Bone morphogenetic protein (BMP-7) και μοριακά δίκτυα δυνητικών stem κυττάρων στο νεφρό

Abstract

BMP-7 is a key molecule during kidney embryogenesis, and later is considered a renoprotective factor. The kidney is characterized by a unique plasticity between epithelial and mesenchymal cells. In developing kidney, the epithelium of the nephron is created through the process of Epithelial to Mesenchymal Transition (MET-Mesenchymal Epithelial Transition). In this process BMP-7 plays a pivotal role. The reverse process - Epithelial to Mesenchymal Transition (EMT) contributes the creation of mesenchymal cells - fibroblasts with the potential outcome of renal fibrosis that manifests clinically as renal failure. Besides the tissue reaction after injury, EMT and MET are actively involved in the process of carcinogenesis and metastasis. Stem cells are involved in tissue homeostasis and regeneration after injury as well. Cells with the unique properties of stem cells (self-renew and plurypotency), are also involved in carcinogenesis and metastasis. These unique stem cell properties are regulated by molecular networks in which the transcription factors OCT4 SOX2, Nanog, Polycomb group proteins (EZH2), and microRNAs, pose a critical role. The BMP signaling pathway participates actively in reprogramming somatic cells into mature cells with stem cells properties (iPS). Former publications have recognized potential stem cells in the kidney. Recent studies suggest the involvement of stem cells during regeneration after renal injury, while the exact molecular events within this process remain to be clarified. The addressed issue of renal stem cells is an open and promising research field. In this study, we investigated with a morphological method (immunohistochemistry) in renal tissue in situ, the possible correlation between the molecules of BMP signaling pathway with known key factors in stem cell molecular networks. We used antibodies against the renoprotective factor BMP-7, its receptor BMRRIB and its antagonist gremlin. Furthermore we used antibodies against the transcription factors OCT4 and SOX2, the member of the Polycomb group proteins EZH2, which is involved in the epigenetic regulation of transcription and a known stem cells marker, aldehyde dehydrogenase ALDH1, which is associated with retinoic acid, which plays a key role in determining stem cell fate. We obtained tissue samples from 49 patients (30 men, 19 women) aged 50 to 79 years. The immunohistochemical technique was applied to the surgical material of renal clear cell carcinoma. The biopsy samples contained both neoplastic and adjacent to the tumor non-neoplastic the renal tissue. In both compartments it is possible to identify locations of potential stem cells (somatic stem cells, cancer stem cells). The expression of the investigated factors was studied in both tumor tissue (tumor cells) and in the counterpart non-neoplastic renal tissue, we assessed the potential distribution in renal epithelial tubular cells, the glomeruli and the interstitium. We also assessed the precise location of the studied factors in the cells (nucleus, cytoplasm, cytoplasmic membrane). The results of the samples from non-malignant renal parenchyma and carcinoma of the same patient were compared with Wilcoxon statistical analysis test. The correlations between the expression of different markers, in the same samples was tested by Spearman Correlation test. For the multiple group comparison, we used the Kruskal Wallis statistical test. The categorized variables were audited by chi square. In all tests, p value <0,05 was considered statistically significant. The statistical analysis was performed with the SPSS program 16.0 for windows. All present indicators are expressed in varying degrees, both in tumor tissue and in the adjacent non-neoplastic kidney tissue. In the counterpart non-neoplastic kidney tissue immunohistochemical nuclear positivity was detected mainly in single tubular cells. BMP-7 expression was week in tubular epithelial cells of the counterpart kidney tissue. Its receptor, BMPRIB was expressed in the cytoplasmic membrane in the cytoplasm and in the nucleus of the tubular epithelial cells. The clear presence of BMPRIB in the proximal tubular cells, may support a possible effect of BMP-7 in these cells. Furthermore, gremlin immunohistochemical positivity was also expressed in the cytoplasm of non-malignant kidney tissue, particularly in areas of fibrosis. In fact, gremlin may exacerbate the decrease in the presence of BMP-7, since it antagonizes BMP-7 expression. Acknowledged positive nuclear expression of EZH2 and OCT4 (cytoplasmic co-expression) was noted in single tubular cells. SOX2, although it is considered a transcription factor, it was found in the cytoplasm and not in the nucleus. Its nuclear expression in the nervous tissue was used as an internal control. Basically, it showed strong cytoplasmic staining in the renal tubuli of the counterpart renal tissue. This probably indicates that SOX2 is not a stem cell marker, but more likely it indicates later stages of cell differentiation in the kidney, maybe final differentiated tubular epithelial cells. ALDH1, showed strong cytoplasmic localization in the tubular epithelial cells, suggesting that its expression is not restricted to stem cells but also occurs in more differentiated tubular epithelial cells. Regarding the neoplastic tissue, there was weak cytoplasmic positivity for BMP-7, but with a clear concomitant positivity for the individual molecules of the signaling pathway of BMP-7 that were studied (BMPRIB and gremlin) and for the potential stem cell markers. Positive staining was recognized for the BMPRIB membrane receptor in the cytoplasm and cytoplasmic membrane of tumor cells, while an interesting and unexpected finding was the nuclear localization of BMPRIB in the tumor tissue, especially in the peripheric region compared to more central areas of the tumor. Gremlin was expressed in the cytoplasm of cancer cells. EZH2 expression was noted in the nuclei of cancer cells, while in terms of topographic localization in the tumor, it showed a higher incidence of positive nuclei in the periphery region compared to more central areas where the positivity was minimal. Notable was the EZH2 positivity in a large percentage of tumor cells in apparent invasive front positions. Both nuclear and cytoplasmic expression for OCT4 was found in neoplastic and non-neoplastic tissue. The tumor cells expressed greater density of nuclear positivity in the periphery region compared to more central areas of the tumor. The immunohistochemical expression of SOX2, was extensively cytoplasmic in the tumor tissue. ALDH1 was widely over-expressed in the cytoplasm of the tumor cells in ccRCC, compared with the other studied factors. Although ALDH1 is been used as a general marker for the identification and isolation of stem cells in various organs, it can be argued that it indicates adult or cancer stem cells in clear cell carcinoma of the kidney. The results from non-malignant renal parenchyma and carcinoma of the same patient were compared using the statistical test Wilcoxon. Nuclear expression of BMPRIB, is increased in the tumor compared with the tubular cells of the adjacent renal parenchyma and the increasion is statistically significant (Wilcoxon p<0,001). Moreover, we found a statistically significant increase between the expression of gremlin in the counterpart kidney tissue in relation to the tumor tissue. (Wilcoxon p<0,001). Regarding the correlation of the studied factors with clinicopathological parameters, this study has not demonstrated a statistically significant difference in the expression of individual factors in the tumor tissue between men and women. The statistical evaluation of the expression of the individual factors in relation to the different tumor grade (Grade) showed a statistically significant correlation only for the factor EZH2 (p<0,007). It was found that with increasing tumor Grade, the immunohistochemical expression of EZH2 in tumor cells was weaker. The statistical Spearman correlation analysis, shower positive correlations between several markers of this study. In the tumor cells, a positive correlation was revealed between OCT4 and BMPRIB (intensity of nuclear expression p<0,015, r=0,48, intensity of cytoplasmic expression p<0,016, r=0,361). Positive correlation was also found in tumor cells between OCT4 and EZH2 (nuclear expression in the periphery of the tumor p<0,037, r=0,382). Moreover, a positive correlation concerning the tumor cells, was found between gremlin and SOX2 (intensity cytoplasmic expression p<0,002, r=0,467, and cytoplasmic expression p<0,019, r=0,334), and between SOX2 and BMPRIB (intensity of cytoplasmic expression p<0,05, r=0,293). Regarding the renal tubular epithelial cells in the counterpart kidney tissue, a positive correlation between SOX2 and BMPRIB (cytoplasmic expression of p<0,035, r=0,338) was found. In terms of the glomeruli in the counterpart kidney tissue, it showed no statistically significant correlation between the studied factors. These statistical correlations were performed in order to investigate any possible "cross-talk" between the selected markers in the present study. The conclusions should be evaluated, having in mind that the immunohistochemical method provides only the information of the coexistence of the studied factors in cancer and in the counterpart renal tissue, and not of the possible functional interactions that may take place. From the present study, we concluded probable "cross-talks" between factors of the BMP-7 signaling pathway, with stem cell markers OCT4 and SOX2. These correlations, combined with the interesting co-expression of BMPRIB, OCT4 and EZH2, mainly at the periphery of the tumor, can probably lead to the conclusion that these markers may act in common signal transduction networks. However, the correlation of the immunohistochemical expression of BMPRIB and the above potential stem cell markers, involves the risk of oversimplification, due to the dense network signaling that these factors are involved. The statistical correlation test results, despite the risk of overestimation, probably suggest an interaction and interdependence of these molecular networks in physiological and pathological processes in the kidney. In the counterpart renal tissue, as mentioned above, the immunohistochemical nuclear positivity for OCT4, EZH2 and BMPRIB that was detected mainly in single tubular cells may suggest a possible presence of potential stem cells in these positions. Regarding the tumor tissue, we detected immunohistochemical nuclear positivity for OCT4, EZH2 and BMRRIB with a noted distribution in the tumor periphery region compared to more central areas. Interesting was the finding of EZH2 positivity in a large percentage of tumor cells in invasive front positions. The unexpected finding of nuclear localisation of the membrane receptor BMRRIB, is consistent with a similar finding for a well-known cytoplasmic protein (beta-catenin) that was found in the nucleus of cells in the invasive edge in colon cancer. These cells have been identified as cancer stem cells, which have undergone EMT process. In the present study, the application of the statistical correlation Spearman test, strengthened the morphological findings on OCT4, EZH2 and BMRRIB, revealing positive correlations between OCT4 and BMRRIB (p<0,015) and OCT4 and EZH2 (p<0,037). All these support the possibility of the presence of potential cancer stem cells in renal clear cell carcinoma.

Η BMP-7 αποτελεί μόριο κλειδί στην νεφρογένεση και την νεφροπροστασία, μέσω ΜΕΤ. Η αντίστροφη διαδικασία EMT συμβάλει στην νεφρική ίνωση. Οι EMT και MET ενέχονται επίσης στην καρκινογένεση και την μετάσταση. Τα stem κύτταρα συμμετέχουν στην ιστική επανόρθωση μετά από βλάβη, επίσης εμπλέκονται στις διαδικασίες καρκινογένεσης και μετάστασης. Οι μοναδικές ιδιότητες των stem κυττάρων (αυτό-ανανέωση και πολυδυναμία) καθορίζονται από μοριακά δίκτυα στα οποία πρωταγωνιστικό ρόλο παίζουν οι μεταγραφικοί παράγοντες Oct4, Sox2 οι πρωτεΐνες της ομάδας Polycomb όπου ανήκει ο EZH2 και microRNAs. Η σηματοδοτική οδός της BMP συμμετέχει κατά τον επαναπρογραμματισμό ώριμων σωματικών κυττάρων σε κύτταρα με ιδιότητες stem κυττάρων (iPS). Πρόσφατες μελέτες εισηγούνται την συμμετοχή stem κυττάρων κατά την αποκατάσταση της νεφρικής βλάβης και την κυτταρική αναγέννηση στον νεφρό. Ένα ανοικτό προς διερεύνηση, κλινικά πολλά υποσχόμενο πεδίο. Διερευνήθηκε με μορφολογική (ανοσοϊστοχημική) μέθοδο σε νεφρικό ιστό από υλικό νεφρεκτομής λόγω διαυγοκυτταρικού καρκινώματος του νεφρού, η πιθανή συσχέτιση μορίων της σηματοδοτικής οδού BMP με παράγοντες κλειδί σε μοριακά δίκτυα stem κυττάρων. Χρησιμοποιήθηκαν αντισώματα για την BMP-7, ΒΜΡR1B, γκρεμλίνη, Oct4, Sox2, EZH2 και ALDH1. Μελετήθηκε η νεοπλασματική μάζα και ο παρακείμενος του νεοπλάσματος, μη νεοπλασματικός νεφρικός ιστός. Όλοι οι υπό εξέταση δείκτες εκφράζονται, σε άλλοτε άλλο βαθμό, τόσο στο νεοπλασματικό όσο και στον παρακείμενο του όγκου νεφρικό ιστό (κυρίως στα επιθηλιακά σωληναριακά κύτταρα). Στον μη νεοπλασματικό ιστό, διαπιστώθηκε ασθενής κυτταροπλασματική έκφραση της BMP-7. Ο ΒΜΡR1B εκφράζεται στην κυτταροπλασματική μεμβράνη, στο κυτταρόπλασμα και στον πυρήνα. Η σαφής παρουσία του στα σωληναρικά κύτταρα, μπορεί να υποστηρίζει την ενδεχόμενη δράση της BMP-7 στα εν λόγω κύτταρα. Αναγνωρίσθηκε θετικότητα για την γκρεμλίνη στο κυτταρόπλασμα σε ινωτικές κυρίως περιοχές, που ίσως επιτείνει την ελάττωση της παρουσίας της BMP-7 αφού ανταγωνίζεται την έκφραση της. Θετική πυρηνική έκφραση EZH2 και Oct4 (εδώ και κυτταροπλασματική) σε μεμονωμένα κύτταρα σωληναρίων και σπειραμάτων. Το Sox2 παρότι ανήκει στους μεταγραφικούς παράγοντες εντοπίστηκε στο κυτταρόπλασμα και δεν αφορά stem κύτταρα στο νεφρό αλλά πιθανόν τελικώς διαφοροποιημένα σωληναριακά κύτταρα. Για την ALDH1 έντονη κυτταροπλασματική εντόπιση στα πιο σωληναριακά κύτταρα. Στο νεόπλασμα, διαπιστώθηκε ασθενής κυτταροπλασματική θετικότητα για την BMP-7 με σαφή θετικότητα για την γκρεμλίνη και τον ΒΜΡR1B και τους δυνητικούς δείκτες stem κυττάρων. Απροσδόκητο το εύρημα της πυρηνικής εντόπισης του ΒΜΡR1B στην περιφέρεια σε σχέση με κεντρικότερες περιοχές του όγκου. Θετικότητα (πυρηνική) για EZH2 και Oct4 σε θέσεις διήθησης. Η ALDH1 υπερ-εκφράζεται σε μεγάλο ποσοστό στο κυτταρόπλασμα στο RCC, και δεν υποδηλώνει θέσεις φυσιολογικών ή καρκινικών stem κυττάρων. Όσο αυξάνεται το grade του νεοπλάσματος, τόσο μειώνεται η έκφραση του EZH2. Η στατιστική ανάλυση ανέδειξε θετική συσχέτιση μεταξύ Oct4 και ΒΜΡR1B. Oct4 και EZH2 (πυρηνική έκφραση στον όγκο). Επιπλέον μεταξύ Sox2 και γκρεμλίνης και Sox2 και ΒΜΡR1B (κυτταροπλασματική έκφραση, όγκος) και μεταξύ Sox2 και ΒΜΡR1B (κυτταροπλασματική έκφραση, παρακείμενος νεφρικός ιστός). Προκύπτει το συμπέρασμα πιθανής διασυνομιλίας του ΒΜΡR1B με τους Oct4 και EZH2 στην περιφέρεια κυρίως του όγκου.