Abstract
Purpose: :
Our aim is to study the role of Klotho family members Klotho-alpha (KL) and Klotho-gamma (lactase-like, LCTL) in the function of Retinal Pigment Epithelia (RPE) and their possible implication in Age-related Macular Degeneration (AMD).
Methods: :
To study the effect of KL and LCTL on human RPE, gene and protein expression was analyzed by qRT-PCR, immunostaining and Western blot analysis. The siRNA technology was used to knock down KL, LCTL and Transient Receptor Potential cation channel subfamily V member 5 (TRPV5) genes in RPE. Phagocytosis assays were performed using fluorescently labeled zymosan. Oxidative stress was induced by tert-butylhydroperoxide (tBH). VEGF secretion was measured by ELISA. Single cell patch clamp electrophysiology technique and calcium imaging were also applied to measure the activity of TRPV5 channel in RPE.
Results: :
Human RPE express both KL and LCTL genes, with LCTL mRNA levels being approximately 10 times higher than KL. Our results demonstrated that KL protein increases phagocytosis and reduces VEGF secretion significantly in RPE. Using Ca2+ imaging we demonstrated that KL protein regulates the activity of TRPV5 in human RPE and increases the Ca2+ entry across the apical membrane. We further demonstrated that both KL and LCTL are down regulated by oxidative stress.
Conclusions: :
Our results show for the first time that KL regulates RPE functions such as phagocytosis and VEGF secretion by increasing Ca+2 entry through TRPV5 channels. Therefore, a decrease in KL expression during aging might be directly implicated in AMD.
Keywords: retinal pigment epithelium • aging • calcium