Protective effect of ischaemic preconditioning on acute and chronic renal damage following ischaemia reperfusion injury: characterisation of fibrosis development after inflammation resolution.

OBJECTIVES: Acute Kidney Injury (AKI) and Chronic Kidney Disease (CKD) are increasingly recognised as one disease continuum, rather than distinct entities, and are associated with a huge burden to healthcare services. The leading cause of AKI worldwide is Ischaemia Reperfusion Injury (IRI), most commonly seen in clinical settings of sepsis-driven hypotension. Ischaemic Preconditioning (IPC) is a strategy aimed at reducing the deleterious effects of IRI. The objectives of this study were to demonstrate an efficacious in vivo model of Kidney IRI, and the protective influence of IPC in attenuating AKI and development of renal fibrosis. METHODS: A rat model of bilateral kidney IRI was used: Male Lewis rats (n=84) were assigned to IRI, sham or IPC. In IRI, renal pedicles were clamped for 45 minutes. IPC groups underwent pulsatile IPC prior to IRI. Kidneys were retrieved at 24 hours, 48 hours, 7 days, 14 days and 28 days, and assessed histologically. RESULTS: IRI led to marked AKI (24-48 h) and renal fibrosis development by 28 days. IPC attenuated this damage, with 66% less fibrosis. Interestingly, at 14-days, the histological appearance of both IRI and IPC kidneys was rather similar, potentially representing an important transitional point at which kidneys commit to either fibrosis or recovery. This may provide a suitable inflexion point for introduction of novel anti-fibrotic therapies. CONCLUSIONS: In conclusion, we have characterised a model of kidney injury from acute to chronic phases, allowing detailed mechanistic understanding and which can be manipulated by effective treatment strategies such as IPC.

Robust Rat and Mouse Models of Bilateral Renal Ischemia Reperfusion Injury.

BACKGROUND/AIM: Acute and chronic kidney diseases are a major contributor to morbidity and mortality worldwide, with no specific treatments currently available for these. To enable understanding the pathophysiology of and testing novel treatments for acute and chronic kidney disease, a suitable in vivo model of kidney disease is essential. In this article, we describe two reliable rodent models (rats and mice) of efficacious kidney injury displaying acute to chronic kidney injury progression, which is also reversible through novel therapeutic strategies such as ischemic preconditioning (IPC). MATERIALS AND METHODS: We utilized adult male Lewis rats and adult male wildtype (C57BL/6) mice, performed a midline laparotomy, and induced warm ischemia to both kidneys by bilateral clamping of both renal vascular pedicles for a set time, to mimic the hypoxic etiology of disease commonly found in kidney injury. RESULTS: Bilateral ischemia reperfusion injury caused marked structural and functional kidney injury as exemplified by histology damage scores, serum creatinine levels, and kidney injury biomarker levels in both rodents. Furthermore, this effect displayed a dose-dependent response in the mouse model. CONCLUSION: These rodent models of bilateral kidney IRI are reliable, reproducible, and enable detailed mechanistic study of the underlying pathophysiology of both acute and chronic kidney disease. They have been carefully optimised for single operator use with a strong track record of training both surgically trained and surgically naïve operators.

Determination of a microRNA signature of protective kidney ischemic preconditioning originating from proximal tubules.

Ischemic preconditioning (IPC) is effective in limiting subsequent ischemic acute kidney injury in experimental models. MicroRNAs are an important class of post-transcriptional regulator and show promise as biomarkers of kidney injury. We evaluated the time- and dose-dependence of benefit from IPC in a rat model of functional (bilateral) ischemia-reperfusion injury (IRI). We found optimal protection from subsequent injury following short, repetitive sequences of preconditioning insult. We subsequently used hybridization array and microRNA sequencing to characterize microRNA signatures of protective IPC and of IRI. These approaches identified a profile of microRNA changes consequent on IRI, that were limited by prior IPC. To localize these signals within the kidney, we used laser capture microdissection and RT-qPCR to measure microRNA abundance in nephron segments, pinpointing microRNA changes principally to glomeruli and proximal tubules. Our data describe a unique microRNA signature for IRI in the rat kidney. Pulsatile IPC reduces kidney damage following IRI and diminishes this microRNA signal. We have also identified candidate microRNAs that may act as biomarkers of injury and therapeutic targets in this context.

A Localized Ischemic Preconditioning Regimen Increases Tumor Necrosis Factor α Expression in a Rat Model of Kidney Ischemia-Reperfusion Injury.

OBJECTIVES: We evaluated a continuous, immediate, localized ischemic preconditioning regimen in a rat model of ischemia-reperfusion injury and assessed whether it attenuated injury at the histologic and molecular levels. MATERIALS AND METHODS: Fifteen adult male Lewis rats received sham operation, left unilateral warm ischemia (45 minutes of cross-clamping of the renal pedicle; ischemia-reperfusion injury group), or 15 minutes of ischemia followed by a 20-minute reperfusion period, 45 minutes of ischemia-reperfusion injury, and subsequent reperfusion (ischemic preconditioning/ischemia-reperfusion injury group). Kidney tissue was retrieved 48 hours later, sectioned, stained with hematoxylin and eosin, and examined. We used RNA extraction and real-time quantitative polymerase chain reaction analysis to assess acute kidney injury markers, cytokines, and microRNA-21. RESULTS: Forty-five minutes of unilateral ischemia-reperfusion injury caused marked changes in histology at 48 hours, characterized by endothelial loss, tubulointerstitial damage (inflammation, cast formation), tubular cell necrosis, and glomerular capsule thickening. The ischemia-reperfusion injury and ischemic preconditioning/ischemia-reperfusion injury groups showed no measurable differences in histology. Expression of the acute kidney injury markers was significantly increased in the ischemia-reperfusion injury versus Sham group; however, no difference was found between the ischemia reperfusion injury and ischemic preconditioning/ischemia-reperfusion injury groups. Similarly, expression of interleukin 17, interleukin 18, and tumor necrosis factor ? was significantly increased in the ischemia-reperfusion injury versus Sham group. No significant difference was found between the ischemia-reperfusion injury and ischemic preconditioning/ischemia-reperfusion injury groups for interleukin 17 and interleukin 18; however, tumor necrosis factor ? expression was significantly increased in the ischemic preconditioning/ischemia-reperfusion injury versus ischemia-reperfusion injury group. CONCLUSIONS: In our ischemic preconditioning model, tumor necrosis factor α expression was increased without altering the sequelae of ischemia-reperfusion injury. The long-term consequences of this augmented early inflammatory response and whether these consequences are altered by variations in ischemic preconditioning or a subsequent injury require further study.

[Child and adolescent abuse recorded at a national referral hospital, 2006 - 2011]. / Maltrato infantil y del adolescente registrado en un hospital de referencia nacional, 2006 - 2011.

OBJECTIVES: To describe the records of child and adolescent abuse of the Instituto Nacional de Salud del Niño (INSN) from January 2006 to September 2011, characterizing the victim and perpetrator. MATERIALS AND METHODS: A secondary sources analysis was performed, based on the domestic violence and child abuse records, from froms administered by Child Abuse and Adolescent Health Unit (MAMIS) at the INSN. The records include data of the victim, offender and characteristics of the aggression. Types of aggression were categorized as: sexual, physical, psychological or neglection. Frequencies and percentages are presented. RESULTS: A total of 1798 records were included. From them 63.9% were girls, and 39.9% were adolescents. Males accounted for 60.6% of the offenders, and 65.8% of assaults occurred at home. Sexual assault was recorded in 48.6%, with more frequency among girls (73.2%) and adolescents (44.4%); and intercourse occurred in 9.6% of the cases. CONCLUSIONS: From the INSN MAMIS records, aggressions to girls was the most frequent type of report, the aggressor was often a male and most of the attacks occurred in the child's home. Sexual assault accounted for almost half of the series.

Maltrato infantil y del adolescente registrado en un hospital de referencia nacional, 2006 - 2011 / Child and adolescent abuse recorded at a national referral hospital, 2006 - 2011

Objetivos. Realizar una descripción de los registros del Instituto Nacional de Salud del Niño (INSN), desde enero de 2006 hasta septiembre de 2011, sobre maltrato infantil y del adolescente, lo cual permite la caracterización del agredido y del agresor. Materiales y métodos. Se realizó un análisis de fuentes secundarias, basado en registros de la "Ficha de evaluación de violencia familiar y maltrato infantil", aplicada por el Módulo de Atención al Maltrato Infantil y del Adolescente en Salud (MAMIS) del INSN. La ficha incluye datos del agredido, del agresor y las características de la agresión. Se diferenció el tipo de agresión como: sexual, física, psicológica o por abandono. Se muestran los resultados en frecuencias y porcentajes. Resultados. Se incluyeron 1798 registros. El 63,9% eran niñas y el 39,9% fueron adolescentes. El 60,6% de los agresores fueron varones y el 65,8% de las agresiones ocurrieron en casa. El 48,6% fueron registros de agresión sexual, que fue más frecuente en niñas (73,2%) y adolescentes (44,4%); en el 9,6% de los casos existió coito. Conclusiones. En los registros del MAMIS del INSN, la agresión en niñas fue la más frecuente; el agresor con frecuencia era un varón y la mayoría de las agresiones ocurrieron en el domicilio del menor. La agresión sexual fue casi la mitad de la serie.

Objectives. To describe the records of child and adolescent abuse of the Instituto Nacional de Salud del Niño (INSN) from January 2006 to September 2011, characterizing the victim and perpetrator. Materials and methods. A secondary sources analysis was performed, based on the domestic violence and child abuse records, from froms administered by Child Abuse and Adolescent Health Unit (MAMIS) at the INSN. The records include data of the victim, offender and characteristics of the aggression. Types of aggression were categorized as: sexual, physical, psychological or neglection. Frequencies and percentages are presented. Results. A total of 1798 records were included. From them 63.9% were girls, and 39.9% were adolescents. Males accounted for 60.6% of the offenders, and 65.8% of assaults occurred at home. Sexual assault was recorded in 48.6%, with more frequency among girls (73.2%) and adolescents (44.4%); and intercourse occurred in 9.6% of the cases. Conclusions. From the INSN MAMIS records, aggressions to girls was the most frequent type of report, the aggressor was often a male and most of the attacks occurred in the child’s home. Sexual assault accounted for almost half of the series.

Maltrato infantil y del adolescente registrado en un Hospital de referencia nacional, 2006 2011 / Child and adolescent abuse recorded at a national referral hospital, 2006 - 2011

Objetivos. Realizar una descripción de los registros del Instituto Nacional de Salud del Niño (INSN), desde enero de 2006 hasta septiembre de 2011, sobre maltrato infantil y del adolescente, lo cual permite la caracterización del agredido y del agresor. Materiales y métodos. Se realizó un análisis de fuentes secundarias, basado en registros de la “Ficha de evaluación de violencia familiar y maltrato infantil”, aplicada por el Módulo de Atención al Maltrato Infantil y del Adolescente en Salud (MAMIS) del INSN. La ficha incluye datos del agredido, del agresor y las características de la agresión. Se diferenció el tipo de agresión como: sexual, física, psicológica o por abandono. Se muestran los resultados en frecuencias y porcentajes. Resultados. Se incluyeron 1798 registros. El 63,9 por ciento eran niñas y el 39,9 por ciento fueron adolescentes. El 60,6 por ciento de los agresores fueron varones y el 65,8 por ciento de las agresiones ocurrieron en casa. El 48,6 por ciento fueron registros de agresión sexual, que fue más frecuente en niñas (73,2por ciento) y adolescentes (44,4 por ciento); en el 9,6 por ciento de los casos existió coito. Conclusiones. En los registros del MAMIS del INSN, la agresión en niñas fue la más frecuente; el agresor con frecuencia era un varón y la mayoría de las agresiones ocurrieron en el domicilio del menor. La agresión sexual fue casi la mitad de la serie.(AU)

The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts.

BACKGROUND: In pig-to-nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay-accelerating factor (hDAF), the main type of rejection is antibody-mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement-regulatory function of the transgene, neutralization of natural antibodies to Galalpha1-3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10). METHODS: Life-supporting hDAF-transgenic kidney transplantation was performed in cynomolgus monkeys, using cyclophosphamide induction, and maintenance immunosuppression with cyclosporin A, mycophenolate sodium, and tapering steroids. Rejection was treated with bolus steroid injections: if not successful animals were terminated. Three groups were studied: in group 1 (n=4) GAS914 (a soluble glycoconjugate comprising Gal on a poly-L-lysine backbone) was added before and after transplantation; group 2 (n=2) received GAS914 as in group 1 and in addition TP10 before and after transplantation; in group 3 (n=4) GAS914 was only given before transplantation and TP10 as in group 2. Monitoring included the regular assessment of anti-porcine antibodies, complement activity (soluble C5b-9), therapeutic drug monitoring, and graft histology. RESULTS: Survival in group 1 was 6, 12, 31 and 37 days, respectively, and in all four cases graft histology showed AHXR. The two animals in groups 2 survived 3 and 15 days, respectively, and similarly showed AHXR in graft histology. In group 3 two animals showed AHXR (10 and 37 days survival, respectively), and two others did not show AHXR (20 and 32 days survival, respectively). The diagnosis AHXR included the deposition of complement activation products in the graft, which were present at lower intensity in animals treated with TP10. In all animals GAS914 effectively neutralized circulating anti-Gal antibody. Antibodies were detectable in the circulation of all animals using porcine erythrocytes in a hemolytic assay, although at lower levels than before transplantation. Soluble C5b-9 was not detectable in the circulation of animals receiving TP10, and circulating TP10 concentrations in these animals were in a presumed pharmacologically active range. CONCLUSIONS: The inclusion of TP10 in the immunosuppressive protocol does not clearly lead to improved xenograft survival. Despite effective neutralization of anti-Gal antibodies and effective inhibition of systemic complement activity, AHXR was apparent in four of six animals under chronic TP10 treatment, including deposits of complement activation products in the graft. Apparently, effective systemic complement inhibition by TP10 in combination with local complement regulation by the hDAF transgene product does not necessarily result in effective inhibition of complement activation at locations in the xenograft upon binding of anti-porcine antibodies to the grafted endothelium.

Maintenance triple immunosuppression with cyclosporin A, mycophenolate sodium and steroids allows prolonged survival of primate recipients of hDAF porcine renal xenografts.

To date, the best results in life-supporting pig-to-primate renal xenotransplantation have been obtained in recipients exposed to long-term immunosuppression with cyclophosphamide. As this agent is frequently associated with side-effects, we have explored the potential of a mycophenolate sodium-based maintenance immunosuppression in this model. Human decay-accelerating factor (hDAF) transgenic kidneys were transplanted into splenectomized and bilaterally nephrectomized cynomolgus monkeys immunosuppressed with mycophenolate sodium, cyclosporin A and steroids, and exposed to a brief induction course with cyclophosphamide (up to four doses). After transplantation, the primates were monitored daily for biochemical and haematological evaluations and for the measurements of haemolytic anti-pig antibodies (APA). A detailed histological analysis of each explanted graft was also performed. All the animals showed very poor initial graft function but survived for up to 51 days. In contrast to our previous studies in xenograft recipients on long-term immunosuppression with cyclophosphamide, minimal or no circulating xeno-directed antibodies, as measured by the evaluation of APA titres, were detected in this series although some degree of acute humoral rejection was observed in all the explanted grafts and was the primary cause of graft failure. Furthermore, in addition to areas of humorally mediated graft damage, we have observed for the first time areas with exclusive and prominent infiltration by CD2+ and CD8+ mononuclear cells presenting patterns compatible with tubulitis, glomerulitis and arteritis, which we have called acute cellular xenograft rejection (ACXR). In addition, CD68+ infiltrating macrophages and CD20+ B-cells were also present. This study demonstrates that a triple maintenance immunosuppression with mycophenolate sodium, cyclosporin A and steroids is a viable alternative to a cyclophosphamide-based immunosuppression to obtain prolonged survival of porcine organs transplanted into primates. However, a more stringent control of antibody forming cells remains essential to further extend the survival of xenografts in this model. In addition, the use of the immunosuppressive regimen reported here in the primate is associated with the occurrence of a new category of cell-mediated xenograft injury (ACXR) whose significance has yet to be clarified.

Estudio clínico e inmunólogico del xenorrechazo en el xenotrasplante ortotópico de hígado de cerdo a babuino / Clinical and immunological study of xenorejection in orthotopic liver xenotransplantation from pig to baboon

Introducción. La experiencia de xenotrasplante hepático (Xtoh) de cerdo a primate no humano es muy limitada. Nuestros objetivos han sido: a) comprobar si el hígado de un cerdo transgénico h-DAF evita el rechazo hiperagudo; b) estudiar las funciones metabólicas del hígado porcino tras el Xtoh; y c) analizar el perfil clínico, bioquímico e inmunológico del rechazo vascular agudo retardado. Animales y métodos. Se realizaron 6 Xtoh de cerdo a babuino, 4 de cerdos no modificados y dos de cerdos transgénicos para h-DAF. Se llevaron a cabo determinaciones hematológicas, de coagulación, de xenoanticuerpos y del complemento. En el babuino que sobrevivió 8 días, se estudiaron durante los mismos las poblaciones linfocitarias y la actividad lítica de los linfocitos. Resultados. Los valores de xIgG e IgM descendieron drásticamente a los 3 min de la reperfusión, sobre todo del CH50, C3 y C4. En los hígados no modificados genéticamente apareció una coagulación intravascular diseminada por rechazo hiperagudo, con una supervivencia inferior a 12 h. Con los hígados h-DAF, la coagulación se normalizó, con una supervivencia de 8 y 4 días, falleciendo ambos por insuficiencia respiratoria, sin rechazo hiperagudo. El babuino que sobrevivió 8 días presentó a las 36 h un rechazo vascular agudo retardado, detectándose una estimulación de las HLA clase I sobre los linfocitos CD3+ y CD19+, que respondió al tratamiento. Conclusiones. El hígado transgénico h-DAAF previene el rechazo hiperagudo y mantiene la coagulación en rangos normales en el babuino. El rechazo vascular agudo provoca el cese en la producción de bilis y un patrón mixto de citólisis y colostasis. Los valores de expresión de HLA clase I en los linfocitos podrían ser útiles para diagnosticarlo (AU)

Production and characterization of a pig line transgenic for human membrane cofactor protein.

A pig line transgenic for human membrane cofactor protein (hMCP) has been established. Offspring from the founder were produced by crossing the founder with pigs heterozygous for the human decay accelerating factor (hDAF) transgene. As a result, pigs transgenic for both hMCP and hDAF have been produced. Ribonuclease protection assay (RPA) indicated that hMCP was expressed in all the tissues analysed. In addition, immunohistochemical results indicated a high level of expression of hMCP on neural tissues and islets where hDAF was absent or weakly expressed. C3 fragment deposition and cytotoxicity assays indicated that hMCP expression alone on pig endothelial cells and peripheral blood lymphocytes (PBLs) provided protection against human complement mediated damage. However, we did not find that porcine endothelial cells expressing both hDAF and hMCP were better protected than those expressing hDAF alone. The expression of hMCP on tissues where hDAF is not expressed could provide these tissues with protection against human complement mediated lysis.

Incidence of hyperacute rejection in pig-to-primate transplantation using organs from hDAF-transgenic donors.

BACKGROUND: Cynomolgus monkeys or baboons received under immunosuppression kidney or heart grafts from pigs transgenic for human decay-accelerating factor (hDAF) or from control pigs. Hyperacute rejection (HAR) is often difficult to differentiate from nonimmunological causes of organ or recipient dysfunction (NIC), and therefore, a thorough pathology review of all cases with 0-4 days survival (inclusive) was conducted. METHODS: Pathology slides were blinded and together with limited clinical data reviewed by two pathologists. After unblinding, data were compared with the original diagnosis made during the course of the program, and a final diagnosis was reached considering the complete clinical dataset. RESULTS: Life-supporting kidney transplantation was performed in 245 cynomolgus monkeys (234 hDAF, 11 controls), of which 102 cases had 0-4 day survival. None of the hDAF cases showed HAR, whereas this occurred in 27% of controls (P<10-6). Heterotopic heart transplantation was performed in 65 monkeys (57 hDAF, 8 controls), of which 41 cases had 0-4 day survival. HAR was observed in 7% of hDAF cases and in 57% of controls (P=0.002). Heterotopic heart transplantation in baboons was performed in 33 animals (28 hDAF, 5 controls), of which 15 cases had 0-4 day survival. HAR was observed in 11% of hDAF cases and in 20% of controls. Sixteen baboons were subjected to orthotopic heart transplantation, all from hDAF donors, out of which eight survived 0-4 days. The incidence of HAR was 6%. CONCLUSIONS: In the largest series of pig-to-primate solid organ transplants performed thus far, the presence of the hDAF transgene fully prevents HAR of cynomolgus monkey kidney transplants and partially inhibits HAR of heart grafts in cynomolgus monkeys or baboons. The incidence of HAR in control grafts is significantly higher.

Histopathology of cardiac xenograft rejection in the pig-to-baboon model.

BACKGROUND: The use of pig organs transgenic for human decay accelerating factor (hDAF) has largely overcome the problems of hyperacute rejection. With improved immunosuppressive protocols, life supporting grafts are showing greater survival times bringing the possibility of clinical xenotransplantation closer. Examination of the histopathology of the rejection process provides insight into the underlying mechanism and may suggest ways in which new immunosuppressive strategies should be directed. METHODS: 44 baboons (Papio anubis) underwent heart transplants of which 39 were from transgenic donors. The transplanted organs were examined histologically and stained for evidence of immunoglobulin and complement deposition as well as cellular infiltrates. RESULTS: In the transgenic animals survival times were 2 to 99 days (mean 23.5) and the heterotopic group and 1 to 39 days (mean 11.7) in the orthotopic group. There were 3 cases of hyperacute rejection between the 2 groups. Rejected organs showed areas of old and recent myocardial infarction associated with vascular thrombosis. There was widespread deposition within vessels of immunoglobulins IgM and IgG together with complement fractions C3 and C5b to 9 in those organs that were rejected. The amount of complement positive in the longer surviving organs was less than those rejecting early. Cellular infiltate was predominantly macrophage with some later appearing T or natural killer cells. CONCLUSIONS: The histopathological changes support the importance of immunoglobulin and complement in delayed xenograft or acute vascular rejection. With time there is an increase in cellular infiltrate predominantly macrophages and these findings suggest an increasingly important role for the cells and the rejection process. The presence of areas of infarction and underlying vascular thrombosis is in keeping with endothelial activation and the establishment of procoagulant phenotype which may be due to immunoglobulin, complement, secreted cytokines and direct cellular effects.

Serum anti-pig antibodies as potential indicators of acute humoral xenograft rejection in pig-to-cynomolgus monkey kidney transplantation.

BACKGROUND: Hyperacute rejection of solid organ pig xenografts in nonhuman primates has been overcome by using donors transgenic for human complement regulatory proteins, but grafts are still susceptible to humoral (antibody-mediated) rejection. We investigated whether circulating xenoreactive antibodies are a useful indicator of this xenograft rejection. METHODS: Five assays were employed in a retrospective analysis on 20 selected cynomolgus monkey recipients of renal xenografts transgenic for human decay-accelerating factor, with survival between 4 and 60 days. The assays included hemolytic and hemagglutination assays and the measurement of immunoglobulin (Ig)G and IgM binding to porcine endothelial cells and leukocytes, and to the Gal alpha 1-3Gal trisaccharide (Gal) antigen. To assess non-Gal-directed antibodies, sera were absorbed with a Gal-coated resin. A predictive value was defined as an increase in antibody levels before a decline in graft failure (>20% increase in creatinine levels) and humoral rejection in graft pathology. RESULTS: Data on hemolytic anti-pig antibody correlated with those on IgM antibody to endothelial cells, leukocytes, and Gal. In absorbed sera IgM and IgG antibody to endothelial cells and leukocytes correlated with each other, indicative for an elicited antibody response to non-Gal antigens. Sixteen animals showed humoral rejection, and in all but two animals one or more assays was considered of predictive value. On the other hand, increased antibody levels were noted in two animals without signs of rejection in graft pathology and in two cases with cellular xenograft rejection. CONCLUSIONS: It is recommended to use multiple assays (preferably hemolytic, anti-Gal, and anti-endothelial cell) to be able to fully monitor the peripheral antibody responses in pig-to-primate xenograft recipients.