Lysosomal cystine accumulation promotes mitochondrial depolarization and induction of redox‐sensitive genes in human kidney proximal tubular cells

Abstract

Cystine is a disulphide amino acid that is normally generated within the lysosomes through lysosomal-based protein degradation and via extracellular uptake of free cystine. In the autosomal recessive disorder, cystinosis, a defect in the CTNS gene results in excessive lysosomal accumulation of cystine, with early kidney failure a hallmark of the disease. Previously, we demonstrated that silencing of the CTNS gene in kidney proximal tubular epithelial cells (PTECs) resulted in an increase in intracellular cystine concentration coupled with a dramatic reduction in the total GSH content. Because of the crucial role of GSH in maintaining the redox status and viability of kidney PTECs, we assessed the effects of CTNS knockdown-induced lysosomal cystine accumulation on intracellular reactive oxygen species (ROS) production, activity of classical redox-sensitive genes, mitochondrial integrity and cell viability. Our results showed that lysosomal cystine accumulation increased ROS production and solicitation to oxidative stress (OS). This was associated with the induction of classical redox-sensitive proteins, NF-κB, NRF2, HSP32 and HSP70. Cystine-loaded PTECs also displayed depolarized mitochondria, reduced ATP content and augmented apoptosis. Treatment of CTNS knockdown PTECs with the cystine-depleting agent cysteamine resulted in the normalization of OS index, increased GSH and ATP content, and preservation of cell viability. Taken together, the alterations observed in cystinotic cells may represent different facets of a cascade leading to tubular dysfunction and, in combination with cysteamine therapy, may offer a novel link for the attenuation of renal injury and preservation of functions of other organs affected in cystinosis.

Inducible nitric oxide synthase inhibitor 1400W increases Na+,K+‐ATPase levels and activity and ameliorates mitochondrial dysfunction in Ctns null kidney proximal tubular epithelial cells

“…Since the ER represents a major storage of intracellular calcium (Ca 2+ ), alterations in ER homeostasis can lead to the efflux of Ca 2+ . Indeed, we have previously shown that intracellular Ca 2+ levels were increased in CTNS knockdown PTECs . Previous studies have implicated Ca 2+ dependency of iNOS activity and iNOS is twice as active in the presence of Ca 2+ as in its absence …”

“…Intracellular NO levels were determined in live cells by fluorescence microscopy (Nikon Instruments, Melville, NY, USA) and flow cytometry (BD Biosciences, San Jose, CA, USA) using the NO‐sensitive dye DAF‐FMDA, as described previously …”

“…The OS index of cells was assessed using the probe 5‐(and‐6)‐carboxy‐2′,7′‐difluorodihydrofluorescein diacetate (carboxy‐H 2 DFFDA) following the procedures previously described …”

“…Western blot was performed following procedures as previously described . The following concentrations of primary antibodies in 5% non‐fat milk/Tris‐base saline buffer (pH 7.4) containing 0.1% Tween‐20 were used: anti‐iNOS (1:1000; Sigma), anti‐eNOS (1:1000, Abcam, Cambridge, MA, USA), anti‐nNOS (1:1000, Abcam), anti‐Na + ,K + ‐ATPase‐α‐1 (1:1000; Abcam), anti‐β‐actin (1:2000; Thermo Fisher Scientific).…”

“…Δψ m was measured by flow cytometry following the procedures previously described . The ratio of J‐monomers to J‐aggregates was used to assess Δψ m .…”