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Published on: September 1, 2015
Novel mechanism for tubular injury in nephropathic cystinosis
Swastika Sur1, Maggie Kerwin1, Silvia Pineda1,2
1Division of Multi-Organ Transplantation, Department of Surgery, University of California, San Francisco, San Francisco, United States.
Abstract:
Understanding the unique susceptibility of the human kidney to pH dysfunction and injury in cystinosis is paramount to developing new therapies to preserve renal function. Renal proximal tubular epithelial cells (RPTECs) and fibroblasts isolated from patients with cystinosis were transcriptionally profiled. Lysosomal fractionation, immunoblotting, confocal microscopy, intracellular pH, TEM, and mitochondrial stress test were performed for validation. CRISPR, CTNS -/- RPTECs were generated. Alterations in cell stress, pH, autophagic turnover, and mitochondrial energetics highlighted key changes in the V-ATPases in patient-derived and CTNS-/- RPTECs. ATP6V0A1 was significantly downregulated in cystinosis and highly co-regulated with loss of CTNS. Correction of ATP6V0A1 rescued cell stress and mitochondrial function. Treatment of CTNS -/- RPTECs with antioxidants ATX induced ATP6V0A1 expression and improved autophagosome turnover and mitochondrial integrity. Our exploratory transcriptional and in vitro cellular and functional studies confirm that loss of Cystinosin in RPTECs, results in a reduction in ATP6V0A1 expression, with changes in intracellular pH, mitochondrial integrity, mitochondrial function, and autophagosome-lysosome clearance. The novel findings are ATP6V0A1's role in cystinosis-associated renal pathology and among other antioxidants, ATX specifically upregulated ATP6V0A1, improved autophagosome turnover or reduced autophagy and mitochondrial integrity. This is a pilot study highlighting a novel mechanism of tubular injury in cystinosis.
Insights
Cystinosis causes kidney injury by reducing ATP6V0A1 expression, impacting pH and mitochondrial function. Antioxidant ATX may restore ATP6V0A1, improving cell health in this rare kidney disease.
Area of Science:
- Nephrology
- Cell Biology
- Biochemistry
Background:
- Cystinosis is a rare genetic disorder causing lysosomal accumulation of cystine.
- Kidney dysfunction, particularly in proximal tubular epithelial cells (RPTECs), is a major complication of cystinosis.
- Understanding the molecular mechanisms of renal injury in cystinosis is crucial for developing effective therapies.
Purpose of the Study:
- To investigate the role of V-ATPases in cystinosis-associated renal pathology.
- To identify molecular targets for preserving renal function in cystinosis patients.
- To explore the therapeutic potential of antioxidants in mitigating cystinosis-induced kidney injury.
Main Methods:
- Transcriptional profiling of patient-derived RPTECs and fibroblasts.
- In vitro validation using techniques such as intracellular pH measurements, mitochondrial stress tests, and confocal microscopy.
- Generation of CRISPR-Cas9 gene-edited CTNS knockout RPTECs for mechanistic studies.
Main Results:
- Loss of Cystinosin (CTNS) in RPTECs led to downregulation of ATP6V0A1, a V-ATPase component.
- Cystinosis was associated with altered intracellular pH, impaired mitochondrial function, and disrupted autophagic turnover.
- Correction of ATP6V0A1 expression or treatment with antioxidant ATX improved cellular stress, mitochondrial integrity, and autophagosome-lysosome clearance in CTNS knockout cells.
Conclusions:
- ATP6V0A1 plays a significant role in the renal pathology of cystinosis.
- Targeting ATP6V0A1 or utilizing antioxidants like ATX represents a potential therapeutic strategy for cystinosis.
- This pilot study reveals a novel mechanism of tubular injury in cystinosis, highlighting ATP6V0A1 as a key player.
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