Identification of metabolic enzymes in renal cell carcinoma utilizing PROTEOMEX analyses

PROTEOMEX, an approach which combines conventional proteome analysis with serological screening, is a powerful tool to separate proteins and identify immunogenic components in malignant diseases. By applying this approach, we characterized nine metabolic enzymes which were differentially expressed in renal cell carcinoma (RCC) cell lines and compared their expression profiles to that of normal kidney epithelium cells. Four of these proteins, superoxide dismutase (SODC), triosephosphatase isomerase (TPIS), thioredoxin (THIO) and ubiquitin carboxyl-terminal hydrolase (UBL1) were further analysed for both their constitutive and interferon (IFN)-gamma inducible protein expression pattern in cell lines or tissue specimens derived from RCC or normal kidney epithelium using Western blot analysis and immunohistochemistry, respectively. With the exception of the RCC cell line MZ1940RC, which completely lacks the expression of UBL1, a heterogeneous and variable expression pattern of the different metabolic enzymes was detected in RCC and normal renal epithelium. The highest differences in the expression levels were found for THIO in the RCC cell lines, which was 2-fold upregulated when compared to autologous normal kidney epithelium. Moreover, IFN-gamma treatment did not influence the constitutive expression of these metabolic enzymes. Thus, PROTEOMEX represents a valuable approach for the identification of metabolic enzymes which might be used as markers for the diagnosis of RCC.     

Identification of markers for the selection of patients undergoing renal cell carcinoma-specific immunotherapy

Renal cell carcinoma (RCC) represents the most common malignant tumor in the kidney and is resistant to conventional therapies. The diagnosis of RCC is often delayed leading to progression and metastatic spread of the disease. Thus, validated markers for the early detection of the disease as well as selection of patients undergoing specific therapy is urgently needed. Using treatment with the monoclonal antibody (mAb) G250 as a model, proteome-based strategies were implemented for the identification of markers which may allow the discrimination between responders and nonresponders prior to application of G250-mediated immunotherapy. Flow cytometry revealed G250 surface expression in approximately 40% of RCC cell lines, but not in the normal kidney epithelium cell lines. G250 expression levels significantly varied thereby distinguishing between low, medium and high G250 expressing cell lines. Comparisons of two-dimensional gel electrophoresis expression profiles of untreated RCC cell lines versus RCC cell lines treated with a mAb directed against G250 and the characterization of differentially expressed proteins by mass spectrometry and/or Edman sequencing led to the identification of proteins such as chaperones, antigen processing components, transporters, metabolic enzymes, cytoskeletal proteins and unknown proteins. Moreover, some of these differentially expressed proteins matched with immunoreactive proteins previously identified by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, a technique called PROTEOMEX. Immunohistochemical analysis of a panel of surgically removed RCC lesions and corresponding normal kidney epithelium confirmed the heterogeneous expression pattern found by proteome-based technologies. In conclusion, conventional proteome analysis as well as PROTEOMEX could be successfully employed for the identification of markers which may allow the selection of patients prior to specific immunotherapy.     

Identification of tumor antigens in renal cell carcinoma by serological proteome analysis

We have investigated the suitability of proteomics for identification of tumor-associated antigens. First, we compared the proteomes of nontumorous kidney and renal cell carcinoma (RCC) by two-dimensional gel electrophoresis (2-DE) and silver staining. Protein patterns were markedly different (approximately 800 spots in RCCs versus approximately 1400 spots in kidney). 2-DE immunoblotting revealed five RCC-specific spots, reproducibly reactive with RCC-patient but not healthy donor control sera. Two of these antigens were isolated by preparative 2-DE, and identified by Edman sequencing of tryptic peptides. The first antigen, smooth muscle protein 22-alpha (SM22-alpha), is an actin-binding protein of unknown function predominantly expressed in smooth muscle cells. In situ hybridization revealed that SM22-alpha is not expressed in the malignant cells but in mesenchymal cells of the tumor stroma. The second antigen represents carbonic anhydrase I (CAI), an isoform usually not expressed in kidney. Interestingly, a different isoform (CAXII) has previously been identified by serological expression cloning as an antigen overexpressed in some RCCs. In additional assays, antibodies to recombinant CAI or SM22-alpha were detected in sera from 3/11 or 5/11 RCC patients, respectively, whereas sera from 13 healthy individuals did not react. In conclusion, serological proteome analysis may be a new tool for the identification of tumor-associated antigens.     

Expression of intermediate-sized filaments in developing and adult human kidney and in renal cell carcinoma.

We studied the distribution of intermediate-sized filaments in developing and adult kidneys and renal cell carcinoma (RCC) by indirect immunohistochemistry, using a pan-cytokeratin mouse monoclonal antibody (MAb), chain-specific anti-cytokeratin MAb, and anti-vimentin and anti-desmin MAb, to resolve controversy concerning intermediate-sized filament expression in the kidney. With the pan-cytokeratin MAb, cytokeratin expression was detectable in all stages of nephron development, starting with expression in the renal vesicles, the progenitors of the glomeruli, proximal tubules, Henle's loop, and part of the distal tubules. Using chain-specific anti-cytokeratin MAb, cytokeratin 8 and 18 expression was demonstrated in all developmental structures of the nephron, whereas cytokeratin 19 expression was more complex. None of the nephrogenic blastema cells from which the renal vesicles arise expressed cytokeratins. Transient expression of vimentin and cytokeratin 19 was observed in differentiating collecting ducts and proximal tubule cells at the S-shaped stage of nephron development, respectively. In RCC, cytokeratin expression closely resembled that of the mature proximal tubule, i.e., RCC cells expressed cytokeratins 8 and 18. However, in a subset of RCC additional cytokeratin 19 expression was noted. In addition, all except one RCC showed co-expression of cytokeratins and vimentin.     

The expression of ketohexokinase is diminished in human clear cell type of renal cell carcinoma

For identification and targeting of tumor-associated marker proteins, the proteome of clear cell type of renal cell carcinoma (RCC) and normal kidney tissues was analyzed by 2-DE. Ketohexokinase (also called fructokinase), which catalyzes the phosphorylation of fructose to fructose 1-phosphate, was identified by MALDI-TOF MS and found to be expressed at low rates in the renal tumor tissues. We found a decreased amount of ketohexokinase mRNA in RCC compared to that observed in the normal kidney tissues by Northern blot. The activity of ketohexokinase in 20 clear cell RCC specimens and the 20 corresponding normal kidneys was investigated, and its activity was shown to be approximately 1.4-fold lower in the RCC specimens than in the normal kidney. Ketohexokinase activity in tumor stage pT3 RCC was 1.5-fold lower than in pT1 RCC. The level of ketohexokinase activity in histological grade 3 RCC was 1.8-fold lower than that in grade 1 cancer. In addition, using in situ hybridization, it was revealed that ketohexokinase in the normal kidney tissue was confined to the proximal tubular epithelial cells, while the expression of ketohexokinase in RCC tissues was extremely low. Our research results show that the expression of human ketohexokinase was diminished in clear cell RCC.     

Heat shock protein expression and anti-heat shock protein reactivity in renal cell carcinoma

Heat shock proteins (HSP) are families of highly conserved proteins which are induced in cells and tissues upon exposure to extreme conditions causing acute or chronic stress. They exhibit distinct functions and have been implicated in the pathogenesis of a number of diseases, including cancer. A causal relationship between HSP expression and immunogenicity has been demonstrated in murine and human tumors and is also associated with the immune response. In order to investigate the correlation of HSP expression and their immunogenic potential in renal cell carcinoma (RCC), we here analyzed (i) the protein expression profile of various members of the HSP family in untreated and interferon (IFN)-gamma treated RCC cell lines as well as normal kidney epithelium, and (ii) the anti-heat shock protein reactivity in sera derived from RCC patients and healthy controls using proteomics-based techniques. A heterogeneous expression pattern of members of the HSP families was demonstrated in RCC cell lines and in cells representing normal renal epithelium. In some cases the expression rate is moderately altered by IFN-gamma treatment. In addition, a distinct anti-heat shock protein reactivity could be detected in autologous and allogeneic sera from RCC patients and healthy controls. These data suggest that HSP play a role in the immunogenicity of RCC and thus might be used for the design of immunization strategies to induce a potent antitumor response in this disease.     

Optimized serological isolation of lung-cancer-associated antigens from a yeast surface-expressed cDNA library

The technique of serological analysis of antigens by recombinant cDNA expression library (SEREX) uses autologous patient sera as a screening probe to isolate tumor-associated antigens for various tumor types. Isolation of tumor-associated antigens that are specifically reactive with patient sera, but not with normal sera, is important to avoid false-positive and autoimmunogenic antigens for the cancer immunotherapy. Here, we describe a selection methodology to isolate patient sera-specific antigens from a yeast surface-expressed cDNA library constructed from 15 patient lung tissues with non-small cell lung cancer (NSCLC). Several rounds of positive selection using patient sera alone as a screening probe isolated clones exhibiting comparable reactivity with both patient and normal sera. However, the combination of negative selection with allogeneic normal sera to remove antigens reactive with normal sera and subsequent positive selection with patient sera efficiently enriched patient sera-specific antigens. Using the selection methodology described here, we isolated 3 known and 5 unknown proteins, which have not been isolated previously, but and potentially associated with NSCLC.     

Cytoskeleton in neuroectodermally derived epithelial and muscle cells of the human iris and ciliary body.

The cytoskeleton of epithelial and muscle cells of the human iris and ciliary body was analyzed by immunohistochemistry in three morphologically normal formalin-fixed, paraffin-embedded eyes and in 34 eyes containing a uveal melanoma. Both layers of the iris epithelium reacted with monoclonal antibodies (MAb) V9 and Vim 3B4 to vimentin, whereas the ciliary epithelia additionally reacted with MAb CAM 5.2, CK5, KS-B17.2, and CY-90, recognizing cytokeratins 8 and 18. The same cytokeratin MAb labeled the retinal pigment epithelium, which lacked vimentin. The muscle portion of the anterior iris epithelium, which forms the dilator muscle, as well as the sphincter and ciliary muscles, reacted with MAb DE-U-10 to desmin and 1A4 to alpha-smooth muscle actin. The dilator and ciliary muscles also reacted with V9 and Vim 3B4 to vimentin, and some dilator fibers were weakly immunopositive for cytokeratin 8 and 18 with CY-90 and CAM 5.2. The antigenic profile of iris and ciliary epithelia infiltrated by melanoma cells remained unchanged. The intraocular epithelia, which are developmentally related but differ in function, and the intraocular muscles, which differ in origin but are functionally related, have distinct cytoskeletal profiles and may provide insights into the functional significance of intermediate filament expression.     

Intermediate filament protein expression in early developmental stages of the mouse

Expression patterns of intermediate filament proteins have been studied during early mouse embryo development. For this purpose, pre-implantation embryos at different stages of development after in vitro fertilization were studied using antibodies to cytokeratins, vimentin and lamins, using the indirect immunofluorescence assay. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies showed that, although the embryos grew in culture, vimentin could not be detected in a filamentous organization. Immunofluorescence for cytokeratins was only positive from the 8-cell stage onwards. In the morula stage an increased level of cytokeratin expression was observed with a transitional staining pattern, combining a filamentous and a diffuse occurrence. In the blastocyst stages profound cytokeratin filaments were seen in trophoblast cells but not in the inner cell mass. When the cytokeratin subtypes were analysed separately, it became apparent that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage and showing a filamentous distribution, were not found as prominently as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected in all developmental stages examined, as a faintly reactive nuclear lamina. In blastocysts both lamin types were detected in trophoblast as well as in inner cell mass.     

Expression of bone morphogenetic protein-7, its receptors and Smad1/5/8 in normal human kidney and renal cell cancer

Bone morphogenetic proteins (BMPs) are cytokines which are important for kidney homeostasis but also have role in the some renal diseases and renal cell carcinoma (RCC). In the last three decades incidence of RCC was constantly increased and the role of different molecular biomarkers in RCC is explored'. We analyzed expression of BMP-7, their receptors (BMPR-IA, BMPR-IB, BMPR-II) and proteins of their signaling pathway (pSmad1/5/8) in sixteen renal cancer samples and paired normal tissue. Tissue samples were analyzed by immunohistochemistry and Western blot. BMP-7, BMP receptors and pSmad1/5/8 were expressed in all structures of normal kidney but dominantly in the proximal tubular cells. In the cancer samples their expression was also noticed. Comparison of BMPs between different tissue showed increased expression of BMPR-IB and pSmad 1/5/8 and decreased expression of BMP-7 and BMPR-II in RCC compared to normal kidney. BMPR-IA was detected with immunohistochemistry but with Western blot attenuated signal was presented. BMP-7, BMP receptors and pSmad1/5/8 were showed in normal kidney and RCC. Detected alterations of BMP-7, BMP receptors and pSmad expression in RCC suggested their possible role in tumorigenesis of kidney cancer.     

Candidate biomarkers in renal cell carcinoma

Although the human genome has been decoded, the knowledge about the pathogenesis of diseases including cancer is still limited. By focusing on renal cell carcinoma (RCC) we here summarize the data of various research groups analyzing the protein/peptide expression profiles of tumor lesions/cell lines or serum obtained from patients and respective controls. Different powerful approaches such as 2-DE, PROTEOMEX/SERPA/SPEARS, and T cell epitope discovery upon elution of MHC class I-bound peptides in combination with MS/LC-MS/MS revealed 500 differentially expressed proteins. The overlap in target recognition limits the pool to 299 unique protein identities, but only few thereof (12%) have been validated. The management, analysis, and interpretation of the distinct data sets derived from 27 publications required bioinformatic restructuring of the results. However, the comprehensive analysis of the results expands the knowledge about the pathophysiology of RCC in particular of the most prominent clear cell subtype by providing information on the differentially expressed proteins, their regulation status in RCC compared to normal kidney epithelium next to additional information on MHC-presented T cell epitopes and on serological targets. Despite the low number of validated differentially expressed proteins some of them might serve as candidate biomarkers for the diagnosis and/or as therapeutic targets.     

Advances in molecular classification of renal neoplasms

Kidney neoplasms are classified by light microscopy using the World Health Organization (WHO) system. The WHO system defines histopathologic tumor subtypes with distinct clinical behavior and underlying genetic mutations. In adults, the common malignant subtypes are variants of renal cell carcinoma (RCC). Histopathologic classification is critical for clinical management of RCC, but is becoming more complex with recognition of novel tumor subtypes, development of procedures yielding small diagnostic biopsies, and emergence of molecular therapies directed at tumor gene activity. Therefore, classification systems based on gene expression are likely to become essential for diagnosis, prognosis and treatment of kidney tumors. Recent DNA microarray studies have shown that clinically relevant renal tumor subtypes are characterized by distinct gene expression profiles, which are useful for discovery of novel diagnostic and prognostic biomarkers. In this review, we summarize the WHO classification system for renal tumors, general applications of microarray technology in cancer research, and specific microarray studies that have advanced knowledge of renal tumor diagnosis, prognosis, therapy and pathobiology.     

Cytoplasmic Expression of Pontin in Renal Cell Carcinoma Correlates with Tumor Invasion,Metastasis and Patients’ Survival

Renal cell carcinoma (RCC) is the most lethal of all genitourinary malignancies. Distant metastasis represents the major cause of death in patients with RCC. Recent studies have implicated the AAA+ ATPase pontin in many cellular activities that are highly relevant to carcinogenesis. In this study, we demonstrate for the first time that pontin was up-regulated in RCC, and plays a previously unknown pro-invasive role in the metastatic progression of RCC through epithelial-to-mesenchymal transition (EMT) pathway. 28 pairs of freshly frozen clear cell RCC samples and the matched normal renal tissues analyzed by quantitative RT-PCR and western blotting demonstrated that pontin was up-regulated in clear cell RCC tissues than in normal renal tissues. In addition, immunohistochemistry was used to evaluate subcellular pontin expression in 95 RCC patients, and found that overexpression of pontin in cytoplasm positively correlated with the metastatic features, predicting unfavorable outcomes of RCC patients. Furthermore, in vitro experiments show pontin was predominantly expressed in cytoplasm of RCC cell lines, and a significant suppression of cell migration and invasion in pontin siRNA treated RCC cell lines was observed. Mechanistic studies show that pontin depletion up-regulated the E-cadherin protein and down-regulated vimentin protein, and decreased nuclear β-catenin expression, suggesting the involvement of EMT in pontin induced metastatic progression. Together, our data suggest pontin as a potential prognostic biomarker in RCC, and provide new promising therapeutic targets for clinical intervention of kidney cancers.     

Ectopic expression of alkaline phosphatase in proximal tubular brush border membrane of human renal cell carcinoma

The present study was conducted to find out any alteration in the expression and activity of alkaline phosphatase in the brush border membrane (BBM) from renal cell carcinoma (RCC) in comparison to normal renal BBM. The specific activity of alkaline phosphatase was drastically reduced in homogenate as well as BBM from RCC kidney when compared to ALP activity in BBM of normal kidney. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (V(max)) and increase in affinity constant (K(m)) of the enzyme. SDS-PAGE studies showed that the BBM proteins having molecular weights ranging from 95 to 170 kDa were poorly expressed in RCC BBM in relative to normal kidney BBM. Incubation of SDS-PAGE gel with BCIP/NBT dye clearly showed that the expression of ALP in tumor renal BBM was markedly reduced as compared to normal kidney. Further, Western blot analysis using anti-alkaline phosphatase antibody also confirmed the reduced expression of ALP in tumor renal BBM. Lipid composition in reference to phospholipids, glycolipids and cholesterol in tumor renal BBM was altered to that of normal renal BBM, indicating alteration in membrane fluidity of tumor renal BBM.     

Heterogeneity of tumor microenvironment is associated with clinical prognosis of non-clear cell renal cell carcinoma: a single-cell genomics study

Non-clear renal cell carcinomas (nccRCCs) are less frequent in kidney cancer with histopathological heterogeneity. A better understanding of the tumor biology of nccRCC can provide more effective treatment paradigms for different subtypes. To reveal the heterogeneity of tumor microenvironment (TME) in nccRCC, we performed 10x sing-cell genomics on tumor and normal tissues from patients with papillary renal cell carcinoma (pRCC), chromophobe RCC (chrRCC), collecting duct carcinoma (CDRCC) and sarcomatoid RCC (sarRCC). 15 tissue samples were finally included. 34561 cells were identified as 16 major cell clusters with 34 cell subtypes. Our study presented the sing-cell landscape for four types of nccRCC, and demonstrated that CD8+ T cells exhaustion, tumor-associated macrophages (TAMs) and sarcomatoid process were the pivotal factors in immunosuppression of nccRCC tissues and were closely correlated with poor prognosis. Abnormal metabolic patterns were present in both cancer cells and tumor-infiltrating stromal cells, such as fibroblasts and endothelial cells. Combined with CIBERSORTx tool, the expression data of bulk RNA-seq from TCGA were labeled with cell types of our sing-cell data. Calculation of the relative abundance of cell types revealed that greater proportion of exhausted CD8+ T cells, TAMs and sarRCC derived cells were correlated with poor prognosis in the cohort of 274 nccRCC patients. To the best of our knowledge, this is the first study that provides a more comprehensive sight about the heterogeneity and tumor biology of nccRCC, which may potentially facilitate the development of more effective therapies for nccRCC.Subject terms: Cancer genomics, Cancer microenvironment, Renal cell carcinoma     

Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.     

Secreted Products of Macrophages Exposed to Calcium Oxalate Crystals Induce Epithelial Mesenchymal Transition of Renal Tubular Cells via RhoA-Dependent TGF-β1 Pathway

Kidney stone disease is associated with renal fibrosis by the unclear mechanisms. We hypothesized that calcium oxalate (CaOx), a major crystalline component of kidney stones, could induce secretion of fibrotic factors from macrophages leading to “epithelial mesenchymal transition/transdifferentiation” (EMT) of renal tubular cells. Western blot analysis revealed an increased level of vimentin (mesenchymal marker) but decreased levels of E-cadherin and cytokeratin (epithelial markers) in MDCK cells treated with “secreted products from CaOx-exposed macrophages” (CaOx-M-Sup). Immunofluorescence study confirmed the increased level of vimentin and decreased level of cytokeratin, and also revealed the increased level of fibronectin (another mesenchymal marker). The data also showed decreased levels and disorganization of F-actin (cytoskeletal marker) and zonula occludens-1 (ZO-1) (tight junction marker) induced by CaOx-M-Sup. ELISA demonstrated the increased level of transforming growth factor-β1 (TGF-β1), the well-defined EMT inducer, in CaOx-M-Sup. Downstream signaling of TGF-β1 was involved as demonstrated by the decreased level of RhoA. Interestingly, pretreatment with a proteasome inhibitor (MG132) could restore RhoA to its basal level, most likely through ubiquitin-proteasome pathway (UPP). Moreover, MG132 successfully sustained cytoskeletal assembly and tight junction, and could prevent the cells from EMT. Altogether, these data demonstrate for the first time that CaOx-M-Sup could induce EMT in renal tubular cells by TGF-β1 signaling cascade via RhoA and UPP. This may be, at least in part, the underlying mechanism for renal fibrosis in kidney stone disease.     

Expression of intermediate filament proteins in fetal and adult human lung tissues

The expression patterns of intermediate filament proteins in fetal and normal or nonpathological adult human lung tissues are described using (chain-specific) monoclonal antibodies. In early stages of development (9-10 weeks and 25 weeks of gestation) only so-called simple cytokeratins such as cytokeratins 7 (minor amounts). 8, 18 and 19 are detected in bronchial epithelial cells. At later stages of development, the cytokeratin expression patterns become more complex. The number of bronchial cells positive for cytokeratin 7 increases, but basal cells in the bronchial epithelium remain negative. These latter cells show, however, expression of cytokeratin 14 in the third trimester of gestation. Developing alveolar epithelial cells express cytokeratins 7, 8, 18 and 19. In adult human bronchial epithelium cytokeratins 4 (varying amounts), 7, 8, 13 (minor amounts), 14, 18 and 19 can be detected, with the main expression of cytokeratins 7, 8, and 18 in columnar cells and the main expression of cytokeratin 14 in basal cells. Vimentin is detected in all mesenchymal tissues. In addition, fetal lung expresses vimentin in bronchial epithelium, however, to a lesser extent with increasing age, resulting in the expression of vimentin in only few scattered bronchial cells at birth. Also in adult bronchial epithelium the expression of vimentin is noticed in part of the basal and columnar epithelial cells. Desmin filaments, present in smooth muscle cells of the lung, appear to alter their protein structure with age. In early stages of development smooth muscle cells surrounding blood vessels are partly reactive with some cytokeratin antibodies and with a polyclonal desmin antibody. At week 9-10 and week 25 of gestation a monoclonal antibody to desmin, however, is not reactive with blood vessel smooth muscle cells but is only reactive with smooth muscle cells surrounding bronchi. With increasing age the reactivity of cytokeratin antibodies with smooth muscle cells in blood vessels decreases, while the reactivity with the monoclonal desmin antibody increases. Our results show that during differentiation profound changes in the intermediate filament expression patterns occur in the different cell types of the developing lung.     

Expression of vimentin and high‐molecular‐weight cytokeratin (clone 34ßE12) in differentiating reactive renal tubular cells from low‐grade urothelial carcinoma cells in voided urine

H. Ohsaki, E. Hirakawa, M. Nakamura, Y. Norimatsu, H. Kiyomoto and R. Haba Expression of vimentin and high‐molecular‐weight cytokeratin (clone 34ßE12) in differentiating reactive renal tubular cells from low‐grade urothelial carcinoma cells in voided urine Objective: Reactive renal tubular cells show features of an atypical repair reaction. Differentiation between reactive renal tubular cells and low‐grade urothelial carcinoma (LG‐UC) cells can therefore be a diagnostic challenge based on morphology alone. In this study, we evaluated the diagnostic utility of vimentin and a high‐molecular‐weight cytokeratin antibody (clone 34ßE12) in differentiating reactive renal tubular cells from LG‐UC. Methods: We evaluated voided urine cytology and surgical specimens from 40 patients with renal disease, and 17 patients with LG‐UC. All slides were stained with vimentin and 34ßE12. Results: In the reactive renal tubular cells in voided urine cytology, vimentin showed strong cytoplasmic staining in 39/40 (97.5%) cases, but all were negative for 34ßE12. LG‐UC cells showed positive staining for 34ßE12 in 3/17 (17.6%) cases, whereas none were positivity for vimentin. The reactive renal tubular cells of histological specimens in the renal disease group demonstrated positive for vimentin in all 40 cases and all were negative for 34ßE12. The LG‐UC group showed abnormal staining for 34ßE12 in 4/17 (23.5%) cases, whereas none were positive for vimentin. Conclusions: Vimentin expression in urine cytology can help to distinguish reactive renal tubular cells from LG‐UC. However, 34ßE12 does not appear to be a useful adjunct to distinguish these two groups in voided urine cytology.     

Localization of NBC-1 variants in human kidney and renal cell carcinoma

The Na(+)-HCO(3)(-) cotransporter (NBC-1) plays a major role in bicarbonate absorption from proximal tubules. However, which NBC-1 variant mediates proximal bicarbonate absorption has not been definitely determined. Moreover, the localization of this cotransporter in human kidney and renal cell carcinoma (RCC) tissues has not been clarified. To clarify these issues, immunohistochemical analysis was performed using the specific antibodies against kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters. In Western blot analysis the expression of kNBC-1 but not of pNBC-1 was detected in both normal human kidney and RCC tissues. In immunofluorescence analysis on normal renal tissues the anti-kNBC-1 antibody strongly and exclusively labeled the basolateral membranes of proximal tubules, which was confirmed by electron microscopic observation. In RCC cells, the anti-kNBC-1 antibody labeled both plasma membranes and intracellular organelles. The labeling by anti-pNBC-1 antibody was not detected in both normal kidney and RCC tissues. These results indicate that kNBC-1 is the dominant variant that mediates bicarbonate absorption from human renal proximal tubules. They also suggest that NBC-1 may have distinct roles in cancer cells.