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Ashay D Bhatwadekar, Iraj Hassan, Sreeparna Majumdar, Vaishnavi Jadhav, Julia V Busik; The Tumor Necrosis Factor α (TNF-α) Disturbs Kir4.1 Rhythm in Muller Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3208.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal Müller cells maintain water homeostasis and K+ concentration by specialized inwardly rectifying Kir4.1 channels. However, the expression of Kir4.1 is decreased in diabetes. We previously reported diabetic retinopathy (DR) as the disease of clock gene dysregulation. The TNF- α, a pro-inflammatory cytokine is known to be involved in the pathogenesis of DR, however, the interplay of TNF-α, Kir4.1 and clock genes in Müller cells remains mostly unknown. For this study, we hypothesized that the Kir4.1 in Müller cells is under the clock regulation and elevated level of the TNF-α is detrimental to Kir4.1 expression.
The Long Evans rats were made diabetic using streptozotocin (STZ) and sacrificed after 6 weeks after induction of the diabetes at the different time intervals. The mRNA expression of Kcnj10 (gene for Kir4.1), TNF-α and clock genes was determined using qRT-PCR. The rat Müller (rMC-1) cells were transfected with siRNA for Per2 and Bmal 1 to determine the Kcnj10 expression. In parallel, the effect of TNF-α (5 to 500 pM) treatment on Kcnj10 and Kir4.1 expression was evaluated. The mechanism of decrease in Kir4.1 expression was determined by immunofluorescence using a co-staining for Kir4.1 channels and synapse associated protein (SAP97).
The mRNA expression of Kcnj10 in rat retina exhibited a diurnal rhythm for control animals with a peak increase during the night. For STZ diabetic rats, there was an overall reduction in Kcnj10 expression. Per2 and Bmal1 siRNA transfected rMC-1 showed a profound decrease in Kcnj10 expression. The TNF-α treatment of rMCs resulted in a profound decrease in Kir4.1 expression, disorganization of the actin cytoskeleton and the dislocalization of Kir4.1 and SAP-97
Our studies suggest that Kir4.1 possesses a diurnal variation with dampening of its amplitude during diabetes. The TNF-α exhibits a diurnal rhythm and a profound increase in TNF-α in diabetes is detrimental to Kir4.1 expression due to altered cytoskeletal signaling.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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