title: Testosterone promotes apoptotic damage in human renal tubular cells.
abstract: BACKGROUND
  Apoptosis is a mode of cell death that participates in the kidney physiologic remodeling processes and is thought to contribute to cell loss and kidney structural damage in chronic renal diseases. Gender is one factor which contributes to accelerated nephron loss, with progression more rapid in men than in women in diabetic and nondiabetic chronic renal diseases. Mechanisms by which androgens may cause higher rate of progression of chronic renal diseases in men are poorly explored.
  
  
  METHODS
  In this study, to investigate the role of androgens on apoptotic damage and its associated mechanisms, we examined the effects of testosterone (T) (0.1 nmol/L to 1 micromol/L) on apoptosis, and apoptosis-related proteins in a proximal human tubule cell line (HK-2 cells). Additional experiments were performed in primary cultures of proximal tubular epithelial cells (PTECs). Cells were grown to subconfluence in normal growth medium, and apoptotic damage was induced by serum deprivation for 24 to 48 hours. Cycloheximide, flutamide (a T-receptor antagonist), 17-beta estradiol, or caspase inhibitors were added to cultures that were successively processed for terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end-labeling (TUNEL) analysis, annexin V/propidium iodide staining, immunofluorescence, or immunoblots to identify effects and apoptotic pathways that could be modulating cell survival.
  
  
  RESULTS
  Both morphologic analysis by annexin V/propidium iodide staining and TUNEL showed that physiologic T levels (1 to 10 nmol/L) induced a significant increase in apoptosis both in HK-2 cells and PTECs. In both types of cell lines pretreatment with the androgen receptor antagonist flutamide prevented the T-induced apoptosis. T-induced apoptosis was enhanced by treatment with cycloheximide and prevented by 17beta-estradiol. Fas, Fas ligand (FasL), and Fas-associating death domain containing protein (FADD) were clearly up-regulated within 48 hours of T treatment in HK-2 cells. Also, T significantly increased the expression of Bax protein (P < 0.01 vs. control) (an effect which was blocked by flutamide), and decreased the expression of Bcl-2. Western blot analysis showed that caspase-3 was activated. Moreover, cleavage into an 85-kD poly(ADP-ribose) polymerase-1 (PARP-1) terminal breakdown product was detectable. The changes in cellular morphology induced by T at 48 hours were no longer observed after the addition of caspase-8, caspase-9, and caspase-3 inhibitors to the culture medium.
  
  
  CONCLUSIONS
  These results indicate that T increases the permissiveness of proximal tubule kidney cells to apoptotic effects by triggering an apoptotic pathway involving caspase activation, Fas up-regulation, and FasL expression, thus potentially interacting with mechanisms of cell loss which have been already shown to be activated in chronic renal diseases. This is consistent with a role for T in promoting renal injury in men.
authors.affiliation: Nephrology Division, Department of Internal Medicine and Urology Division, University of Genoa, Genoa, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Verzola
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Gandolfo
authors.forename: Francesca
authors.initials: F
authors.lastname: Salvatore
authors.forename: Barbara
authors.initials: B
authors.lastname: Villaggio
authors.forename: Fabio
authors.initials: F
authors.lastname: Gianiorio
authors.forename: Paolo
authors.initials: P
authors.lastname: Traverso
authors.forename: Giacomo
authors.initials: G
authors.lastname: Deferrari
authors.forename: Giacomo
authors.initials: G
authors.lastname: Garibotto
mesh_terms: D000728:Androgens / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D029681:Arabidopsis Proteins / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation; D053222:Fas Ligand Protein; D044943:Fatty Acid Desaturases / Q000378:metabolism; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013739:Testosterone / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000378:metabolism
chemical_list: D000728:Androgens; D029681:Arabidopsis Proteins; C494826:BAX protein, human; D061945:Caspase Inhibitors; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D013739:Testosterone; D044943:Fatty Acid Desaturases; C084083:Fad7 protein, Arabidopsis; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases