AMPK mediates regulation of glomerular volume and podocyte survival
Khadija Banu, Qisheng Lin, John M. Basgen, Marina Planoutene, Chengguo Wei, Anand C. Reghuvaran, Xuefei Tian, Hongmei Shi, Felipe Garzon, Aitor Garzia, Nicholas Chun, Arun Cumpelik, Andrew D. Santeusanio, Weijia Zhang, Bhaskar Das, Fadi Salem, Li Li, Shuta Ishibe, Lloyd G. Cantley, Lewis Kaufman, Kevin V. Lemley, Zhaohui Ni, John Cijiang He, Barbara Murphy, Madhav C. Menon
Khadija Banu, Qisheng Lin, John M. Basgen, Marina Planoutene, Chengguo Wei, Anand C. Reghuvaran, Xuefei Tian, Hongmei Shi, Felipe Garzon, Aitor Garzia, Nicholas Chun, Arun Cumpelik, Andrew D. Santeusanio, Weijia Zhang, Bhaskar Das, Fadi Salem, Li Li, Shuta Ishibe, Lloyd G. Cantley, Lewis Kaufman, Kevin V. Lemley, Zhaohui Ni, John Cijiang He, Barbara Murphy, Madhav C. Menon
View: Text | PDF
Research Article Cell biology Nephrology

AMPK mediates regulation of glomerular volume and podocyte survival

Abstract

Herein, we report that Shroom3 knockdown, via Fyn inhibition, induced albuminuria with foot process effacement (FPE) without focal segmental glomerulosclerosis (FSGS) or podocytopenia. Interestingly, knockdown mice had reduced podocyte volumes. Human minimal change disease (MCD), where podocyte Fyn inactivation was reported, also showed lower glomerular volumes than FSGS. We hypothesized that lower glomerular volume prevented the progression to podocytopenia. To test this hypothesis, we utilized unilateral and 5/6th nephrectomy models in Shroom3-KD mice. Knockdown mice exhibited less glomerular and podocyte hypertrophy after nephrectomy. FYN-knockdown podocytes had similar reductions in podocyte volume, implying that Fyn was downstream of Shroom3. Using SHROOM3 or FYN knockdown, we confirmed reduced podocyte protein content, along with significantly increased phosphorylated AMPK, a negative regulator of anabolism. AMPK activation resulted from increased cytoplasmic redistribution of LKB1 in podocytes. Inhibition of AMPK abolished the reduction in glomerular volume and induced podocytopenia in mice with FPE, suggesting a protective role for AMPK activation. In agreement with this, treatment of glomerular injury models with AMPK activators restricted glomerular volume, podocytopenia, and progression to FSGS. Glomerular transcriptomes from MCD biopsies also showed significant enrichment of Fyn inactivation and Ampk activation versus FSGS glomeruli. In summary, we demonstrated the important role of AMPK in glomerular volume regulation and podocyte survival. Our data suggest that AMPK activation adaptively regulates glomerular volume to prevent podocytopenia in the context of podocyte injury.

Authors

Khadija Banu, Qisheng Lin, John M. Basgen, Marina Planoutene, Chengguo Wei, Anand C. Reghuvaran, Xuefei Tian, Hongmei Shi, Felipe Garzon, Aitor Garzia, Nicholas Chun, Arun Cumpelik, Andrew D. Santeusanio, Weijia Zhang, Bhaskar Das, Fadi Salem, Li Li, Shuta Ishibe, Lloyd G. Cantley, Lewis Kaufman, Kevin V. Lemley, Zhaohui Ni, John Cijiang He, Barbara Murphy, Madhav C. Menon

×

Figure 5

Shroom3 or Fyn knockdown increases cellular AMPK activation.

Options: View larger image (or click on image) Download as PowerPoint
Shroom3 or Fyn knockdown increases cellular AMPK activation. (A) WBs of ...
(A) WBs of lysates from Scramble-1, SHROOM3-shRNA (top panel), and Scramble-2 FYN-shRNA podocytes (top panel) probed for SHROOM3 or FYN and phosphorylated and total MTOR, phosphorylated and total AMPK, and actin. Representative WBs of lysates from Scramble-1, SHROOM3-shRNA (bottom panel), and Scramble-2 FYN-shRNA podocytes (bottom panel) probed for SHROOM3 or FYN, phosphorylated and total EF2, phosphorylated and total ULK1, LC3-I and II, and actin. (B) Box plots show respective relative band intensity (normalized to actin) (n ≥ 3 sets). (C) Representative immunofluorescence images (left panels = 20×) of SHROOM3-shRNA and Scramble-1 cells stained for LC3 (TRITC). Right panels show confocal images obtained after 24 hours of bafilomycin treatment. (D) Dot plots quantify LC3-positive vacuoles per podocyte (n = 2 sets). (E) Representative immunofluorescence images show glomerular staining for phosphorylated AMPK (top row) and merge with DAPI (bottom row) in Shroom3-KD and control mice. (F) Box plots quantify intensity of phosphorylated AMPK/per glomerular outline per group (30 glomeruli/animal; n = 5 each). (G) Representative WBs of glomerular lysates from Shroom3-KD mice and controls probed for Shroom3, phosphorylated and total Ampk, phosphorylated and total Ef2 and actin, and (H) respective relative band intensities are shown (normalized to actin; n > 4 each). WB, Western blot. In the box-and-whisker plot, the box represents the middle quartiles, the lines indicate the median, and the whiskers denote the 5th–9th percentile. Line and whiskers indicate mean ± SEM; unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.