Possible role for glomerular-derived angiotensinogen in nephrotic syndrome

Background and objective: Renin–angiotensin system (RAS) inhibitors reduce glomerular injury and proteinuria, indicating that angiotensin II (Ang II) is involved in glomerular diseases. Although the local RAS is reported to play an essential role in maintaining local tissue functions, the role of the local RAS in regulating glomerular function is not well evaluated. In this study, we analyzed the glomerular expression of RAS components in nephrotic models and the effect of Ang II receptor blockers (ARB) on the expression of angiotensinogen (AGT). Methods: The levels of glomerular expression of RAS components were analyzed in two nephrotic models: anti-nephrin antibody-induced nephropathy and PAN nephropathy, a mimic of human minimal change nephrotic syndrome. The effect of the ARB irbesartan on the expression of AGT in the nephrotic model was analyzed. Results: Glomerular expression of AGT and the receptors for Ang II was clearly increased in the nephrotic models, while the expression levels of renin, ACE and ACE2 were decreased. ARB treatment suppressed the increase of glomerular expression of AGT in the nephrotic model. Conclusion: It is conceivable that the promoted local RAS action participated in the glomerular dysfunction, and that ARB treatment ameliorated slit diaphragm injury by inhibiting the positive feedback loop of the activated local Ang II action

The number of mitophagic cells is not affected by co-expression of ATP13A2-Halo and Tom20.

HeLa cells stably overexpressing ATP13A2-Halo were additionally transfected with plasmids carrying Tom20 and Su9-mCherry-GFP. Two days after transfection, the cells were analyzed by confocal microscopy. The mitophagic cells are defined as those exhibiting mCherry-positive and GFP-negative particles. White arrow heads indicates representative mitophagic particles. As a positive control condition, cells were treated with 50 μM 3-chlorophenylhydrazone (CCCP). (B) The numbers of mitophagic cells were counted and shown as frequencies (%). Values are average ± S.E.M. (n = 50 cells). Statistical analysis was performed using Student’s t test: *p p p < 0.001, “n.d.” difference is not significant.</p

ATP13A2-dependent sorting of mCherry-Tom20-N is sensitive to MPP⁺ treatment.

(A) HeLa cells were cotransfected with mCherry-TOM20-N and ATP13A2-Halo. One day after transfection, MPP+ was added to the culture medium and cells were further incubated for 24 hours, followed by immunostaining. After MPP+ treatment, no colocalization was observed for ATP13A2 and TOM20. Scatter plots are also shown in S6 Fig (B) Pearson correlation coefficients of ATP13A2-Halo and mCherry-TOM20-N are shown. Data represent averages of n = 10 samples with S.E.M. (bars). Statistical analysis was performed using Student’s t test: *p p p (C) Together with mCherry-TOM20-N, mito Grx1-roGFP2 was transfected for assessing mitochondrial shapes in the presence of MPP+.</p

Full documentation of the immunostaining data (ATP13A2-Halo).

Only representative images are shown in the main Fig 2. (PDF)</p

Scatter plots of the images used in the main Fig 1A and 1B.

Representative 3 plots for each experimental condition are shown. A, ATP13A2-Halo-OregonG vs LysoTracker. B, ATP13A2-Halo-OregonG vs MitoTracker. (PDF)</p

Construction and expression of ATP13A2-Halo.

Halo tag was C-terminally fused with human ATP13A2. Pilot expression in HEK cells produced expected size of ATP13A2-Halo protein (164K). Stable cells were harvested and microsomes were prepared. ATP13A2-Halo was specifically labeled ex vivo with TMR-ligand (left) or Oregon green (right), followed by SDS-PAGE and fluorescence imaging.</p

List of organelle markers used in the study and summary of the results.

List of organelle markers used in the study and summary of the results.</p

ATP13A2 overexpression altered the intracellular distribution of mCherry-TOM20-N (mitochondrial outermembrane, MOM).

(A) MOM protein, mCherry-TOM20-N, was overexpressed in HeLa cells and mitochondrial localization was confirmed by immunostaining; TOM20 exhibited complete colocalization with the endogenous mitochondrial protein, TUFM. (B) Mitochondria was visualized by mitochondrial intermembrane space (IMS)/matrix marker, mito Grx1-roGFP2. Upon co-overexpression of ATP13A2, substantial portion of mCherry-TOM20-N appeared as particulate structures. Scatter plots are also shown in S3 Fig (C) Pearson correlation coefficients of mCherry-TOM20-N and mito Grx1-roGFP2 are shown. Data represent averages of n = 10 samples with S.E.M. (bars). Statistical analysis was performed using Student’s t test: *p p p (D) mCherry-TOM20-N and ATP13A2-Halo were coexpressed and their localizations were analyzed by live-cell imaging. TOM20 showed mixed localization pattern with mitochondria-like tubular shapes and particulate structures. The latter signals substantially merge with ATP13A2-positive vesicles. Catalytically dead mutant of ATP13A2 (D513A) produced no effect on the localization pattern of mCherry-TOM20-N. Scatter plots are also shown in S4 Fig (E) Pearson correlation coefficients of indicated proteins are shown. Data represent averages of n = 10 samples with S.E.M. (bars). Statistical analysis was performed using Student’s t test: *p p p < 0.001.</p

Scatter plots (Mito Grx1-roGFP2 vs mCherry-Tom20-N) of the images used in the main Fig 3B and 3C.

Representative 3 plots for each experimental condition are shown. Cells were co-transfected with ATP13A2-Halo (B), resulting in partial segregation of Tom20 signals from Mito Grx1-roGFP2 signals (dashed circles). Without ATP13A2 co-transfection (A), Mito Grx1-roGFP2 and mCherry-Tom20-N signals produced practically perfect overlap. (PDF)</p

Original images of the membranes shown in the main Fig 6.

Nonspecific bands are marked with asterisks. (PDF)</p