Intracellular Mechanisms of Cyclosporin A–Induced Tubular Cell Apoptosis

Abstract

ABSTRACT. Tubular cell apoptosis contributes to the pathogenesis of renal injury. However, the intracellular pathways that are active in tubular epithelium are poorly understood. The lethal pathways activated by cyclosporin A (CsA), a nephrotoxin that induces caspase-dependent apoptosis in tubular epithelium, were explored. Fas expression, caspase activation, and mitochondrial injury were assessed by Western blot, flow cytometry, and microscopy in cultured murine tubular epithelial cells exposed to CsA. The influence of FasL antagonists, Bax antisense oligodeoxynucleotides, and caspase inhibitors on cell survival was explored. Tubular cells constitutively express FasL. CsA increased the expression of Fas. However, Fas had no role in CsA-induced apoptosis, as CsA did not sensitize to FasL-induced apoptosis, caspase-8 activity was not increased, and neither blocking anti-FasL antibodies nor caspase-8 inhibition prevented CsA-induced apoptosis. Apoptosis induced by CsA is associated with the translocation of Bax to the mitochondria and Bax antisense oligodeoxynucleotides protected from CsA-induced apoptosis. CsA promoted a caspase-independent release of cytochrome c and Smac/Diablo from mitochondria. CsA also led to a caspase-dependent loss of mitochondrial membrane potential. Caspase-2, caspase-3, and caspase-9 were activated, and specific caspase inhibitor prevented apoptosis and increased long-term survival. Evidence for endoplasmic reticulum stress, such as induction of GADD153, was also uncovered. However, endoplasmic reticulum-specific caspase-12 was not activated. CsA induces changes in several apoptotic pathways. However, the main lethal apoptotic pathway in CsA-exposed tubular epithelial cells involves mitochondrial injury.

Mechanisms of Renal Apoptosis in Health and Disease

“…65 CsA increases Fas expression in tubular cells in culture and in vivo 66,67 ; however, neither neutralizing anti-FasL antibodies nor caspase-8 inhibitors decrease apoptosis induced by CsA. 67 Similar observations were made with acetaminophen. 68 This suggests some changes in apoptosis-related molecules are epiphenomena not directly related to cell death.…”

“…By contrast, Bax-mediated mitochondrial injury and caspase activation are key events in CsA-induced apoptosis of tubular cells. 67,69,70 CsA induces Bax aggregation and translocation to mitochondria, causing permeabilization of the outer mitochondrial membrane, re-lease of cytochrome c and SMAC/DIA-BLO, and activation of caspase-9 and -3. Initiator caspase-2 is also activated and may lead to mitochondrial injury.…”

Role of mitochondrial dysfunction in cellular responses to S-(1,2-dichlorovinyl)-l-cysteine in primary cultures of human proximal tubular cells

“…Taking into account the amount of cells and volumes used in the assays with CsA and that 1 × 10 6 hPT cells has about 1 mg of protein [30,32], the concentration of CsA used here to assess the role of the MPT in DCVC-induced cytotoxicity was approximately 0.8 μM. In contrast, Justo et al [38] demonstrated renal tubular cell apoptosis at CsA concentrations of 0.8 to 8 mM, which is three-to four-orders of magnitude higher than the concentration used here. Hence, CsA-induced cytotoxicity was not a confounding effect in the present studies.…”

“…Thus, cyclosporine A (CsA) was used to block the mitochondrial membrane permeability transition (MPT), ruthenium red (RR) was used to inhibit the calcium uniporter, thereby preventing calcium ion cycling, and PK11195 [1-(2-chlorophenyl)-N-(1-methylpropyl)-3-isoquinoline-carboxamide] was used as an antagonist of the mitochondrial or peripheral benzodiazepine receptor (mBzR), which is a component of the permeability transition pore. Whereas protection from mitochondrial dysfunction and cell injury by CsA has been used as a marker for involvement of the MPT for many years [37], CsA is also known to cause renal tubular cell apoptosis [38]. In the present study, as in the original work demonstrating inhibition of the MPT [37], we used a CsA concentration of 0.5 nmol/mg protein.…”

PPAR-γ agonist rosiglitazone protects peritoneal membrane from dialysis fluid-induced damage

“…Apoptosis was quantified assessing the DNA content by flow cytometry using Cellquest software (BD Biosciences). 36,37 Statistical Analysis Results are presented as 25th and 75th percentiles, median, minimum, and maximum values in box plots graphics of Figures 1, 2, 4-7. In Figures 8-10, bar graphics depict mean ± s.e.…”