Klotho Depletion Contributes to Increased Inflammation in Kidney of the db/db Mouse Model of Diabetes

OBJECTIVE—Klotho is an antiaging hormone present in the kidney that extends the lifespan, regulates kidney function, and modulates cellular responses to oxidative stress. We investigated whether Klotho levels and signaling modulate inflammation in diabetic kidneys. RESEARCH DESIGN AND METHODS—Renal Klotho expres-sion was determined by quantitative real-time PCR and immuno-blot analysis. Primary mouse tubular epithelial cells were treated with methylglyoxalated albumin, and Klotho expression and inflammatory cytokines were measured. Nuclear factor (NF)-kB activation was assessed by treating human embryonic kidney (HEK) 293 and HK-2 cells with tumor necrosis factor (TNF)-a in the presence or absence of Klotho, followed by immunoblot anal-ysis to evaluate inhibitor of kB (IkB)a degradation, IkB kinase (IKK) and p38 activation, RelA nuclear translocation, and phos-phorylation. A chromatin immunoprecipitation assay was per-formed to analyze the effects of Klotho signaling on interleukin-8 and monocyte chemoattractant protein-1 promoter recruitment of RelA and RelA serine (Ser)536. RESULTS—Renal Klotho mRNA and protein were significantly decreased in db/db mice, and a similar decline was observed in the primary cultures of mouse tubule epithelial cells treated with methylglyoxal-modified albumin. The exogenous addition of sol-uble Klotho or overexpression of membranous Klotho in tissue culture suppressed NF-kB activation and subsequent production of inflammatory cytokines in response to TNF-a stimulation. Klotho specifically inhibited RelA Ser536 phosphorylation as well as promoter DNA binding of this phosphorylated form of RelA without affecting IKK-mediated IkBa degradation, total RelA nuclear translocation, and total RelA DNA binding. CONCLUSIONS—These findings suggest that Klotho serves as an anti-inflammatory modulator, negatively regulating the pro-duction of NF-kB–linked inflammatory proteins via a mechanism that involves phosphorylation of Ser536 in the transactivation domai