Figure 4.
No evidence for tubular obstruction or ectopic cell ablation in the glomerulus. (A–F) To test if tubular obstruction occurs as a result of PEC ablation, experimental animals were injected with gold-labeled BSA 15 minutes before sacrifice (A–D, 9-µm thick sections). In control animals, gold-labeled BSA reabsorption occurred in the proximal part of proximal tubules throughout the entire cortex (A, arrows). (B) The same was true for experimental animals 4 days after induction with doxycycline. (C) On day 9, gold-labeled BSA was observed as a brown color in proximal tubules throughout the entire cortex similar to controls (black arrows). Because of proteinuria, gold-labeled BSA was also observed as a gray color in other parts of the tubular system (white arrows). (D) As a positive control for focal tubular obstruction by protein casts, a Thy1.1 transgenic mouse is shown 1 day after injection of Thy1.1 antiserum, which induces nephrotic-range proteinuria within minutes. Reabsorption of gold-labeled BSA can only be seen in some proximal tubuli (black arrow), whereas other tubuli are unstained (white arrows). (E) On semi-thin sections, gold-labeled BSA was still present within the capillary lumen (arrowheads) and reabsorbed in proximal tubuli (arrows). (F) In the Thy1.1 transgenic mouse with tubular obstruction, gold-labeled BSA–containing tubules (white arrow) were found next to protein-filled tubules without any gold-labeled BSA but instead with signs of cellular degeneration (black arrow). Some tubular lumina were blocked by protein casts (pc). All animals in A–F were 16-week-old male mice. (G and H) Absolute podocyte number per glomerular cross-section (n=150 in three animals of each time point as indicated; G, female). (I) To test whether leaky expression of the PEC-rtTA transgene occurs, an additional transgene tetO7-HIST1H2BJ/GFP was introduced that expresses histone-eGFP in a doxycycline-inducible fashion. (J and J′) When doxycycline was administered in triple transgenic mice that only lacked the ROSADTA176 transgene (suicide transgene), histone-eGFP was expressed specifically in almost all PECs (arrowheads) and no trace was observed elsewhere in the glomerulus (arrow). (K and K′) When doxycycline was administered in quadruple transgenic mice, histone-eGFP labeled PECs were observed much less frequently (arrowhead, 5 days after induction). No labeling of other glomerular cells, specifically of podocytes was observed (mice in J–K′ are male).