1. Volume 86, Issue 4, Pages 712-725 (October 2014)
Histone deacetylase 4 selectively contributes to podocyte injury in diabetic nephropathy 
Xiaojie Wang, Jiang Liu, Junhui Zhen, Chun Zhang, Qiang Wan, Guangyi Liu, Xinbing Wei, Yan Zhang, Ziying Wang, Huirong Han, Huiyan Xu, Chanchan Bao, Zhenyu Song, Xiumei Zhang, Ningjun Li, Fan Yi 
Kidney International 
Volume 86, Issue 4, Pages (October 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Expression patterns of histone deacetylases (HDACs) in the kidneys of streptozotocin (STZ)-induced diabetic rats and diabetic db/db mice. (a) Relative mRNA levels of HDAC1-11 in the kidneys of STZ-induced diabetic rats on real-time RT-PCR analysis. (b) Representative western blot gel documents and summarized data showing the relative protein levels of HDAC1-11 in the kidneys of STZ-induced diabetic rats. (c) Representative photomicrographs of HDAC2, HDAC4, and HDAC5 immunohistochemical staining in the kidneys of STZ-induced diabetic rats. Negative control by omission of the corresponding primary antibodies demonstrated no nonspecific staining. (d) Representative western blot gel documents and summarized data showing the relative protein levels of HDAC2, HDAC4, and HDAC5 in the kidneys of diabetic db/db mice and their genetic control db/+ mice. *P<0.05 versus control (n=10).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

3. Figure 2
Renal cortical expressions of histone deacetylase (HDAC)2, HDAC4, and HDAC5 in human diabetic nephropathy (DN) biopsies. (a) Representative photomicrographs of HDAC2, HDAC4, and HDAC5 staining in human renal cortical tissue from normal subjects (n=11) and from patients with diabetic nephropathy (DN) (n=8) or diabetic patients without nephropathy (DM-NN) (n=8). (b) Relative mRNA levels of HDAC2 in the renal biopsies from different patients. (c) Negative correlation between HDAC2 mRNA levels and estimated glomerular filtration rate (GFR) in all subjects. (d) Relative mRNA levels of HDAC4 in the renal biopsies from different patients. (e) Negative correlation between HDAC4 mRNA levels and estimated GFR in all subjects. (f) Relative mRNA levels of HDAC5 in the renal biopsies from different patients. (g) Negative correlation between HDAC5 mRNA levels and estimated GFR in all subjects. (h) Relative mRNA levels of autophagy-related genes in the renal biopsies from different patients. *P<0.05 versus normal subjects.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

4. Figure 3
The effects of different stimuli on the expressions of histone deacetylase (HDAC)2, HDAC4, and HDAC5 in podocytes. (a) Representative western blot gel documents and summarized data showing the protein levels of HDAC2, HDAC4, and HDAC5 in conditionally immortalized human podocytes treated with different concentrations of high glucose (HG, final concentration 20 or 40mmol/l in medium), advanced glycation end product (AGE, 50–200μg/ml), or transforming growth factor-β (TGF-β, 2–8ng/ml) for 24h. (b) Representative confocal microscopic images showing HDAC2, HDAC4, or HDAC5 expression in the kidneys of streptozotocin (STZ)-induced diabetic rats; synaptopodin was used as podocyte marker. *P<0.05 versus control (n=6).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

5. Figure 4
Gene silencing of histone deacetylase (HDAC)4 reduced inflammatory response and apoptosis in podocytes with high-glucose treatment. (a) Representative western blot gel documents and summarized data showing the efficiency of HDAC4 knockdown by shRNA-NOD2 transfection. (b) Representative western blot gel documents and summarized data showing the overexpression of HDAC4 by pCMV6-HDAC4 transfection. (c) The effects of gene silencing of HDAC4 on the levels of proinflammatory mediators in podocytes with high-glucose treatment (HG, final concentration 40mmol/l in medium) for 24h. (d) Summarized data showing podocyte apoptosis determined by flow cytometric analysis. *P<0.05 versus control, #P<0.05 versus vehicle of high-glucose treatment (n=6).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

6. Figure 5
Histone deacetylase (HDAC)4 contributed to high glucose–inhibited basal autophagy in podocytes. (a) Representative western blot gel documents and summarized data showing the levels of autophagy-related proteins in podocytes with different treatments. (b) Representative electronic micrographs and summarized data showing the number of autophagosomes/cell in different groups (a random number of 30 cells were selected for each group) including high glucose (HG, final concentration 40mmol/l in medium, 24h)–treated podocytes with or without HDAC4 knockdown. (c) Representative images showing LC3 staining in different groups of podocytes infected with GFP-RFP-LC3 adenovirus for 24h. *P<0.05 versus control, #P<0.05 versus vehicle of high-glucose treatment (n=6).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

7. Figure 6
Histone deacetylase (HDAC)4-inhibited autophagy reduced the expressions of podocyte slit diaphragm proteins and disrupted the podocyte architectural integrity. (a) Representative western blot gel documents and summarized data showing the relative protein levels of podocin and nephrin in podocytes treated with rapamycin (1μmol/l) in response to high glucose (HG, final concentration 40mmol/l in medium, 24h). (b) The effects of HDAC4 knockdown on the expressions of podocin and nephrin in podocytes. (c) Summarized data showing podocyte apoptosis determined by flow cytometric analysis. (d) Representative images showing that HDAC4-inhibited autophagy was associated with changes in cytoskeleton distribution as evidenced by rearrangement of the actin cytoskeleton as indicated. *P<0.05 versus control, #P<0.05 versus vehicle of high-glucose treatment (n=6).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

8. Figure 7
Activation and translocation of signal transducers and activators of transcription factor 1 (STAT1) were associated with histone deacetylase (HDAC4)-mediated deacetylation in response to hyperglycemia. (a) Representative western blot gel documents and summarized data showing the effects of high glucose (HG, final concentration 40mmol/l in medium, 24h) on the expression of STAT1 in podocytes. (b) HG enhanced the interaction of HDAC4 and STAT1 by immunoprecipitation. (c) Representative western blot gel documents and summarized data showing the acetylation of STAT1 in podocytes with different treatments by immunoprecipitation. (d) Subcellular location of STAT1 was determined by immunofluorescence microscopy in podocytes. Nuclei were revealed using 4',6-diamidino-2-phenylindole (DAPI) staining. *P<0.05 versus control, #P<0.05 versus vehicle of high-glucose treatment (n=6).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

9. Figure 8
In vivo gene silencing of histone deacetylase (HDAC)4 by intrarenal lentiviral gene delivery in rats. (a) Representative renal fluorescent photomicrographs of the rats 1 week after intrarenal delivery of lentivirus pGLV3-shRNA2-HDAC4 encoding green fluroscent protein (GFP) demonstrating predominant localization of GFP expression in the cortex (c) and corticomedullary junction (cmj); relatively weak GFP expression was observed in the medullary areas (m). (b) Representative confocal microscopic images showing GFP expression in podocytes from the kidney 1 week after intrarenal delivery of lentivirus pGLV3-shRNA2-HDAC4; synaptopodin was used as podocyte marker. (c) Relative mRNA levels of HDAC4 at different time points after pGLV3-shRNA2-HDAC4 was delivered into the rat kidney by means of intraparenchymal injections. *P<0.05 versus control.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

10. Figure 9
Gene silencing of histone deacetylase (HDAC)4 ameliorated renal injury in streptozotocin (STZ)-induced diabetic rats. (a) Relative mRNA levels of HDAC4 in the kidney from different groups of rats after 3-month pGLV3-shRNA2-HDAC4 transfection. (b) Representative western blot gel documents and summarized data showing HDAC4 protein levels in the kidney from different groups of rats after 3-month pGLV3-shRNA2-HDAC4 transfection. (c) Photomicrographs showing typical glomerular structural changes in different groups of rats. (d) Morphological changes in the podocyte foot process on electron microscopic analysis in different groups of rats. (e) The levels of proinflammatory mediators in the kidneys from different groups of rats. (f) Representative western blot gel documents and summarized data showing the levels of autophagy-related proteins in the kidney from different groups of rats. (g) Representative western blot gel documents and summarized data showing the levels of nephrin in the kidney from different groups of rats. *P<0.05 versus control, #P<0.05 versus STZ-induced diabetic rats (n=10).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions