microRNA (miR)-299a-5p Promotes Apoptosis of Tubular Cells Through Erk5 Inhibition
- Diabetic Kidney Disease: Basic - I
November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Diabetic Kidney Disease
- 701 Diabetic Kidney Disease: Basic
- Nmecha, Ifeanyi Kennedy/k, McMaster University Faculty of Health Sciences, Hamilton, Ontario, Canada
- Gao, Bo, McMaster University Faculty of Health Sciences, Hamilton, Ontario, Canada
- Macdonald, Melissa, McMaster University Faculty of Health Sciences, Hamilton, Ontario, Canada
- Krepinsky, Joan C., McMaster University Faculty of Health Sciences, Hamilton, Ontario, Canada
Diabetic kidney disease (DKD) is the leading cause of end stage renal failure in North America. Tubular epithelial cell apoptosis was shown to contribute to the pathogenesis of DKD by promoting interstitial fibrosis. The mechanism involves the release of cytokines and growth factors that promote interstitial inflammation and myofibroblast activation, which leads to the increased production and accumulation of extracellular matrix proteins in the interstitium. Previously, we showed that microRNA (miR) 299a-5p promotes the fibrotic response in mesangial cells. Here we studied the role of this miR in regulating tubular apoptosis.
Immortalized human kidney proximal tubular cells (HK2) were treated with high glucose (HG); miR299a-5p was detected by qPCR. HK2 cells were transfected with miR overexpression and inhibition plasmids using Transfectamine reagent. Apoptosis was detected using the Annexin V kit. Immunoblotting and immunohistochemistry were used to assess protein expression.
HG increased the expression of miR299a-5p in HK2 cells. A bioinformatics screen of four databases showed that miR299a-5p targets MAP3K2, a protein kinase primarily expressed in the tubules. MAP3K2 is a major activator of extracellular signal-regulated kinase 5 (ERK 5), a known anti-apoptotic and anti-inflammatory kinase. miR299a-5p overexpression reduced the protein expression of MAP3K2 and phosphorylation of ERK5, while augmenting the expression of apoptotic markers and endoplasmic reticulum stress in response to HG. Conversely, miR299a-5p inhibition restored MAP3K2 expression and ERK5phosphorylation to basal levels, while attenuating HG-induced apoptosis. In type-1 diabetic Akita mice overexpressing TGFβ1, a model which accelerates DKD, miR299a was increased in association with reduced expression of MAP3K2 and phosphorylated ERK5.
These data support an important role for miR299a-5p in regulating tubular cell apoptosis in HG. Future studies will determine whether inhibition of this miR in vivo attenuates apoptosis and reduces tubulointerstitial fibrosis in DKD. We will also explore whether ERK5 phosphorylation may be a potential therapeutic for interstitial fibrosis.
- Government Support – Non-U.S.