Characterization of a Trpc6 Transgenic Mouse Associated with Early Onset FSGS.

Rationale: Mutations in Transient Receptor Potential Channel 6 (TRPC6) gene are associated with autosomal dominant focal and segmental glomerulosclerosis (FSGS). The majority of the identified mutations affect the ion channel function. Since calcium channels are promising candidate drug targets, there is an an urgent need for a mouse model to assess new therapeutic drugs and to help delineate the pathogenic process leading to FSGS. We have previously reported the generation of three independent transgenic mouse lines carrying different Trpc6 mutations that display a glomerular disease comparable to the phenotype presented by individuals with FSGS. However, the utility of these models for drug testing is dampened by the late-onset of the presentation and the mild phenotypic manifestations. Methodology: In order to obtain a time-effective mouse model for Trpc6-associated FSGS we generated a new transgenic mutant Trpc6 mouse model emulating the amino acid change carried by the first pediatric patient of FSGS associated with a TRPC6 mutation: M132T. Results: Mice carrying the orthologous Trpc6 M131T transgene showed early onset proteinuria and early signs of FSGS. When exploring molecular consequences of the overexpression of this mutated form of Trpc6 in podocytes, differences in expression levels of Axin2 and β-catenin were found in glomeruli from transgenic Trpc6 M131T mice. These data supports the proposed molecular mechanisms related to the activation of calcineurin-NFAT/Wnt signaling, as outcome of the increased calcium influx caused by the mutated form of Trpc6. Conclusion: Given that the Trpc6 M131T mouse develops an early onset of FSGS-like phenotypes it represents a promising model for studying the pathogenesis of FSGS caused by TRPC6, facilitating the assessment of new drugs as treatments and allowing further studies to understand underlying molecular pathways involved in the development of the TRPC6 mediated disease.

Valor crítico da citrulina para as complicações do enxerto no transplante de intestino / Critical value of citrulline for complications of intestinal transplant graft

OBJETIVO: Analisar, numa ampla amostra, o valor crítico da citrulina que confirma a presença das principais complicações do enxerto: rejeição e infecção. MÉTODOS: Foram coletadas 2135 amostras de citrulina sérica, na forma de gota de sangue seca, de 57 doentes submetidos a transplante de intestino/multivisceral no Jackson Memorial Hospital na Universidade de Miami, de março de 2004 a abril de 2006. Todas as amostras são do pós-operatório três meses em diante, passada a conhecida curva de elevação da citrulina após a recuperação das lesões causadas pela isquemia e reperfusão do pós-transplante. RESULTADOS: Utilizando um valor limite menor que 13 µmoles/L, a sensibilidade da citrulina foi de 96,4 por cento para detectar rejeicão celular aguda (RCA) moderada ou grave. A especificidade para as complicações mais freqüentes, rejeição e infecção foi de 54 por cento-74 por cento nas crianças e 83 por cento-88 por cento nos adultos, e o valor preditivo negativo (VPN) foi > 99 por cento. CONCLUSÃO: A citrulina pode ser utilizada como método não-invasivo para avaliar a evolução do enxerto intestinal após três meses do TI. Os episódios de RCA moderado e grave podem ser afastados quando o valor da citrulina for maior que 13 µmoles/L devido ao alto valor preditivo negativo.

OBJECITIVE: A biochemical marker for detection of acute cellular rejection following small intestine transplantation has been sought. Citrulline, a non- protein amino acid synthesized mainly by functioning enterocytes, has been proposed. Trial sensitivity has been reportedly high but with low specificity. Thus, the goal was to determine, in a sufficiently large analysis, the significant value of citrulline level in the post-transplant setting, which would correlate with complications such as rejection and infection. METHODS: Since March, 2004 2,135 dried blood spot (DBS) citrulline samples were obtained from 57 small intestine transplant recipients three months or more after post-transplant, i.e., once the expected period of recovery in the citrulline levels had occurred. RESULTS: Using a <13 vs. > 13 µmoles/L cut off point, sensitivity of DBS citrulline for the detection of moderate or severe ACR was extremely high (96.4 percent). Furthermore, specificity estimates (given the absence of ACR and these particular infections), while controlling for time-to-DBS sample were reasonably high (54 percent-74 percent in children and 83 percent-88 percent in adults), and the negative predictive value (NPV) was >99 percent. CONCLUSION: Citrulline is a non-invasive marker to evaluate problems of the intestinal graft after three months post-transplant. Due to the high NPV, a moderate or severe ACR can be ruled out, based exclusively on knowledge of a high value for DBS citrulline.

Graft versus host disease in a rat small bowel transplant model after T-cell depleted donor specific bone marrow infusion

Low cytoreductive regimen of irradiation associated to unmodified bone marrow infusion (UBM) does not prevent the occurrence of graft versus host disease (GVHD) after transplant. PURPOSE: In this study we evaluated the potential advantages of a long-term immunossupression and T-cell depleted bone marrow infusion (TCDBMI) in preventing the occurrence of GVHD after small bowel transplantation (SBTx). METHODS: Heterotopic SBTX was performed with Lewis rats as recipients and DA as donors and distributed into 5 groups according to the irradiation, duration of immunossupression and the use of UBM or TCDBMI: G1 (n=6), without irradiation and G2 (n=9), G3 (n=4), G4 (n=5) and G5 (n=6) was given 250 rd of irradiation. Groups 1,2,4 and G3 and 5 were infused with 100 x 106 UBM and TCDBM respectively. Animals in G1, 2, 3 were immunossupressed with 1mg/ FK506/Kg/IM for 5 days and G4 and G5 for 15 days. Anti CD3 monoclonal antibodies and immunomagnetic beads were used for T-cell depletion.Animals were examined for rejection, GVHD, chimerism characterization and ileal and skin biopsies. RESULTS: Minimal to mild rejection was observed in all groups; however, GVHD were present only in irradiated groups. Long-term immunossupression changed the severity of GVHD in G4 and G5. Rejection was the cause of death in G1 while GVHD in G2, 3, 4 and 5, not avoided by the use of TCDBMI. Total chimerism and T-cell chimerism was statistically higher in irradiated groups when compared to G1. CONCLUSION: Extended immunossupression associated to low dose of irradiation decrease the severity of GVHD, not avoided by the use of TCDBMI.

Chimerism induction by nonmyeloablactive preconditioning and bone marrow infusion in rat small bowel transplantation

ABSTRACT: In our previous work we demonstrated that the use of donor specific bone marrow infusions ( DSBMI ) after small bowel transplantation did not improve the graft survival after a short course of immunossupression. PURPOSE: In the current study, we evaluated whether recipient preconditioning with different regimens of radiation combined with DSBMI may enhance small bowel allograft survival with minimum recipient morbidity. METHODS: Heterotopic small bowel transplantation (SBTx) was performed with Lewis rats as recipients and DA rats as donors, which were immunossupressed with a short course of tacrolimus (FK 506 ) at 1mg/Kg/day for 5 days and distributed in 4 groups: group 1 (n= 4) without both irradiation and DSBMI; Groups 2 (n= 6), 3 (n= 9) and 4 (n= 6) received 100 x 106 DSBM cells at the time of the transplant. Groups 3 and 4 were irradiated with 250 and 400 rd respectively. Animals were examined daily for clinical signs of rejection or GVHD. Blood samples were taken weekly for chimeric studies by FC and intestinal biopsies were performed every 2 weeks. RESULTS: Animals in G1 and G2 had minimal rejection at day 15 after SBTx while GVHD was clinically and histologically characterized in G 3 and G 4. Total chimerism and T-cell chimerism was higher in irradiated groups when compared to non-irradiated groups. With exception of G1 and 2 where rejection was the cause of death, all animals in G3 and 4 died of GVHD. CONCLUSION:We concluded that low cytoreductive of irradiation can successfully decrease the graft rejection but not prevent the occurrence of GVHD.