Intragraft transcriptional profiling of renal transplant patients with tubular dysfunction reveals mechanisms underlying graft injury and recovery.

BACKGROUND: Proximal tubular dysfunction (PTD) is associated with a decreased long-term graft survival in renal transplant patients and can be detected by the elevation of urinary tubular proteins. This study investigated transcriptional changes in biopsies from renal transplant patients with PTD to disclose molecular mechanisms underlying graft injury and functional recovery. METHODS: Thirty-three renal transplant patients with high urinary levels of retinol-binding protein, a biomarker of PTD, were enrolled in the study. The initial immunosuppressive scheme included azathioprine, cyclosporine, and steroids. After randomization, 18 patients (group 2) had their treatment modified by reducing cyclosporine dosage and substituting azathioprine for mycophenolate mofetil, while the other 15 patients (group 1) remained under the initial scheme. Patients were biopsied at enrollment and after 12 months of follow-up, and paired comparisons were performed between their intragraft gene expression profiles. The differential transcriptome profiles were analyzed by constructing gene co-expression networks and identifying enriched functions and central nodes in each network. RESULTS: Only the alternative immunosuppressive scheme used in group 2 ameliorated renal function and tubular proteinuria after 12 months of follow-up. Intragraft molecular changes observed in group 2 were linked to autophagy, extracellular matrix, and adaptive immunity. Conversely, gene expression changes in group 1 were related to fibrosis, endocytosis, ubiquitination, and endoplasmic reticulum stress. CONCLUSION: These results suggest that molecular networks associated with the control of endocytosis, autophagy, protein overload, fibrosis, and adaptive immunity may be involved in improvement of graft function.

Transcriptome analysis of renal ischemia/reperfusion injury and its modulation by ischemic pre-conditioning or hemin treatment.

Ischemia/reperfusion injury (IRI) is a leading cause of acute renal failure. The definition of the molecular mechanisms involved in renal IRI and counter protection promoted by ischemic pre-conditioning (IPC) or Hemin treatment is an important milestone that needs to be accomplished in this research area. We examined, through an oligonucleotide microarray protocol, the renal differential transcriptome profiles of mice submitted to IRI, IPC and Hemin treatment. After identifying the profiles of differentially expressed genes observed for each comparison, we carried out functional enrichment analysis to reveal transcripts putatively involved in potential relevant biological processes and signaling pathways. The most relevant processes found in these comparisons were stress, apoptosis, cell differentiation, angiogenesis, focal adhesion, ECM-receptor interaction, ion transport, angiogenesis, mitosis and cell cycle, inflammatory response, olfactory transduction and regulation of actin cytoskeleton. In addition, the most important overrepresented pathways were MAPK, ErbB, JAK/STAT, Toll and Nod like receptors, Angiotensin II, Arachidonic acid metabolism, Wnt and coagulation cascade. Also, new insights were gained about the underlying protection mechanisms against renal IRI promoted by IPC and Hemin treatment. Venn diagram analysis allowed us to uncover common and exclusively differentially expressed genes between these two protective maneuvers, underscoring potential common and exclusive biological functions regulated in each case. In summary, IPC exclusively regulated the expression of genes belonging to stress, protein modification and apoptosis, highlighting the role of IPC in controlling exacerbated stress response. Treatment with the Hmox1 inducer Hemin, in turn, exclusively regulated the expression of genes associated with cell differentiation, metabolic pathways, cell cycle, mitosis, development, regulation of actin cytoskeleton and arachidonic acid metabolism, suggesting a pleiotropic effect for Hemin. These findings improve the biological understanding of how the kidney behaves after IRI. They also illustrate some possible underlying molecular mechanisms involved in kidney protection observed with IPC or Hemin treatment maneuvers.

Identificação de mpléculas de citotoxicidade e de padrão de resposta celular TH1, em células de sedimento urináro de pacientes transplantados renais, com possíveis biomarcadores de rejeição aguda / Expression of perforin and granzyme-B genes in peripheral blood lynphocytes of renal allograft recipients

0 desenvolvimento de um teste preciso e não invasivo para rejeição aguda que forneça informações sobre os mecanismos poderá melhorar a evolução do transplante renal. Rejeição aguda é um evento Th1 relacionado, onde fatores celulares e humorais estão presentes. Nós exploramos a utilidade clínica do perfil de mRNA de células urinárias no diagnóstico de rejeição aguda do aloenxerto renal. Sessenta amostras de urina de 60 pacientes foram analisadas para os níveis mRNA de granzima-B, perforina, Fas-L, γ-IFN e TIM-3. mRNA quantificado utilizando-se RT-PCR em tempo real Assays-by- design/MGB Primer específico e foram expressos em mRNA 2DDCT/GAPDH. Nossos dados demonstraram que os níveis de mRNAr de granzima-B, perforina, Fas-L, y-IFN e especialmente TIM-3, estão altamente expressos em células urinárias de pacientes com rejeição aguda (RA) comparado com sem rejeição aguda (SAR). TIM-3, um membro da superfamília das Imunoglobulinas preferencialmente expresso em células diferenciadas Th-1, apresentou a maior expressão nos pacientes com RA, sendo uma excelente molécula candidata a biomarcador de rejeição aguda. Nossos dados mostraram que o perfil de mRNa de células urinárias pode detectar imune-ativação nos aloenxertos e permitir a identificação de novos alvos para terapêuticas disponíveis.

Correlaçao dos resultados da pesaquisa de anticorpos anti-HIV pelos metodos de enzimaimunoensaio (ELISA) e Western Blot / Correlation of the results of antibodies to human immunodeficiency virus (HIV) detected by the enzyme-linked-immunosorbent assay and the Western Blot methods

Varios testes sorologicos tem sido utilizados para a detecçao de anticorpos anti-HIV (Virus da Imunodeficiencia Humana), sendo os metodos enzimaimunoensaio (ELISA) e Western Blot os mais sensiveis e especificos. Os autore, objetivando correlacionar os resultados por estes metodos, analisam 7.946 exames realizados no Setor de Imunologia Clinica do Hospital Universitario Regional Norte do Parana (HURNPR),no periodo de agosto de 1987 a agosto de 1990. Destes 134 apresentaram-se reagentes pelo metodo ELISA, segundo recomendaçoes da Organon Teknika e Abbott Laboratories e foram posteriormente correlacionados com metodos de Western Blot. Os resultados permitiram discordar da interpretaçao proposta pelo fabricante do kit de ELISA com relaçao relaçao aos limites estabelecidos para se considerar os resultados como reagentes e nao reagentes.