Single-Cell RNA Sequencing Analysis Reveals Inhibition of Proximal Tubule mTORC1 in Myoinositol Oxygenase Knockout Mice: A Possible Mechanism for Ameliorating CKD
- 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
- Sharma, Isha, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
- Zhou, Yalu, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
- Maekawa, Hiroshi, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
- Quaggin, Susan E., Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
MIOX is a proximal tubular specific enzyme and it catalyzes the rate limiting step of myoinositol metabolism. MIOX overexpression has been implicated in the progression of chronic kidney disease. Conversely its genetic deletion shows significant amelioration in progression of chronic kidney disease (CKD) in mouse models of diabetes.
Kidneys harvested from 6-week-old male C57BL/6J (WT) and MIOX-KO mice, fed with normal or high fat diet (HFD, 60% calories from fat) for six weeks, were analyzed. Change in body weight, blood glucose level was estimated. Single cell RNA sequence (scRNA seq) analysis was performed on libraries prepared from whole kidneys of six-week ND (normal diet) and HFD mice. We assessed pS6 level in kidneys of MIOX-KO and WT mice via immunoblotting.
UMAP showed retrieval of 27 clusters which included cells of proximal tubules, loop of Henle, distal convoluted tubules, collecting duct, podocytes, stroma, vasculature, and immune cells. Pathway enrichment analysis with all cells highlighted mTORC1 signaling pathway was substantially downregulated in MIOX-KO HFD mice as compared to MIOX-KO ND mice. Conversely, mTORC1 pathway was significantly upregulated in cells from WT-HFD. Further we narrowed down the pathway enrichment analysis only to proximal tubules (PT) as MIOX is only expressed in PT cells. Beside endoplasmic reticulum stress, hypoxia, apoptosis, mTORC1 pathway was significantly downregulated in MIOX-KO-HFD mice as compared to WT-HFD and MIOX-KO ND.
Immunoblotting analysis showed downregulation of p-mTORC1 in MIOX-KO mice fed with normal chow as compared to WT mice. In addition, scRNA showed that mitophagy, which is modulated by mTORC1 signaling, is induced in PT cells of MIOX-KO mice.
MIOX-KO reduced mTORC1 activation compared to WT following HFD. We posit that decreased activation of mTORC1 is one mechanism whereby MIOX inhibition/loss protects the kidney from injury in diabetes.
- NIDDK Support