanki MH, Chatterjee PK, Gupta M, Xue X, Plagov A, Metz MH, Mintz R, Singhal PC, Metz CN. Am J Physiol Renal Physiol 307: F369-F384, 2014). This study investigates the role of magnesium in cisplatin-induced AKI using a human ovarian tumor (A2780) xenograft model in mice and the effect of magnesium status on tumor growth and the chemotherapeutic efficacy of cisplatin in vivo. Tumor progression was unaffected by magnesium status in saline-treated mice. Cisplatin treatment reduced tumor growth in all mice, irrespective of magnesium status. In fact, cisplatin-treated magnesium-supplemented mice had reduced tumor growth after 3 wk compared with cisplatin-treated controls. While magnesium status did not interfere with tumor killing by cisplatin, it significantly affected renal function following cisplatin. Cisplatin-induced AKI was enhanced by magnesium deficiency, as evidenced by increased blood urea nitrogen, creatinine, and other markers of renal damage. This was accompanied by reduced renal mRNA expression of the cisplatin efflux transporter Abcc6. These effects were significantly reversed by magnesium replacement. On the contrary, magnesium status did not affect the mRNA expression of cisplatin uptake or efflux transporters by the tumors in vivo. Finally, magnesium deficiency enhanced platinum accumulation in the kidneys and renal epithelial cells, but not in the A2780 tumor cells. These findings demonstrate the renoprotective role of magnesium during cisplatin AKI, without compromising the chemotherapeutic efficacy of cisplatin in an ovarian tumor-bearing mouse model.

Chronic Antibody-Mediated Rejection in Nonhuman Primate Renal Allografts: Validation of Human Histological and Molecular Phenotypes

Molecular testing represents a promising adjunct for the diagnosis of antibody‐mediated rejection (AMR). Here, we apply a novel gene expression platform in sequential formalin‐fixed paraffin‐embedded samples from nonhuman primate (NHP) renal transplants. We analyzed 34 previously described gene transcripts related to AMR in humans in 197 archival NHP samples, including 102 from recipients that developed chronic AMR, 80 from recipients without AMR, and 15 normal native nephrectomies. Three endothelial genes (VWF, DARC, and CAV1), derived from 10‐fold cross‐validation receiver operating characteristic curve analysis, demonstrated excellent discrimination between AMR and non‐AMR samples (area under the curve = 0.92). This three‐gene set correlated with classic features of AMR, including glomerulitis, capillaritis, glomerulopathy, C4d deposition, and DSAs (r = 0.39–0.63, p < 0.001). Principal component analysis confirmed the association between three‐gene set expression and AMR and highlighted the ambiguity of v lesions and ptc lesions between AMR and T cell–mediated rejection (TCMR). Elevated three‐gene set expression corresponded with the development of immunopathological evidence of rejection and often preceded it. Many recipients demonstrated mixed AMR and TCMR, suggesting that this represents the natural pattern of rejection. These data provide NHP animal model validation of recent updates to the Banff classification including the assessment of molecular markers for diagnosing AMR.