Date of Award
Doctor of Philosophy
Dr. Ahmed Bettaieb
Dr. Brynn Voy, Dr. Dallas Donohoe, and Dr. Jay Whelan
Background: Renal diseases are major health concerns and among the top ten leading causes of death in the US. A large number of these diseases are characterized by deterioration in glomerular structure and function, leading to reduced filtration capacity and proteinuria. Glomerulus podocytes are epithelial cells that maintain glomerular integrity and act as a defense mechanism against proteinuria. Recent advances in renal research suggested a novel role of glycolysis and its related enzymes, pyruvate kinase M2 (PKM2) in particular, in the progression of renal diseases. However, the precise role of PKM2 in podocyte homeostasis and its contribution to glomerular function under normal and pathological conditions remains to be determined.
Methods: In this project, we evaluated the role of PKM2 in podocyte differentiation and homeostasis, using shRNA-mediated PKM2 knockdown in murine podocytes. Next, we examined the clinical significance of PKM2 deficiency to renal function using the Cre-LoxP technology to generate mice that specifically lack PKM2 in podocytes. Then, lipopolysaccharide (LPS), an endotoxin agent, was used to induce renal injury. We also used various genetic approaches and pharmaceutical compounds to decipher the molecular mechanisms mediating PKM2 action.
Results: The genetic depletion of PKM2 increased podocyte differentiation markers and protected against LPS induced albumin permeability in vitro. These effects were concomitant with enhanced activation of autophagy, AMPK, and mTORC1 but reduced AKT phosphorylation. On the other hand, the prolonged pharmacological inhibition of AKT or activation of AMPK recapitulated the effects of PKM2 deficiency on autophagy induction, podocyte differentiation, and albumin loss. In vivo, the deletion of PKM2 preserved podocyte integrity and protected against LPS induced proteinuria and nephrin loss. Further analysis revealed that PKM2 deficiency was associated with reduced inflammatory cytokines, inflammation, ER stress, and β-catenin level but sustained Wilms’ Tumor 1 (WT1) expression after LPS challenge. Additionally, PKM2 deficiency enhanced podocyte survival and ameliorated LPS-induced podocytes cell death. Mechanistic studies revealed that PKM2 interacts with β-catenin to promote LPS induced podocytes cell death.
Conclusion: Our data elucidate a novel role of PKM2 in podocyte homeostasis and propose PKM2 as a potential therapeutic target to halt renal injury progression.
Alquraishi, Mohammed, "Novel Insights into The Role of Pyruvate Kinase M2 in Podocyte Homeostasis and Function. " PhD diss., University of Tennessee, 2021.