Normal Renal Epithelium Research Articles

Abstract PR002: Injury-associated transcriptional state transitions in early renal carcinogenesis

Abstract Most epithelial cells with cancer driver mutations fail to form tumors. This has been particularly well established by genetic analysis of the renal epithelium, where cells with tumor-initiating mutations in the von Hippel-Lindau tumor suppressor (VHL) only rarely develop into clear cell renal carcinomas (ccRCC). The progression of VHLmutant cells to malignancy can take decades, offering a wide window of opportunity for preventative intervention. However, the cellular programs that facilitate the malignant transformation of VHL null ccRCC precursor clones remain elusive. Using a genetically engineered mouse model that allows conditional inactivation of Vhl and Polybromo 1 (Pbrm1), the second most commonly mutated ccRCC gene after VHL, in the renal epithelium of adult mice, we have analyzed the phenotypic evolution of the earliest stages of renal carcinoma formation over months at single cell resolution. Transcriptomic analysis by single cell RNA-sequencing (scRNA-seq) revealed that the acute activation of the hypoxia signaling response, a predicted consequence of genetic Vhl inactivation, is followed by distinct transcriptomic state transitions that precede the emergence of early carcinoma cells. These transitions are associated with constitutive activation of transcriptional programs that normally mediate acute and transient responses to renal injury, and are progressively activated at a low level in aging normal renal epithelium. Analysis of DNA accessibility by the single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) identified widespread opening of chromatin regions in association with progression towards malignancy, revealing a chromatin footprint of tissue repair pathway activation. Analysis of human ccRCC samples, including early-stage cases from VHL mutation carriers, validated our observations at the transcriptomic and protein level. Furthermore, genetic perturbation experiments in transplantable ccRCC models demonstrated the functional role of the renal injury pathway in tumor re-initiation. These results are compatible with a model whereby activation of the renal epithelial injury pathway facilitates the early progression of mutant ccRCC precursor clones towards malignancy. That the same repair pathway is increasingly activated over time in normal renal epithelium points at a link between ageing and ccRCC development. An understanding of the causes of ageing-associated renal damage could provide opportunities for early intervention in ccRCC, especially in high-risk individuals. Citation Format: Shoko Hirosue, Jianfeng Ge, Leticia Castillon, Paulo Rodrigues, Dóra Bihary, Alyson Speed, Ludovic Wesolowski, Veronica Caraffini, Anne Y. Warren, Grant D. Stewart, Sarah J. Welsh, Thomas J. Mitchell, Athena Matakidou, Fatemeh S. Seyednasrollah, Shamith A. Samarajiwa, Sakari Vanharanta. Injury-associated transcriptional state transitions in early renal carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR002.

Tribocorrosion and cytotoxicity of FeB-Fe2B layers on AISI 316 L steel

New results about the tribocorrosion resistance and cytotoxicity of FeB-Fe2B layer developed by the powder-pack boriding process on the surface of AISI 316 L steel are presented in this work. Initially, the tribocorrosion tests in the borided AISI 316 L steel and the untreated material (AISI 316 L steel) were performed in Hank's solution, using a ball-on-flat configuration, which was connected with a three electrode-chemical cell. The sliding tests, in the presence or absence of corrosion, were performed under 20N normal force using an alumina ball as a counter body. For the overall experimental conditions, the material loss for each material was estimated according to the ASTM G119 procedure. Further, the in vitro cytocompatibility of FeB-Fe2B layer on AISI 316 L steel and the untreated material was evaluated by the indirect contact method, in which cell lines of immortalized human fibroblast (CHON-002) and normal renal epithelium of Cercopithecus aethiops (Vero) were used.The results showed that the presence of FeB-Fe2B layer on the AISI 316 L steel improves the tribocorrosion resistance 1.5 times than that of the untreated material, with a presence of a wear-corrosion degradation mechanism in contrast with a wear-dominated regime for the untreated material. Finally, the cytotoxicity tests revealed satisfactory properties in terms of effects on survival and proliferative activity of human fibroblasts and Vero cells on the surface of the borided AISI 316 L steel.

Stabilization of Hypoxia Inducible Factor by Cobalt Chloride Can Alter Renal Epithelial Transport

Around 600,000 people in the United States alone have polycystic kidney disease (PKD) which is characterized by the formation of fluid‐filled pouch‐like structures, called cysts. These cysts tend to expand over the years, finally leading to kidney failure. Previously, kidney cysts were reported to be hypoxic [1]. Cyst expansion, stagnant fluid accumulation, and insufficient vascular supply can result in localized chronic ischemia‐hypoxia in cysts and as well as in normal epithelia adjacent to a cyst. We hypothesize that in normal epithelia near a cyst, the stabilization of Hypoxia Inducible Factor 1α (HIF1α), a major regulator of cellular response to hypoxia, can cause altered paracellular and transcellular transport in normal renal epithelia, transforming the normal absorptive epithelia to a secretory and leaky phenotype, leading to cyst expansion.Using cobalt chloride (CoCl2), HIF1α was stabilized in cellular nucleus of a mouse cortical collecting duct cell line (mCCD 1296 (d)), which resulted in an increased level of erythropoietin, an effector molecule of HIF1α. The mCCD monolayers have high transepithelial resistance (TER) value around 3000 Ω‐cm2 and around 95% amiloride sensitive voltage value. Equivalent current (Ieq) was calculated to compare active ion transport and FITC‐conjugated dextran molecules (3 and 70 kDa) were used to quantify transepithelial transport in terms of permeability values. MTT assay was used in order to make sure CoCl2 treated and untreated groups maintained same levels of metabolically active cells. Our results showed that TER values decreased significantly after 48 and 72 hours of HIF stabilization by CoCl2. The decrease of TER value was consistent with the increase in the permeability of large‐size 70 kDa FITC‐dextran molecules, supporting HIF stabilization actually altered paracellular transport. Western blot analysis showed HIF stabilization caused a significant decrease in the protein level of one of the major tight junction proteins, zonula occludin 1 (ZO1). Decrease in the ZO1 protein level was consistent with the decreased TER value and the increased paracellular permeability. HIF stabilization by CoCl2 for 48 hours caused almost complete loss of active sodium ion transport, and very interestingly, 72 hours of HIF stabilization caused a switch in the direction of active ion transport. Western blot analysis showed that HIF stabilization caused a decrease in the protein level of sodium‐potassium‐ATPase (Na+‐K+‐ATPase) α1 subunit, the catalytic subunit of the enzyme responsible for active sodium ion transport. Decreased level of Na+‐K+‐ATPase α1 subunit due to HIF stabilization was consistent with the loss of active sodium ion transport. Thus, HIF stabilization by CoCl2 can affect Na+‐K+‐ATPase α1 subunit and ZO1 protein levels, leading to alterations of transcellular and paracellular transport through normal kidney epithelial monolayers. Thus, our results indicate that localized ischemia‐hypoxia can transform a normal absorptive epithelia to a secretory and leaky epithelia and may contribute to cyst expansion.Support or Funding InformationThis research was supported by the Dr. John Vitullos Pilot and Bridge Funding Program at the Center for Gene Regulation in Health and Disease (GRHD), NIH DK092716 award (both to AR) and Cleveland State University Dissertation Research Award to SN.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal Epithelial Function Can Be Altered by Hypoxia Inducible Mechanisms

Polycystic Kidney Disease (PKD) is characterized by fluid filled cysts which interfere with renal function and can lead to kidney failure. In cystic epithelia, hypoxia inducible factor 1α (HIF1α) and HIF2α were reported to be elevated [1]. Cyst expansion, stagnant fluid accumulation, and insufficient vascular supply may result in localized chronic ischemia‐hypoxia in cysts and as well as in normal epithelia adjacent to a cyst. We hypothesize that in normal epithelia near a cyst, the stabilization of HIF1α, a major regulator of cellular response to hypoxia, can cause altered paracellular and transcellular epithelial transport. Thus, increased level of HIF1α can be a triggering factor in the transformation of normal renal epithelia from an absorptive to a secretory phenotype, leading to cyst expansion.Cobalt chloride (CoCl2) was used at a concentration of 100 μM to stabilize HIF1α in the cellular nucleus. Using western blots, we confirmed that increased HIF1α levels in the nuclear extracts by CoCl2 also increased cellular erythropoietin (EPO) levels. Application of a HIF1α inhibitor, methyl 3‐[[2‐[4‐(2‐adamantyl)phenoxy]acetyl]amino]‐4‐hydroxybenzoate was found to suppress the increased level of EPO expression by CoCl2. Transcellular and paracellular transport studies were performed in a mouse cortical collecting duct cell line (mCCD 1296 (d)) which has a high monolayer transepithelial resistance (TER) value around 3000 Ω‐cm2 and a detectable 95% amiloride sensitive voltage value. A few paracellular transport studies were also carried out in Mardin‐Darby Canine Kidney (MDCK) epithelial cell line which has relatively low monolayer TER value around 400 Ω‐cm2 with no detectable voltage value. Short circuit current (Isc) was calculated using Ohm's law (I = V/R) to compare active ion transport. TER and voltage values were recorded using an Endohm chamber. Higher TER values in mCCD cells can reflect reduced paracellullar permeability which was verified by permeability assay using FITC‐conjugated dextran molecules (3 and 70 kDa). MTT assay was used in order to make sure CoCl2 treated and untreated groups maintained same level of cellular viability. Cells exposed to CoCl2 showed altered active ion transport in mCCD cells. Treatment with CoCl2 caused increased 70 kDa FITC‐dextran paracellular permeability both in mCCD and MDCK monolayers. In MDCK cell line, we performed a paracellular permeability experiment in which we used the HIF1α inhibitor along with CoCl2. The HIF1α inhibitor was found to block the effect of CoCl2 treatment on FITC‐dextran permeability to a certain extent. Thus, CoCl2 can alter epithelial transport most likely due to hypoxia inducible mechanisms. Currently, we are trying to strengthen the connection between HIF1α levels and renal epithelial function using HIF1α inhibitors in active and passive transport experiments in the presence of CoCl2 or another HIFα stabilizer, the iron chelator desferrioxamine.Support or Funding InformationThe project is supported by National Institute of Diabetes and Digestive and Kidney Diseases 1R15DK092716 to AR and Cleveland State University Dissertation Research Award to SN.

Simultaneous targeted activation of Notch1 and Vhl-disruption in the kidney proximal epithelial tubular cells in mice.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, representing approximately 75% of all renal neoplasms. ccRCC is known to be strongly associated with silencing of the von Hippel Lindau (VHL) tumor suppressor gene, yet VHL deficiency alone does not seem to be sufficient to drive the oncogenic transformation of normal renal epithelium and induce renal tumorigenesis. We, and others, have previously suggested that constitutive activation of the Notch signaling pathway, alongside with VHL loss, contribute to the oncogenic features of ccRCC. Here we report a prevailing hyperactivation of the Notch1 receptor in human ccRCC relative to the healthy counterpart. To explore the consequences of the elevated Notch1 signaling observed in ccRCC patient material, we made use of a conditional mouse model based on concurrent ectopic expression of constitutively active Notch1 (NICD1) and deletion of the Vhl gene. Histological examination of the kidneys of the conditional mice demonstrate the existence of nests of dysplastic cells with a clear cytoplasm as a consequence of lipid accumulation, thus displaying a one important hallmark of human ccRCC.

Transient potential receptor channel 4 controls thrombospondin‐1 secretion and angiogenesis in renal cell carcinoma

Angiogenic switch in renal cell carcinoma (RCC) is attributed to the inactivation of the von Hippel-Lindau tumor suppressor, stabilization of hypoxia inducible factor-1 transcription factor and increased vascular endothelial growth factor. To evaluate the role of an angiogenesis inhibitor, thrombopsondin-1 (TSP1), we compared TSP1 production in human RCC and normal tissue and secretion by the normal renal epithelium (human normal kidney, HNK) and RCC cells. Normal and RCC tissues stained positive for TSP1, and the levels of TSP1 mRNA and total protein were similar in RCC and HNK cells. However, HNK cells secreted high TSP1, which rendered them nonangiogenic, whereas RCC cells secreted little TSP1 and were angiogenic. Western blot and immunostaining revealed TSP1 in the cytoplasm of RCC cells on serum withdrawal, whereas, in HNK cells, it was rapidly exported. Seeking mechanisms of defective TSP1 secretion, we discovered impaired calcium uptake by RCC in response to vascular endothelial growth factor. In HNK cells, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, a calcium chelator, simulated TSP1 retention, mimicking the RCC phenotype. Further analysis revealed a profound decrease in transient receptor potential canonical ion channel 4 (TRPC4) Ca(2+) channel expression in RCC cells. TRPC4 silencing in HNK cells caused TSP1 retention and impaired secretion. Double labeling of the secretory system components revealed TSP1 colocalization with coatomer protein II (COPII) anterograde vesicles in HNK cells. In contrast, in RCC cells, TSP1 colocalized with COPI vesicles, pointing to the retrograde transport to the endoplasmic reticulum caused by misfolding. Our study indicates that TRPC4 loss in RCC leads to impaired Ca(2+) intake, misfolding, retrograde transport and diminished secretion of antiangiogenic TSP1, thus enabling angiogenic switch during RCC progression.

Evaluation of S100A10, annexin II and B‐FABP expression as markers for renal cell carcinoma

This study aimed to analyze expression of S100A10, annexin II and B-FABP genes in renal cell carcinoma (RCC) and their potential value as tumor markers. Furthermore, any correlation between the gene expression and prognostic indicators of RCC was analyzed. Expression of each gene was estimated by RT-PCR in the non-neoplastic (normal) and tumorous parts of resected kidney samples. Also, each antigen was immunostained in RCC and normal kidney tissues. Expression of the S100A10 gene averaged 2.5-fold higher in the tumor than that in the normal tissues (n = 47), after standardization against that of beta-actin. However, expression of annexin II, a natural ligand of S100A10, was only 1.64-fold higher. In the tissue sections of RCC, S100A10 and annexin II were immunostained in membranes. In the normal renal epithelia, however, both antigens were stained in the Bowman's capsule and the tubules from Henle's loop through the collecting duct system, but not in the proximal tubules, from where most RCC are derived. In contrast, expression of the B-FABP gene was 20-fold higher in the tumor. No B-FABP was immunohistochemically detected in normal kidney sections, but it was stained in the cytoplasm of RCC tissue sections. S100A10 and B-FABP genes were overexpressed regardless of nuclear grade and stage of RCC. Immunopositivity in RCC tissues (n = 13) was 100% for S100A10 and annexin II, and 70% for B-FABP; however, no clear relationship was observed in either antigen with nuclear grade and stage. It was found that all three performed well as RCC markers. B-FABP was most specific to RCC, as it was expressed little in normal kidney tissues.

Ontogeny and Oncogenesis Balance the Transcriptional Profile of Renal Cell Cancer

Global transcript analysis is increasingly used to describe cancer taxonomies beyond the microscopic reach of the eye. Diagnostic and prognostic portraits are formulated by ranking cancers according to transcriptional proximity. However, the role that distinct biological factors play in defining these portraits remains undefined. It is likely that the transcriptional repertoire of cancers depends, on one hand, on the anamnestic retention of their ontogenesis and, on the other, on the emergence of novel expression patterns related to oncogenesis. We compared the transcriptional profile of primary renal cell cancers (RCCs) with that of normal kidney tissue and several epithelial cancers of nonrenal origin to weigh the contribution that ontogeny and oncogenesis make in molding their genetic profile. Unsupervised global transcript analysis demonstrated that RCCs retain transcriptional signatures related to their ontogeny and cluster close to normal renal epithelium. When renal lineage-associated genes are removed from the analysis and cancer-specific genes are analyzed, RCCs segregate with other cancers with limited lineage specificity underlying a predominance of the oncogenic process over lineage specificity. However, a RCC-specific set of oncogenesis-related genes was identified and surprisingly shared by sarcomas. In summary, the transcriptional portrait of primary RCCs is largely dominated by ontogeny. Genes responsible for lineage specificity may represent poor molecular targets for immune or drug therapy. Most genes associated with oncogenesis are shared with other cancers and may represent better therapeutic targets. Finally, a small subset of genes is associated with lineage-specific oncogenesis, and these may provide information regarding the biological behavior of RCCs and facilitate diagnostic classification of RCCs.

Epithelial cell adhesion molecule (KSA) expression: pathobiology and its role as an independent predictor of survival in renal cell carcinoma.

Epithelial cell adhesion molecule (EpCAM) is a widely expressed adhesion molecule in epithelial cancers. The purpose of this study is to determine the protein expression patterns of EpCAM in renal cell carcinoma (RCC) using tissue arrays linked to a clinicopathological database to evaluate both its predictive power in patient stratification and its suitability as a potential target for immunotherapeutic treatment strategies. The University of California, Los Angeles kidney cancer tissue microarray contains specimens from 417 patients treated with nephrectomy. EpCAM protein expression in tumors and matched morphologically normal renal tissues was evaluated using anti-EpCAM immunohistochemistry. The resultant expression reactivity was correlated with clinicopathological variables. EpCAM is consistently expressed in the distal nephron on normal renal epithelium. Clear cell RCCs show minimal and infrequent EpCAM expression, whereas chromophobe and collecting duct RCCs both demonstrate intense and frequent expression. Of 318 clear cell carcinomas used in the analysis, 10% were EpCAM positive in > or = 50% of cells, and 8% of patients would be considered candidates for EpCAM-based therapy, based on high expression [> or = moderate intensity and frequent (> or = 50%) expression] and the need for systemic treatment. EpCAM expression was an independent prognostic factor for improved disease-specific survival, with a multivariate hazard ratio of 0.63 (P = 0.017; 95% confidence interval, 0.43-0.92). EpCAM is a novel prognostic molecular marker in RCC patients, and its positive expression is an independent predictor associated with improved survival. However, high expression in morphologically normal renal tissues and minimal or absent expression in clear cell carcinomas will likely limit the utility of this epithelial marker in targeted treatments of this most common RCC type.

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)-γ 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-γ 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.

Targeting of tumor associated antigens in renal cell carcinoma using proteome-based analysis and their clinical significance.

The suitability of proteome-based strategies for the targeting of tumor-associated markers along with further analysis regarding their clinical significance were investigated in human renal cell carcinoma (RCC). The immunogenic protein expression profile of normal kidney and RCC cell lines was studied by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, also termed PROTEOMEX. Employing this approach, a series of proteins reactive with either RCC patient sera and/or reactive with control sera were identified by microanalysis of tryptic peptides. Some of these candidate antigens represent members of the cytoskeletal family, such as cytokeratins, in particular cytokeratin 8, cytoskeletal tropomyosin, F-actin capping protein, gamma-actin, stathmin, tubulin-alpha, tubulin-beta and vimentin. The expression pattern and clinical significance of three of these antigens, namely cytokeratin 8, stathmin and vimentin, were further analyzed in a large series of surgically removed RCC lesions of distinct subtypes. A heterogeneous expression pattern of cytokeratin 8, stathmin and vimentin was demonstrated in the different RCC subtypes. All epithelial cells of the autologous normal kidney showed a strong cytokeratin 8 staining pattern, whereas they totally lack vimentin expression. Stathmin was expressed in 10% of tubule cells. In conclusion, PROTEOMEX could be employed for the identification of tumor-associated antigens of the cytoskeleton which are differentially expressed in RCC of distinct subtypes as well as in normal renal epithelium.

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.

FREQUENTLY DELETED LOCI ON CHROMOSOME 9 MAY HARBOR SEVERAL TUMOR SUPPRESSOR GENES IN HUMAN RENAL CELL CARCINOMA

Loss of various loci on chromosome 9 has been reported in various cancers. To determine the frequency of deletions at different loci of chromosome 9 in renal cell carcinoma microdissected samples of normal renal epithelium and carcinoma from the same patients were analyzed. DNA was isolated from microdissected sections of normal and tumor cells of 60 renal specimens, amplified by polymerase chain reaction and analyzed for loss of heterozygosity on chromosome 9 using the 16 microsatellite markers D9S178, D9S157, D9S274, D9S168, D9S285, D9S156, D9S1839, D9S162, IFNA, D9S736, D9S171, D9S1749, D9S273D9S270, D9S153 and D9S170. Loss of heterozygosity was analyzed by a polymerase chain reaction based technique developed at our laboratory. This study showed a high incidence of loss of heterozygosity on chromosome 9 in renal cell carcinoma. Of 60 cases 44 (73%), 24 (40%) and 14 (23%) showed loss of heterozygosity at a minimum of 1, at a minimum of 3 and at 4 or more loci, respectively. The main deletion was found on the 9p21 region at loci DS171 in 38% of cases, D9S1749 in 42% and DS270 in 14%. Overall deletion on chromosome 9p21 was noted in 57% of renal cancer cases. Other deleted regions were on chromosome 9p'0022 to 23 at loci D9S157 in 37% of cases, D9S274 in 20%, D9S168 in 27%, D9S285 in 20%, D9S156 in 12%, D9S1839 in 17% and D9S162 in 24%. Overall deletion at chromosome 9q32 to 33 was noted in 46% of renal cell carcinoma cases. Chromosome 9q32 to 33 also showed deletion at locus D9S170 in 22% of renal cell carcinoma cases. When we compared the incidence of deletion at various loci on chromosome 9 according to renal cell carcinoma grade, we found a higher rate of deletion in advanced grades of renal cell carcinoma. A candidate target tumor suppressor gene, p16 (MTS-1/CDKN2), has been identified within the 9p21 deleted region in various cancers. In our study the expression of p16 protein was absent or low in renal cell cancer samples, suggesting that loss of the p16 gene may be involved in renal cell carcinogenesis. Our study demonstrates a high incidence of loss of heterozygosity on chromosome 9, mainly 9p21 and 9p22 to 23, in renal cell carcinoma, suggesting several putative tumor suppressor genes on these regions. The identification of other tumor suppressor genes on the 9p21 and 9p22 to 23 regions warrants further studies.

Mechanisms of epithelial development and neoplasia in the metanephric kidney.

Recent studies on the mechanisms of normal epithelial development in the kidney, and on the aetiology of renal neoplasms, are converging to reveal remarkably close relationships between the phenotypes and behaviours of normally-developing and neoplastic cells. Normal renal epithelia arise from two sources; those of the collecting duct system develop by arborisation of an initially-unbranched ureteric bud, in a manner similar to the development of other glandular organs, while epithelial nephrons develop via an unusual mesenchyme-to-epithelial transition. Both types of development require controlled proliferation, cell-cell and cell-matrix interactions, protease activity etc., but of the two tissues, the development of the nephrons is arguably the more complex. It includes many defined stages, signals and checkpoints that ensure that events happen at the right time, and that processes such as proliferation, apoptosis and differentiation are properly balanced. Detailed investigation of renal neoplasms has revealed some to be caused by mutations in molecules with known roles in normal nephrogenesis (e.g. Wilms' tumour and the WT-1 gene, renal cell carcinoma and the c-met receptor tyrosine kinase gene), some to be caused by mutations in genes expressed during normal development (e.g. renal cell carcinoma and the TSC-2 gene, renal cell carcinoma of the clear cell variety and the VHL gene). Furthermore, these and other tumours of unknown aetiology re-express genes such as Pax-2 that are expressed during the normal mesenchyme-to-epithelium transition but are shut off during terminal differentiation. Their re-appearance in tumours suggests that the cells have 'regressed' in an ontogenic sense, and their biology may therefore be understood most clearly by reference to the properties of normal developing cells rather than cells of a mature kidney.

Attenuated expression of epithelial cell adhesion molecules in murine polycystic kidney disease.

Polycystic kidney disease is an inherited disorder of parenchymal structure that leads to renal failure. Cysts begin as focal dilations in proximal tubules and collecting ducts, giving rise to cyst walls lined by a phenotypically disturbed epithelium that expresses dysfunctional transport and matrix proteins. We used an mRNA search protocol to probe efficiently for tissue-specific disturbances that might underlie the formation of cysts. This search assessed the relative abundance of transcripts encoding a variety of growth factors (transforming growth factor-beta 1, interleukin-6, tumor necrosis factor, and endothelin-1), structural proteins (collagen IV, nidogen, fibronectin, and laminins A and B1), and cell adhesion molecules (CAMs; E-cadherin, N-CAM, laminin receptor, and fibronectin receptor) in the cystic kidneys of cpk/cpk mice and uncovered a previously unrecognized early reduction in mRNA encoding N-CAM (54%) and E-cadherin (56%) (n = 5; P less than 0.001). Levels of transcripts for growth factors, structural proteins, and for fibronectin and laminin receptors in normal and cystic kidneys were generally similar. The reduction in transcripts for N-CAM and E-cadherin in kidneys from cystic mice was not observed in autologous liver. The immunofluorescent staining of cystic kidneys confirmed that the decrease in N-CAM and E-cadherin was generally confined to regions abundant in developing cystic epithelium. The presence of both N-CAM and E-cadherin appears to guide the sequential differentiation and polarization of normal renal epithelium, and their attenuated expression in the kidney of cpk/cpk mice may be a material factor contributing to the pathogenesis of cyst formation.