Vitamin D-VDR (vitamin D receptor) regulates defective autophagy in renal tubular epithelial cell in streptozotocin-induced diabetic mice via the AMPK pathway

Abstract

Diabetic nephropathy (DN) has become a major cause of end-stage renal disease, and autophagy disorder is implicated in the pathogenesis of DN. Our previous studies found that vitamin D (VD) and VDR (vitamin D receptor) played a renoprotective role by inhibiting inflammation and fibrosis. However, whether VD-VDR regulates autophagy disorders in DN remains unclear. In this study, we established a streptozotocin (STZ)-induced diabetic model in <i>vdr</i> knockout (<i>vdr</i>-KO) mice and VDR specifically overexpressed in renal proximal tubular epithelial cells (<i>Vdr</i>-OE) mice. Our results showed that paricalcitol (an activated vitamin D analog) or <i>Vdr</i>-OE could alleviate STZ-induced ALB (albumin) excretion, renal tubule injury and inflammation, while these were worsened in <i>vdr</i>-KO mice. Defective autophagy was observed in the kidneys of STZ mice, which was more pronounced in <i>vdr</i>-KO mice and could be partially restored by paricalcitol or <i>Vdr</i>-OE. In high glucose-induced HK-2 cells, defective autophagy and decreased PRKAA1/AMPK phosphorylation was observed, which could be partially restored by paricalcitol in a VDR-dependent manner. AMPK inhibitor abolished paricalcitol-induced autophagy activation, and AMPK activator restored the defective autophagy in high glucose-induced HK-2 cells. Furthermore, paricalcitol-mediated AMPK activation was abrogated by CAMKK2/CaMKKβ inhibition, but not by <i>STK11/LKB1</i> knockout. Meanwhile, paricalcitol rescued the decreased Ca²⁺ concentration induced by high glucose. In conclusion, VD-VDR can restore defective autophagy in the kidney of STZ-induced diabetic mice, which could be attributed to the activation of the Ca²⁺-CAMKK2-AMPK pathway in renal tubular epithelial cells.<b>Abbreviations:</b> ACTB/β-actin: actin beta;AGE: advanced glycation end-products;AMPK: AMP-activated protein kinase;CAMKK2/CaMKKβ: calcium-calmodulin dependent protein kinase kinase 2;CQ: chloroquine;DN: diabetic nephropathy;HG: high levels of glucose;KO: knockout;LG: low levels of glucose;MAP1LC3/LC3: microtubule associated protein 1 light chain 3;NOD2: nucleotide binding oligomerization domain containing 2;OE: overexpression;PAS: periodic acid Schiff; Pari: paricalcitol;PTECs: proximal renal tubule epithelial cells;RT: room temperature;SQSTM1/p62: sequestosome 1;STK11/LKB1: serine/threonine kinase 11;STZ: streptozotocin;TEM: transmission electron microscopy;VD: vitamin D;VDR: vitamin D receptor;WT: wild-type.