Impact of atypical mitochondrial cyclic-AMP level in nephropathic cystinosis

Francesco Bellomo, Anna Signorile, Grazia Tamma, Marianna Ranieri, Francesco Emma, Domenico De Rasmo

Research output: Contribution to journalArticle

Abstract

Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H+ symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS-/- conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS-/-) or with compound heterozygous loss-of-function mutations (mutCTNS-/-). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS-/- ciPTEC by 56% ± 10.5, P < 0.0001; mutCTNS-/- by 26% ± 4.3, P < 0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.

Original languageEnglish
Pages (from-to)3411-3422
Number of pages12
JournalExperientia
Volume75
Issue number18
DOIs
Publication statusPublished - Sep 2018

Fingerprint

Cystinosis
Cystine
Cyclic AMP
Fanconi Syndrome
Symporters
Cysteamine
Mitochondria
Gene encoding
Epithelial Cells
Mitochondrial Diseases
Mutation
Active Biological Transport
Mitochondrial Membrane Potential
Rare Diseases
Lysosomes
Cytosol
Membranes
Proteins
Therapeutics
Genes

Keywords

  • Amino Acid Transport Systems, Neutral/antagonists & inhibitors
  • Cell Line
  • Cyclic AMP/metabolism
  • Cystinosis/metabolism
  • Electron Transport Chain Complex Proteins/metabolism
  • Epithelial Cells/cytology
  • Humans
  • Kidney Tubules, Proximal/cytology
  • Membrane Potential, Mitochondrial
  • Mitochondria/metabolism
  • RNA Interference
  • RNA, Small Interfering/metabolism
  • Sirtuin 3/metabolism

Cite this

Impact of atypical mitochondrial cyclic-AMP level in nephropathic cystinosis. / Bellomo, Francesco; Signorile, Anna; Tamma, Grazia; Ranieri, Marianna; Emma, Francesco; De Rasmo, Domenico.

In: Experientia, Vol. 75, No. 18, 09.2018, p. 3411-3422.

Research output: Contribution to journalArticle

Bellomo, Francesco ; Signorile, Anna ; Tamma, Grazia ; Ranieri, Marianna ; Emma, Francesco ; De Rasmo, Domenico. / Impact of atypical mitochondrial cyclic-AMP level in nephropathic cystinosis. In: Experientia. 2018 ; Vol. 75, No. 18. pp. 3411-3422.
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abstract = "Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H+ symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS-/- conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS-/-) or with compound heterozygous loss-of-function mutations (mutCTNS-/-). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS-/- ciPTEC by 56{\%} ± 10.5, P < 0.0001; mutCTNS-/- by 26{\%} ± 4.3, P < 0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.",
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AU - Tamma, Grazia

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AU - Emma, Francesco

AU - De Rasmo, Domenico

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AB - Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H+ symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS-/- conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS-/-) or with compound heterozygous loss-of-function mutations (mutCTNS-/-). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS-/- ciPTEC by 56% ± 10.5, P < 0.0001; mutCTNS-/- by 26% ± 4.3, P < 0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.

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KW - Humans

KW - Kidney Tubules, Proximal/cytology

KW - Membrane Potential, Mitochondrial

KW - Mitochondria/metabolism

KW - RNA Interference

KW - RNA, Small Interfering/metabolism

KW - Sirtuin 3/metabolism

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