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Abstract
Disruption of the insulin-PI3K-Akt signalling pathway in kidney podocytes causes endoplasmic reticulum (ER) stress, leading to podocyte apoptosis and proteinuria in diabetic nephropathy. We hypothesised that by improving insulin sensitivity we could protect podocytes from ER stress. Here we use established activating transcription factor 6 (ATF6)- and ER stress element (ERSE)-luciferase assays alongside a novel high throughput imaging-based C/EBP homologous protein (CHOP) assay to examine three models of improved insulin sensitivity. We find that by improving insulin sensitivity at the level of the insulin receptor (IR), either by IR over-expression or by knocking down the negative regulator of IR activity, protein tyrosine-phosphatase 1B (PTP1B), podocytes are protected from ER stress caused by fatty acids or diabetic media containing high glucose, high insulin and inflammatory cytokines TNFα and IL-6. However, contrary to this, knockdown of the negative regulator of PI3K-Akt signalling, phosphatase and tensin homolog deleted from chromosome 10 (PTEN), sensitizes podocytes to ER stress and apoptosis, despite increasing Akt phosphorylation. This indicates that protection from ER stress is conferred through not just the PI3K-Akt pathway, and indeed we find that inhibiting the MEK/ERK signalling pathway rescues PTEN knockdown podocytes from ER stress.
Highlights
Diabetic nephropathy (DN) is the leading global cause of end-stage renal disease[1,2], accounting for nearly 50% of patients in the United States requiring dialysis or a kidney transplant[3]
Insulin sensitivity was increased by insulin receptor (IR) over-expression, protein tyrosine-phosphatase 1B (PTP1B) knockdown, and phosphatase and tensin homolog deleted from chromosome 10 (PTEN) knockdown, confirmed by measuring the insulin-stimulated phosphorylation of Akt
Stable podocyte cell lines were made through transduction of lentiviruses to stably express the endoplasmic reticulum (ER) stress-specific promoter sequences activating transcription factor 6 (ATF6) or ER stress response element (ERSE) driving the firefly luciferase reporter, together with renilla luciferase expressed independently as an internal control
Summary
Diabetic nephropathy (DN) is the leading global cause of end-stage renal disease[1,2], accounting for nearly 50% of patients in the United States requiring dialysis or a kidney transplant[3]. Herp (encoded by the HERPUD1 gene; homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1) is a member of the ERAD pathway, and by interacting with components of the ubiquitin family it helps to target irrevocably misfolded proteins to the proteasome for degradation[13]. If these adaptive responses are unable to bring this ER stress under control and reverse it within a certain timeframe, apoptosis is initiated to eliminate the damaged cells. We conclude that the relationship between insulin sensitivity and ER stress is more complex than anticipated; the protection from ER stress conferred by improving insulin sensitivity is clearly not communicated through the PI3K-Akt pathway alone
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