Volume 86, Issue 1, Pages 103-117 (July 2014) Activation of the receptor for advanced glycation end products induces nuclear inhibitor of protein phosphatase-1 suppression  Marita Liebisch, Tzvetanka Bondeva, Sybille Franke, Christoph Daniel, Kerstin Amann, Gunter Wolf  Kidney International  Volume 86, Issue 1, Pages 103-117 (July 2014) DOI: 10.1038/ki.2014.3 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Expression of nuclear inhibitor of protein phosphatase-1 (NIPP1) in podocytes treated with control-bovine serum albumin (co-BSA) and advanced glycation end product-BSA (AGE-BSA). (a) Real-time polymerase chain reaction (PCR) analysis of NIPP1 mRNA expression. Treatment of podocytes with AGE-BSA reduced the expression of NIPP1 mRNA, compared with co-BSA-treated cells. The data are quantified as described in the Materials and Methods section and expressed as the percentage of co-BSA. ***P<0.001 versus co-BSA, n=18. (b) Detection of NIPP1 protein by western blot analyses in lysates from co-BSA- and AGE-BSA-treated cells. The equal loading was monitored by β-actin protein expression. Left margins show the molecular weight of the proteins in kDa. Lysate expressing NIPP1DDK-tagged protein was used as a positive control for NIPP1 expression. Representative western blot analysis are shown. NIPP1 protein level was decreased after AGE-BSA addition. (c) Quantification of NIPP1 protein expression. The expression of NIPP1 protein was normalized to β-actin and presented in percentage relative to co-BSA. *P<0.05 versus co-BSA, n=9. (d) Immunological analysis of NIPP1 protein expression. Representative images of NIPP1 detection in co-BSA- and AGE-BSA-treated podocytes, as well as merge image of NIPP1 and 4',6-diamidino-2-phenylindole (DAPI) (nuclear staining), are shown. The staining demonstrates mainly nuclear localization and only a weak staining for NIPP1 into the cytoplasm of the podocytes. Bars=20μm. (e) Densitometry quantification of NIPP1 immunoreactivity. The relative intensity of NIPP1 in the cell nucleus and cytoplasm is presented in percentage relative to co-BSA. ***P<0.001 versus co-BSA, n=6. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Expression and activity of protein phosphatase-1 (PP1) in podocytes treated with control-bovine serum albumin (co-BSA) or advanced glycation end product-BSA (AGE-BSA). (a) Real-time polymerase chain reaction (PCR) analysis of PP1 mRNA expression. Addition of AGE-BSA to podocytes did not affect the expression of PP1 mRNA. The data are quantified as described in Materials and Methods and expressed as the percentage of co-BSA, n=18. (b) Detection of PP1 protein by western blot analysis in lysates from co-BSA- and AGE-BSA-treated cells. The membrane was reprobed with β-actin antibody to confirm equal protein loading. Left margins show the molecular weight of the proteins in kDa. A recombinant His-PP1 protein was used as a positive control for PP1 detection. Representative western blot analysis are shown. (c) Quantification of PP1 protein expression from western blot analysis. PP1 protein is presented in percentage relative to co-BSA. The difference between AGE-BSA- and co-BSA-treated cells was not significant, n=9. (d) Immunostaining analysis of PP1 protein expression. Representative images of co-BSA- and AGE-BSA-treated podocytes stained with anti-PP1 antibody are shown. PP1 protein is present in the nucleus and the cytoplasm of co-BSA-treated podocytes, whereas AGE-BSA slightly reduced the nuclear staining and induced a perinuclear accumulation of the protein (arrow). The nuclear staining is also shown in merged PP1+4',6-diamidino-2-phenylindole (DAPI) images. The images were analyzed using Axioplan light microscope and the AxioVision Rel.4.6 software. Original magnification, × 400. (e) Densitometry quantification of PP1 immunofluorescence. The relative intensity of PP1 in the cell nucleus and cytoplasm are presented in percentage relative to co-BSA. Bars=20μm, n=6. (f) PP1 phosphatase activity assay. The podocytes were treated with either co-BSA or AGE-BSA for 24h, cells were lysed, total protein was isolated, and 300μg of total protein was subjected to the phosphatase assay. AGE-BSA treatment significantly increased PP1 phosphatase activity. *P<0.05 versus co-BSA, n=9. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Suppression of nuclear inhibitor of protein phosphatase-1 (NIPP1) expression by transient transfection of NIPP1 small interfering RNA (siRNA) increased PP1 enzymatic activity. Podocytes were transiently transfected with either NIPP1 siRNA or control nonsilencing siRNA for 24h. NIPP1 mRNA and protein expression were then analyzed. (a) Detection of NIPP1 and PP1 mRNA expression by real-time polymerase chain reaction (PCR). NIPP1 downregulation specifically suppressed NIPP1 mRNA, but not PP1 mRNA. The mRNA levels are presented in percentage relative to control-siRNA (co-siRNA)-transfected cells. ***P<0.001 versus co-siRNA, n=18. (b) Western blot analysis of NIPP1 and PP1 protein expression in co-siRNA- and NIPP1 siRNA–transfected cells. An equal protein loading was controlled by β-actin protein expression. Left margins show the molecular weight of the proteins in kDa. The corresponding NIPP1 (NIPP1DDK)- and PP1 (His-PP1)-positive controls are also presented on the western blot analysis. Representative western blot analysis are shown. NIPP1 inhibition suppressed only NIPP1 protein expression, but did not influence PP1 or β-actin protein expression, n=3. (c) PP1 phosphatase activity in protein lysates from co-siRNA- and NIPP1 siRNA–transfected podocytes. NIPP1 downregulation elevates PP1 phosphatase activity relative to co-siRNA. ***P<0.001 versus co-siRNA, n=12. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Nuclear inhibitor of protein phosphatase-1 (NIPP1) is not involved in apoptosis or necrosis of podocytes. (a) Detection of apoptosis in cells transfected with either NIPP1 or co-small interfering RNAs (control-siRNAs) by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. For signal conversion, the POD (antibody-conjugated peroxidase) substrate diaminobenzidine was used. Quantification was performed by measuring the relative intensity of at least 200 nuclei per slide and is expressed in percentage relative to co-siRNA. Co-siRNA TUNEL staining was arbitrarily considered as 100%, n=10. (b) TUNEL assay was performed as in a, but podocytes were stimulated for 24h with co-BSA or advanced glycation end product-bovine serum albumin (AGE-BSA). Neither AGE-BSA nor NIPP1 siRNA induced apoptosis, as detected by TUNEL assays, suggesting that apoptosis is independent of NIPP1. (c) Detection of early apoptosis and necrosis via the Annexin-V assay. The cells were transiently transfected with either co-siRNA or NIPP1 siRNA, or stimulated with 1μmol/l staurosporine (positive control), or grown in RPMI 1640 medium with 10% fetal calf serum (FCS). At 24h after transfection, cells were subjected to fluorescence-activated cell sorting (FACS) analysis using an Annexin V-FLUOS assay. A total of 1 × 104 podocytes were counted for each treatment and the percentage of vital, apoptotic, and necrotic cells was analyzed using FACSCalibur. The graphic represents the percentage of vital, necrotic, and apoptotic cells for 10% FCS-grown cells, for co-siRNA- and NIPP1-siRNA-transfected podocytes, as well as for 1μmol/l staurosporine-treated cells. The podocytes transfected with NIPP1 siRNA show only a low percentage of apoptotic or necrotic cells, as did cells treated with the 10% FCS cells. The positive control (staurosporine) clearly reduced the percentage of vital podocytes by inducing necrosis. ***P<0.001 versus 10% FCS, n=12. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Nuclear inhibitor of protein phosphatase-1 (NIPP1) suppression inhibits proliferation and induces cell cycle arrest in podocytes. (a) Proliferation assay. The podocytes were transfected with either co-small interfering RNA (siRNA) or NIPP1 siRNA and were subjected to 5-bromo-2-deoxyuridine (BrdU) incorporation assay to monitor cell proliferation. Analyses were performed 24, 48, and 72h after transfection. NIPP1 suppression inhibits podocyte proliferation relative to co-siRNA-transfected cells. **P<0.01 versus co-siRNA, ***P<0.001 versus co-siRNA, n=6. (b) Cell cycle analysis. Cells were transfected and subjected to cell cycle analyses 24 and 48h after transfection. The percentage of the cells in a particular cell cycle phase was determined by FACS analysis of the DNA content. The graph represents the percentage of cells in G0/G1 cell cycle phase. Suppression of NIPP1 expression increases the number of cells in the G0/G1 phase. **P<0.01 versus co-siRNA, ***P<0.001 versus co-siRNA, n=8. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Nuclear inhibitor of protein phosphatase-1 (NIPP1) inhibition induces hypertrophy of podocytes. (a) Determination of cellular hypertrophy by measuring the ratio of protein content per 103 podocytes. The cells transfected with control-small interfering RNA (co-siRNA) or NIPP1 siRNA were lysed 24h after transfection and cell hypertrophy. Hypertrophy is presented in percentage relative to co-siRNA-transfected podocytes. *P<0.05 versus co-siRNA, n=9. (b) Determination of the extent of hypertrophy by measurement of the cell size of podocytes transfected with either co-siRNA or NIPP1 siRNA. The cell size of at least 200 transfected cells was measured and presented in percentage relative to co-siRNA. NIPP1 suppression significantly increased the size of the cells compared with the co-siRNA-transfected cells. **P<0.01 versus co-siRNA, n=10. (c) Detection of p27Kip1 protein expression in lysates from co-siRNA- and NIPP1 siRNA–transfected podocytes. NIPP1 downregulation stimulated the expression of p27Kip1. Left margins show the molecular weight of the proteins in kDa. As a positive control for p27Kip1 expression, a recombinant His-p27Kip1 was used. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 Nuclear inhibitor of protein phosphatase-1 (NIPP1) downregulation induced receptor for advanced glycation end product (RAGE) protein expression and nuclear factor-κB (NF-κB) transcriptional activity. (a) Relative intensity of RAGE expression in podocytes stained with anti-RAGE antibody in cells treated with control-bovine serum albumin (co-BSA) and AGE-BSA. ***P<0.001 versus co-BSA, n=4. (b) Representative images of RAGE expression in podocytes transfected with either co-small interfering RNA (siRNA) or NIPP1 siRNA. Nuclear staining is shown in the merge RAGE+4',6-diamidino-2-phenylindole (DAPI) images, respectively. Bars=20μm. (c) Quantification of the immunofluorescence intensity. ***P<0.001 versus co-siRNA, n=4. (d) Nuclear factor-κB (NF-κB) luciferase assay. NF-κB reporter plasmid was co-transfected with either NIPP1 siRNA or co-siRNA, and the cell lysates were subjected to luciferase assay to determine NF-κB transcriptional activity. NIPP1 suppression induced NF-κB activity. *P<0.01 versus co-siRNA, n=10. (e) Tumor necrosis factor-α (TNF-α) mRNA expression in NIPP1 siRNA– and co-siRNA-transfected podocytes relative to co-siRNA. NIPP1 suppression significantly increased the expression of TNF-α. **P<0.01 versus co-siRNA, n=6. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 8 Detection of nuclear inhibitor of protein phosphatase-1 (NIPP1) in kidney glomeruli of diabetic db/db and db/+ mice. (a) Nε-carboxy-methyl-lysine (CML) serum concentrations in diabetic (db/db) and non-diabetic db/+ mice. CML levels were significantly elevated in diabetic mice (db/db) compared with db/+ mice. *P<0.05, n=5. (b) Detection of NIPP1 and synaptopodin (SYN) protein expression in kidney sections of diabetic (db/db) and non-diabetic control mice (db/+). The mice were perfused with saline to remove blood cells, and the kidneys were fixed and embedded in paraffin. Upper panel: staining of non-diabetic db/+ kidney sections. Lower panel: staining of diabetic (db/db) mice. Representative images of NIPP1 and SYN expression are shown. NIPP1 staining is significantly reduced in podocytes from diabetic (db/db) kidney sections compared with the db/+ mice. NIPP1 podocyte expression is visualized through its colocalization with SYN shown on the merge images for diabetic db/db and db/+ mice. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). The nuclear localization of NIPP1 (white arrows) in db/+ mice is also detectable on the merge images, which is significantly decreased in db/db mice. Kidney sections were analyzed using the Axioplan fluorescent microscope and the AxioVision Rel.4.6 software. Bars=20μm. (c) Relative expression of NIPP1 in glomeruli from diabetic (db/db) and db/+ mice. At least 10 glomeruli were analyzed per kidney section. ***P<0.005 versus db/+, n=5. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 9 Correlation between nuclear inhibitor of protein phosphatase-1 (NIPP1) podocyte expression and Nε-carboxy-methyl-lysine (CML) plasma concentrations in db/db mice. (a) The plot represents a correlation between NIPP1 expression in podocytes and CML plasma concentration of diabetic (db/db) mice aged 6, 12, 27, 39, and 47 weeks. The NIPP1 expression in glomerular podocytes was analyzed by densitometry in double-positive cells (NIPP1 and synaptopodin, a podocyte-specific protein) in 5-μm paraffin kidney sections. The NIPP1 expression is presented as average arbitrary units (AU) of the fluorescent intensity measured from at least 10 glomeruli per kidney section. The CML concentrations in blood plasma were determined by enzyme-linked immunosorbent assay (ELISA) in duplicate. The Spearman correlation coefficient between NIPP1 and CML was ρ=-0.9, P<0.05, which demonstrates a decreasing monotonic trend between NIPP1 and CML levels in db/db mice. (b) The plot represents a correlation between the NIPP1 expression in podocytes and PP1 phosphatase activity measured in total kidney protein lysates of diabetic (db/db) mice aged 6, 12, 27, 39, and 47 weeks. The NIPP1 expression in glomerular podocytes was analyzed by densitometry in double-positive cells (NIPP1 and synaptopodin) in 5-μm paraffin kidney sections. The NIPP1 expression is presented as average AU of the fluorescent intensity quantified from at least 10 glomeruli per kidney section. PP1 phosphatase activity was measured in vitro from protein lysates and is presented as fluorescent light units (FLUs). (c) Expression of NIPP1 protein in db/db and age-matched db/+ mice. Double immunofluorescence for NIPP1 and synaptopodin (SYN) was performed on 5-μm paraffin kidney sections from mice aged 6, 12, 27, 39, and 47 weeks old. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). In the analysis, two mice per time point and at least 10 glomeruli per kidney section were used. Representative images are shown. Bars=20μm. The quantification of the staining for NIPP1 expression is shown in Table 1. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 10 Effect of soluble receptor for advanced glycation end products (RAGE) on nuclear inhibitor of protein phosphatase-1 (NIPP1) expression in diabetic mice. The experiment was performed to test whether NIPP1 expression is affected in vivo from the RAGE blockade. A proof-of-concept experiment was performed using two groups of mice: diabetic db/db (n=2) and age-matched non-diabetic db/+ (n=1) mice per group were injected intraperitoneally (i.p.) with 20μg per day of soluble His-RAGE (sRAGE) or an equimolar concentration of mouse serum albumin (MSA), 40μg per day for 14 days. (a) Immunological detection of NIPP1 expression in kidney section of mice treated with 40μg per day MSA. The sections were double-stained with synaptopodin (SYN). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Merge image of the staining is also shown. Bars=20μm. Representative images from each kidney staining used in the analysis are shown. NIPP1 expression is reduced in db/db mice compared with the non-diabetic mice. (b) Immunological detection of NIPP1 expression in kidney section of mice i.p. injected with 20μg per day of sRAGE. The sections were stained as in a. Merged images of the stainings are shown. Bars=20μm. Representative images from each kidney staining for analysis are shown. Injection of sRAGE for 14 days increased the NIPP1 expression in podocytes in vivo. (c) Densitometry of the NIPP1 staining intensity in db/db and db/+ mice treated with sRAGE and mouse serum albumin (MSA). At least 10 glomeruli per kidney section were quantified. The NIPP1 expression in podocytes is presented in percentage relative to the NIPP1 expression measured in db/+ mice treated with MSA. db/db mice, n=20 glomeruli; db/+ mice, n=20 glomeruli. (d) PP1 phosphatase activity in protein lysates isolated from kidneys of db/db and db/+ mice treated with sRAGE and MSA. PP1 activity was measured in duplicate from each lysate. db/+ mice, n=2; db/db mice, n=4. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 11 Expression of nuclear inhibitor of protein phosphatase-1 (NIPP1) in human kidney biopsies. Analysis of NIPP1 and podocin expression in double-stained, paraffin-embedded human kidney biopsies. (a) Detection of NIPP1 and podocin in 4-μm paraffin human kidney sections in control kidney biopsies. At least 10 glomeruli were analyzed per kidney section. Representative image is shown. Bar=100μm. Biopsies used in the analysis, n=18. (b) Detection of NIPP1 and podocin in 4-μm paraffin kidney sections in control, healthy kidney biopsies. Bar=50μm. (c) Detection of NIPP1 and podocin in 4-μm paraffin kidney sections of patients diagnosed with diabetic nephropathy (DN). At least 10 glomeruli were analyzed per kidney section. Representative image is shown. Bar=100μm. Biopsies used in the analysis, n=21. (d) Semiquantitative analysis of podocyte NIPP1 expression using a scoring method. NIPP1 expression in podocytes of biopsies of patients with DN is significantly decreased compared with control kidney tissue. Control biopsies, n=18; DN biopsies, n=21. *P<0.001. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 12 Expression of protein phosphatase-1 (PP1) in human biopsies. Analysis of PP1 and podocin expression in double immunohistochemistry staining of human kidney biopsies. (a) PP1 and podocin protein expression detected in control kidney biopsies. At least 10 glomeruli were analyzed per kidney section. Representative image is shown. Bar=100μm. Biopsies used in the analysis, n=18. (b) Detection of PP1 and podocin in kidney biopsies of patients diagnosed with diabetic nephropathy (DN). At least 10 glomeruli were analyzed per kidney section. Representative image is shown. Bar=100μm. Biopsies used in the analysis n=21. (c) Quantification of podocyte PP1 expression using a scoring method. The scores are set as follows: 0—no double-positive podocytes; 1—up to 3 double-positive podocytes per glomerular cross-section (gcs); 2—more than 3 double-positive podocytes per gcs; 3—more than 6 double-positive podocytes per gcs; 4—nearly all podocytes were double positive per gcs. There is no significant difference in the PP1 expression between control and DN kidney biopsies. Normal kidneys, n=18; biopsies with DN, n=21. Kidney International 2014 86, 103-117DOI: (10.1038/ki.2014.3) Copyright © 2014 International Society of Nephrology Terms and Conditions