Protective Role of Kynurenine 3-Monooxygenase in Allograft Rejection and Tubular Injury in Kidney Transplantation.

Renal tubular epithelial cells (TECs) are the primary targets of ischemia-reperfusion injury (IRI) and rejection by the recipient's immune response in kidney transplantation (KTx). However, the molecular mechanism of rejection and IRI remains to be identified. Our previous study demonstrated that kynurenine 3-monooxygenase (KMO) and kynureninase were reduced in ischemia-reperfusion procedure and further decreased in rejection allografts among mismatched pig KTx. Herein, we reveal that TEC injury in acutely rejection allografts is associated with alterations of Bcl2 family proteins, reduction of tight junction protein 1 (TJP1), and TEC-specific KMO. Three cytokines, IFN γ , TNFα, and IL1ß, reported in our previous investigation were identified as triggers of TEC injury by altering the expression of Bcl2, BID, and TJP1. Allograft rejection and TEC injury were always associated with a dramatic reduction of KMO. 3HK and 3HAA, as direct and downstream products of KMO, effectively protected TEC from injury via increasing expression of Bcl-xL and TJP1. Both 3HK and 3HAA further prevented allograft rejection by inhibiting T cell proliferation and up-regulating aryl hydrocarbon receptor expression. Pig KTx with the administration of DNA nanoparticles (DNP) that induce expression of indoleamine 2,3-dioxygenase (IDO) and KMO to increase 3HK/3HAA showed an improvement of allograft rejection as well as murine skin transplant in IDO knockout mice with the injection of 3HK indicated a dramatic reduction of allograft rejection. Taken together, our data provide strong evidence that reduction of KMO in the graft is a key mediator of allograft rejection and loss. KMO can effectively improve allograft outcome by attenuating allograft rejection and maintaining graft barrier function.

Differential Expression of Prostaglandin E2 Receptors in Porcine Kidney Transplants.

BACKGROUND: Acute rejection of a kidney allograft results from adaptive immune responses and marked inflammation. The eicosanoid prostaglandin E2 (PGE2) modulates the inflammatory response, is generated by cyclooxygenase 2 (COX-2), and binds to 1 of the 4 G protein-coupled E prostanoid cell surface receptors (EP1-4). Receptor activation results in in proinflammatory (EP1 and EP3) or anti-inflammatory (EP2 and EP4) responses. We theorized that expression of the components of the COX-PGE2-EP signaling pathway correlates with acute rejection in a porcine model of allogeneic renal transplantation. METHOD: COX-2 enzyme and EP receptor protein expression were quantitated with western blotting and immunohistochemistry from allotransplants (n = 18) and autotransplants (n = 5). Linear regression analysis was used to correlate EP receptor expression with the Banff category of rejection. RESULTS: Pigs with advanced rejection demonstrated significant increases in serum PGE2 metabolites, while pigs with less rejection demonstrated higher tissue concentrations of PGE2 metabolites. A significant negative correlation between COX-2 expression and Banff category of rejection (R = -0.877) was shown. Rejection decreased expression of EP2 and EP4. For both receptors, there was a significant negative correlation with the extent of rejection (R = -0.760 and R = -0.891 for EP2 and EP4, respectively). Rejection had no effect on the proinflammatory receptors EP1 and EP3. CONCLUSION: Downregulation of COX-2 and the anti-inflammatory EP2 and EP4 receptors is associated with acute rejection in unmatched pig kidney transplants, suggesting that the COX-2-PGE2-EP pathway may modulate inflammation in this model. Enhancing EP2 and/or EP4 activity may offer novel therapeutic approaches to controlling the inflammation of acute allograft rejection.

Regulation of indoleamine 2,3 dioxygenase and its role in a porcine model of acute kidney allograft rejection.

In kidney transplantation acute allograft rejection is the most common cause of late allograft loss. Changes in indoleamine 2,3 dioxygenase (IDO) activity, which catabolizes the degradation of tryptophan to kynurenine, may predict rejection. However, exogenous IDO is immunosuppressive in rodent kidney transplantation. Thus, the increase in IDO activity observed in acute allograft rejection is insufficient to prevent rejection. To address this question, we assessed the regulation of IDO and its role in acute rejection in a porcine model of kidney transplant. In tissue samples from rejecting kidney allografts, we showed a 13-fold increase in IDO gene transcription and 20-fold increase in IDO enzyme activity when compared with autotransplanted kidneys. Allografts also demonstrated an over fourfold increase in tissue interferon (IFN)-γ, with marked increases in tumor necrosis factor (TNF)-α, TNF-ß and interleukin 1ß. Gene transcription and protein levels of kynurenine 3-monooxygenase (KMO) were decreased. KMO generates the immunosuppressive kynurenine, 3-hydroxykynurenine. The results of these studies demonstrate a clear association between rejection and increased allograft IDO expression, likely driven in part by IFN-γ and facilitated by other cytokines of the allogeneic response. Moreover, the loss of downstream enzymatic activity in the IDO metabolic pathway may suggest novel mechanisms for the perpetuation of rejection.

A model of acute renal allograft rejection in outbred Yorkshire piglets.

Pigs represent a desirable animal model for the study of rejection in kidney transplantation with inbred Yucatan miniature swine (YMS) the most commonly studied strain due to well defined swine leukocyte antigen (SLA) genotypes. However, limitations to YMS may include cost and availability. Outbred Yorkshire pigs are widely available and significantly cheaper than YMS. Recent advances in SLA genotyping have allowed its application to outbred strains. On this basis, we theorized that Yorkshire pigs would be a viable alternative to YMS for the study of rejection in kidney transplantation. To address this question, we performed auto (Auto) and allotransplants (Allo) in 24 Yorkshire pigs, and assessed SLA genotypes and acute rejection after 72h. At sacrifice, and when compared to autotransplants, allotransplants had significant elevations in serum creatinine (8.4±1.3 vs 2.8±2.0mg/dL for Allo vs autotransplants, respectively) and BUN (61±9 vs 19.2±15mg/dL for Allo vs autotransplants, respectively). Warm ischemia times between the two groups did not differ (24±2.3 vs 26.4±1.4min for Auto vs Allo, respectively). There were 16 distinct SLA haplotypes identified from pigs undergoing allotransplantion, no matched donor-recipient pairs, and all allografts demonstrated rejection. Type IIA cellular rejection (Banff) was the most common. One allograft demonstrated hyperacute rejection due a blood group incompatibility. Histologically, the expression of regulatory Tcells and dendritic cells was increased in allografts. These data suggest that Yorkshire pigs may be a useful model for the study of acute rejection in experimental kidney transplantation.

Indoleamine 2,3-dioxygenase inhibition alters the non-coding RNA transcriptome following renal ischemia-reperfusion injury.

BACKGROUND: Indoleamine 2,3 dioxygenase (IDO) degrades the essential amino acid tryptophan and has been shown to minimize rejection in animal models of renal transplantation. Ischemia-reperfusion injury (IRI) is unavoidable in renal transplantation and correlates with shorter graft survival times. Despite its favorable effects on rejection, there is evidence that IDO may facilitate renal IRI. Differentiating the negative impact of IDO on IRI from its pro-tolerant effects in allograft rejection is of clinical relevance. In these studies we hypothesized that constitutive IDO activity may influence renal genes associated with recovery from IRI, and that IDO inhibition may unmask these effects. METHODS: We examined the renal transcriptome in a rat model of IRI with and without IDO inhibition with 1-methyl-d-tryptophan (1-MT), and assessed for alterations in the gene expression signature. RESULTS: These studies demonstrated that during recovery from renal IRI, pre-treatment with 1-MT alleviated alterations in 105 coding sequences associated with IRI, and in turn triggered new changes in 66 non-coding transcripts, the majority of which were represented by small nucleolar RNA. CONCLUSION: These results suggest a biologic role for non-coding, IDO-dependent genes in regulating the early response to IRI.

Transcriptome profiling reveals differentially expressed transcripts between the human adrenal zona fasciculata and zona reticularis.

CONTEXT: The human adrenal zona fasciculata (ZF) and zona reticularis (ZR) are responsible for the production of cortisol and 19-carbon steroids (often called adrenal androgens), respectively. However, the gene profiles and exact molecular mechanisms leading to the functional phenotype of the ZF and ZR are still not clearly defined. In the present study, we identified the transcripts that are differentially expressed in the ZF and ZR. OBJECTIVE: The objective of the study was to compare the transcriptome profiles of ZF and ZR. DESIGN AND METHODS: ZF and ZR were microdissected from 10 human adrenals. Total RNA was extracted from 10 ZF/ZR pairs and hybridized to Illumina microarray chips. The 10 most differentially expressed transcripts were studied with quantitative RT-PCR (qPCR). Immunohistochemistry was also performed on four zone-specific genes. RESULTS: Microarray results demonstrated that only 347 transcripts of the 47 231 were significantly different by 2-fold or greater in the ZF and ZR. ZF had 195 transcripts with 2-fold or greater increase compared with its paired ZR, whereas ZR was found to have 152 transcripts with 2-fold or greater higher expression than in ZF. Microarray and qPCR analysis of transcripts encoding steroidogenic enzymes (n = 10) demonstrated that only 3ß-hydroxysteroid dehydrogenase, steroid sulfotransferase, type 5 17ß-hydroxysteroid dehydrogenase, and cytochrome b5 were significantly different. Immunohistochemistry and qPCR studies confirmed that the ZF had an increased expression of lymphoid enhancer-binding factor 1 and nephroblastoma overexpressed, whereas ZR showed an increased expression of solute carrier family 27 (fatty acid transporter) (SLC27A2), member 2 and TSPAN12 (tetraspanin 12) CONCLUSION: Microarray revealed several novel candidate genes for elucidating the molecular mechanisms governing the ZF and ZR, thereby increasing our understanding of the functional zonation of these two adrenocortical zones.

Radical nephrectomy and inferior vena caval thrombectomy: outcomes in a lower volume practice.

INTRODUCTION: Surgical volume correlates with improved outcomes for some complex urologic procedures. We reviewed the outcomes of a lower volume practice (1-2 cases per year) experience with radical nephrectomy with infra/retrohepatic vena caval thrombectomy (RNCT). METHODS: We retrospectively reviewed 10 patients who underwent RNCT performed by a single surgeon at a single state institution over 7 years (2002-2009). Patient demographics, presenting symptoms, preoperative imaging, intraoperative findings, pathology, hospital course, outcomes, level of caval involvement, renal artery embolization, liver mobilization, blood loss, transfusion requirements and follow up times were recorded. RESULTS: Median patient BMI (n = 8) was 25.7 (18.3-31.9). Eight patients underwent renal artery embolization prior to RNCT. A vascular or liver surgeon assisted in all 10 RNCT cases. Six thrombi were infrahepatic and four were retrohepatic requiring liver mobilization. Median operative time was 340 minutes (220-480) with a median vena cava clamp time of 17 minutes (11-22). Eight (80%) patients required intraoperative transfusion. Median pathologic tumor size was 9.5 cm (range 6-21). Median hospital stay was 7.5 days (5-15). Four patients had complications including colonic mesenteric rent (n = 2), abscess (n = 1), retroperitoneal hematoma (n = 1), distal pancreatic injury (n = 1), and splenic capsular tear (n = 1). One patient had postoperative liver metastasis. Two patients died from postoperative metastasis, at 5 months and 11 months. CONCLUSIONS: RNCT can be performed, with the assistance of a vascular/liver transplant surgeon, for an infrahepatic or retrohepatic thrombus satisfactorily in a lower volume practice.

An initial experience and evolution of laparoscopic hepatic resectional surgery.

BACKGROUND: The use of minimally invasive procedures has revolutionized modern surgery. Only recently has laparoscopy been introduced for use in hepatic surgery. METHODS: Patient demographics, tumor characteristics, and outcomes were evaluated for all initial cases of laparoscopic hepatic resection. RESULTS: Twenty-one resections were performed in 17 patients; 5 were performed for malignancy, of which 3 had underlying cirrhosis, and the remaining 12 for benign symptomatic disease. Mean patient age was 55.4 (range, 24-82 years). The mean number of lesions was 1.4 (range, 1-5), having an average size of 7.6 cm (range, 2-30 cm). Mean operative time was 2.8 hours (range, 2-5 hours) hours. Most resections involved 1 or more Couinaud segments. Mean blood loss was 288 cc (range, 50-150 cc). Complications included re-operation for hemorrhage (n=2), biliary leakage (n=1), and death from hepatic failure (n=1). Mean length of stay was 2.9 days (range, 1-14). When compared with our series of 100 patients who underwent open hepatic resection for benign tumors, significantly greater means ( P <.05) were noted for blood loss (485 cc), operative time (4.5 hours), and length of stay (6.5 days). CONCLUSIONS: Laparoscopic hepatic surgery, though complex, can be performed safely and efficaciously. Minimally invasive surgery appears to provide several distinct advantages over traditional open hepatic surgery. However, techniques for the laparoscopic control of bleeding and bile leak remain in their infancy.