Part 3:Calpastatin Prevents Angiotensin Il-mediated Podocyte Injury Through Maintenance Of Autophagy

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Figure 8| Calpastatin overexpression prevents angiotensin Il (Angel)+ high-salt diet (HSD)-induced autophagy downregulation in podocytes. (a,b)Representative immunofluorescence images of the expression of P62 (green) and Podocalyxin (PODXL; red)in glomeruli from green fluorescent protein (GFP)-LC3 and CST'9(calpastatin transgenic) GFP-LC3 mice after 6 weeks of Angel +HSD. Figure subparts with prime indicate higher magnification. Nuclei were stained with Hoechst (blue).Bars=50 um. (c,d) Quantification of P62+ area per glomerular section.n=9 wild-type (WT) mice and n=7 CST'9 mice in (c), and n = 16 GFP-LC3 mice and n=16 CST'9 GFP-LC3 mice in (d).Values are presented as individual plots and mean ± SEM. Mann-Whitney test:**P=0.0033 in (c),**P=0.0011 in (d),(e,f) Representative

immunofluorescence images of the expression of GFP(green) and P62 (red) in glomeruli from GFP-LC3 and CST'9GFP-LC3 mice after 6 weeks of Angel + HSD. Figure subparts with prime indicate higher magnification. The arrowheads indicate GFP+ P62+ autophagosomes. (g)Quantification of the number of LC3+ P62+dots per podocyte.n =11 GFP-LC3 mice and n =16 CST19 GFP-LC3 mice, Values are presented as individual plots and mean ± SEM. Unpaired t-test with equal SD: P=0.1014. To optimize viewing of this image, please see the online version of this article at www. kidney-international org.

confirmed the effect of angle on podocyte injury and podocyte-specific gene targeting of AT1 demonstrated that activation of AT1 receptors in the glomerulus in experimental lupus nephritis is sufficient to accelerate kidney injury in the absence of hypertension. Calpain-mediated autophagy dysregulation in our model could be linked to direct AnglI-AT1 signaling on podocytes or to be a consequence of hypertension. We may provide an early answer to this question. Indeed, in the DOCA-salt model, we also found P62 accumulation in podocytes from hypertensive mice (Supplementary Figure S3). This suggests that autophagy blockade occurs in podocytes in this model, which is supposed to be independent of angle. Further studies evaluating podocyte injury pertaining to calpain activity and autophagic flux in mice deficient for AT1 in podocytes selectively would be required to delineate if such regulation of podocyte autophagy depends on direct or indirect effects of Angi.

Calpain-1 and calpain-2 are ubiquitous pro-inflammatory proteases, whose activity is controlled by calpastatin, their specific inhibitor. Indeed, calpastatin selectively inhibits cal-pains and no other proteases to date. Calpain activation has been linked recently to kidney injury in several pathological contexts."The calcium channel transporter transient receptor potential channel C6 was found to activate calpain-1 in podocytes via Ca²f/calcineurin activation. Kidneys of patients with focal and segmental glomerulosclerosis had increased transient receptor potential channel C6 expression, increased calpain and calcineurin activity, and reduced expression of the calpain target talin-1, which is critical for podocyte cytoskeletal stability.' Transient receptor potential channel C6 also directly binds to calpain-1 and calpain-2. This interaction is crucial for the regulation of talin-1 cleavage and control motility of podocytes.2

Transgenic mice overexpressing calpastatin are protected against vascular remodeling and AngII-dependent inflammation; against inflammation in models of glomerulonephritis, sepsis, or allograft rejection; and against aged-related inflammation./"Podocyte injury in these mice has not been explored. Peltier et al. showed that overexpression of calpastatin prevented Angl-dependent perivascular inflammation in kidneys. Thus, kidney protection in CST mice could be mediated, at least partially, by an anti-inflammatory mechanism. We evaluate macrophages and lymphocytes infiltration in our model and found no significant difference in kidney inflammation between CST and control mice when analyzing global kidney leukocyte infiltration (Supplementary Figure S6).

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Calpain was involved recently in autophagy regulation (reviewed recently in Weber et al.)with interesting endo-atheroprotective properties of calpain inhibition in diabetic context through the restoration of autophagy. Here, we

Figure 9] Glomerular endoplasmic reticulum (ER) stress and oxidative stress. (a) Quantitative polymerase chain reaction analysis of the mRNA expression of genes of the ER stress, oxidative stress, and apoptosis pathway by using a Qiagen quantitative polymerase chain reaction array in glomeruli from wild-type (WT), Nphs2.cre Atg5, and CST'9 mice after 6 weeks of angiotensin Il (Angel)+ high-salt diet (HSD). Data are presented as a heatmap of the Log2 fold change in gene expression.n=5 mice per genotype. (b)Representation of the genes significantly upregulated or downregulated in Nphs2.cre Atg5 versus WT mice. (c)Representation of the genes significantly upregulated or downregulated in CSTl9 versus WT mice. (b,c) Unpaired t-test with equal SD: P<0.05.

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reported that calpain inhibition through calpastatin over-expression(i)prevented podocyte injury during hypertension and (ii)restored autophagy in podocytes, thus highlighting a novel deleterious role of calpain activation during hypertension through the inhibition of autophagy.

Nearly all ATGproteins were shown to be cleaved by calpains in vitro. Here we postulated that autophagy maintenance in hypertensive CST'8 mice was mediated by calpain inhibition. To support our hypothesis, we showed that podocytes from CSTTg have a decreased calpain activity when challenged with AnglI

in vitro(Figure 3c). We found increased ATG5 protein level in podocytes from CST'号 mic， which suggests that calpastatin overexpression prevented calpain-mediated ATG5 cleavage in this context (Figure 5a). In contrast, it was demonstrated that calpastatin-mediated calpain inhibition could be independent of the inhibition of their protease activity; thus, we could not exclude regulation of autophagy by calpastatin independent of calpain enzymatic activity.

In summary, these findings revealed a previously unrecognized role of calpastatin in the regulation of podocyte autophagy and provided a lead for the investigation of novel therapeutic strategies to enhance podocyte survival during hypertensive nephropathies.

DISCLOSURE

All the authors declared no competing interests.

ACKNOWLEDGMENTS

This work was supported by the Institut National de la Santé Et de la Recherche Médicale (Inserm) and Université de Paris.IB was

supported by a graduate fellowship from the Ministere de IEducation Nationale, de la Recherche et de la Technologie. OL was funded through a European Foundation for the Study of Diabetes (EFSD)award supported by the EFSD/Novo Nordisk Programme for Diabetes Research in Europe and a grant from the Société Francophone du Diabetes (SFD).BR and CH were funded by Starting Grant 107037 from European Research Council and the European Union (P-LT). YS was supported by a graduate fellowship from the Fondation de France.

We thank Elizabeth Huc, Nicolas Perez, Corina Suldac, and the ERI U970 team (Université de Paris, PARCC, Inserm, Paris, France) for assistance in animal care and handling, Nicolas Sorhaindo for biochemical measurements(ICB-IFR2, Laboratoire de Biochimie, Hopital Bichat, Paris, France), and Alain Schmitt and Jean-Marc Masse for transmission electron microscopy (Institut Cochin, Paris, France). We thank Morgane Le Gall (Cochin proteomic facility 3P5, Paris, France) for help in silico analysis. We acknowledge administrative support from Véronique Oberweis, Bruno Pillard, and Cyrille Mahieux (Université de Paris, PARCC, Inserm, Paris, France).

SUPPLEMENTARY MATERIAL

Supplementary File (PDF)

Figure S1.Primary podocyte culture expresses podocyte markers. Western blot analysis of the expression of the podocyte markers NPHS1 and NPHS2 in primary podocyte culture from WT and CST mice. Tubulin (TUBA) serves as a loading control. Representative of n =4 mice per genotype.

Figure S2. The high basal level of podocyte autophagy. (A, B)

Representative immunofluorescence images of the expression of GFP (green) and NPHS1(red) in glomeruli from GFP-LG mice treated or not with CQ(80mg/kg)4 hours before killing. Arrowheads show GFP+autophagosomes.(C, D)Representative immunofluorescence images of the expression of GFP(green) and P62(red) in glomeruli from GFP-LC3 mice treated or not with CQ (80mg/kg)4 hours before killing. Arrow-heads show GFP+P62+ autophagosomes.(A-D)()represent higher magnification. Nuclei were stained with Hoechst （blue）.Bar = 50 μm. N=4 mice per condition (E-H)Representative immunofluorescence images of the expression of GFP (green) and P62(red) in primary podocyte from GFP-LC3 mice treated (F, H) or not (E, G)with Bafilomycin A1 (100 nM) for 4 hours.N=5 mice per condition.

Figure S3.P62 accumulates in podocytes in the DOCA-salt model of hypertension. (A-C)Representative immunofluorescence images of the expression of P62(green) and PODXL (red) in glomeruli from WT mice after 2 to 6 weeks of DOCA-salt model. (')represent higher magnification. Nuclei were stained with Hoechst （blue）.Bar= 50 μm. (D) Quantification of P62 area per podocyte area (%).N=5-6 mice per condition.One-way analysis of variance: time P= 0.0059, Sidak's multiple comparison test: D42 versus D14*P=0.0055, D28 versus D14P=0.8590.

Figure S4. Podocyte autophagy is dispensable for podocyte development. (A)Systolic blood pressure, (B) urine albumin-to-creatinine ratio, and(C) blood urea nitrogen levels in Atgsloxlbx and Nphs2.cre Atg5oxloxmice. N =5-6 mice per genotype. Values are presented as individual plots and means ± SEM. Mann-Whitney test:P=0.7273(A),P=0.4286(B), and P=0.6623(C). (D-E)Representative images of Masson's trichrome-stained sections of glomeruli from Atg5oxloxand Nphs2.cre Atgsloxlbx mice. (F-G)Representative immunofluorescence images of the expression of PODXL(green) and WT1(red) in Atg5bxlox and Nphs2.cre Atgsboxlo mice. Nuclei were stained with Hoechst (blue). (D-G)Bar=50 um.N=6 mice per genotype. (H-I) Representative photomicrographs of transmission electron microscopy sections of glomeruli from Atgslox/ox and Nphs2.cre Atgsox/o mice. Bar = 1 um. N=3 mice per genotype. Figure S5. Calpastatin overexpression does not influence kidney function at baseline. (A)blood urea nitrogen and (B) plasma albumin levels in 12-week-old GFP-LC3and CST'9 GFP-LC3mice.N=5 GFP-LC3 and N=6CST9 GFP-LC3.Values are presented as individual plots and means ± SEM. Mann-Whitney test: P=0.6623 (A) and P=0.9307(B). (C,D) Representative images of Masson's trichrome-stained sections of glomeruli from GFP-LC3 and CST19 GFP-LC3 mice.(E-H) Representative immunofluorescence images of the expression of PODXL(E, F)and NPHS1(G, H) in GFP-LC3 and CSr9 GFP-LC3 mice. (C-H)Bar =50 um. (UJ) Associated quantification of PODXL and NPHS1 area per glomerular section. N=5 GFP-LG3 and N=6 CST19 GFP-LC3.Values are presented as individual plots and means ± SEM. Mann-Whitney test: P=0.1898(A) and P= 0.8413 (B).

Figure S6.Calpastatin overexpression does not influence global kidney inflammation. Representative immunohistochemistry of the expression of F4/80(A, B) and CD3 (D-E) in GFP-LG3 and CS79 GFP-LC3 mice after 6 weeks of Angiotensin I +HSD.Bar=200 um. (C, F)Associated quantification of F4/80 and CD3 area per kidney section. N=7 CST19 GFP-LG3 and N=8 GFP-LC3 mice. Values are presented as individual plots and means± SEM.Mann-Whitney test: P=0.3969 (C) P=0.3357(F).

Figure S7.Podocyte autophagy deficiency does not induce ER stress or oxidative stress in young adults at baseline.qPCR analysis of the mRNA expression of genes of the ER stress, oxidative stress, and apoptosis pathway by Qiagen qPCR array in glomeruli from WT and Nphs2.cre Atgsoxlomice (A) and WT and CSr9 mice (B).N=4 mice per genotype. For Nox3, Ct >33.

Table S1.In silico prevision of calpain cleavage sites. Calpain cleavage sites were predicted in podocyte-related and autophagy-related proteins with GPS-CCD(http://ccdbiocuckoo.org), CaMPDB(http://calpain.org), and DeepCalpain Predict (http://deepcalpain.cancerbio. info/help.php).

Supplementary File (Excel)

Supplementary Table S2. The full file of in silico prevision of calpain cleavage sites. Calpain cleavage sites were predicted in podocyte-related and autophagy-related proteins with GPS-CCD(http://ccd. biocuckoo.org), CaMPDB(http://calpain.org),and DeepCalpain Predict (http://deepcalpain.cancerbio.info/help.php).Prediction cleavage sites are resumed for each protein for GPS-CCD and DeepCalpain Predict.

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