Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jul 8;10(7):524.
doi: 10.1038/s41419-019-1754-3.

PGRN acts as a novel regulator of mitochondrial homeostasis by facilitating mitophagy and mitochondrial biogenesis to prevent podocyte injury in diabetic nephropathy

Affiliations

PGRN acts as a novel regulator of mitochondrial homeostasis by facilitating mitophagy and mitochondrial biogenesis to prevent podocyte injury in diabetic nephropathy

Di Zhou et al. Cell Death Dis. .

Abstract

Mitochondrial dysfunction is considered as a key mediator in the pathogenesis of diabetic nephropathy (DN). Therapeutic strategies targeting mitochondrial dysfunction hold considerable promise for the treatment of DN. In this study, we investigated the role of progranulin (PGRN), a secreted glycoprotein, in mediating mitochondrial homeostasis and its therapeutic potential in DN. We found that the level of PGRN was significantly reduced in the kidney from STZ-induced diabetic mice and patients with biopsy-proven DN compared with healthy controls. In DN model, PGRN-deficient mice aggravated podocyte injury and proteinuria versus wild-type mice. Functionally, PGRN deficiency exacerbated mitochondrial damage and dysfunction in podocytes from diabetic mice. In vitro, treatment with recombinant human PGRN (rPGRN) attenuated high glucose-induced mitochondrial dysfunction in podocytes accompanied by enhanced mitochondrial biogenesis and mitophagy. Inhibition of mitophagy disturbed the protective effects of PGRN in high glucose-induced podocytotoxicity. Mechanistically, we demonstrated that PGRN maintained mitochondrial homeostasis via PGRN-Sirt1-PGC-1α/FoxO1 signaling-mediated mitochondrial biogenesis and mitophagy. Finally, we provided direct evidence for therapeutic potential of PGRN in mice with DN. This study provides new insights into the novel role of PGRN in maintaining mitochondrial homeostasis, suggesting that PGRN may be an innovative therapeutic strategy for treating patients with DN.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. The level of PGRN was significantly reduced in the kidney under diabetic conditions.
a Representative photomicrographs and semiquantitation of PGRN immunohistochemical staining in human renal cortical tissue from normal subjects (n = 8), patients with diabetic nephropathy (DN) (n = 8), or diabetic patients without nephropathy (DM-NN) (n = 7). *P < 0.05 vs. Normal, #P < 0.05 vs. DM-NN. b Representative photomicrographs of PGRN immunohistochemical staining in the kidney from wild-type (WT) and PGRN-deficient (Grn−/−) mice (n = 6). Negative control by omission of the corresponding primary antibodies demonstrated no nonspecific staining. c Representative western blot gel documents and summarized data showing the relative protein levels of PGRN in human podocytes treated with high glucose (HG, final concentration 20 or 40 mmol/l in medium) for 48 h. *P < 0.05 vs. NG treatment. d Representative confocal microscopic images showing the expression of PGRN in podocytes of kidney from STZ-induced diabetic mice (n = 6), synaptopodin was used as a podocytes marker. The arrows indicate representative podocytes
Fig. 2
Fig. 2. PGRN deficiency exacerbated podocyte injury and proteinuria in DN.
a Urine albumin-to-creatinine ratio (UACR) in different groups of mice. b Representative images of periodic-acid-Schiff (PAS) staining of kidney sections in different groups of mice. c Quantification of Mesangial Matrix Index of glomerulus from different groups of mice. d Representative transmission electron microscopy (TEM) images showing morphological changes in the podocyte foot process in different groups of mice. e Quantitative assessment of glomerular filtration barrier integrity, including glomerular basement membrane (GBM) thickness, foot process width and the number of foot processes/μm GBM. *P < 0.05 vs. sham-operated mice, #P < 0.05 vs. WT diabetic mice (n = 6). f Representative microscopic images showing the expressions of podocin and nephrin in the kidney from different groups of mice
Fig. 3
Fig. 3. The cell death and mitochondrial damage were further exacerbated in podocytes of Grn−/− diabetic mice.
a In situ terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling (TUNEL) assays were performed to assess the cell death in glomerulus. Nuclei were revealed using 4′,6-diamidino-2-phenylindole (DAPI) staining. Quantitative assessment of the number of cell death (number of TUNEL-positive cells per glomerulus). b Double immunofluorescence staining for cleaved-caspase 3 (green) and synaptopodin (red) in the kidney from different groups of mice. Quantitative assessment of the number of cleaved-caspase 3-positive podocytes in glomerulus. c Representative TEM images of glomeruli demonstrating mitochondrial morphology in podocytes of kidney sections and quantification of percentage of altered mitochondria characterized by mitochondria swelling, vacuolization and cristae fragmentation in podocytes from different groups of mice. Red arrows indicate representative mitochondria. d Representative photomicrographs and semiquantitation of cytochrome C oxidase (complex IV) subunit IV isoform 1 (COXIV) immunohistochemical staining in the kidney from different groups of mice. *P < 0.05 vs. sham-operated mice, #P < 0.05 vs. WT diabetic mice (n = 6)
Fig. 4
Fig. 4. PGRN protected against HG-induced mitochondrial damage and dysfunction, and restored HG-reduced mitochondrial biogenesis in podocytes.
a Representative images for MitoTracker green staining showing mitochondrial morphology and quantification of mitochondrial length in live cultured podocytes with different treatments. The results were normalized to the mitochondrial length of NG-treated podocytes. b Representative TEM images showing mitochondrial morphology and quantification of the percentage of altered mitochondria and mitochondria diameter of live cultured podocytes with different treatments. c Representative images of podocytes stained with JC-1 and quantification of JC-1 fluorescence (red-to-green ratio) showing changes in fluorescence intensity in podocytes with different treatments. JC-1 fluorescence was normalized with the red-to-green ratio of NG-treated podocytes. d Relative mitochondrial DNA content (mtDNA:nDNA) in podocytes with different treatments. The results were normalized to the ratio of mtDNA:nDNA of NG-treated podocytes. e Representative western blot gel documents and summarized data showing the expression levels of PGC-1α and TFAM in podocytes with different treatments. *P < 0.05 vs. NG treatment, #P < 0.05 vs. HG treatment
Fig. 5
Fig. 5. PGRN rescued HG-reduced mitophagy in podocytes.
a Representative western blot gel documents and summarized data showing the protein levels of PARK2 in kidney from different groups of mice. *P < 0.05 vs. sham-operated mice, #P < 0.05 vs. WT diabetic mice (n = 6). b Representative western blot gel documents and summarized data showing the relative protein levels of PINK1 and PARK2 in podocytes with different treatments. c Representative confocal microscopic images of double immunofluorescence staining for TOMM20 (green) and LC3 (red) in podocytes with different treatments. Nuclei were revealed using DAPI staining. White arrows indicate colocalization of autophagosomes (LC3) and mitochondria (TOMM20). d Quantitative analysis of mitophagosome formation represented by colocalization of autophagosomes (LC3) and mitochondria (TOMM20). e Representative TEM images demonstrating mitophagosomes (mitophagosomes engulfing mitochondria) in podocytes with different treatments. Red arrows indicate representative mitophagosomes. f Quantification of the numbers of mitophagic vacuoles of each group. *P < 0.05 vs. NG treatment, #P < 0.05 vs. HG treatment. g Representative western blot gel documents and summarized data showing the relative protein level of PARK2 in siRNA-NC or siRNA-PARK2-transfected podocytes. *P < 0.05 vs. siRNA-NC. h Representative western blot gel documents and summarized data showing the expression levels of cleaved-PARP1 in podocytes with different treatments. i Representative flow cytometry analysis depicting the detection of apoptosis in podocytes with different treatments stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI). j Quantitative data expressing overall percentage of cell death (annexin V-FITC-positive), including the amount of apoptotic and necrotic cells determined by flow cytometric analysis in podocytes with different treatments. *P < 0.05 vs. NG + siRNA-NC, #P < 0.05 vs. HG + siRNA-NC, §P < 0.05 vs. HG + rPGRN + siRNA-NC
Fig. 6
Fig. 6. PGRN activated Sirt1-PGC-1α/FoxO1 pathway in HG-treated podocytes.
a Representative western blot gel documents of Sirts in podocytes with different treatments. b Summarized data showing the expression levels of Sirts in podocytes with different treatments. *P < 0.05 vs. NG treatment, #P < 0.05 vs. HG treatment. c Representative western blot gel documents and summarized data showing the expression levels of Sirt1 in kidney from different groups of mice. *P < 0.05 vs. sham-operated mice, #P < 0.05 vs. WT diabetic mice (n = 6). d Representative western blot gel documents and summarized data showing the levels of acetylated-FoxO1 in podocytes with different treatments. e Representative western blot gel documents and summarized data showing the levels of acetylated-PGC-1α in podocytes with different treatments. The acetylated proteins were immunoprecipitated with an antibody to acetylated lysine and the presence of PGC-1α in the immune-complex was assessed by western blot with the antibody to PGC-1α. f Relative mRNA levels of PGC-1α, TFAM, PINK1, and PARK2 in podocytes with different treatments. *P < 0.05 vs. NG treatment, #P < 0.05 vs. HG treatment
Fig. 7
Fig. 7. Inhibition of Sirt1 expression counteracted the protective effects of PGRN in podocytes with HG treatment.
a Representative western blot gel documents of acetylated-FoxO1, FoxO1, PINK1, and PARK2 in podocytes with different treatments. b Representative western blot gel documents of acetylated-PGC-1α and PGC-1α in podocytes with different treatments. c Summarized data showing the levels of acetylated-FoxO1 and acetylated-PGC-1α relative to FoxO1 and PGC-1α, and the expression levels of PINK1, PARK2, and PGC-1α in podocytes with different treatments. d Representative confocal microscopic images of double immunofluorescence staining for TOMM20 (green) and LC3 (red) in podocytes with different treatments. Nuclei were revealed using DAPI staining. e Quantitative analysis of mitophagosome formation represented by colocalization of autophagosomes (LC3) and mitochondria (TOMM20). f Relative mitochondrial DNA content (mtDNA:nDNA) in podocytes with different treatments. The results were normalized to the ratio of mtDNA:nDNA of NG-treated podocytes with siRNA-NC transfection. g Representative images of podocytes stained with JC-1 in live cultured podocytes with different treatments. h Quantification of JC-1 fluorescence (red-to-green ratio) showing changes in fluorescence intensity in podocytes with different treatments. JC-1 fluorescence was normalized with the red-to-green ratio of NG-treated podocytes with siRNA-NC transfection. i Representative flow cytometry analysis depicting the detection of apoptosis in podocytes with different treatments stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI). j Quantitative data expressing overall percentage of cell death (annexin V-FITC-positive), including the amount of apoptotic and necrotic cells determined by flow cytometric analysis in podocytes with different treatments. *P < 0.05 vs. NG treatment, #P < 0.05 vs. HG treatment, §P < 0.05 vs. siRNA-NC
Fig. 8
Fig. 8. Administration of recombinant human PGRN (rPGRN) protected against podocyte injury in mice with DN.
a A representative figure showing the procedure of rPGRN treatment in this study. b Urine albumin-to-creatinine ratio (UACR) in different groups of mice. c Representative images of PAS staining of kidney sections in different groups of mice. d Quantification of Mesangial Matrix Index of glomerulus from different groups of mice. e Representative TEM images showing morphological changes in the podocyte foot process in different groups of mice. Quantitative assessment of glomerular filtration barrier integrity, including GBM thickness and the number of foot processes/μm GBM. f In situ TUNEL assays were performed to assess the cell death in glomerulus from different groups of mice. Nuclei were revealed using DAPI staining. Quantitative assessment of the number of cell death (number of TUNEL-positive cells per glomerulus). g Representative TEM images of glomeruli demonstrating mitochondrial morphology in podocytes of kidney sections. Quantification of the percentage of altered mitochondria in podocytes from different groups of mice. h Representative western blot gel documents and summarized data showing the expression levels of Sirt1, PGC-1α, and PINK2 in kidney from different groups of mice. *P < 0.05 vs. sham-operated mice, #P < 0.05 vs. WT diabetic mice (n = 6). i Representative photomicrographs of PINK1 immunohistochemical staining in the kidney from different groups of mice. j Schematic representation showing that PGRN plays an important role in maintaining mitochondrial homeostasis to prevent podocyte injury in diabetic nephropathy by regulating Sirt1-PGC-1α/FoxO1 signaling-mediated mitochondrial biogenesis and mitophagy

References

    1. Pagtalunan ME, et al. Podocyte loss and progressive glomerular injury in type II diabetes. J. Clin. Investig. 1997;99:342–348. doi: 10.1172/JCI119163. - DOI - PMC - PubMed
    1. Steffes MW, Schmidt D, McCrery R, Basgen JM, International Diabetic Nephropathy Study Group Glomerular cell number in normal subjects and in type 1 diabetic patients. Kidney Int. 2001;59:2104–2113. doi: 10.1046/j.1523-1755.2001.00725.x. - DOI - PubMed
    1. Susztak K, Raff AC, Schiffer M, Bottinger EP. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes. 2006;55:225–233. doi: 10.2337/diabetes.55.01.06.db05-0894. - DOI - PubMed
    1. Kume S, Yamahara K, Yasuda M, Maegawa H, Koya D. Autophagy: emerging therapeutic target for diabetic nephropathy. Semin. Nephrol. 2014;34:9–16. doi: 10.1016/j.semnephrol.2013.11.003. - DOI - PubMed
    1. Bhargava P, Schnellmann RG. Mitochondrial energetics in the kidney. Nat. Rev. Nephrol. 2017;13:629–646. doi: 10.1038/nrneph.2017.107. - DOI - PMC - PubMed

Publication types

MeSH terms