Increased Apoptosis in Cystinotic Fibroblasts and Renal Proximal Tubule Epithelial Cells Results from Cysteinylation of Protein Kinase Cδ

Park, Margaret A.; Pejovic, Vojislav; Kerisit, Kathryn G.; Junius, Sacha; Thoene, Jess G.

doi:10.1681/asn.2006050474

Cited by 75 publications

(65 citation statements)

References 38 publications

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“…40,42,46 Apoptosis is thought to involve the activation of caspases as well as a stereotyped pattern of mitochondrial alterations, namely release of cytochrome c and mitochondrial membrane potential dissipation that contribute to the acquisition of the apoptotic morphology. Apoptosis rate determined by caspases-3 activity in cystinotic RPTE cells has been reported to be significantly increased both with and without an apoptosis trigger.…”

Section: Discussionmentioning

confidence: 99%

“…It has been shown that protein kinase C␦ forms disulfide bonds specifically with cystine that is released from lysosomes in cultured cystinotic fibroblasts and RPTE cells during apoptosis. 46 Also, loss of mitochondria integrity, as measured by cytochrome c release, has been shown to occur downstream of lysosomal permeability in cystinotic cells. 46 Accumulating evidence suggests that different intracellular organelles contribute synergistically to the initiation of apoptosis by specific stress inducers.…”

Section: Discussionmentioning

confidence: 99%

“…46 Also, loss of mitochondria integrity, as measured by cytochrome c release, has been shown to occur downstream of lysosomal permeability in cystinotic cells. 46 Accumulating evidence suggests that different intracellular organelles contribute synergistically to the initiation of apoptosis by specific stress inducers. It is evident from the electron micrographs that the mitochondria in cystinotic cells are abnormal in structure and vary in size.…”

Section: Discussionmentioning

confidence: 99%

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Mitochondrial Autophagy Promotes Cellular Injury in Nephropathic Cystinosis

Sansanwal¹,

Yen

Gahl

et al. 2010

Journal of the American Society of Nephrology

127

113

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The molecular and cellular mechanisms underlying nephropathic cystinosis, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. Renal biopsies from patients with this disorder can reveal abnormally large mitochondria, but the relevance of this and other ultrastructural abnormalities is unclear. We studied the ultrastructure of fibroblasts and renal proximal tubular epithelial cells from patients with three clinical variants of cystinosis: Nephropathic, intermediate, and ocular. Electron microscopy revealed the presence of morphologically abnormal mitochondria and abnormal patterns of mitochondrial autophagy (mitophagy) with a high number of autophagic vacuoles and fewer mitochondria (P Ͻ 0.02) in nephropathic cystinosis. In addition, we observed increased apoptosis in renal proximal tubular epithelial cells, greater expression of LC3-II/LC3-I (microtubule-associated protein 1 light chain 3), and significantly more autophagosomes in the nephropathic variant. The autophagy inhibitor 3-methyl adenine rescued cell death in cystinotic cells. Cystinotic cells had increased levels of beclin-1 and aberrant mitochondrial function with a significant decrease in ATP generation and an increase in reactive oxygen species. This study provides ultrastructural and functional evidence of abnormal mitophagy in nephropathic cystinosis, which may contribute to the renal Fanconi syndrome and progressive renal injury.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Mitochondrial Autophagy Promotes Cellular Injury in Nephropathic Cystinosis

Sansanwal¹,

Yen

Gahl

et al. 2010

Journal of the American Society of Nephrology

127

113

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“…Cystinotic cells present alterations in glutathione metabolism (10 -13), which can result in enhanced oxidative cell damage. Furthermore, increased cysteinylation of proapoptotic protein kinase C␦, make cystinotic cells more sensitive for apoptotic stimuli (14). An increase in mitochondrial and cytosolic reactive oxygen species decreased intracellular ATP and increased sensitivity to apoptosis may all cooperate in the pathogenesis of cystinosis in a complex mechanism.…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial Complex V Expression and Activity in Cystinotic Fibroblasts

et al. 2008

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Alterations in ATP metabolism have been proposed to be involved in the pathogenesis of cystinosis, the most common form of inherited Fanconi syndrome. A recent study showed normal activity of respiratory chain complexes I-IV with decreased ATP levels in cystinotic fibroblasts. Here, we show normal complex V expression and activity in mitochondria of cystinotic fibroblasts. This indicates that alterations in mitochondrial oxidative phosphorylation enzymes are not responsible for ATP decrease in cystinotic fibroblasts. T he autosomal recessive disorder cystinosis is caused by mutations in the CTNS gene (17p13.3), resulting in the defective lysosomal cystine transporter cystinosin (1). The consequential elevated cystine levels in the lysosomes lead to renal tubular dysfunction by mechanisms that are still elusive. Based on results of studies in cells loaded with cystine dimethyl ester, it was hypothesized that defects in mitochondrial ATP generating capacity are involved, resulting in decreased Na, K-ATPase activity (2,3). This ATP-driven proton pump generates a transmembrane Na ϩ gradient, required for tubular reabsorption of essential solutes such as amino acids, phosphate, and glucose in proximal tubules of the kidney. From this perspective, mitochondrial dysfunction is a possible cause of renal Fanconi syndrome in cystinosis (3). Recently our group extensively studied ATP metabolism in cystinotic fibroblasts and demonstrated slightly decreased intracellular ATP levels, but an intact Na, K-ATPase activity, hormone stimulated mitochondrial ATP production and normal activity of respiratory chain complexes I-IV (4). The activity of mitochondrial enzyme complex V, the ATP synthase complex of the oxidative phosphorylation system, could not be measured at the time of these studies, because lack of sensitivity of the assay. At present, we complete this study in cystinotic fibroblasts by measuring complex V activity using an enzymatic assay. In addition, we examined the expression and assembly status of complex V by Western blot analysis in a blue native gel. MATERIALS AND METHODSFibroblast culture. Patient (n ϭ 8) and control (n ϭ 9) fibroblasts were obtained after informed consent and subsequently cultured as described previously (4). The study was approved by the Institutional Review Board of Radboud University Medical Centre. All patients were diagnosed with cystinosis by measuring elevated cystine levels in isolated polymorphonuclear cells (Ͼ0.5 nmol cystine/mg protein) by high performance liquid chromatography (HPLC) and confirmed by molecular analysis. Previous data showed elevated levels of cystine in cystinotic fibroblasts compared with controls (4.3 Ϯ 1.1 versus 0.2 Ϯ 0.1 nmol/mg protein) and decreased ATP levels in cystinotic fibroblasts (mean 35.9 (Ϯ16.7) versus 51.7 (Ϯ4.5) nmol/mg protein) (4). Approximately 10 ϫ 10 6 cells were harvested using trypsin and divided in two samples for protein expression analysis and enzyme activity assay, respectively.Expression of complex V. A mitochondrial enriche...

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“…Studies using a cystinotic mouse model have shown decreased expression of the multi-ligand receptors megalin and cubilin in proximal tubules; this was associated with cell dedifferentiation and apoptosis (5,8). Also, enhanced cell death due to activation of proapoptotic signals probably occurs in other tissues (9)(10)(11).…”

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confidence: 99%

Cystinosin-LKG rescues cystine accumulation and decreases apoptosis rate in cystinotic proximal tubular epithelial cells

et al. 2016

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Background: Nephropathic cystinosis is a lysosomal storage disease that is caused by mutations in the CTNS gene encoding a cystine/proton symporter cystinosin and an isoform cystinosin-LKG which is generated by an alternative splicing of exon 12. We have investigated the physiological role of the cystinosin-LKG that is widely expressed in epithelial tissues. Methods: We have analyzed the intracellular localization and the function of the cystinosin-LKG conjugated with DsRed (cystinosin-LKG-RFP) in Madin-Darby canine kidney cells (MDCK II) and in proximal tubular epithelial cells carrying a deletion of the CTNS gene (cystinotic PTEC), respectively. results: Cystinosin-LKG-RFP colocalized with markers of lysosomes, late endosomes and was also expressed on the apical surface of polarized MDCK II cells. Moreover, immuneelectron microscopy images of MDCK II cells overexpressing cystinosin-LKG-RFP showed stacked lamellar membranes inside perinuclear lysosomal structures. To study the role of LKG-isoform, we have investigated cystine accumulation and apoptosis that have been described in cystinotic cells. Cystinosin-LKG decreased cystine levels by approximately 10-fold similarly to cystinosin-RFP. The levels of TNFα-and actinomycin D-inducted apoptosis dropped in cystinotic cells expressing LKG-isoform. This effect was also similar to the main isoform. conclusion: Our results suggest that cystinosin-LKG and cystinosin move similar functional activities in cells.n ephropathic cystinosis (NC) is a rare lysosomal storage disease. It is caused by defective activity of cystinosin, a proton/cystine symporter that is ubiquitously expressed on lysosomal membranes, which leads to cystine accumulation into lysosomes and crystal formations in many tissues (1). One of the first symptoms of NC is renal Fanconi syndrome, secondary to a global dysfunction of proximal tubular cells. During their lifetimes, patients with NC develop several others symptoms in particular, if they are not well treated with cysteamine (2,3).Recent works have demonstrated an involvement of cystinosin in lysosomal kinetics and exocytosis (4), vesicle trafficking (4,5), autophagy, (6) and in sensing cell oxidative state (7). Studies using a cystinotic mouse model have shown decreased expression of the multi-ligand receptors megalin and cubilin in proximal tubules; this was associated with cell dedifferentiation and apoptosis (5,8). Also, enhanced cell death due to activation of proapoptotic signals probably occurs in other tissues (9-11).Cystinosin (accession number: NP_004928) is composed of seven transmembrane domains that bear two lysosomal targeting motifs (GYDQL and YFPQA) and seven N-glycosylation sites in the amino-terminal region (12). The aceview database reports various alternative splicing transcripts for the CTNS gene that encodes the cystinosin (http://www.ncbi.nlm.nih. gov/IEB/Research/Acembly). In 2008, we have identified a 400 amino acid cystinosin isoform that derives from alternative splicing of CTNS gene exon 12 (accession number: NP_001026...

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Increased Apoptosis in Cystinotic Fibroblasts and Renal Proximal Tubule Epithelial Cells Results from Cysteinylation of Protein Kinase Cδ

Cited by 75 publications

References 38 publications

Mitochondrial Autophagy Promotes Cellular Injury in Nephropathic Cystinosis

Mitochondrial Autophagy Promotes Cellular Injury in Nephropathic Cystinosis

Mitochondrial Complex V Expression and Activity in Cystinotic Fibroblasts

Cystinosin-LKG rescues cystine accumulation and decreases apoptosis rate in cystinotic proximal tubular epithelial cells

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