ROMANIAN ACADEMY INSTITUTE OF CELLULAR BIOLOGY AND PATHOLOGY „NICOLAE SIMIONESCU” THESIS Cellular and molecular mechanisms involved in the morphological and functional alterations of the renal glomerulus in aging and hypertension

The thesis entitled "Cellular and molecular mechanisms involved in themorphological and functional alterations of the renal glomerulus in aging andhypertension" is aimed to identify new molecular mechanisms associated with thedevelopment of cellular senescence of the renal glomerular cells in physiological agingand hypertension. The content of this thesis is structured in two parts: "The current knowledge" and “The original Contributions".Worldwide the proportion of people over 60 years is growing faster than anyother group. According to the World Health Organization (2012), by 2050 the populationof people over 60 will double, as compared with the year 2000. Aging is not a disease,but advancing age is associated with increased vulnerability to chronic diseases, suchas hypertension, atherosclerosis, heart failure. The health care needs and comorbidhealth problems of the elderly require a special focus, and impose a substantialpressure on publicly-funded healthcare system. The subject of the thesis research is important because the understanding of the mechanisms of aging will contribute todeveloping novel strategies for increasing the quality of life of the elderly, in order tomaximize their health and functional capacity, as well as their social participation.In physiological aging, morphological and functional changes of the kidneycontribute to the development of chronic kidney disease. The accumulation ofsenescent cells has been implicated in driving aging-related tissue alterations. Thus,targeting of senescent cells during the course of normal aging is expected to improvethe tissue milieu, thereby preventing the malfunction of the remaining nonsenescentcells. Recently, using a progeroid mouse model it has been shown that p16-positivesenescent cells drive age-related pathologies, and that selective removal of these cellscan delay tissue dysfunction and the aging phenotype (Baker et al., 2011). Studies onrenal cell senescence could lead to development of new therapeutic approaches fordelaying age-associated dysfunctions of the aging kidneys, extending the health span.The 1st part of the thesis is consisted of three chapters. Chapter I containsgeneral concepts on the kidney anatomy and physiology, with special emphasis on therenal glomerular anatomy and physiology, covering the architecture of the glomerularfiltration barrier, and the mechanism of glomerular ultrafiltration. Chapter II refers to thepathological changes of glomerular cells and related glomerular dysfunction. The alterations of the glomerular endothelial cells, such as alterations of their fenestratedphenotype, changes of the glycocalyx, or cellular alterations due to the blood flow stress are detrimental for the glomerular filtration. Also, the dysfunctional glomerular filtration barrier is caused by: i) structural changes in the glomerular basement membrane,ii) structural and functional alterations of podocytes (such as the effacement of theirpedicels, or the hypertrophy, or even the cells loss), and iii) morpho-functionalalterations in the mesangial region (the proliferation, or apoptosis of the mesangial cells,and mesangial hyperplasia). In Chapter II are highlighted the morphological andfunctional alterations of the renal glomeruli in aging and hypertension. Assuming that one of the leading cause of changes in the glomerular basement membranearchitecture, and of the podocytes’ alterations in the aging kidney is represented by theaccumulation of the senescent cells, in the Chapter III are revealed those proteinsinvolved in intracellular signaling responsible for the senescent phenotype: the markersand effectors of cellular senescence. In this chapter is highlighted the relevance ofcellular senescence in the physiological aging process. In its "early" stage, the cellular senescence is a protective mechanism against oncogenic conversion of the cells, butexacerbation of the cell senescence-associated secretory phenotype has negative impact on the neighboring non-senescent cells. The second part, named Original contributions, comprises the original resultsobtained in three studies focused on: 1) identifying senescent cells in renal glomeruli from 24 months old Golden Syrian hamsters (aged hamsters), or from hamsters withL-NAME-induced hypertension; 2) deciphering the cellular and molecular mechanisms involved in the setting of the cellular senescence in renal glomeruli, and 3) theimprovement of the oxidative stress induced alterations in renal cortex, by Zofenopriladministration to L-NAME-induced hypertension rats.In the first study, named "Cellular and molecular mechanisms involved in cellsenescence in the kidney glomerulus in aging", we have investigated the molecularmechanisms involved in the onset of cellular senescence in physiological aging,targeting the development of new therapeutic strategies to delay the occurrence ofchronic renal disease for the improvement of the quality of life of older adults. We usedas experiment model the Golden Syrian hamsters from which we harvested the kidneys.The experiments were performed on 24 months old (considered aged hamsters), and4 months old (young) hamsters, respectively, and the renal cortex was processed for:i) light microscopy (on frozen and paraffin sections), and electron microscopy, foridentifying glomerular cells alterations in aging, mitochondrial dysfunctions,endoplasmic reticulum stress, and the autophagy; ii) the activity of the lysosomal β-galactosidase, known as senescence-associated β-galactosidase (SAβ-gal), at pH 6; iii) the immunochemical labeling of the proteins of interest, either by Western blotting,or by immunohistochemistry (fluorescence microscopy); iv) estimation of the reactiveoxygen species production, related to the oxidative modification of proteins, and thestress-activated signaling pathways involved in the regulation of the cell senescence.Histological examination of the kidney sections stained with hematoxylin-eosin,PAS, Masson’s trichrome, or methenamine silver, revealed the presence of the focal and segmental glomerulosclerosis, the excessive accumulation of type IV collagen andthe thickening of the basement membrane in the glomeruli from aged hamsters. Theactivity of SA β-gal at pH 6 revealed the presence of senescent cells in vivo, in renalglomeruli from aged hamsters (Fig. 1, left). So far, there are only few data reporting thepresence of senescent cells in the kidney. Senescent cells are irreversible growth-arrested in the G1phase of the cell cycle, and p16, a cyclin-dependent kinaseinhibitor, enforces growth arrest, being recognized as a biomarker for senescence. Theincreased expression of p16 in the renal cortex from aged hamsters was evidenced by Western blotting, and by immunofluorescence microscopy (Fig. 1, right). Fig.1. Senescent cellsin renal glomeruli fromaged hamsters. Left:blue color is indicatingthe senescent cellspositive for SA β-galactivity at pH 6. Right:p16 positive cells(green; labeled withAlexa 488). Senescent cells usually adopt a flattened appearance in vitro, and, while not dividing, remain metabolically active, displaying characteristic changes in geneexpression. These cells are characterized by a pro-inflammatory phenotype, possiblyleading to detrimental effects on tissue homeostasis (Passos et al., 2010). Theincreased expression of p27 and p21 (the Cip / Kip family of cell cycle inhibitorswhich get in the way of the cell cycle G1 phase complexes arresting the cell cycle beforeS phase) has revealed the permanent growth arrest of the renal cells from agedhamsters in the G1 phase of the cell cycle, figuring the senescence of these renal cells.Also, the Western blot studies indicate the tissue accumulation of CDK4 in the renalcortex of the aged hamsters. Senescence is a cellular response to various stress factors that induce the DNA damages, and post-translational modifications of the protein, governing the activation ofthe two main signaling pathways mediated by ARF/p53/p21 and p16/pRb.Determination of the reactive oxygen species (ROS) production in the renal cortexhomogenates from young and old hamsters showed increased generation of ROS in oldhamsters that could be in charge for the launching of cellular senescence in renalglomeruli. The rising of the oxidative stress in the kidney of the aged hamsters may be the cause of the increased protein expression of p53, a redox-sensitive transcriptionfactor involved in the cell decision to become senescent.The alarmin HMGB1 ("high mobility group box 1") is another protein recentlyidentified as a mediator of senescent phenotype due to the role it plays in the chromatinremodeling and for its property to enable the transcription factors to bind the nuclearDNA, (Davalos et al. , 2013). In early phases of senescence, HMGB1 translocate fromthe nucleus to the cytosol, subsequently being secreted outside the cell, bothtranslocation of HMGB1 from the nucleus, and its redistribution into the extracellular space depends on p53 activation. Taking into account the involvement of HMGB1 insenescence, we showed the reduction of the HMGB1 protein expression in the renalcortex from the aged hamsters (Fig. 2, left, Western blot) and the translocation ofHMGB1 from the nucleus to the cytosol of the glomerular cells from aged hamsters (Fig.2, right, immunofluorescence ).