Fabry disease is a rare X-linked genetic lysosomal disorder caused by mutations in the
Renal fibrosis was triggered by unilateral ureteral obstruction (UUO) in mice with Fabry disease to investigate the pathogenic mechanism leading to fibrosis in diseased kidneys.
Compared to kidneys of wild-type mice, lamellar inclusion bodies were recognized in proximal tubules of mice with Fabry disease. Sirius red and trichrome staining revealed significantly increased fibrosis in all UUO kidneys, though it was more prominent in obstructed Fabry kidneys. Renal messenger RNA levels of inflammatory cytokines and profibrotic factors were increased in all UUO kidneys compared to sham-operated kidneys but were not significantly different between UUO control and UUO Fabry mice. Protein levels of Nox2, Nox4, NQO1, catalase, SOD1, SOD2, and Nrf2 were not significantly different between UUO control and UUO Fabry kidneys, while the protein contents of LC3-II and LC3-I and expression of Beclin1 were significantly decreased in UUO kidneys of Fabry disease mouse models compared with wild-type mice. Notably, TUNEL-positive cells were elevated in obstructed kidneys of Fabry disease mice compared to wild-type control and UUO mice.
These findings suggest that impaired autophagy and enhanced apoptosis are probable mechanisms involved in enhanced renal fibrosis under the stimulus of UUO in Fabry disease.
Fabry disease (OMIM #301500) is a rare X-linked lysosomal storage disorder resulting from an error in glycosphingolipid metabolism caused by a lack of alpha-galactosidase A (α-Gal A) [
Despite marked advances in patient care and improved overall outlook on Fabry disease [
The autophagy-lysosome pathway could be another important signaling pathway contributing to the onset and progression of Fabry disease in involved cells and tissues [
Fabry disease mice, a rodent model with α-Gal A gene-disruption, were bred to produce sufficient numbers. Heterozygous female mice were bred with control males to maintain the mouse colony. Mating between mutant males and females was performed to generate littermates with α-Gal A deficiency [
To genotype each mouse, polymerase chain reaction (PCR) was performed as described previously [
To evaluate the severity of tubulointerstitial fibrosis, 4% phosphate-buffered paraformaldehyde-fixed kidney sections were stained with Masson’s trichrome or with sirius red following the manufacturer’s protocols (Merck KGaA, Darmstadt, Germany) [
According to the manufacturer's manual, total renal RNA was isolated using TRIzol reagents (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative reverse transcription (qRT) PCR assays were performed using SYBR Premix (Takara Bio Inc., Otsu, Japan). Primer sequences for each gene used in PCR are listed in
Total proteins from harvested kidneys were extracted using a PRO-PREP Protein Extraction Kit (iNtRON Biotechnology, Seongnam, Korea). Protein concentrations were determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). After electrophoresis, proteins in gels were transferred into nitrocellulose membranes and incubated with primary antibodies at 4°C overnight. Antibodies against the following proteins were used: NAD(P)H:quinone oxidoreductase 1 (NQO1; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), catalase (Abcam, Cambridge, UK), superoxide dismutase 1 (SOD1; Enzo Life Science, Inc., Farmingdale, NY, USA), SOD2 (Abcam), heme oxygenase-1 (HO-1; Thermo Fisher Scientific), nuclear factor-erythroid-2-related factor 2 (Nrf2; Santa Cruz Biotechnology Inc.), Nox2 (BD Bioscience, San Jose, CA, USA), Nox4 (Cell Signaling Technology, Danvers, MA, USA), LC3B (Novus Biologicals, Littleton, CO, USA), Beclin1 (Novus), β-actin (Sigma-Aldrich, St. Louis, MO, USA), β-tubulin (Sigma-Aldrich), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Sigma-Aldrich). After washing with PBS, all blots were incubated with a secondary antibody conjugated with horseradish peroxidase. Immunoreactive proteins were detected by enhanced chemiluminescence reagents. Densities of protein bands were measured and normalized to those of respective housekeeping proteins in the same sample.
For electron microscopic examination, small sections of kidney tissues were fixed in 2.5% glutaraldehyde in phosphate buffer. After washing and dehydrating in serial alcohol and propylene oxide, tissues were infiltrated with a series of graded ethyl alcohol solutions and embedded in Epon substitute. Thin sections were prepared and stained with uranyl acetate and lead citrate. Samples were observed under a transmission microscope to identify representative lesions in glomeruli and proximal tubules.
To examine DNA fragmentation indicative of apoptosis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Millipore, Billerica, MA, USA) was performed according to the manufacturer’s protocol. TUNEL-positive cells were evaluated in 20 randomly selected fields for each section using ImageJ 1.53a software.
Quantitative data are expressed as mean ± standard error of mean. All statistical analyses were performed using GraphPad Prism (GraphPad Software LLC, La Jolla, CA, USA). In all analyses, p-values less than 0.05 were considered to indicate a statistically significant difference.
Compared with sham-operated kidneys, obstructed kidneys induced by UUO showed renal tubulointerstitial fibrosis, as indicated by Masson’s trichrome and sirius red staining (
Results of qRT-PCR revealed that renal mRNA levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNFα) were significantly increased on day 7 post-UUO compared to those of sham-operated kidneys (
Levels of matrix metalloproteinase (MMP)2 and MMP9 mRNA were significantly enhanced in obstructed kidneys of both UUO control and UUO Fabry mice without significant difference between the two groups (
On day 7 post-UUO, obstructed kidneys showed suppressed expression of NQO1, catalase, SOD1, and SOD2 proteins (
Results of Western blot analysis revealed that protein level of Nox2, one of the NADPH oxidases in the Nox family, was significantly increased in UUO control and tended to be increased in UUO Fabry without a statistically significant difference from that of the sham group (
It has been reported that autophagy is involved in the development and progression of renal fibrosis induced by UUO [
In TUNEL assay analyzing renal apoptosis, while a few TUNEL-positive cells were detected in sham-operated kidneys, there was a significant increase in the number of TUNEL-positive cells in UUO control (
This study demonstrated that UUO kidneys could induce renal tubulointerstitial fibrosis, inflammation, EMT, oxidative stress, autophagy, and apoptosis in similar patterns as described in previous reports [
Chronic immune system stimulation in Fabry disease has been reported [
Oxidative stress has been implicated as one of the major underlying mechanisms behind the pathogenesis of obstructive nephropathy [
Since homeostatic processes are particularly active in renal proximal tubular cells, in which reabsorption and transport properties are the most active in the kidney, autophagy-mediated turnover of damaged mitochondria would be required for protecting proximal tubules from renal injury [
Autophagy can interact with the apoptotic machinery by acting upstream of apoptosis, converging with the apoptotic pathway, or mediating steps downstream of apoptosis. Autophagy can prevent apoptosis by selectively removing damaged mitochondria that might otherwise accumulate under stress conditions [
Although it has been suggested that ERT could slow the progression of renal involvement in Fabry disease, its limited effect in specific situations has been observed [
Sungjin Chung is a deputy editor of
This study was supported from The Catholic Medical Center Research Foundation 2019.
Conceptualization: SC, HSK
Data curation: SC, MS, YC, SO, ESK
Formal analysis: SC, YKK, SJS, SWP, SCJ, HSK
Funding acquisition: SC
Investigation: SC, MS, YC, SO, ESK
Writing–original draft: SC, HSK
Writing–review & editing: All authors
All authors read and approved the final manuscript.
The authors would like to thank Dr. Jong Hee Chung (Department of Statistics, Yonsei University, Seoul, Republic of Korea) for providing statistical advice.
Representative micrographs after Masson’s trichrome (A) and sirius red (B) staining for kidneys from different groups (×200 in all micrographs). (C) Quantitative analysis for Masson’s trichrome staining showing significantly aggravated fibrotic lesions by unilateral ureteral obstruction (UUO) with greater enhancement in Fabry disease mice (*p = 0.001 vs. Sham, **p < 0.001 vs. Sham, p = 0.006 vs. UUO Cont). (D) More enhanced renal fibrotic areas in obstructed kidneys of Fabry disease mice as indicated by sirius red staining (*p = 0.009 vs. Sham, **p < 0.001 vs. Sham, p = 0.02 vs. UUO Cont).
Cont, control; Sham, sham-operated wild-type mice.
(A) Quantitative reverse transcription polymerase chain reaction analysis showing the increased expression level of renal interleukin (IL)-1β on day 7 after unilateral ureteral obstruction (UUO), without significant difference between UUO control (Cont) and UUO Fabry (*p = 0.05 vs. Sham, **p = 0.02 vs. Sham). (B) Increased IL-6 mRNA level in UUO kidneys (*p = 0.02 vs. Sham, **p = 0.04 vs. Sham). (C) Similarly increased tumor necrosis factor-α (TNFα) mRNA level in both UUO Cont and UUO Fabry (*p = 0.05 vs. Sham, **p = 0.05 vs. Sham). (D) Significantly enhanced matrix metalloproteinase (MMP)2 mRNA level in the obstructed kidneys of all UUO groups (*p = 0.03 vs. Sham, **p = 0.04 vs. Sham). (E) Increased renal MMP9 mRNA level observed in all UUO groups (*p = 0.01 vs. Sham, **p = 0.02 vs. Sham). (F) No significant increase in epithelial cadherin (E-cadherin) mRNA level in all UUO groups compared to Sham. (G) Significantly increased renal mRNA level of vascular E-cadherin (VE-cadherin) in UUO Cont (*p = 0.03 vs. Sham). (H) Significantly enhanced renal α-smooth muscle actin (α-SMA) mRNA level in UUO Cont (*p = 0.009 vs. Sham). (I) Increased fibronectin mRNA level in all UUO groups (*p = 0.006 vs. Sham, **p = 0.05 vs. Sham). (J) Increased renal vimentin mRNA level in all UUO groups (*p = 0.002 vs. Sham, **p = 0.004 vs. Sham). (K) Similarly enhanced mRNA level of renal transforming growth factor (TGF)-β in all UUO groups (*p = 0.01 vs. Sham, **p = 0.007 vs. Sham). (L) Increased collagen type IVα1 chain (COL4A1) mRNA level on day 7 after UUO in obstructed kidneys of all UUO groups (*p = 0.004 vs. Sham, **p = 0.04 vs. Sham).
Sham, sham-operated wild-type mice.
(A) Representative western blot results for NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase, superoxide dismutase (SOD)-1, heme oxygenase-1 (HO-1), total nuclear factor-erythroid-2-related factor 2 (Nrf2), and SOD2. (B) Increased renal protein expression of NQO1 after UUO in both wild-type and Fabry disease mice, without significant difference between the two UUO groups (*p < 0.001 vs. Sham, **p < 0.001 vs. Sham). (C) Similarly increased catalase expression after UUO in UUO control (Cont) and UUO Fabry (*p < 0.001 vs. Sham, **p < 0.001 vs. Sham). (D) Decreased expression of SOD1 in both UUO Cont and UUO Fabry groups, with UUO Fabry having a slightly higher level than UUO Cont (*p < 0.001 vs. Sham, **p < 0.001 vs. Sham, p = 0.02 vs. UUO Cont). (E) Renal HO-1 expression significantly increased in all UUO groups (*p = 0.003 vs. Sham, **p = 0.02 vs. Sham). (F) Significantly enhanced total Nrf2 expression in all UUO groups (*p < 0.001 vs. Sham, **p = 0.002 vs. Sham). (G) Lower renal SOD2 expression in both UUO Cont and UUO Fabry than in Sham (*p < 0.001 vs. Sham, **p <0.001 vs. Sham).
Sham, sham-operated wild-type mice.
(A) Representative western blot results for Nox2 and Nox4. The β-tubulin image was reused because the membrane for immunoblot analyses of superoxide dismutase-1, heme oxygenase-1, and total nuclear factor-erythroid-2-related factor 2 in
Sham, sham-operated wild-type mice.
(A) Western blot results representing microtubule-associated protein light chain 3 (LC3) and Beclin1. The β-actin image was reused because the membrane for immunoblot analysis of NAD(P)H:quinone oxidoreductase 1 and catalase in
Sham, sham-operated wild-type mice.
Representative electron micrographs of glomeruli (A, B) and proximal tubules (C, D). (Sham A–D) Sham-operated alpha-galactosidase A wild-type mice had normal glomerulus and proximal tubule appearance. (Unilateral ureteral obstruction [UUO] control [Cont] A–D) Following UUO, disruption of glomerular and tubular barriers, irregular cytoplasmic processes, and autophagic vacuoles were shown. (UUO Fabry A–D) Inclusion bodies containing globotriaosylceramide were observed in renal proximal tubular cells but not in podocytes of UUO-operated Fabry disease mice.
Scale bars: 2 μm for A and C, 0.2 μm for B and D.
(A) Representative image of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (×200). (B) Quantification of renal TUNEL-positive cells showing increased apoptosis in wild-type mice with unilateral ureteral obstruction (UUO) and more highly enhanced apoptosis in Fabry disease mice with UUO (*p = 0.003 vs. Sham, **p < 0.001 vs. Sham, p = 0.002 vs. UUO control [Cont]).
Sham, sham-operated wild-type mice.