Development of a multivariable gene-expression signature targeting T-cell-mediated rejection in peripheral blood of kidney transplant recipients validated in cross-sectional and longitudinal samples.

BACKGROUND: Acute T-cell mediated rejection (TCMR) is usually indicated by alteration in serum-creatinine measurements when considerable transplant damage has already occurred. There is, therefore, a need for non-invasive early detection of immune signals that would precede the onset of rejection, prior to transplant damage. METHODS: We examined the RT-qPCR expression of 22 literature-based genes in peripheral blood samples from 248 patients in the Kidney Allograft Immune Biomarkers of Rejection Episodes (KALIBRE) study. To account for post-transplantation changes unrelated to rejection, we generated time-adjusted gene-expression residuals from linear mixed-effects models in stable patients. To select genes, we used penalised logistic regression based on 27 stable patients and 27 rejectors with biopsy-proven T-cell-mediated rejection, fulfilling strict inclusion/exclusion criteria. We validated this signature in i) an independent group of stable patients and patients with concomitant T-cell and antibody-mediated-rejection, ii) patients from an independent study, iii) cross-sectional pre-biopsy samples from non-rejectors and iv) longitudinal follow-up samples covering the first post-transplant year from rejectors, non-rejectors and stable patients. FINDINGS: A parsimonious TCMR-signature (IFNG, IP-10, ITGA4, MARCH8, RORc, SEMA7A, WDR40A) showed cross-validated area-under-ROC curve 0.84 (0.77-0.88) (median, 2.5th-97.5th centile of fifty cross-validation cycles), sensitivity 0.67 (0.59-0.74) and specificity 0.85 (0.75-0.89). The estimated probability of TCMR increased seven weeks prior to the diagnostic biopsy and decreased after treatment. Gene expression in all patients showed pronounced variability, with up to 24% of the longitudinal samples in stable patients being TCMR-signature positive. In patients with borderline changes, up to 40% of pre-biopsy samples were TCMR-signature positive. INTERPRETATION: Molecular marker alterations in blood emerge well ahead of the time of clinically overt TCMR. Monitoring a TCMR-signature in peripheral blood could unravel T-cell-related pro-inflammatory activity and hidden immunological processes. This additional information could support clinical management decisions in cases of patients with stable but poor kidney function or with inconclusive biopsy results.

Chronic histological changes in deceased donor kidneys at implantation do not predict graft survival: a single-centre retrospective analysis.

The use of preimplantation kidney biopsies (PIKBs) to aid deceased donor kidney utilization decisions is controversial. Outcomes of transplants that had been biopsied after the decision had been made to implant were analysed, in order to determine the association between chronic histological changes at implantation and graft outcomes. A retrospective analysis of transplants between the year range 2006-2015 was performed. Karpinski scores on biopsies were collected, and graft outcomes were analysed using univariate and multivariable techniques. Also, Karpinski scores from single and dual kidney transplants from older donors were examined to determine if knowledge of the score preoperatively would have altered utilization. Four hundred and eight single kidneys were transplanted. Although kidneys with scores >4 had lower 1- and 3-year median (IQR) estimated glomerular filtration rates (eGFRs) than those scoring 0-4 (51 (37-66) vs. 35 (26-52) ml/min/1.73 m2 , P < 0.001, and 52 (34-64) vs. 35 (24-52) ml/min/1.73 m2 , P < 0.001, respectively), there was no significant association between Karpinski score and death-censored graft survival on univariate or multivariable analyses. The utilization analysis (75 single and 25 dual kidney transplant recipients) suggested that systematic use of PIKBs would have resulted in 29% fewer patients being transplanted. This analysis does not support the systematic use of PIKBs to determine deceased donor kidney utilization.

Outcomes in Third and Fourth Kidney Transplants Based on the Type of Donor.

BACKGROUND: An increasing number of patients are requiring multiple retransplants. We assessed outcomes of third and fourth kidney transplants, to aid decision making on the most suitable donor type. METHODS: Data were collected retrospectively for 2561 transplants, including 69 third and 8 fourth, performed from 2000 to 2017. Demographics and outcomes for the combined third/fourth group were compared to first and second transplants. Within the third/fourth kidney transplant group, comparisons were made between deceased donors (n = 39), live donor HLA-compatible (n = 23) and -incompatible (n = 13) transplants, as well as between standard (n = 25) and extended-criteria (n = 14) deceased donor transplants. RESULTS: Patient survival did not differ significantly by transplant number (P = 0.532), whereas death-censored graft survival declined progressively, from 89% at 5 years in first, 85% in second and 74% in the third/fourth transplant group (P < 0.001). Within the combined third/fourth transplant subgroup, 5-year graft survival was found to be 100% in recipients of HLA-compatible live donors, compared to 75% in deceased donors and 53% in HLA-incompatible live donors, although this difference did not reach statistical significance (P = 0.083). No significant difference in patient survival (P = 0.356) or complication rates (P = 0.757) were detected between these groups. For recipients of deceased donors in the third/fourth transplant group, there were no significant differences between standard versus extended-criteria donors for any of the outcomes considered. CONCLUSIONS: Despite variable functional outcomes, third and fourth kidney transplant recipients experience comparable patient survival rates to first and second transplants, regardless of the donor type. In selected patients, HLA-incompatible live donors and extended-criteria deceased donors should be considered.

Perioperative administration of high-dose recombinant human erythropoietin for delayed graft function prevention in kidney transplantation: a meta-analysis.

Delayed graft function (DGF) due to ischemia-reperfusion injury is a major early complication of kidney transplantation (KT). Recombinant human erythropoietin (rHuEPO) has been shown to exert nephroprotective action in animal models. We conducted a meta-analysis to explore the impact of rHuEPO on DGF in KT. Eligible studies comparing perioperative high-dose rHuEPO with placebo or no therapy for prevention of DGF were identified through MEDLINE, CENTRAL, and Transplant Library. Their design and data were assessed by two independent reviewers. Among 737 examined studies, four randomized controlled trials, involving 356 recipients of kidney allografts from deceased donors, fulfilled inclusion criteria. Statistical heterogeneity across studies was not significant (P = 0.98, I(2) = 0%). In a random effects model, no significant difference was found in the occurrence of DGF (odds ratio: 0,74, 95% CI: 0.47-1.18, P = 0.21). At 4 weeks after KT, the rHuEPO group exhibited higher systolic blood pressure (mean difference: 6.47 mmHg, 95% CI: 1.25-11.68, P = 0.02). Perioperative, high-dose rHuEPO administration does not prevent DGF in deceased donor KT. Furthermore, it is associated with higher systolic blood pressure leading to safety concerns. Nonerythropoietic rHuEPO derivatives, designed for nephroprotective action without increasing cardiovascular risk, might prove an alternative but still are at early stages of development.

Effect of Smad pathway activation on podocyte cell cycle regulation: an immunohistochemical evaluation.

BACKGROUND: Mature podocytes are in cell cycle arrest and their inability to proliferate successfully is a consequence of negative cell-cycle regulators' expression, such as p57. Phosphorylated smad2/smad3 (pSmad2/3) is an intracellular heteromeric mediator of transforming growth factor beta (TGF-ß) signals and, together with co-activators such as P300, regulates gene transcription, including cell cycle regulator proteins. METHODS: In order to investigate Smad pathway activation and podocyte cell cycle regulation in glomerular injury, we studied the glomerular immunohistochemical expression of p57, pSmad2/3 and P300 in samples from 67 patients with various types of glomerulonephritis (GN) and 10 normal kidney tissue specimens. RESULTS: pSmad2/3 and p300 expression were found significantly increased in all glomerular cell types in both proliferative and nonproliferative GN, while a significant reduction in p57-positive podocytes was observed when compared to controls. Staining for p57 was found to inversely correlate to pSmad2/3 suggesting that glomerular Smad pathway activation is related to down-regulation of p57 expression in proliferative glomerulonephritis. To our knowledge, this is the first study that indicates a relation between the TGF-beta/Smad signalling pathway and the cell cycle regulatory protein p57 in human GN. CONCLUSION: The increased pSmad2/3 staining together with the reduced p57 expression found in biopsy specimens with intense interstitial inflammation, indicate a possible relation between interstitial inflammation, glomerular Smad pathway activation and podocyte cell-cycle deregulation.

Statins and prostate cancer: molecular and clinical aspects.

The field of the potential applications of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) beyond their unambiguous cardiovascular beneficial effects is steadily increasing. In this regard, statins have also been shown to possess anti-inflammatory, immunomodulatory, antioxidant and growth inhibitory properties. Regarding their role in carcinogenesis, both preclinical and clinical studies report conflicting results. Intriguingly, accumulating evidence suggests that statins may relate to decreased prostate cancer incidence and recurrence risk. However, data from clinical studies seem to be still weak and are confounded by several factors. Nonetheless, preclinical data suggest that statins might exert a chemopreventive role against prostate cancer by inhibiting the proliferation and inducing apoptosis of prostate cancer cells and also inhibiting angiogenesis, inflammation and metastasis. Cholesterol lowering as well as statin pleiotropy through inhibition of the synthesis of isoprenoids have both been implicated in their anticancer properties. In this review, we discuss the preclinical and clinical evidence supporting the preventive or potentially harmful effects of statins on prostate tumourigenesis and conclude that statins should not be recommended for the prevention of prostate cancer development or progression based on the current data.

Transcription factor Sp1 expression is upregulated in human glomerulonephritis: correlation with pSmad2/3 and p300 expression and renal injury.

BACKGROUND: Sp1 is a ubiquitous transcription factor that mediates the fibrogenic factor transforming growth factor beta (TGF-beta) signals through cooperation with Smad proteins. The transcriptional coactivator p300 is also suggested to play a role in Smad signal transduction. METHODS: We investigated the immunohistochemical expression of Sp1 as well as the expression of pSmad2/3 and the coactivator p300 in 157 renal biopsy specimens from patients with various types of glomerulonephritis (GN). Correlations between immunohistochemical, clinical, and histologic parameters were performed. RESULTS: Sp1 exhibited an increased glomerular and proximal tubular expression in all forms of GN compared to controls. The proximal tubular expression of Sp1 was significantly increased in proliferative GNs (p = 0.025), whereas in secondary GNs, there was a significant increase in the molecule's glomerular expression (p = 0.008). Sp1 correlated positively with pSmad2/3 and p300 expression in proximal tubules (r = 0.241, p = 0.018 and r = 0.244, p = 0.014, respectively), while in proliferative GNs, its expression correlated positively with pSmad2/3 expression in glomeruli (r = 0.32, p = 0.028). Sp1 glomerular and proximal tubular immunostaining correlated positively with serum creatinine levels (r = 0.265, p = 0.02 and r = 0.306, p = 0.006, respectively), while its proximal tubular expression showed a similar correlation with interstitial fibrosis (r = 0.213, p = 0.025). Sp1 was constantly detected in hyperplastic lesions and cellular crescents (each 100%), and very often in micro adhesions (94%) and segmentally or globally sclerotic areas (each 83%). CONCLUSIONS: This study documents the upregulation of Sp1 expression in glomeruli and proximal tubules of GN specimens. Our findings suggest a possible cooperation of Sp1 with pSmad2/3 and p300 in mediating renal injury as well as a possible role for this molecule in the pathogenesis and the progression of human GN.

Anaemia, microcytosis and sirolimus--is iron the missing link?

BACKGROUND: Sirolimus (SRL) has been implicated in the causation of post-transplantation anaemia (PTA). It also induces profound red blood cell (RBC) microcytosis, which is poorly understood. METHODS: We conducted a retrospective study of SRL-induced anaemia and microcytosis [mean corpuscular volume (MCV) <80 fl] with specific reference to iron homeostasis in 93 renal transplant patients treated with SRL for at least 3 months. RESULTS: While mean haemoglobin (Hb) and use of erythropoiesis-stimulating agents increased on SRL, RBC MCV underwent a significant decline throughout the whole study period of 24 months (P < 0.001) with the percentage of microcytosis rising from 2.2% at the start of SRL therapy to 40.7% after 24 months of therapy. An association between dMCV (MCV change on SRL) and SRL levels was shown at 3, 6, 12 and 24 months post-SRL (P = 0.015, P = 0.037, P = 0.002 and P = 0.001, respectively). Intravenous (IV) iron administration was an independent predictor of dMCV at 12 and 24 months on SRL (P = 0.031 and P = 0.048, respectively). All patients who, after starting SRL and seeing a fall in MCV, then went on to receive IV iron therapy, showed a marked increase in MCV; this did not happen to patients given oral iron therapy. CONCLUSIONS: SRL is associated with mild anaemia, but marked RBC microcytosis-these phenomena are correlated with SRL levels and the use of IV iron. Functional iron deficiency and impaired gastrointestinal absorption of iron seem likely explanations.

Prognostic significance of TGFbeta-1 and pSmad2/3 in breast cancer patients with T1-2,N0 tumours.

BACKGROUND: The transforming growth factor beta (TGFbeta) signaling pathway has been shown to exert divergent effects and to cross-talk with estrogen pathways in mammary gland tumorigenesis. TGF signaling in early stage breast cancer was investigated by examining the expression of TGFbeta-1 and the signaling mediators pSmad2/3 and Smad4. Their association with oestrogen and progesterone receptors, as well as with clinical and pathological features was also analyzed. PATIENTS AND METHODS: Sixty-one tumor specimens from surgically treated patients with primary T12,N0 breast cancer were examined. The expression of TGFbeta-1, pSmad2 and Smad4 was assessed implementing immunohistochemical assays. RESULTS: TGFbeta-I, pSmad2/3 and Smad4 were expressed in 50.9%, 74.0% and 61.0% of specimens, respectively. The degree of expression of the three molecules was significantly associated with each other. Loss of pSmad2/3 expression indicated a shorter disease-free survival in all patients, including those with oestrogen receptor-positive tumors. Patients not expressing TGFbeta-1 were 4.6 times more likely to experience distant recurrence. CONCLUSION: Our results demonstrate that pSmad2/3 and TGFP-1 may be promising novel prognostic markers for T1.2,N0 breast carcinomas.

Immunohistochemical evaluation of phosphorylated SMAD2/SMAD3 and the co-activator P300 in human glomerulonephritis: correlation with renal injury.

BACKGROUND: Smad2 and Smad3 are transcription factors that mediate transforming growth factor beta (TGF-beta) signals. Upon their activation, phosphorylated Smad2/Smad3 (pSmad2/Smad3), translocate to the nucleus and associate with co-activators such as p300, regulating the transcription of genes that contribute to the fibrotic processes. METHODS: We investigated the immunohistochemical expression of pSmad2/Smad3 and the co-activator p300 in 152 renal biopsy specimens from patients with various types of glomerulonephritides (GNs) and in 15 normal kidney specimens. Patients' clinical data (serum creatinine levels and proteinuria) had been collected. RESULTS: There was a dramatic increase in the expression of pSmad2/3 and p300 in all glomerular cell types in all GNs. pSmad2/3 expression was increased in all tubular segments (except for the proximal tubules in nonproliferative GNs), while p300 expression was significantly increased only in the proximal tubular cells in all GNs. Glomerular and tubular pSmad2/Smad3 and p300 were significantly increased in proliferative GNs (compared to the nonproliferative), particularly in the secondary group. The expression profile of p300 correlated positively with the expression of pSmad2/Smad3 in the diseased glomeruli and proximal tubules. pSmad2/3 and p300 were very often detected in segmental hyperplastic lesions, cellular crescents, microadhesions and segmental or global sclerotic areas. Glomerular and proximal tubular pSmad2/Smad3 was positively correlated with serum creatinine levels, while distal and collecting tubular pSmad2/3 and p300 correlated positively with tubular atrophy. Glomerular and proximal tubular pSmad2/3 expression and glomerular p300 expression correlated positively with lupus nephritis activity. CONCLUSION: Our results suggest that pSmad2/3-p300 pathway may play a pivotal role in the pathogenesis and progression of human glomerulonephritis.