Aging-Related Mitochondrial Dysfunction Is Associated With Fibrosis in Benign Prostatic Hyperplasia

Abstract

Background: Age is the greatest risk factor for lower urinary tract symptoms attributed to benign prostatic hyperplasia (LUTS/BPH). Although LUTS/BPH can be managed with pharmacotherapy, treatment failure has been putatively attributed to numerous pathological features of BPH (eg, prostatic fibrosis, inflammation). Mitochondrial dysfunction is a hallmark of aging; however, its impact on the pathological features of BPH remains unknown.

Methods: Publicly available gene array data were analyzed. Immunohistochemistry examined mitochondrial proteins in the human prostate. The effect of complex I inhibition (rotenone) on a prostatic cell line was examined using quantitative polymerase chain reaction, immunocytochemistry, and Seahorse assays. Oleic acid (OA) was tested as a bypass of complex I inhibition. Aged mice were treated with OA to examine its effects on urinary dysfunction. Voiding was assessed longitudinally, and a critical complex I protein measured.

Results: Mitochondrial function and fibrosis genes were altered in BPH. Essential mitochondrial proteins (ie, voltage-dependent anion channels 1 and 2, PTEN-induced kinase 1, and NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial [NDUFS3]) were significantly (p < .05) decreased in BPH. Complex I inhibition in cultured cells resulted in decreased respiration, altered NDUFS3 expression, increased collagen deposition, and gene expression. OA ameliorated these effects. OA-treated aged mice had significantly (p < .05) improved voiding function and higher prostatic NDUFS3 expression.

Conclusions: Complex I dysfunction is a potential contributor to fibrosis and lower urinary tract dysfunction in aged mice. OA partially bypasses complex I inhibition and therefore should be further investigated as a mitochondrial modulator for treatment of LUTS/BPH. Hypotheses generated in this investigation offer a heretofore unexplored cellular target of interest for the management of LUTS/BPH.

Keywords: Complex I; Lower urinary tract symptoms; Oxidative phosphorylation; Prostate; Urology.

Figure 1.

Reduced accumulation of various markers of mitochondrial function in BPH prostate. VDAC1/2 (brown), PINK1 (red), and NDUFS3 (green) were all measured using IHC on normal adjacent and BPH prostate tissue samples (BPT = 48, BPH = 24). Using the Inform software, tissues were segmented into epithelial and stroma (Supplementary Material), scored to determine a positivity threshold, and calculated as percent positive of total cells (DAPI). Total expression was calculated by averaging the epithelial and stromal percent positivity of patients. Images were taken at 20× magnification, the scale bar is 100 microns (*p < .05; ****p < .0001). BPH = benign prostatic hyperplasia; BPT = benign prostatic tissue; IHC = immunohistochemistry; NDUFS3 = NADH dehydrogenase [ubiquinone] iron–sulfur protein 3, mitochondrial; PINK1 = PTEN-induced kinase 1; VDAC1/2 = voltage-dependent anion channels 1 and 2; DAPI = 4′,6-diamidino-2-phenylindole.

Figure 2.

Pharmacologic inhibition of complex I in prostate stromal cells promotes measures of fibrosis. Inhibition of complex I results in increased collagen expression and decreased cellular respiration. (A) ICC analysis of extracellular COL1A1 (green) was increased in 25 nM rotenone-treated BHPrS1 cells, an increase that was not seen when 100 nM oleic acid was also present. Nuclei were stained with DAPI (blue). Images were taken at 20× magnification. Green intensity was measured in ImageJ and normalized to the number of cells per field of view (*p < .05). (B) COL1A1 and COL3A1 gene expression was upregulated in rotenone-treated BHPrS1 cells and this was mitigated by 100 nM oleic acid as determined by qPCR (*p < .05, **p < .01, ns, not significant). (C) Seahorse mito stress test analysis of BHPrS1 cells showed a decrease in oxygen consumption rate for rotenone-treated cells, which was mitigated by methylene blue (MB). Oleic acid was able to mitigate the decrease as effectively as methylene blue, the known bypass agent (*p < .05 for DMSO vs 25 nM rotenone, #p < .05 for DMSO vs 25 nM rotenone + 100 nM oleic acid, & p < .05 for DMSO vs 25 nM Rotenone + 5 ng/mL MB). DMSO = dimethyl sulfoxide; ICC = immunocytochemistry; DAPI = 4′,6-diamidino-2-phenylindole.

Figure 3.

Oleic acid mitigates mitochondrial and urinary dysfunction seen in aged mice. (A) Percent positive NDUFS3 (green), cells a complex I marker, was decreased in aged mouse anterior prostate (A). This change was partially rescued by a 4-week treatment of oleic acid. (B) Images of the urogenital tract of young, aged, and oleic-acid-treated aged mice. (C) Oleic acid treatment did not affect the body weight at the time of euthanasia. (D) Bladder mass was significantly decreased in oleic acid treated mice. (E) Anterior prostate mass was not significantly altered in the oleic acid treated mice. (F) Void spot count was not different between the aged mice prior to treatment. After treatment, void spot count was significantly different between the treated and untreated group, as well as comparing the pretreatment and posttreatment counts of the oleic-acid-treated mice (*p < .05, ***p < .001, ns, not significant). AP = anterior prostate; DLP = dorsolateral prostate; SV = seminal vesicles; VP = ventral prostate; UGT = urogenital tract.