Autophagic Vacuolization as a Mechanism for Proteinuria-Induced Proximal Tubulopathy in the PEXTKO Mouse.
Delecia R LaFrance, George Rhodes, Bruce Molitoris, Deborah J McCarthy, Kevin J McCarthy. LSU Health Sciences Center, Shreveport, LA; Indiana University Purdue University at Indianpolis
Background: Albuminuria in glomerular diseases has been attributed to the loss of glomerular basement membrane (GBM) heparan sulfate (HS), which is recapitulated in the PEXTKO mouse model (Kidney Int. 74:289-99), whose GBM lack significant amounts of HS. Although they have a normal lifespan, PEXTKO mice develop vacuolated proximal tubule epithelium (PTE) and microalbuminuria. Similar PTE vacuoles have been described in diabetes, light chain disease and other stress-inducing nephropathies; however, the mechanism for vacuole formation remains elusive. The purpose of this study was to investigate the pathophysiology of proteinuria-induced proximal tubulopathy.
Design: Paraffin-embedded kidney sections (6 PEXTKO, 6 controls) were evaluated for apoptosis and autophagy using TUNEL assay (Promega, Madison, WI) and anti-LC3 (Novus biologicals, Littleton, CO), respectively. Additionally, multiphoton microscopy studies were conducted by labeling kidneys of anesthetized PEXTKO mice with Hoechst 33342 nuclear stain, followed by a bolus of Texas Red-labeled albumin. Kidneys were imaged with a 60x water objective lens; albumin delivery and uptake into the proximal tubules was monitored using time lapse-z step image collection.
Results: Cytoplasmic vacuoles within PTE of HS-deficient mice were identified on light microscopy.
Multiphoton microscopy studies documented the inability of vacuolated proximal tubule cells to absorb albumin as compared to non-vacuolated PTE in the same animals. TUNEL results indicated no significant increase in apoptosis of PEXTKO tubules (15.17 vs. 11.02 apoptotic cells/mm2 tubular area; p=0.11). Vacuolated PTE were positive for cytoplasmic LC3 suggesting autophagy as a mechanism of vacuole formation.
Conclusions: Protein overload eventually results in proximal tubulopathy evidenced by vacuole formation. The development of microalbuminuria (and vacuole formation) in HS-deficient PEXTKO mice is not due to loss of proximal tubule cells through apoptosis. Rather, our data suggests that proximal tubulopathy may be due to formation of autophagic vacuoles as a result of increased cell stress in response to proteinuria.
Wednesday, March 2, 2011 9:30 AM
Poster Session V # 225, Wednesday Morning