Normal Renal Tissue Research Articles

High Macrophage Densities in Native Kidney Biopsies Correlate With Renal Dysfunction and Promote ESRD

Macrophages and monocytes are main players in innate immunity. The relevance of mononuclear phagocyte infiltrates on clinical outcomes remains to be determined in native kidney diseases. Our cross-sectional study included 324 patients with diagnostic renal biopsies comprising 17 disease entities and normal renal tissues for comparison. All samples were stained for CD68+ macrophages. Selected groups were further subtyped for CD14+ monocytes and CD163+ alternatively activated macrophages. Using precise pixel-based digital measurements, we quantified cell densities as positively stained areas in renal cortex and medulla as well as whole renal tissue. Laboratory and clinical data of all cases at the time of biopsy and additional follow-up data in 158 cases were accessible. Biopsies with renal disease consistently revealed higher CD68+-macrophage densities and CD163+-macrophage densities in cortex and medulla compared to controls. High macrophage densities correlated with impaired renal function at biopsy and at follow-up in all diseases and in diseases analyzed separately. High cortical CD68+-macrophage densities preceded shorter renal survival, defined as requirement of permanent dialysis. CD14+ monocyte densities showed no difference compared to controls and did not correlate with renal function. Precise quantification of macrophage densities in renal biopsies may contribute to risk stratification to identify patients with high risk for end-stage renal disease (ESRD) and might be a promising therapeutic target in renal disease.

Increased expression of HMMR in renal cell carcinoma is an independent prognostic factor.

The present study investigated how abnormal expression of hyaluronan-mediated mobility receptor (HMMR) in renal cell carcinoma (RCC) tissue affects the growth of RCC cells and the association between expression of HMMR and pathological staging and prognosis of patients with RCC. Reverse transcription-quantitative PCR was used to measure the expression of HMMR mRNA in RCC tissue and cell lines. For the prediction of HMMR gene expression, The Cancer Genome Atlas online database was utilized to compare differential gene expression of HMMR in normal renal and RCC tissue. Cell Counting Kit-8 assay was used to examine cell proliferation. The expression of HMMR increased in RCC tissue and renal cancer cell lines. The expression of HMMR was an independent prognostic factor for 5-year and disease-free survival in patients with RCC. The silencing of HMMR expression decreased the expression of Cyclin B1 and inhibited the proliferation of RCC cells. Overexpression of HMMR promoted the expression of Cyclin B1 and cell proliferation. The present study demonstrated that the expression of HMMR was significantly upregulated in RCC and was an independent prognostic factor for RCC. HMMR may be involved in the occurrence of RCC by regulating Cyclin B1.

A Computationally Constructed lncRNA-Associated Competing Triplet Network in Clear Cell Renal Cell Carcinoma.

Long noncoding RNAs (lncRNAs) are revealed to be involved in the tumorigenesis and progression of human malignancies mediated by microRNA (miRNA) via the competing endogenous RNA (ceRNA) mechanism, a newly proposed "RNA language." However, the lncRNA-associated competing triplet (lncACT) network among ceRNA transcripts in clear cell renal cell carcinoma (ccRCC) is currently lacking. We carried out differential expression analysis to identify aberrantly expressed lncRNAs, miRNAs, and mRNAs by analyzing the RNA-seq data of 420 ccRCC tissues and 71 noncancerous kidney tissues obtained from The Cancer Genome Atlas (TCGA). Then, a ccRCC-specific ceRNA network was built using computational algorithms, including miRcode, TargetScan, miRanda, and miRTarBase. In total, 1491 dysregulated lncRNAs were found between normal renal tissues and ccRCC (fold change > 4 and false discovery rate < 0.01). A ceRNA network that comprised of 46 DElncRNAs, 11 DEmiRNAs, and 55 DEmRNAs was established by integrating the lncRNA/miRNA and miRNA/mRNA interactions into lncACTs. Several lncRNAs were identified to be significantly associated with clinical features of ccRCC patients. Notably, four key lncRNAs (TCL6, HOTTIP, HULC, and PCGEM1) were tightly correlated with both patients' clinical characteristics and overall survival (log-rank P < 0.05), indicating their potential important roles in ccRCC. HOTTIP may be a potential prognostic and therapeutic molecular marker for ccRCC patients. Collectively, our results provide a comprehensive view of the lncRNA-associated ceRNA regulatory network for a better understanding of the mechanisms and prognosis biomarkers for ccRCC.

Expression and correlation of PBRM1 and P53 in clear cell carcinoma of kidney

Prevalence of clear cell renal cell carcinoma (ccRCC) among human population though common among adults, it occurs in children and young adults as well. The prognostic value of P53 expression in ccRCC is well known. Recently, PBRM1 has also acquired attention for its prognostic and predictive value in ccRCC. Here, we investigated the expression and correlation of PBRM1 and P53 in ccRCC. Renal tissues were collected from 70 patients who have undergone radical nephrectomy for clear cell carcinoma of the kidney in our hospital and 24 healthy volunteers for the study. We used immunohistochemical approach to determine the expression of PBRM1 and P53 in clear cell carcinoma of the kidney and normal kidney tissues and to analyze the correlation between them. Clinicopathological parameters and prognosis of patients were also studied. The positive expression rate of PBRM1 in clear renal cell carcinoma tissues was significantly higher (62.86%) compared to the normal renal tissues 8.33%. Similarly, positive expression rate of P53 in clear renal cell carcinoma tissues was 40%, while it was no expression in normal renal tissues. The expression level of PBRM1 was correlated with pathological grade and clinical stage of ccRCC patients, but not with age, sex and tumor size. P53 and expression levels were independent of age, sex, tumor size, pathological grade, and clinical stage of patients with clear cell carcinoma of the kidney. The 5-year survival rate of PBRM1 positive expression patients was 40.91% significantly lower than that of PBRM1 negative expression patients (84.62%), whereas in P53 it was 50 and 61.90%, respectively. Clinical stage, pathological grade and PBRM1 were all independent risk factors affecting the prognosis of patients with clear cell carcinoma of the kidney. Overall, the results suggest that PBRM1 is positively correlated with P53 in clear cell carcinoma of kidney (r=0.781, P=0.012). PBRM1 and P53 are both highly expressed in ccRCC and play an important role in the development of the disease. PBRM1 can also be used as an independent risk factor affecting the prognosis of ccRCC patients.

Upregulation of Metallothionein 1 G (MT1G) Negatively Regulates Ferroptosis in Clear Cell Renal Cell Carcinoma by Reducing Glutathione Consumption.

Ferroptosis is characterized by lipid peroxidation and iron accumulation, closely associated with clear cell renal cell carcinoma (ccRCC). It is of great significance for prognostic prediction and treatment of ccRCC to find biomarkers related to ferroptosis. We conducted several bioinformatic analyses using the transcriptome data and clinical information derived from online databases. Firstly, we identified the differentially expressed target genes in ccRCC. Then, t test and COX analysis were used to determine whether it was an independent prognostic factor combined with clinical information. String and gene set enrichment analysis (GSEA) were used to predict its function. Finally, we used ccRCC cells: 769-P and KAKI-1 in vitro to verify the regulation of target genes on cell proliferation apoptosis, iron metabolism, and GSH metabolism, which were used to judge the effect of target genes on ferroptosis. The study showed that MT1G is downregulated in ccRCC tissues compared with normal renal tissues. However, the ccRCC patients with higher expression relatively had higher malignancy and advanced stages. MT1G is an independent adverse factor for the prognosis of ccRCC. The protein interaction network analysis and GSEA showed that MT1G was closely related to GSH metabolism-related proteins (GSR) and lipid oxidation-related proteins (PLA2G2A). Samples with high expression of MT1G were enriched in “glutathione metabolism,” “oxidative phosphorylation,” and “proteasome,” whose function was involved in GSH metabolism and lipid peroxidation. The term associated with the occurrence and development of tumors included “P53 signaling pathway.” Furthermore, in vitro experiments showed that MT1G partially blocked ferroptosis induced by erastin and sorafenib-induced ccRCC cell lines (769-P and CAKI-1). The mechanism may be that MT1G affects ferroptosis by regulating GSH consumption in ccRCC cells. MT1G may be a negative regulator of ferroptosis in ccRCC cells and a biomarker of poor prognosis.

Effect of AKT silence on malignant biological behavior of renal cell carcinoma cells

BackgroundAs the most common malignant tumor of primary renal tumor, renal cell carcinoma (RCC) is the highly invasive disease with high mortality. AKT is a serine/threonine kinase that play a critical role in the phosphoinositide 3-kinase (PI3K) signaling pathway, and it is an attractive target for RCC treatment. The aim of present study was to investigate the effect of AKT silence on malignant behavior of renal cell carcinoma cells.MethodsAKT expression was quantified by immunohistochemistry in tumor tissues and normal tissues. The human RCC cell lines Caki-2 cell were chosen for this study. The optimal silencing siRNA was subsequently selected by RT-qPCR and western blot. The effect of AKT silence on RCC cells was investigated by CCK8 assay, transwell assay, scratch test and flow cytometry. The AKT1 expression in human renal cell carcinoma tissue was detected by immunohistochemical staining.ResultsThe AKT in Caki-2 cells was silenced successfully. The results shown AKT silence could inhibit cell proliferation, invasion, and, migration. In addition, AKT silence could promote Caki-2 cell apoptosis with prevention of RCC cells move from G1 phase to S phase. Immunohistochemical staining revealed significant difference of expression of AKT1 in RCC tissues and normal renal tissues. Taken together, AKT family members might involve in malignant growth of RCC, and might be a potential therapeutic target.ConclusionOur data show that AKT silence inhibited cell proliferation, invasion, and, migration of Caki-2 cell, and promoted Caki-2 cell apoptosis. Moreover, AKT silence prevented RCC cells move from G1 phase to S phase. Therefore, AKT may act as an effective therapeutic target for RCC.

TSC/MTOR -associated Eosinophilic Renal Tumors Exhibit a Heterogeneous Clinicopathologic Spectrum : A Targeted Next-generation Sequencing and Gene Expression Profiling Study.

Several TSC1/2- or MTOR -mutated eosinophilic renal tumor subsets are emerging, including eosinophilic solid and cystic renal cell carcinoma (ESC RCC), eosinophilic vacuolated tumors (EVTs) and low-grade oncocytic tumors (LOTs). "Unclassified renal tumors with TSC/MTOR mutations" ( TSC -mt RCC-NOS) do not meet the criteria for other histomolecular subtypes. Whether these tumors represent a continuum of 1 TS C/ MTOR -mutation-associated disease is unknown. We evaluated the clinicopathologic and IHC profiles of 39 eosinophilic renal tumors with targeted DNA sequencing-confirmed TSC/MTOR mutations. Twenty-eight of these, plus 6 ChRCC, 5 RO, 5 ccRCC, 7 MiT RCC and 6 normal renal tissues, were profiled transcriptionally by RNA-seq. The 39 cases were reclassified based on morphological and IHC features as ESC RCC (12), EVT (9), LOT, (8) and TSC -mt RCC-NOS (10). The mutation profiles demonstrated consistency; ESC RCCs (12/12) had TSC mutations, and most LOTs (7/8) had MTOR mutations. Ten TSC -mt RCC-NOSs exhibited heterogeneous morphology, arising a differential diagnosis with other renal tumors, including MiT RCC, PRCC and epithelioid PEComa. RNA sequencing-based clustering segregated ESC RCC, EVT and LOT from each other and other renal tumors, indicating expression profile-level differences. Most TSC- mt RCC-NOSs (6/7) formed a mixed cluster with ESC RCC, indicating similar expression signatures; one TSC- mt RCC-NOS with unusual biphasic morphology clustered with EVT. We expanded the TSC/MTOR -associated eosinophilic renal tumor morphologic spectrum, identified gene mutation characteristics, and highlighted differential diagnosis challenges, especially with MiT RCC. ESC RCC, EVT, and LOT having distinct expression profiles. TSC -mt RCC-NOS may cluster with recognized TSC/MTOR -associated entities.

An integrative pan-cancer analysis revealing the difference in small ring finger family of SCF E3 ubiquitin ligases.

The SCF (Skp1-cullin-F-box proteins) complex is the largest family of E3 ubiquitin ligases that mediate multiple specific substrate proteins degradation. Two ring-finger family members RBX1/ROC1 and RBX2/RNF7/SAG are small molecular proteins necessary for ubiquitin ligation activity of the multimeric SCF complex. Accumulating evidence indicated the involvement of RBX proteins in the pathogenesis and development of cancers, but no research using pan-cancer analysis for evaluating their difference has been directed previously. We investigated RBX1/2 expression patterns and the association with clinicopathological features, and survivals of cancer patients obtained from the TCGA pan-cancer data. The binding energies of RBX1/2-CUL1 complexes were preliminarily calculated by using molecular dynamics simulations. Meanwhile, we assessed their immune infiltration level across numerous databases, including TISIDB and Timer database. High expression levels of RBX1/2 were observed in most cancer types and correlated with poor prognosis of patients analyzed. Nonetheless, exceptions were observed: RBX2 expression in KICH was higher than normal renal tissues and played a detrimental role in KICH. The expression of RBX1 was not associated with the prognostic risk of KICH. Moreover, the combination of RBX1 and CUL1 expression is more stable than that of RBX2 and CUL1. RBX1/2 expression showed their own specific characteristics in tumor pathological stages and grades, copy number variation and immune components. These findings not only indicated that the difference of RBX1/2 might result in varying degrees of tumor progression, but also suggested that they might serve as biomarkers for immune infiltration in cancers, shedding new light on therapeutics of cancers.

APOA1 mRNA and protein in kidney renal clear cell carcinoma correlate with the disease outcome

Renal cancer is one of the most common malignant tumors with high mortality, and kidney renal clear cell carcinoma (KIRC) is the most common type of renal cancer. We attempted to evaluate the clinical and prognostic significance of Apolipoprotein A1 (APOA1) mRNA and protein in KIRC patients. Clinical data along with RNA-sequencing data were downloaded from UCSC Xena. The Human Protein Atlas database was searched to reveal APOA1 protein expression profiles in KIRC and normal renal tissues. The TIMER database was applied to determine the correlations of APOA1 with immune cells and PD-1 and PD-L1 in KIRC. Ninety-one cases of KIRC patients and 93 healthy controls from our hospital were enrolled for clinical validation. Levels of APOA1 mRNA in KIRC tissues (N = 535) are not only lower than the levels in normal renal tissues (N = 117), but also in paired normal renal tissues (N = 72). High expression of APOA1 mRNA at the time of surgery was correlated with worse overall survival (OS) (HR 1.66; p = 0.037) and disease-free survival (DFS) (HR 1.65; p = 0.047), and APOA1 DNA methylation was linked to worse OS (HR 2.1; p = 0.001) rather than DFS (HR 1.12; p = 0.624) in KIRC patients. Concentrations of preoperative serum APOA1 protein were markedly decreased in KIRC patients compared to healthy controls (p < 0.01), and low levels of APOA1 protein predicted less favorable OS than those with high levels (HR = 2.84, p = 0.0407). APOA1 negatively correlated with various immune cell infiltrates and PD-L1 expression (r = − 0.283, p = 2.74e−11) according to the TIMER database. Low levels of APOA1 mRNA at the time of surgery predict favorable survival in KIRC patients. Our results provide insights to identify a novel prognostic index with great clinical utility.

Bioinformatics Analysis of the Mechanisms of Diabetic Nephropathy via Novel Biomarkers and Competing Endogenous RNA Network.

Diabetic nephropathy (DN) is one of the common chronic complications of diabetes with unclear molecular mechanisms, which is associated with end-stage renal disease (ESRD) and chronic kidney disease (CKD). Our study intended to construct a competing endogenous RNA (ceRNA) network via bioinformatics analysis to determine the potential molecular mechanisms of DN pathogenesis. The microarray datasets (GSE30122 and GSE30529) were downloaded from the Gene Expression Omnibus database to find differentially expressed genes (DEGs). GSE51674 and GSE155188 datasets were used to identified the differentially expressed microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), respectively. The DEGs between normal and DN renal tissues were performed using the Linear Models for Microarray (limma) package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to reveal the mechanisms of DEGs in the progression of DN. The protein–protein interactions (PPI) of DEGs were carried out by STRING database. The lncRNA–miRNA–messenger RNA (mRNA) ceRNA network was constructed and visualized via Cytoscape on the basis of the interaction generated through the miRDB and TargetScan databases. A total of 94 significantly upregulated and 14 downregulated mRNAs, 31 upregulated and 121 downregulated miRNAs, and nine upregulated and 81 downregulated lncRNAs were identified. GO and KEGG pathways enriched in several functions and expression pathways, such as inflammatory response, immune response, identical protein binding, nuclear factor kappa b (NF-κB) signaling pathway, and PI3K-Akt signaling pathway. Based on the analysis of the ceRNA network, five differentially expressed lncRNAs (DElncRNAs) (SNHG6, KCNMB2-AS1, LINC00520, DANCR, and PCAT6), five DEmiRNAs (miR-130b-5p, miR-326, miR-374a-3p, miR-577, and miR-944), and five DEmRNAs (PTPRC, CD53, IRF8, IL10RA, and LAPTM5) were demonstrated to be related to the pathogenesis of DN. The hub genes were validated by using receiver operating characteristic curve (ROC) and real-time PCR (RT-PCR). Our research identified hub genes related to the potential mechanism of DN and provided new lncRNA–miRNA–mRNA ceRNA network that contributed to diagnostic and potential therapeutic targets for DN.

GRAMD1A Is a Biomarker of Kidney Renal Clear Cell Carcinoma and Is Associated with Immune Infiltration in the Tumour Microenvironment.

Background GRAM structural domain-containing protein 1A (GRAMD1A) is upregulated in a variety of human cancer tissues and is closely associated with tumourigenesis and progression. Methods Patient RNA-sequencing data and clinicopathological information were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. The expression of GRAMD1A in kidney cancer cell lines and KIRC patients was analysed by quantitative polymerase chain reaction (qPCR). Receiver Operator Characteristic (ROC) curves, nomograms, Kaplan-Meier analysis, forest plots, and COX analysis were used to assess the diagnostic and prognostic value of GRAMD1A in KIRC, and gene set enrichment analysis (GSEA) was used to explore its potential signalling pathways. In addition, the Sangerbox website, Kaplan-Meier plotter database, and TISIDB and TIMER databases were used to further analyse the correlation of GRAMD1A with microsatellite instability (MSI), tumour mutational burden (TMB), immune checkpoint genes, and tumour-infiltrating lymphocytes (TILs). Results GRAMD1A was significantly highly expressed in KIRC and associated with shorter overall survival and relapse-free survival (P < 0.05). The AUC value of the ROC curve to identify KIRC and normal renal tissues was 0.942. Forest plot and COX analysis visualized that GRAMD1A could be an independent prognostic factor in KIRC patients (P < 0.01), and nomograms to determine the overall survival (OS) of KIRC patients also showed good efficacy (C-index: 0.776). Moreover, Spearman correlation analysis showed a positive correlation between GRAMD1A and MSI, TMB (P < 0.01). On the other hand, GRAMD1A was also found to be closely associated with immune checkpoint genes. Meanwhile, patients with KIRC with high GRAMD1A expression had a relatively low hazard ratio (HR) of death when B lymphocytes, natural killer T cells, CD4+ T lymphocytes, CD8+ T lymphocytes, and macrophages were enriched in the tumour microenvironment (TME), and a greater HR of death when regulatory T lymphocytes with tumour-specific immunosuppressive effects were significantly enriched. Last, GSEA shows that GRAMD1A is closely associated with the regulation of energy metabolism in KIRC. Conclusions GRAMD1A is a promising diagnostic and prognostic biomarker for patients with KIRC, and its biological function correlates to some extent with immune infiltration in TME.

Decreased GM3 correlates with proteinuria inminimal change nephrotic syndrome and focal segmental glomerulosclerosis.

Glycolipids on cell membrane rafts play various roles by interacting with glycoproteins. Recently, it was reported that the glycolipid GM3 is expressed in podocytes and may play a role in podocyte protection. In this report, we describe the correlation between changes in GM3 expression in glomeruli and proteinuria in minimal change nephrotic syndrome (MCNS) and focal segmental glomerulosclerosis (FSGS) patients. We performed a case-control study of the correlation between nephrin/GM3 expression levels and proteinuria in MCNS and FSGS patients who underwent renal biopsy at our institution between 2009 and 2014. Normal renal tissue sites were used from patients who had undergone nephrectomy at our institution and gave informed consent. Both MCNS and FSGS had decreased GM3 and Nephrin expression compared with the normal (normal vs. MCNS, FSGS; all p < 0.01). Furthermore, in both MCNS and FSGS, GM3 expression was negatively correlated with proteinuria (MCNS: r = -0.61, p < 0.01, FSGS: r = -0.56, p < 0.05). However, nephrin expression had a trend to correlate with proteinuria in FSGS (MCNS: r = 0.19, p = 0.58, FSGS: r = -0.48, p = 0.06). Furthermore, in a simple linear regression analysis, GM3 expression also correlated with proteinuric change after 12months of treatment (MCNS: r = 0.40, p = 0.38, FSGS: r = 0. 68, p < 0.05). We showed for the first time that decreased GM3 expression correlates with proteinuria in MCNS and FSGS patients. Further studies are needed on the podocyte-protective effects of GM3.

SMOC2 promotes an epithelial-mesenchymal transition and a pro-metastatic phenotype in epithelial cells of renal cell carcinoma origin

Renal Cell Carcinoma (RCC) is the most common form of all renal cancer cases, and well-known for its highly aggressive metastatic behavior. SMOC2 is a recently described non-structural component of the extracellular matrix (ECM) that is highly expressed during tissue remodeling processes with emerging roles in cancers, yet its role in RCC remains elusive. Using gene expression profiles from patient samples, we identified SMOC2 as being significantly expressed in RCC tissue compared to normal renal tissue, which correlated with shorter RCC patient survival. Specifically, de novo protein synthesis of SMOC2 was shown to be much higher in the tubular epithelial cells of patients with biopsy-proven RCC. More importantly, we provide evidence of SMOC2 triggering kidney epithelial cells into an epithelial-to-mesenchymal transition (EMT), a phenotype known to promote metastasis. We found that SMOC2 induced mesenchymal-like morphology and activities in both RCC and non-RCC kidney epithelial cell lines. Mechanistically, treatment of RCC cell lines ACHN and 786-O with SMOC2 (recombinant and enforced expression) caused a significant increase in EMT-markers, -matrix production, -proliferation, and -migration, which were inhibited by targeting SMOC2 by siRNA. We further characterized SMOC2 activation of EMT to occur through the integrin β3, FAK and paxillin pathway. The proliferation and metastatic potential of SMOC2 overexpressing ACHN and 786-O cell lines were validated in vivo by their significantly higher tumor growth in kidneys and systemic dissemination into other organs when compared to their respective controls. In principle, understanding the impact that SMOC2 has on EMT may lead to more evidence-based treatments and biomarkers for RCC metastasis.

Near-Infrared Fluorescence Imaging of Renal Cell Carcinoma with ASP5354 in a Mouse Model for Intraoperative Guidance.

Renal cell carcinoma is a prevalent disease associated with high morbidity and mortality rates. Partial nephrectomy is a first-line surgical option because it allows the preservation of renal function. Clear differentiation between normal and cancerous tissues is critical for increasing the negative margin rates. This study investigated the capability of the near-infrared (NIR) fluorescent imaging agent ASP5354 for in vivo fluorescence imaging of renal cell carcinoma. ASP5354 at a single dose of 12 nmol (0.037 mg)/kg body weight was intravenously administered to healthy and orthotopic renal cell carcinoma mice under anesthesia. NIR images of the abdominal cavity were obtained using a near-infrared fluorescence (NIRF) camera system. In addition, the cancerous kidneys were harvested, and the NIRF in their sections was measured using an NIRF microscope. Normal renal tissue emitted strong NIRF but the cancer tissue did not. The difference in NIRF intensity between the normal and cancer tissues clearly presented the boundary between the normal and cancer tissues in macro and micro NIRF imaging. ASP5354 can distinguish cancer tissue from normal tissue using NIRF. Thus, ASP5354 is a promising agent for renal cell carcinoma tissue imaging in partial nephrectomy for renal cell carcinoma patients.

Decoding the Immune Microenvironment of Clear Cell Renal Cell Carcinoma by Single-Cell Profiling to Aid Immunotherapy.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, and it is the major cause of kidney cancer death. Understanding tumor immune microenvironments (TMEs) is critical in cancer immunotherapies. Here, we studied the immune characterization at single-cell resolution by integrating public data of ccRCC across different tissue types, and comparing the transcriptome features and tumor TME differences in tumors, normal adjacent tissue, and peripheral blood. A total of 16 different types of cell components of ccRCC were identified. We revealed that there is an overall increase in T-cell and myeloid populations in tumor-infiltrated immune cells compared to normal renal tissue, and the B-cell population in the tumor showed a sharp decrease, which indicates that the cells in tumor tissue undergo strong immune stress. In addition, the cell–cell communication analysis revealed specific or conserved signals in different tissue types, which may aid to uncover the distinct immune response. By combining and analyzing publicly available ccRCC bulk RNA-seq datasets, 10 genes were identified as marker genes in specific cell types, which were significantly associated with poor prognosis. Of note, UBE2C, which may be a good indicator of tumor proliferation, is positively associated with reductions in overall survival and highly associated with tumor grade. Our integrated analysis provides single-cell transcriptomic profiling of ccRCC and their TME, and it unmasked new correlations between gene expression, survival outcomes, and immune cell-type components, enabling us to dissect the dynamic variables in the tumor development process. This resource provides deeper insight into the transcriptome features and immune response of ccRCC and will be helpful in kidney cancer immunotherapy.

Analysis of clinicopathological and molecular features of ELOC(TCEB1)-mutant renal cell carcinoma

ObjectiveThis study aimed to investigate the clinicopathological and molecular characteristics of ELOC(TCEB1)-mutant renal cell carcinoma. MethodsSanger sequencing was used to assess 32 cases originally diagnosed as clear cell renal cell carcinoma with CK7 positive and/or fibromyomatous stroma. Of these, 4 patients with ELOC(TCEB1) gene mutation were screened, and their clinicopathological data were collected for histomorphological observation, immunohistochemical staining, and follow-up, and relevant pieces of literature were reviewed. ResultsThe 4 patients with ELOC(TCEB1) mutations were all males and aged between 57 and 64 years (median age: 59 years old). The tumor was located in the renal cortex, with a diameter of 2–3.5 cm. The cross-section was grayish-yellow and grayish brown, solid and nodular, and clearly demarcated from the surrounding tissues. Of the 4 patients, 3 harbored a thick fibrous pseudocapsule rich in smooth muscle and were separated from the surrounding normal renal tissue, and 2 of them showed focal invasion into the pseudocapsule, whereas 1 patient had no capsule but had focal invasion into the surrounding renal parenchyma. The tumor tissues mainly exhibited elongated or branched aciniform or tubular structures, commonly accompanied by interspersed small cystic and focal clustered short papillary structures. The cytoplasm of the tumor cells was rich and lightly stained, and the nuclear grading ranged from 1 to 2. All patients showed loose edema in the stroma, and 2 patients showed a small number of interspersed smooth muscle bundles. All 4 patients showed EMA, CA9, AMACR, and TCEB1 expression, and TCEB1 was mainly located in the nucleus. Vimentin, CK7, and CD10 expressions were observed in most cases; CD117, TFE3, HMB45, and melanA were not expressed in all tumors; the expression rate of Ki67 was 3%− 8%. All 4 patients had a point mutation in ELOC(TCEB1) Y79C. The patients were followed up for 24–93 months (mean 49 months), and all of them survived to date without recurrence or metastasis. ConclusionELOC(TCEB1)-mutant renal cell carcinoma is a rare type of renal cell carcinoma, which tends to occur in middle-aged and elderly men. The main characteristics of this tumor are the branching alveolar or tubular structure with clustered short papillae, presence of fibromyomatous stroma, and the expression of CK7, CA9, CD10, and AMACR. Positive TCEB1 nuclear staining may be an important marker and the Sanger sequencing method is helpful for the diagnosis of this type of RCC. Most patients harbor tumors exhibiting low nuclear grade and inert clinical behavior, and a few tumors exhibit high nuclear grade and aggressive characteristics.

IL-37 Expression in Patients with Abdominal Aortic Aneurysm and Its Role in the Necroptosis of Vascular Smooth Muscle Cells.

Background Our previous studies have shown that interleukin- (IL-) 37 plays a protective role in patients and animal models with coronary artery disease. However, the role of IL-37 in patients with abdominal aortic aneurysm (AAA), another artery disease, is yet to be elucidated. Methods and Results AAA tissues and plasma samples were obtained from patients with or without surgical intervention. Normal renal aortic tissues were collected from kidney transplant donors. Our findings established that in AAA, IL-37 was distributed in endothelial cells, macrophages, and vascular smooth muscle cells (VSMCs) and that it was chiefly concentrated in VSMCs. Furthermore, the expression was found to be downregulated compared with that in normal artery tissues. Immunofluorescence showed that, unlike normal arteries, IL-37 was translocated to the nucleus of VSMCs in AAA. Moreover, in patients with AAA, the expressions of IL-37, IL-6, and tumor necrosis factor- (TNF-) α were increased in the plasma in comparison with the healthy controls. Correlation analysis revealed that IL-37 was positively correlated with IL-6, TNF-α, age, aneurysm diameter, and blood pressure. Furthermore, human aortic vascular smooth muscle cells (HASMCs) were stimulated with angiotensin II (AngII) in vitro to simulate smooth muscle cell (SMC) damage in AAA. A decrease in IL-37 expression and an increase in receptor-interacting serine/threonine-protein kinase 3 (RIPK3) expression were observed in HASMCs stimulated with AngII. On this basis, inhibition of RIPK3 with GSK'872 significantly attenuated necroptosis. Moreover, the necroptosis rates were significantly lowered in HASMCs treated with recombinant IL-37, whereas the rates were enhanced when the cells were depleted of the interleukin. Immunoblotting results showed that both exogenous and endogenous IL-37 could affect the expressions of RIPK3, NLRP3, and IL-1β. Also, the phosphorylation of RIPK3 and p65 was affected. Meanwhile, IL-37 promoted the transition of SMC from proliferative type to contractile type. Conclusions The expression of IL-37 in VSMCs decreases in patients with AAA, whereas IL-37 supplementation suppresses RIPK3-mediated necroptosis and promotes the transition of VSMCs from proliferative to contractile type.

Lox as the Potential Key Gene of Clear Cell Renal Carcinoma

Clear cell renal carcinoma (CCRC) is the most common type of renal carcinoma. We hope to find out the potential key genes playing important roles in CCRC genesis and progression by analysing the recent expression profiling by array from 2014 to 2016. In order to find out the differentially expressed genes (DEGs) between CCRC and normal renal tissue. Gene Ontology (GO) and Kyoto Encyclopedia of Genes, and Genomes (KEGG) pathway enrichment were carried out by the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8. Protein-Protein Interaction (PPI) Networks Functional Enrichment Analysis of these DEGs was analyzed using the Search Tool for Recurring Instances of Neighbouring Genes (STRING). The results were then visualized by the software Cytoscape. The authors also used the online tool of Kaplan-Meier plotter survival analysis to assess the significance of the top ten genes in the prognosis of CCRC. A total of 192 DEGs were identified and the top ten key genes were picked out by the software Cytoscape. FN1, CXCR4, LOX, and PLG were then further screened out based on the overall survival analysis; SLC12A1 and LOX were screened out after the recurrence-free survival analysis. LOX was finally believed to be the most reliable prognostic factor since it has prognostic value for both overall survival and recurrence-free survival analysis. Our analysis suggests that LOX is the most reliable prognostic factor for CCRC patients. Key Words: Renal clear cell carcinoma, Microarray datasets, Bioinformatics approach, Prognostic factor, LOX.

MP50-01 PROGNOSTIC IMPACT OF PATHOLOGICAL RENAL PARENCHYMAL INFILTRATION IN NON-METASTATIC CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 May 2022MP50-01 PROGNOSTIC IMPACT OF PATHOLOGICAL RENAL PARENCHYMAL INFILTRATION IN NON-METASTATIC CLEAR CELL RENAL CELL CARCINOMA Hajime Tanaka, Yuki Fukawa, Kouhei Yamamoto, Shohei Fukuda, Sho Uehara, Soichiro Yoshida, Minato Yokoyama, Yoh Matsuoka, Steven C. Campbell, and Yasuhisa Fujii Hajime TanakaHajime Tanaka More articles by this author , Yuki FukawaYuki Fukawa More articles by this author , Kouhei YamamotoKouhei Yamamoto More articles by this author , Shohei FukudaShohei Fukuda More articles by this author , Sho UeharaSho Uehara More articles by this author , Soichiro YoshidaSoichiro Yoshida More articles by this author , Minato YokoyamaMinato Yokoyama More articles by this author , Yoh MatsuokaYoh Matsuoka More articles by this author , Steven C. CampbellSteven C. Campbell More articles by this author , and Yasuhisa FujiiYasuhisa Fujii More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002625.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous studies highlighted the existence of heterogeneity in survival outcomes of patients with non-metastatic clear cell renal cell carcinoma (nmCCRCC), suggesting that further exploration is required to define high-risk patients. In this study, we investigated the prognostic impact of renal parenchymal infiltration (RPI) in nmCCRCC based on the pathological analysis with focus on the tumor interface with normal renal parenchyma. METHODS: We retrospectively analyzed 333 patients with nmCCRCC treated with radical nephrectomy. Two pathologists reviewed surgical specimens and determined the presence/absence of RPI, which was defined as tumor infiltration into the renal parenchyma by involving normal renal tissue and/or by forming small nests that were separate from the main lesion. Pathological TN stage (UICC version 8), microvascular invasion, and WHO/ISUP grade were also evaluated and the impacts of those parameters on recurrence-free survival (RFS) were analyzed. RESULTS: The median age was 64 and 232 patients (70%) were male. The pathological analysis revealed invasions into perirenal fat, sinus fat, collecting system, and renal vein in 30 (9%), 33 (10%), 30 (9%), and 60 (18%) patients, respectively, resulting in 107 patients (32%) with ≥pT3a. Microvascular invasion was identified in 199 patients (60%) and 138 (41%) had WHO/ISUP grade 3-4. Overall 40 patients (12%), including 24 with ≥pT3a and 16 with pT1-2, were diagnosed with RPI.During the median follow-up of 6.0 years, 67 patients (20%) developed recurrence. In the multivariable analysis, ≥pT3a, microvascular invasion, WHO/ISUP grade 3-4, and RPI were independently associated with shorter RFS. The hazard ratio was highest for RPI (5.41 [95% CI: 3.10-9.44]) followed by microvascular invasion (3.04 [1.26-7.30]), WHO/ISUP grade (2.16 [1.20-3.87]), and ≥pT3a (2.13 [1.23-3.70]). According to pT stage and RPI, 5-year RFS was 17%, 40%, 76%, and 94% in the patients with ≥pT3a and RPI(+), pT1-2 and RPI(+), ≥pT3a and RPI(-), and pT1-2 and RPI(-), respectively (p<0.001). CONCLUSIONS: RPI is the primary determinant of poor prognosis in nmCCRCC among pathological parameters related to stage and grade. The presence of RPI predicts a high risk of recurrence early after surgery and adjuvant therapy may be considered for this population. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e863 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hajime Tanaka More articles by this author Yuki Fukawa More articles by this author Kouhei Yamamoto More articles by this author Shohei Fukuda More articles by this author Sho Uehara More articles by this author Soichiro Yoshida More articles by this author Minato Yokoyama More articles by this author Yoh Matsuoka More articles by this author Steven C. Campbell More articles by this author Yasuhisa Fujii More articles by this author Expand All Advertisement PDF DownloadLoading ...

Synthesis, Characterization, and In Vivo Study of Some Novel 3,4,5-Trimethoxybenzylidene-hydrazinecarbothioamides and Thiadiazoles as Anti-Apoptotic Caspase-3 Inhibitors.

The present study aims to discover novel derivatives as antiapoptotic agents and their protective effects against renal ischemia/reperfusion. Therefore, a series of new thiadiazole analogues 2a–g was designed and synthesized through cyclization of the corresponding opened hydrazinecarbothioamides 1a–g, followed by confirmation of the structure via spectroscopic tools (NMR, IR and mass spectra) and elemental analyses. The antiapoptotic activity showed alongside decreasing of tissue damage induced by I/R in the kidneys of rats using N-acetylcysteine (NAC) as an antiapoptotic reference. Most of the cyclized thiadiazoles are better antiapoptotic agents than their corresponding opened precursors. Particularly, compounds 2c and 2g were the most active antiapoptotic compounds with significant biomarkers. A preliminary mechanistic study was performed through caspase-3 inhibition. Compound 2c was selected along with its corresponding opened precursor 1c. An assay of cytochrome C revealed that there is an attenuation of cytochrome C level of about 5.5-fold, which was better than 1c with a level of 4.1-fold. In caspases-3, 8 and 9 assays, compound 2c showed more potency and selectivity toward caspase-3 and 9 compared with 1c. The renal histopathological investigation indicated normal renal tissue for most of the compounds, especially 2c and 2g, relative to the control. Finally, a molecular docking study was conducted at the caspase-3 active site to suggest possible binding modes.