KCa3.1 mediates dysfunction of tubular autophagy in diabetic kidneys via PI3k/Akt/mTOR signaling pathways

Chunling Huang; Mike Z Lin; Delfine Cheng; Filip Braet; Carol A Pollock; Xin-Ming Chen

doi:10.1038/srep23884

KCa3.1 mediates dysfunction of tubular autophagy in diabetic kidneys via PI3k/Akt/mTOR signaling pathways

Sci Rep. 2016 Mar 31:6:23884. doi: 10.1038/srep23884.

Authors

Chunling Huang¹, Mike Z Lin¹, Delfine Cheng², Filip Braet^{2

3}, Carol A Pollock¹, Xin-Ming Chen¹

Affiliations

¹ Kolling Institute of Medical Research, Sydney Medical School, University of Sydney, Royal North Shore Hospital, St Leonards, Sydney, NSW 2065, Australia.
² School of Medical Sciences (Discipline of Anatomy and Histology) - The Bosch Institute, University of Sydney, NSW 2006, Australia.
³ Australian Centre for Microscopy &Microanalysis, Madsen Building, University of Sydney, NSW 2006, Australia.

Abstract

Autophagy is emerging as an important pathway in many diseases including diabetic nephropathy. It is acknowledged that oxidative stress plays a critical role in autophagy dysfunction and diabetic nephropathy, and KCa3.1 blockade ameliorates diabetic renal fibrosis through inhibiting TGF-β1 signaling pathway. To identify the role of KCa3.1 in dysfunctional tubular autophagy in diabetic nephropathy, human proximal tubular cells (HK2) transfected with scrambled or KCa3.1 siRNAs were exposed to TGF-β1 for 48 h, then autophagosome formation, the autophagy marker LC3, signaling molecules PI3K, Akt and mTOR, and oxidative stress marker nitrotyrosine were examined respectively. In vivo, LC3, nitrotyrosine and phosphorylated mTOR were examined in kidneys of diabetic KCa3.1+/+ and KCa3.1-/- mice. The results demonstrated that TGF-β1 increased the formation of autophagic vacuoles, LC3 expression, and phosphorylation of PI3K, Akt and mTOR in scrambled siRNA transfected HK2 cells compared to control cells, which was reversed in KCa3.1 siRNA transfected HK2 cells. In vivo, expression of LC3 and nitrotyrosine, and phosphorylation of mTOR were significantly increased in kidneys of diabetic KCa3.1+/+ mice compared to non-diabetic mice, which were attenuated in kidneys of diabetic KCa3.1-/- mice. These results suggest that KCa3.1 activation contributes to dysfunctional tubular autophagy in diabetic nephropathy through PI3K/Akt/mTOR signaling pathways.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Autophagy / genetics
Cell Line, Transformed
Diabetes Mellitus, Experimental / chemically induced
Diabetes Mellitus, Experimental / genetics*
Diabetes Mellitus, Experimental / metabolism
Diabetes Mellitus, Experimental / pathology
Diabetic Nephropathies / chemically induced
Diabetic Nephropathies / genetics*
Diabetic Nephropathies / metabolism
Diabetic Nephropathies / pathology
Epithelial Cells / drug effects
Epithelial Cells / metabolism
Epithelial Cells / pathology
Gene Expression Regulation
Humans
Intermediate-Conductance Calcium-Activated Potassium Channels / antagonists & inhibitors
Intermediate-Conductance Calcium-Activated Potassium Channels / genetics*
Intermediate-Conductance Calcium-Activated Potassium Channels / metabolism
Kidney Tubules, Proximal / metabolism
Kidney Tubules, Proximal / pathology
Male
Mice
Mice, Knockout
Phagosomes / metabolism
Phosphatidylinositol 3-Kinases / genetics*
Phosphatidylinositol 3-Kinases / metabolism
Proto-Oncogene Proteins c-akt / genetics*
Proto-Oncogene Proteins c-akt / metabolism
RNA, Small Interfering / genetics
RNA, Small Interfering / metabolism
Signal Transduction
Streptozocin
TOR Serine-Threonine Kinases / genetics*
TOR Serine-Threonine Kinases / metabolism
Transforming Growth Factor beta1 / pharmacology

Substances

Intermediate-Conductance Calcium-Activated Potassium Channels
KCNN4 protein, human
RNA, Small Interfering
TGFB1 protein, human
Transforming Growth Factor beta1
Streptozocin
MTOR protein, human
Proto-Oncogene Proteins c-akt
TOR Serine-Threonine Kinases