Introduction: extended-criteria donors (ECD) are known to have worse long term results than standard criteria donor (SCD) kidneys.The profile of cytokine expression in pretransplantation biopsies of ECD donor kidneys has yet to be elucidated. Objective: To characterize the profile of inflammatory and anti-inflammatory cytokines in protocol pre-implantation biopsies (BxT0) of ECD and SCD kidneys. Materials and Methods: Seventy three BxT0 (ECD/SCD n= 26/47) were analyzed to measure expression levels of Treg-related gene Forkhead Box P3 (FOXP3) and cytokines Interleukin-10 (IL-10), Transforming Growth Factor Beta (TGF-β), Regulated upon Activation Normal T-cell Expressed and Secreted (RANTES) and Monocyte Chemoattractant Protein-1 (MCP-1) in a blinded fashion by qRT-PCR. Results: demographic characteristics of donors did not differ between the ECD/SCD except by a higher age in the ECD group. Proinflammatory cytokines RANTES and MCP-1 and the anti-inflammatory IL-10 were highly expressed in kidneys ECD when compared with SCD kidneys (figure 1). Increased expression of RANTES, MCP-1 and IL-10 was found in 69% (18/26), 81% (21/26) and 92% (24/26) of ECD BxT0, respectively. Although FOXP3 expression was twofold higher in ECD group it did not reach statistical significance and TGF-β was similar in both groups (fig. 1). Molecular profile did not correlate with any histological finding (Banff 07). Conclusion: We found high expression levels of RANTES, MCP-1 and IL-10 in ECD kidneys. This cytokine profile may represent “inflammatory environment” of ECD kidneys with a compensatory anti-inflammatory response. Characterization of molecular phenotype of ECD kidneys could provide new insights to explain the worse long term outcomes of these kidneys.Figure: No Caption available.
Abstract Introduction. Non-melanoma skin cancer (NMSC) is the most common malignancy following renal transplantation and leads to significant years of potential life lost. Dermatoscopy use in the routine clinical setting has shown promise in increasing the diagnosis of NMSC. Objectives. 1) To assess the prevalence of NMSC in renal transplant recipients (RTx) using the dermatoscopy (DC); 2) to identify associated risk factors; 3) to compare the accuracy of dermatoscopy with histopathological results. Methods. A prospective study was conducted from January 2022 to January 2023 and 100 RTx from the outpatient clinic were randomly referred for dermatological examination and then divided into two groups: with NMSC (NMSC; n=39) and without NMSC (non-NMSC; n=61). RTx variables related to habits, tumor characteristics, and transplantation were analyzed. Results: Clinical dermatological evaluation identified 23 squamous cell carcinoma (SCC) (59%) and 16 basal cell carcinoma (BCC) (41%). Factors associated with NMSC included: male gender (OR=2); age over 50 years (OR=5.4); occupational sun exposure (OR=2.5); recreational sun exposure (OR=4.9); actinic keratosis (OR=6.5); family history of NMSC (OR=7.6); and induction with thymoglobulin (OR=2.4). The accuracy rate between clinical diagnosis and dermatoscopy compared to the final histological result after biopsy was 81.25% for suspected NMSC cases. Conclusions: NMSC occurred in 39% of the evaluated RTx cases, with a family history and actinic keratosis being the main risk factors. Dermatoscopy diagnosed NMSC in 81%, and therefore, we suggest that this examination should be incorporated into routine evaluation.
The therapeutic effect of induced pluripotent stem cells (iPSs) on the progression of chronic kidney disease (CKD) has not yet been demonstrated. In this study, we sought to assess whether treatment with iPSs retards progression of CKD when compared with bone marrow mesenchymal stem cells (BMSCs). Untreated 5/6 nephrectomized rats were compared with CKD animals receiving BMSCs or iPSs. Renal function, histology, immunohistochemistry, and gene expression were studied. Implanted iPSs were tracked by the SRY gene expression analysis. Both treatments minimized elevation in serum creatinine, significantly improved clearance, and slowed down progression of disease. The proteinuria was reduced only in the iPS group. Both treatments reduced glomerulosclerosis, iPSs decreased macrophage infiltration, and TGF- β was reduced in kidneys from the BMSC group. Both types of treatments increased VEGF gene expression, TGF- β was upregulated only in the iPS group, and IL-10 had low expression in both groups. The SRY gene was found in 5/8 rats treated with iPSs. These 5 animals presented tumors with histology and cells highly staining positive for PCNA and Wilms’ tumor protein antibody characteristics of Wilms’ tumor. These results suggest that iPSs may be efficient to retard progression of CKD but carry the risk of Wilms’ tumor development.
Breast cancer (BC) has a high mortality rate, which is attributed to the absence of effective treatment markers. Doxorubicin (DOX) was evaluated by molecular docking in vitro in cultured BC spheroids and its association with genes involved in the PI3K/AKT/PTEN signaling pathway. Spheroids were obtained from a primary BC. The selected compound was used for molecular docking experiments. Spheroids were treated with DOX for 1 (D1) and 9 (D9) days. qPCR was used to evaluate PIK3CA, HIF-1α, VEGF-A, PTEN expression. Treatment with DOX (1 µM) significantly increased the number of spheroids (D1), whereas exposure to chemotherapy at 2 µM on D9 was more effective. DOX treatment resulted in significantly higher expression of VEGF-A, HIF-1α and PIK3CA by D1 and HIF-1α and PTEN were upregulated by D9. Compared to treatment on D1 with D9 (1 μM) had significantly higher PTEN and lower PIK3CA gene expression. The genes HIF-1α and PTEN were more expressed with 2 μM of DOX while VEGF-A was downregulated. D1 vs. D9 exhibited reduced VEGF-A, HIF-1α, and PIK3CA expression and upregulation of PTEN expression. DOX effects at the molecular mechanisms can be involved the modulation of genes related to angiogenesis cell proliferation and tumor growth in BC tissue spheroids.
Background: The significance of the factor forkhead box (FOXP3) expression in the peripheral blood of renal transplant recipients (RTx) of extended (ECD) or standard (SCD) criteria donor kidneys is yet to be determined. Aim: To investigate the clinical significance of FOXP3 level in relation to the type of kidney donor, immunosuppressive protocol (ISS) and graft function in stable ECD RTx. Methods: Prospective, open label study with 36 stable RTx randomized according to the type of donor to receive tacrolimus (TAC; n= 19) or everolimus (EVL; n=17) based protocols combined to mycophenolate sodium, prednisone and induction therapy with basiliximab. Using qRT-PCR we measured FOXP3 expression in the peripheral blood of patients (pts) at the 3rd month after Tx. Results: Greater expression of FOXP3 was observed in recipients of ECD when compared to SCD (ECD=1.98 ± 2.8 vs SCD= 0.63 ± 0.71; p=0.02). EVL based immunosuppression (EVL= 1.49 ± 2.1 vs TAC= 0.51 ± 0.57; p=0.03) and absence of DGF post-Tx (DGF- = 1.94 ± 2.5 vs DGF+ = 0.54 ± 0.6; p=0.014) were also associated with high levels of FOXP3 expression. Cold ischemia time, acute rejection episodes and serum creatinine (sCr) at 3rd month were not associated with changes in FOXP3 expression. Multivariate logistic regression analysis showed that ECD (p= 0.025) absence of DGF (0.015) were independently associated with high levels of FOXP3. Correlation between sCr and FOXP3 was inverse in TAC treated pts and positive in those taking EVL (r= 0.74, P = 0.007). FOXP3 expression was low in kidneys with mild or no chronic lesions (IFTA score < 2) regardless the ISS. Patients treated with EVL presenting IFTA score ≥ 2 had increased expression of FOXP3 (p=NS vs TAC). Conclusions: Results suggest that recipients of ECD kidneys without having undergone DGF, treated with EVL presented an increased expression of FOXP3 in their peripheral blood. The higher FOXP3 levels associated with more chronic histological lesions may represent an anti-inflammatory response elicited by ongoing chronic allograft damage. DISCLOSURES:Abbud-Filho, M.: Grant/Research Support, Novartis. Dias, C.: Grant/Research Support, Novartis. Caldas, H.: Grant/Research Support, Novartis. Nunes, R.: Grant/Research Support, Novartis. Mazeti, C.: Grant/Research Support, Novartis. Fernandes-Charpiot, I.: Grant/Research Support, Novartis. Baptista, M.: Grant/Research Support, Novartis. Abbud-Filho, M.: Grant/Research Support, Novartis.
NSDHL, for NAD(P)H steroid dehydrogenase-like, encodes a sterol dehydrogenase or decarboxylase involved in the sequential removal of two C-4 methyl groups in post-squalene cholesterol biosynthesis. Mutations in this gene are associated with human CHILD syndrome (congenital hemidysplasia with ichthyosiform nevus and limb defects), an X-linked, male lethal disorder, as well as the mouse mutations bare patches and striated. In the present study, we have investigated the subcellular localization of tagged proteins encoded by wild-type and selected mutant murine Nsdhl alleles using confocal microscopy. In addition to an ER localization commonly found for enzymes of post-squalene cholesterol biosynthesis, we have identified a novel association of NSDHL with lipid droplets, which are endoplasmic reticulum (ER)-derived cytoplasmic structures that contain a neutral lipid core. We further demonstrate that trafficking through the Golgi is necessary for ER membrane localization of the protein and propose a model for the association of NSDHL with lipid droplets. The dual localization of NSDHL within ER membranes and on the surface of lipid droplets may provide another mechanism for regulation of the levels and sites of accumulation of intracellular cholesterol.
Abstract Background: Chronic Kidney Disease (CKD) is a global public health problem. Cell therapy using pluripotent stem cells represents an attractive therapeutic approach for the treatment of CKD. Methods: We transplanted Mitomycin C (MMC)-treated human induced pluripotent stem cells (hiPSCs) and renal progenitor cells (RPCs) into a CKD rat model system. The RPCs and hiPSCs cells were characterized by immunofluorescence and qRT-PCR. Untreated 5/6 nephrectomized rats were compared to CKD animals receiving the same amount of MMC-treated hiPSCs or RPCs. Renal function, histology and immunohistochemistry were evaluated 45 days post-surgery. Results: We successfully generated hiPSCs from peripheral blood and differentiated them into RPCs expressing renal progenitor genes (PAX2, WT1, SIX2, and SALL1) and podocyte-related genes (SYNPO, NPHS1). RPCs also exhibited reduced OCT4 expression, confirming the loss of pluripotency. After cell transplantation into CKD rats, the body weight change was significantly increased in both hiPSC and RPC groups, in comparison with the control group. Creatinine clearance (CCr) was preserved only in the hiPSC group. Similarly, the number of macrophages in the kidneys of the hiPSC group reached a statistically significant reduction, when compared to control rats. Both treatments reduced positive staining for the marker α-smooth muscle actin. Histological features showed decreased tubulointerstitial damage (interstitial fibrosis and tubular atrophy) as well as a reduction in glomerulosclerosis in both iPSC and RPC groups. Conclusions: In conclusion, we describe that both MMC-treated hiPSCs and RPCs exert beneficial effects in attenuating CKD progression. Both cell types were equally efficient to reduce histological damage and weight loss caused by CKD. hiPSCs seems to be more efficient than RPCs, possibly due to a paracrine effect triggered by hiPSCs. These results demonstrate that the use of MMC-treated hiPSCs and RPCs improve clinical and histological CKD parameters, avoided tumor formation, and therefore may be a promising cell therapy strategy for CKD.
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