Toll-like receptor 3 ligand, polyIC, induces proteinuria and glomerular CD80, and increases urinary CD80 in mice - PubMed (original) (raw)

Toll-like receptor 3 ligand, polyIC, induces proteinuria and glomerular CD80, and increases urinary CD80 in mice

Takuji Ishimoto et al. Nephrol Dial Transplant. 2013 Jun.

Abstract

Background: We have reported that children with biopsy-proven minimal change disease (MCD) express CD80 (also known as B7.1) in their podocytes and excrete high levels of CD80 in their urine during active nephrotic syndrome. We also reported that polyIC, a Toll-like receptor 3 ligand, increases CD80 mRNA and protein expression in cultured human podocytes dose-dependently, with actin re-organization and a reduction in synaptopodin expression.

Methods: To determine the effect of polyIC in the kidney, we subjected mice to systemic injection of polyIC or phosphate buffered saline.

Results: Mice injected with polyIC developed significant proteinuria with increased urinary CD80 excretion. Glomeruli from mice injected with polyIC were normal by light microscopic examination, but showed increased CD80 production in podocytes by immunofluorescence staining. In isolated glomeruli from mice injected with polyIC, expressions of CD80 and interleukin 10 significantly increased with a mild non-significant increase in CTLA-4, and synaptopodin expression decreased significantly.

Conclusions: Our study demonstrates that systemically administered polyIC can induce transient proteinuria and urinary CD80 excretion with an increase in CD80 production in podocytes, increased glomerular CD80 and reduced synaptopodin expression. These findings may be relevant to the pathogenesis of proteinuria in MCD.

Keywords: CD80; animal models; minimal change disease; nephrotic syndrome; proteinuria.

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Figures

FIGURE 1:

FIGURE 1:

PolyIC-LMW increases CD80 on cultured human podocytes. Cultured human podocytes were stimulated with various concentrations of polyIC-LMW. FBS 1% was used as a control. (a) Total protein was extracted from the cells and processed for western blotting for human CD80. The results represent the means ± SEM (_n_= 3 wells) **P < 0.01; ***P < 0.001 versus 1% FBS. (b) Total RNA was extracted from the cells and processed for real-time PCR for human CD80. The results represent the means ± SEM (_n_= 3 wells). *P < 0.05 versus 1% FBS. GAPDH was used as an internal control.

FIGURE 2:

FIGURE 2:

Intravenous injection of polyIC-LMW induces transient albuminuria in mice. Mice were intravenously injected with polyIC-LMW or PBS. (a) Twenty-four-hour urine was collected at 24 h (0–24 h), 1 week and 2 weeks. Urinary albumin-to-creatinine ratio was measured. The results represent the means ± SEM (n = 5). **P < 0.01 versus uninjected, PBS and 2 weeks. (b) Six-hour urine (0–6 h) or 18-h (6–24 h) urine were collected at 6 or 24 h, respectively. Urinary albumin-to-creatinine ratio was measured. The results represent the means ± SEM (n = 5). *P < 0.05 versus PBS and 6–24 h. (c and d) Histological sections of the kidneys (at 6 and 24 h) of mice injected with polyIC-LMW or PBS were stained with PAS. Bar, 50 μm in (c) and 20 μm in (d). (e) Electron microscopy of the kidneys of mice injected with polyIC-LMW or PBS. Bar, 1 μm.

FIGURE 3:

FIGURE 3:

PolyIC-LMW induces urinary CD80 excretion and CD80 production in kidneys. Mice were intravenously injected with polyIC-LMW or PBS. (a) Twenty-four-hour urine was collected at 24 h and 1 week. Urinary concentration of CD80 protein was determined by ELISA, and was corrected by creatinine concentration. The results represent means ± SEM (n = 5). *P < 0.05 versus PBS and 1 week. (b) The kidney samples at 6 and 24 h after the injection were homogenized and then centrifuged. The amount of CD80 in each supernatant was measured by ELISA. The results represent means ± SEM (n = 5). *P < 0.001 versus uninjected, PBS and 1 week. (c) Total RNA was extracted from the kidney samples at 6 and 24 h after the injection. The expression of CD80 was assessed by real-time PCR. The results represent the means ± SEM (n = 5). *P < 0.001 versus uninjected and PBS. #P < 0.01 versus 6 h. β-actin was used as an internal control.

FIGURE 4:

FIGURE 4:

The expressions of TLRs in murine glomeruli. Isolated murine glomeruli from mice injected with PBS were subjected to real-time PCR for murine TLRs. The results represent the means ± SEM (n = 3). β-actin was used as an internal control.

FIGURE 5:

FIGURE 5:

The effect of polyIC-LMW in glomeruli. Mice were intravenously injected with polyIC-LMW or PBS. Glomeruli were isolated at 6 h, and then subjected to real-time PCR for CD80 (a), TLR3 (b), synaptopodine (c), CTLA-4 (d), IL-10 (e) and angiopoietin-like 4 (f). The results represent means ± SEM (n = 5). **P < 0.01, *P < 0.05 versus PBS. N.S., not significant. β-actin was used as an internal control.

FIGURE 6:

FIGURE 6:

The examinations using CD80 null mice. (a and b) WT mice and CD80 null mice were intravenously injected with polyIC-LMW. (a) Twenty-four-hour urine was collected at 24 h (0–24 h). Control urine was collected 1 week prior to the injection of polyIC-LMW. Urinary albumin-to-creatinine ratio was measured. The results represent means ± SEM (n = 6). **P < 0.01 versus respective control. (b) Urinary concentration of CD80 protein was determined by ELISA, and was corrected by creatinine concentration. The results represent the means ± SEM (n = 5). (c) CD80 contents in the kidney and spleen of WT mice and CD80 null mice measured by ELISA. The results represent means ± SD (n = 3). (d) Serum CD80 concentration measured by ELISA. The results represent the means ± SD (n = 3). (e) Representative images of western blotting for CD80 using the kidney of WT mice and CD80 null mice.

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