Molecular Patterns of Subclinical and Clinical Rejection of Kidney Allograft: Quantity Matters

Background/Aims: Subclinical rejection diagnosed from protocol biopsies is thought to be a risk factor of long- term allograft dysfunction. The reason why in some patients subclinical rejection does not represent risk for progression is not fully understood. Methods: The intragraft expression of 376 target genes involved in chemokine defense, apoptosis, inflammation, tolerance and TGF-β signalling pathways was measured using quantitative real-time RT-PCR (2-∆∆Ct) method in subclinical inflammation (SCI, n=10), clinical inflammation in acute T-cell mediated rejection (CI, n=10) and no rejection samples (n=9). Results: Clinical inflammation group showed a increased expression of genes for chemotaxis mediating cytokines (CCL1, CCL17, CCL24, CCL25, CCL26), cytokine receptors (CCR1, CCRL2, IL1RAPL2, CXCR5), proinflammatory cytokines (IL12A, LTA), inflammatory mediator (PTAFR), complement protein C3, executioner protein of apoptosis (CASP7), growth factor (TGFA), colony stimulating factor (CSF-2), proteins involved in dendritic cells differentiation and interaction (CD209, LAMP3), regulation of immune response (LILRB2, LILBRB4). The quantitative difference in transcripts signature between SCI and CI is consistent with stronger proinflammatory setting of CI. Prostaglandin E2 receptor gene expression was independently associated with lower risk of further graft function deterioration (OR 0.11, CI 0.01-0.78, pConclusion: Subclinical acute kidney inflammation has transcriptional profile of immune injury of lower extend compared to clinical acute inflammation

Intrarenal Complement System Transcripts in Chronic Antibody-Mediated Rejection and Recurrent IgA Nephropathy in Kidney Transplantation

Background: The complement system activation and regulation have been linked to post-transplant pathologies including chronic antibody mediated rejection (cAMR) and the recurrence of IgA nephropathy (ReIgAN) but distinct mechanisms remain to be elucidated.Methods: In this retrospective single center study, the outcome of kidney transplantation was studied in 150 patients with late histological diagnosis to be either cAMR or ReIgAN, 14 stable kidney grafts at 3 months and finally 11 patients with native kidney IgAN nephropathy. To study a role of complement cascade and regulation in cAMR and ReIgAN, the RNA was extracted from available frozen kidney biopsy samples and using RT-qPCR transcripts of 11 target genes along with clinical data were determined and compared with stable grafts at 3 months protocol biopsies or IgAN native kidney nephropathy. Immunohistologically, CD46 (MCP), and C5 proteins were stained in biopsies.Results: Interestingly, there were no differences in kidney graft survival between cAMR and ReIgAN since transplantation. cAMR was associated with significantly higher intragraft transcripts of C3, CD59, and C1-INH as compared to ReIgAN (p < 0.05). When compared to normal stable grafts, cAMR grafts exhibited higher C3, CD55, CD59, CFH, CFI, and C1-INH (p < 0.01). Moreover, ReIgAN was associated with the increase of CD46, CD55, CD59 (p < 0.01), and CFI (p < 0.05) transcripts compared with native kidney IgAN. Rapid progression of cAMR (failure at 2 years after biopsy) was observed in patients with lower intrarenal CD55 expression (AUC 0.77, 78.6% sensitivity, and 72.7 specificity). There was highly significant association of several complement intrarenal transcripts and the degree of CKD regardless the diagnosis; C3, CD55, CFH, CFI, and C1-INH expressions positively correlated with eGFR (for all p < 0.001). Neither the low mRNA transcripts nor the high mRNA transcripts biopsies were associated with distinct trend in MCP or C5 proteins staining.Conclusions: The intrarenal complement system transcripts are upregulated in progressively deteriorated kidney allografts

The Effect of Butyrate-Supplemented Parenteral Nutrition on Intestinal Defence Mechanisms and the Parenteral Nutrition-Induced Shift in the Gut Microbiota in the Rat Model

Butyrate produced by the intestinal microbiota is essential for proper functioning of the intestinal immune system. Total dependence on parenteral nutrition (PN) is associated with numerous adverse effects, including severe microbial dysbiosis and loss of important butyrate producers. We hypothesised that a lack of butyrate produced by the gut microbiota may be compensated by its supplementation in PN mixtures. We tested whether i.v. butyrate administration would (a) positively modulate intestinal defence mechanisms and (b) counteract PN-induced dysbiosis. Male Wistar rats were randomised to chow, PN, and PN supplemented with 9 mM butyrate (PN+But) for 12 days. Antimicrobial peptides, mucins, tight junction proteins, and cytokine expression were assessed by RT-qPCR. T-cell subpopulations in mesenteric lymph nodes (MLN) were analysed by flow cytometry. Microbiota composition was assessed in caecum content. Butyrate supplementation resulted in increased expression of tight junction proteins (ZO-1, claudin-7, E-cadherin), antimicrobial peptides (Defa 8, Rd5, RegIIIγ), and lysozyme in the ileal mucosa. Butyrate partially alleviated PN-induced intestinal barrier impairment and normalised IL-4, IL-10, and IgA mRNA expression. PN administration was associated with an increase in Tregs in MLN, which was normalised by butyrate. Butyrate increased the total number of CD4+ and decreased a relative amount of CD8+ memory T cells in MLN. Lack of enteral nutrition and PN administration led to a shift in caecal microbiota composition. Butyrate did not reverse the altered expression of most taxa but did influence the abundance of some potentially beneficial/pathogenic genera, which might contribute to its overall beneficial effect

Effect of APAP and SLM on lipid peroxidation in the liver.

<p>Data are given as mean ± SEM, n = 7. *p < 0.05 APAP vs control; ^#p < 0.05, ^##p < 0.01 APAP + SLM vs APAP.</p

Effect of APAP and SLM on reactive oxygen species production from submitochondrial particles in vitro.

<p>(A) ROS production from NADH -/+ AA; (B) ROS production from succinate -/+ AA. Results are expressed as a percent of basal levels (without substrate). Data are presented as a mean ± SEM, n = 7. *p < 0.05, ***p < 0.001 APAP vs control (succinate + AA); ^† p < 0.05 APAP + SLM vs APAP (only succinate); ^#p < 0.05, ^## p < 0.01 APAP + SLM vs APAP (succinate + AA).</p

Silymarin prevents acetaminophen-induced hepatotoxicity in mice - Fig 3

<p><b>Effect of APAP and SLM on CYP2E1 activity in vivo (A), mRNA expression (B), protein content (C) and CYPE1 activity in vitro (D, E).</b> Results represent mean ± SEM, n = 7. *p < 0.05, **p < 0.01, ***p < 0.001 APAP vs control. ^#p < 0.05, ^##p < 0.01 APAP + SLM vs APAP.</p

Effect of APAP and SLM on neutrophil infiltration.

<p>Tissue sections were stained with hematoxylin and eosin, black arrows indicate infiltrating neutrophiles. Original magnification x 600.</p

Effect of APAP and SLM on RIP-3 expression.

<p>The liver homogenate was separated by SDS-ELFO, immunoblotted and the proteins of interest were detected using specific antibody.</p