Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models

Journal name:
Nature Medicine
Volume:
21,
Pages:
601–609
Year published:
DOI:
doi:10.1038/nm.3843
Received
Accepted
Published online

Abstract

Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to cross-link actin microfilaments into higher-order structures has been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the substantial regenerative potential of injured glomeruli and identifying the oligomerization cycle of dynamin as an attractive potential therapeutic target to treat CKD.

At a glance

Figures

  1. Dynamin oligomerization is essential for kidney function.
    Figure 1: Dynamin oligomerization is essential for kidney function.

    (a) Phenotype of zebrafish larvae injected with either scrambled (control MO) or dynamin-2–specific morpholino (dnm2 MO) 120 h post fertilization. Scale bar, 2 mm. (b) Survivorship curves of zebrafish larvae injected with either control MO or dnm2 MO. Curves represent 180 animals for control MO and 245 animals for dnm2 MO. Error bars, mean ± s.d. (log-rank: P < 0.0001 for comparison of mean survival time). (c) Representative image of the fluorescence of circulating eGFP-DBP in the retinal vessel plexus of the fish eye 96 h post-fertilization and injected with either control MO or dnm2 MO (left) (n = 128 images for control MO, and n = 94 images for dnm2 MO animals). Scale bars, 100 μm. Transmission electron micrographs of glomeruli analyzed in zebrafish larvae 120 h post-fertilization and injected with either control MO or dnm2 MO (right). Scale bars, 0.5 μm. (d) Intensity of circulating eGFP-DBP (AU, arbitrary units) in the retinal vessel plexus of the fish eye 96 h post-fertilization and treated with the indicated MO and/or expression construct and with Bis-T-23 (1 ng per larvae) or with DMSO as vehicle (20% per larvae). For groups 1–6, 16, and 20, n = 100–150; for all other groups n = 40–100. Black lines represent median intensity in each group (**P ≤ 0.01, ***P ≤ 0.001, unpaired t-test). NS, not statistically significant. (e) A schematic diagram indicating the domain structures of dynamin: GTPase, Middle, PH (pleckstrin homology), GED (GTPase effector domain) and PRD (proline/arginine-rich domain). Indicated mutations: K/E (K-to-E mutations of the indicated amino acid residues in black), E/K (E-to-K mutations of the indicated residues in red) and I690K. (f) A schematic diagram indicating that dimers of dynamin (DynDIMER) and tetramers of dynamin (DynTETRA) exhibit basal rate of GTP hydrolysis. Oligomerized dynamin (DynOLIGO), whose formation is promoted by Bis-T-23 (structure shown at right) or through indicated mutations, exhibits increased rate of GTP hydrolysis. DynOLIGO induces actin polymerization and cross-linking of F-actin, which in turn regulates the structure and function of podocytes. The small arrows in e and f indicate the effect of the mutations on dynamin's propensity to oligomerize.

  2. Dynamin oligomerization ameliorates transient proteinuria.
    Figure 2: Dynamin oligomerization ameliorates transient proteinuria.

    (a) Plasma pharmacokinetics of Bis-T-23 after injection (40 mg/kg) in C57BL/6J mice (n = 3) as measured by mass spectrometry. (b) Proteinuria of C57BL/6J mice determined by spot urine test before injection (0 h) and at the indicated time after injection of the indicated concentrations of Bis-T-23. NS, not statistically significant (unpaired t-test; n = 5 mice per concentration). (c–e) Inulin clearance (c), urine volume (d) and para-aminohippurate (PAH) clearance (e) of C57BL/6J mice determined after 8 consecutive days of treatment with DMSO (1%, vehicle) or Bis-T-23 (40 mg/kg). Error bars, mean ± s.d. (n = 6 mice per condition). (f) The systolic (SYS) or diastolic (DIA) blood pressure of 129X1/SvJ mice measured invasively using a carotid catheter after 8 consecutive days of treatment with DMSO (1%, vehicle) or Bis-T-23 (40 mg/kg) (n = 3 mice per condition). (g) Proteinuria of BALB/c mice determined by spot urine test at indicated times after two consecutive doses of LPS. As indicated, animals were injected with either DMSO (1%, vehicle) or Bis-T-23 (40 mg/kg) (n = 10 mice per condition). Error bars, mean ± s.d. (*P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001, unpaired t-test). (h) Proteinuria of Sprague-Dawley rats treated with PAN and determined by spot urine test. Rats were treated once a day starting 12 d after PAN with DMSO (1%, vehicle) or Bis-T-23 (20 mg/kg) for 6 consecutive days (n = 6 rats per condition). Error bars, mean ± s.d. (*P ≤ 0.05; ***P ≤ 0.001, unpaired t-test).

  3. Dynamin oligomerization in podocytes protects against proteinuria.
    Figure 3: Dynamin oligomerization in podocytes protects against proteinuria.

    (a) Domain structure of dynamin (top). R725A mutation is situated in the GED, which renders dynamin prone to oligomerize. A schematic diagram (bottom) indicating that the human gene DNM1 carrying R725A mutation (DNM1R725A) was placed under the regulation of a tetracycline-responsive promoter element (TRE; tetO). This transgenic mouse (Tg2) was subsequently bred to a second transgenic strain expressing the reverse tetracycline-transactivator (rtTA) protein under the control of a podocin promoter to allow for podocyte-specific gene expression (Tg1). Expression of DNM1R725A was induced by administration of the tetracycline analog, doxycycline. (b) RT-PCR of DNM1 from wild-type mice (WT), podocin-Cre only transgenic mice (empty) fed with doxycycline and homozygous DNM1R725A/R725A transgenic mice (R725A) fed with doxycycline. Neg, negative control with water as a template; Pos, positive control with plasmid encoding DNM1R725A. Nephrin (Nphs1) was used as a positive control. Stand., DNA size standard. (c) Representative electron micrographs of glomeruli (n = 5 or 6 glomeruli per genotype) from empty and R725A transgenic mice fed with either a normal diet (− doxycycline) or doxycycline diet (+ doxycycline). Rows 1 (scale bars, 10 μm) and 2 (scale bars, 1 μm) show scanning electron microscopy. Rows 3 and 4 (scale bars, 1 μm) show transmission electron microscopy (TEM) images. (d) Length of foot processes (FP) determined by analyzing images in c. Doxy, doxycycline. Error bars, mean ± s.d. (***P ≤ 0.001; unpaired t-test). (e) Proteinuria determined by analysis of spot urine samples at indicated times and in the indicated genotypes. Mice were fed with doxycycline (doxy) diet before they were injected with LPS (n = 6 mice per condition). Error bars, mean ± s.d. (***P ≤ 0.001, unpaired t-test). (f) Representative TEM images of glomeruli (n = 5 or 6 glomeruli per genotype) from empty and R725A transgenic mice 24 h after LPS injection. Animals were fed with either a normal diet (− doxycycline) or doxycycline diet (+ doxycycline). Scale bars, 2 μm (top row) and 1 μm (bottom row).

  4. Dynamin oligomerization targets actin cytoskeleton in podocytes.
    Figure 4: Dynamin oligomerization targets actin cytoskeleton in podocytes.

    (a) Proteinuria in wild-type and ACTN4 mice (without treatment or with treatment with either DMSO (1%, vehicle) or with Bis-T-23 (40 mg/kg)) as determined by spot urine test at indicated time points (n = 8 mice per condition). Error bars, mean ± s.d. (**P ≤ 0.01, unpaired t-test). (b,c) Proteinuria in ACTN4 mice determined by spot urine test before and after double injection of a podocin-driven expression vector encoding DNM1R725A mutant protein. Animals were grouped by protein levels before treatment (n = 3 for >1,000 μg/ml ACR; n = 7 for 500–1,000 μg/ml ACR). Individual animals from b are shown in c. Red arrows indicate reduction of proteinuria to control levels. Error bars, mean ± s.d. (**P ≤ 0.01, unpaired t-test). CON, control. (d) Proteinuria in CD2APKO mice determined by spot urine test over several days during which animals were treated daily with DMSO (1%, vehicle) or Bis-T-23 (40 mg/kg), starting at postnatal day (P) 18 (n = 5 mice per condition). Error bars, mean ± s.d. (*P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001, unpaired t-test). (e) Coomassie blue staining of SDS-PAGE gel showing protein bands from 2 μl of mouse spot urine at day 22 in d. BSA was used as a standard. (f) Line graph depicting number of live CD2APKO mice (black lines, n = 20 mice) and CD2APKO mice injected daily with Bis-T-23 (40 mg/kg) (red lines, n = 7 mice) at indicated time points. Animals exhibited a statistically significant difference in survival rate (log-rank: P < 0.0163).

  5. Dynamin oligomerization has beneficial effect on kidney morphology in PKC[epsiv]KO mice.
    Figure 5: Dynamin oligomerization has beneficial effect on kidney morphology in PKCεKO mice.

    (a) Proteinuria in PKCεKO mice without treatment or treated with either DMSO (1%, vehicle) or with Bis-T-23 (40 mg/kg) once daily for 8 consecutive days (n = 6 mice per condition) as determined by analysis of the spot urine. Animals were 12 weeks old at the beginning of the treatment (day 0, D0). Error bars, mean ± s.d. (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; unpaired t-test). (b) Total of 150 glomeruli from different animals per group (n = 3) were scored in a blind manner: I, normal glomerulus; II, mild mesangial expansion; III, moderate mesangial expansion; IV, advanced mesangial matrix expansion. −, no treatment. (c) Representative images of PAS-stained glomeruli (n = 150 glomeruli per condition) of 14-week-old untreated animals treated with DMSO (1%) or Bis-T-23 (40 mg/kg). Scale bars, 20 μm. (d) Representative TEM images of glomeruli (n = 10 glomeruli per group) from wild-type and PKCεKO mice harvested after 8 d of injection with either DMSO (1%, vehicle) or Bis-T-23 (40 mg/kg). Scale bars, 2 μm.

  6. Dynamin oligomerization ameliorates proteinuria due to diabetic nephropathy.
    Figure 6: Dynamin oligomerization ameliorates proteinuria due to diabetic nephropathy.

    (a) Proteinuria in 129X1/SvJ mice determined by analysis of spot urine samples after STZ-induced diabetes. Sixteen weeks after STZ injection (D0), animals were injected with either DMSO (1%, vehicle) or Bis-T-23 (20 mg/kg) once a day for 8 consecutive days (D8) (n = 8 male mice per condition). Error bars, mean ± s.d. (**P ≤ 0.01; unpaired t-test). (b) Representative images of glomeruli stained with PAS (n = 50 glomeruli), stained with toluidine blue (n = 15 glomeruli), by TEM (n = 15 glomeruli) and by immunofluorescence using anti-collagen IV (n = 50 glomeruli; Col IV). Glomeruli were from untreated control animals or diabetic animals treated with either DMSO (1%, vehicle) or Bis-T-23 (40 mg/kg) for 8 d, D8 in a. Scale bars, 20 μm (images showing PAS, toluidine blue and collagen IV staining) and 5 μm (TEM images). (c) Glomerular collagen IV expression in glomeruli from the indicated mice quantified in a blind manner: 1, very mild; 2, mild; 3, moderate; 4, intense; 5, very intense. Each symbol represents one mouse. Error bars, mean ± s.d. (**P ≤ 0.01; unpaired t-test). (d) Blood glucose levels in animals used in a. Error bars, mean ± s.d. ***P ≤ 0.001; unpaired t-test. NS, not statistically significant.

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Author information

  1. These authors contributed equally to this work.

    • Mario Schiffer,
    • Beina Teng &
    • Changkyu Gu

Affiliations

  1. Department of Nephrology, Hannover Medical School, Hannover, Germany.

    • Mario Schiffer,
    • Beina Teng,
    • Nils Hanke,
    • Song Rong,
    • Faikah Gueler,
    • Patricia Schroder,
    • Irini Tossidou,
    • Joon-Keun Park,
    • Lynne Staggs &
    • Hermann Haller
  2. Mount Desert Island Biological Laboratory, Salsbury Cove, Maine, USA.

    • Mario Schiffer,
    • Patricia Schroder,
    • Lynne Staggs &
    • Hermann Haller
  3. Department of Medicine, Harvard Medical School and Division of Nephrology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Changkyu Gu,
    • Valentina A Shchedrina,
    • Marina Kasaikina,
    • Vincent A Pham &
    • Sanja Sever
  4. Department of Cardiology, Hannover Medical School, Hannover, Germany.

    • Sergej Erschow &
    • Denise Hilfiker-Kleiner
  5. Department of Medicine, Rush University Medical Center, Chicago, Illinois, USA.

    • Changli Wei,
    • Chuang Chen,
    • Nicholas Tardi &
    • Jochen Reiser
  6. Pathology Department, University Medical Center Göttingen, Göttingen, Germany.

    • Samy Hakroush
  7. Pathology Department, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Martin K Selig
  8. Department of Biomedical Sciences, NYIT COM, Old Westbury, New York, USA.

    • Aleksandr Vasilyev
  9. Division of Nephrology and Hypertension, Department of Medicine, Leonard M. Miller School of Medicine, Miami, Florida, USA.

    • Sandra Merscher

Contributions

M.S., H.H., J.R. and S.S. designed the research; B.T., C.G., V.A.S., M.K., N.H., P.S., L.S., I.T., J.-K.P., S.E., D.H.-K., C.W., S.M., C.C., N.T., S.H., S.R., M.K.S., A.V. and F.G. performed the research. B.T., C.G., M.K., M.S., and S.S. analyzed the data. M.S., B.T., C.G. and S.S. wrote the manuscript.

Competing financial interests

S.S. and J.R. have pending or issued patents on novel kidney-protective therapies that have been out-licensed to Trisaq Inc. in which they have financial interest. In addition, they stand to gain royalties from their commercialization. The remaining authors report no conflicts.

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