The transcription factor nuclear factor erythroid-2–related factor 2 (Nrf2) plays a critical role in reducing oxidative stress by promoting the expression of antioxidant genes. Both individuals with diabetes and preclinical diabetes models exhibit evidence of a defect in retinal Nrf2 activation. We recently demonstrated that increased expression of the stress response protein regulated in development and DNA damage 1 (REDD1) is necessary for the development of oxidative stress in the retina of streptozotocin-induced diabetic mice. In the present study, we tested the hypothesis that REDD1 suppresses the retinal antioxidant response to diabetes by repressing Nrf2 function. We found that REDD1 ablation enhances Nrf2 DNA-binding activity in the retina and that the suppressive effect of diabetes on Nrf2 activity is absent in the retina of REDD1-deficient mice compared with WT. In human MIO-M1 Müller cell cultures, REDD1 deletion prevented oxidative stress in response to hyperglycemic conditions, and this protective effect required Nrf2. REDD1 suppressed Nrf2 stability by promoting its proteasomal degradation independently of Nrf2's interaction with Kelch-like ECH-associated protein 1 (Keap1), but REDD1-mediated Nrf2 degradation required glycogen synthase kinase 3 (GSK3) activity and Ser-351/Ser-356 of Nrf2. Diabetes diminished inhibitory phosphorylation of glycogen synthase kinase 3β (GSK3β) at Ser-9 in the retina of WT mice but not in REDD1-deficient mice. Pharmacological inhibition of GSK3 enhanced Nrf2 activity and prevented oxidative stress in the retina of diabetic mice. The findings support a model wherein hyperglycemia-induced REDD1 blunts the Nrf2 antioxidant response to diabetes by activating GSK3, which, in turn, phosphorylates Nrf2 to promote its degradation.

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Clinical studies support a role for the protein regulated in development and DNA damage response 1 (REDD1) in ischemic retinal complications. To better understand how REDD1 contributes to retinal pathology, we examined human single cell sequencing datasets and found specificity of REDD1 expression that was consistent with markers of retinal Müller glia. Thus, we investigated the hypothesis that REDD1 expression specifically in Müller glia contributes to diabetes-induced retinal pathology. The retina of Müller glia specific REDD1 knockout (REDD1 mgKO) mice exhibited dramatic attenuation of REDD1 transcript and protein expression. In the retina of streptozotocin-induced diabetic control mice, REDD1 protein expression was enhanced coincident with an increase in oxidative stress. In the retina of diabetic REDD1 mgKO mice there was no increase in REDD1 protein expression and oxidative stress was reduced as compared to diabetic control mice. In both Müller glia within the retina of diabetic mice and human Müller cell cultures exposed to hyperglycemic conditions, REDD1 was necessary for increased expression of the gliosis marker glial fibrillary acidic protein. The effect of REDD1 deletion in preventing gliosis was associated with suppression of oxidative stress and required the antioxidant transcription factor Nrf2. In contrast to diabetic control mice, diabetic REDD1 mgKO mice did not exhibit retinal thinning, increased markers of neurodegeneration within the retinal ganglion cell layer, or deficits in visual function. Overall, the findings support a key role for Müller glial REDD1 in the failed adaptive response of the retina to diabetes that includes gliosis, neurodegeneration, and impaired vision.

Activation of the immune costimulatory molecule cluster of differentiation 40 (CD40) in Müller glia has been implicated in the initiation of diabetes-induced retinal inflammation. Results from previous studies support that CD40 protein expression is elevated in Müller glia of diabetic mice; yet, the mechanisms responsible for this increase have not been explored. Here, we evaluated the hypothesis that diabetes augments translation of the Cd40 mRNA. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation and increased Cd40 mRNA translation. TMG administration also promoted Cd40 mRNA association with Müller-cell specific ribosomes isolated from the retina of RiboTag mice. Similar effects on O-GlcNAcylation and Cd40 mRNA translation were also observed in the retina of a mouse model of type 1 diabetes. In cultured cells, TMG promoted sequestration of the cap-binding protein eukaryotic translation in initiation factor 4E (eIF4E) by eIF4E-binding protein 1 (4E-BP1) and enhanced cap-independent Cd40 mRNA translation as assessed by a bicistronic reporter that contained the 5′-UTR of the Cd40 mRNA. Ablation of 4E-BP1/2 prevented the increase in Cd40 mRNA translation in TMG-exposed cells, and expression of a 4E-BP1 variant that constitutively sequesters eIF4E promoted reporter activity. Extending on the cell culture results, we  found that in contrast to wild-type mice, diabetic 4E-BP1/2-deficient mice did not exhibit enhanced retinal Cd40 mRNA translation and failed to upregulate expression of the inflammatory marker nitric oxide synthase 2. These findings support a model wherein diabetes-induced O-GlcNAcylation of 4E-BP1 promotes Cd40 mRNA translation in Müller glia.

Cyclosporine A (CsA) is a nephrotoxicant that causes fibrosis via induction of epithelial–mesenchymal transition (EMT). The flavonoid chrysin has been reported to have anti-fibrotic activity and inhibit signaling pathways that are activated during EMT. This study investigated the nephroprotective role of chrysin in the prevention of CsA-induced renal fibrosis and elucidated a mechanism of inhibition against CsA-induced EMT in proximal tubule cells. Treatment with chrysin prevented CsA-induced renal dysfunction in Sprague Dawley rats measured by blood urea nitrogen (BUN), serum creatinine and creatinine clearance. Chrysin inhibited CsA-induced tubulointerstitial fibrosis, characterized by reduced tubular damage and collagen deposition. In vitro, chrysin significantly inhibited EMT in LLC-PK1 cells, evidenced by inhibition of cell migration, decreased collagen expression, reduced presence of mesenchymal markers and elevated epithelial junction proteins. Furthermore, chrysin co-treatment diminished CsA-induced TGF-β1 signaling pathways, decreasing Smad 3 phosphorylation which lead to a subsequent reduction in Snail expression. Chrysin also inhibited activation of the Akt/ GSK-3β pathway. Inhibition of both pathways diminished the cytosolic accumulation of β-catenin, a known trigger for EMT. In conclusion, flavonoids such as chrysin offer protection against CsA-induced renal dysfunction and interstitial fibrosis. Chrysin was shown to inhibit CsA-induced TGF-β1-dependent EMT in proximal tubule cells by modulation of Smad-dependent and independent signaling pathways.

Na+-dependent glucose reabsorption in the renal proximal tubule is dynamically regulated by changes in blood glucose levels. There is, however, a disparity in reports studying the relationship between hyperglycemia and Na+-glucose-linked transporter (SGLT) function and expression. Similarly, manipulation of the glucose content in growth media of cultured renal cells has been shown to influence SGLT activity. In this investigation, SGLT activity was significantly lower in proximal tubule LLC-PK1 cells cultured in medium containing 17.5 than 5 mM glucose. α-Methyl d-glucopyranoside (AMG) transport kinetics showed reduced apparent Vmax and Km in cells grown in 17.5 mM glucose. SGLT2 was identified as the isoform responsible for glucose transport, and protein expression analyses showed decreased apical membrane localization of SGLT2 in cells grown in 17.5 mM glucose, explaining the reduced activity. Multiple signaling pathways have been implicated in regulation of SGLT activity and trafficking. Elevated media glucose decreased intracellular cAMP and PKA activation, leading to decreased SGLT2 trafficking into the plasma membrane, which was reversed after treatment with 1 µM forskolin. The effects of media glucose on SGLT activity were found to be dependent on p38 MAPK activation due to PKA-mediated signaling. Glucose-modulated AMG uptake is reversible and was associated with altered SGLT2 membrane trafficking and cAMP alterations. In summary, elevated glucose concentrations in culture medium decrease SGLT activity in LLC-PK1 cells by reducing membrane trafficking of SGLT2 via decreasing intracellular cAMP, resulting in a lowered PKA-dependent phosphorylation of p38 MAPK.

Endoplasmic reticulum (ER) stress and inflammation are hallmarks of myocardial impairment. Here we investigated a role for the stress response protein REDD1 as a molecular link between ER stress and inflammation in cardiomyocytes. In mice fed a high-fat high-sucrose (HFHS, 42% kcal fat, 34% sucrose by weight) diet for 12 weeks, REDD1 expression in the heart was increased in coordination with markers of ER stress and inflammation. In human AC16 cardiomyocytes exposed to either hyperglycemic conditions or the saturated fatty acid palmitate, REDD1 expression was increased coincident with ER stress and upregulated expression of the pro-inflammatory cytokines IL-1β, IL-6, and TNF⍺. In cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions, pharmacological inhibition of the ER kinase PERK or knockdown of the transcription factor ATF4 prevented the increase in REDD1 expression. REDD1 deletion reduced pro-inflammatory cytokine expression in both cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions and in the hearts of obese mice. Overall, the findings support a model wherein HFHS diet contributes to the development of inflammation in cardiomyocytes by promoting REDD1 expression via activation of a PERK/ATF4 signaling axis.