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. 2008 May;19(5):915-22.
doi: 10.1681/ASN.2007070745. Epub 2008 Feb 6.

Preconditioning with endoplasmic reticulum stress ameliorates mesangioproliferative glomerulonephritis

Reiko Inagi  1 , Takanori Kumagai, Hiroshi Nishi, Takahisa Kawakami, Toshio Miyata, Toshiro Fujita, Masaomi Nangaku
Affiliations

Preconditioning with endoplasmic reticulum stress ameliorates mesangioproliferative glomerulonephritis

Reiko Inagi et al. J Am Soc Nephrol. 2008 May.

Abstract

Accumulating evidence suggests a pathophysiologic role of endoplasmic reticulum (ER) stress in kidney disease. This study investigated the potential of therapeutic approaches targeting ER stress in the anti-Thy1 model of mesangioproliferative glomerulonephritis in rats. Immunohistochemistry and Western blotting showed a time-dependent increase in the expression of the ER stress-inducible chaperones glucose-regulated protein 78 (GRP78) and oxygen-related protein 150 in isolated glomeruli, especially in the glomerular epithelial cells and mesangial cells, after induction of anti-Thy1 nephritis. For evaluation of whether preconditioning with ER stress ameliorates the severity of disease, rats were pretreated with a subnephritogenic dose of the ER stress inducer tunicamycin or thapsigargin for 4 d before disease was induced. Although preconditioning with ER stress had no effect on the degree of disease induction, it strongly ameliorated the manifestations of disease, evidenced by marked reductions in microaneurysm formation, mesangial proliferation, and adhesion of Bowman's capsule to the glomerular tuft. This improvement in histologic damage was associated with reduced proteinuria (39.4 +/- 10.5 versus 126.1 +/- 18.1 mg/d; P < 0.01) and with attenuated increases in glucose-regulated protein 78 and oxygen-related protein 150 expression. Of note, pretreatment with tunicamycin or thapsigargin decreased the excessive ER stress-induced intracellular signaling observed in anti-Thy1 nephritis. In conclusion, preconditioning with ER stress ameliorates the severity of disease in rats with anti-Thy1 nephritis. These findings suggest the possibility of therapeutic approaches targeting ER stress in mesangioproliferative glomerulonephritis.

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Figures

Figure 1.
Figure 1.
Induction of ER stress–inducible proteins in rats with anti-Thy1 nephritis. Periodic acid-Schiff staining (top) and immunohistochemical detection of GRP78 (middle) or ORP150 (bottom) in the kidney of an untreated healthy control rat; a rat with anti-Thy1 nephritis at day 1, day 3, or day 7; and a rat that was administered an injection of nonspecific mouse IgG in place of anti-Thy1 mouse mAb OX-7 are shown. Expression of GRP78 and ORP150 is markedly augmented in glomeruli of rats with anti-Thy1 nephritis as compared with the control rats. Magnification, ×400.
Figure 2.
Figure 2.
Quantitative analysis of ER stress–inducible protein expression in rats with anti-Thy1 nephritis. (A) Computer-assisted morphometry of the renal paraffin-embedded section stained immunohistochemically with antibody to GRP78 or ORP150 shown in Figure 1. Renal sections of 10 rats each derived from the respective groups were examined, and averages were expressed as means ± SD. Expression level of GRP78 or ORP150 was notably higher in glomeruli of rats with anti-Thy1 nephritis at day 7, which corresponds to the stage of mesangial hypercellularity, than at day 1, or early onset of glomerulonephritis. *P < 0.05 versus untreated control rats; **P < 0.001 versus untreated control rats; #P < 0.05 versus rats with anti-Thy1 nephritis at day 3. D1, day 1; D3, day 3; D7, day 7. (B) Proteins from isolated glomeruli from control rats (n = 3) and rats with anti-Thy-1 nephritis (day 7, n = 5) were analyzed by Western blot analysis for GRP78 or ORP150 detection. Representative pictures from three independent experiments are shown in the top panel. Quantitative densitometry of the bands was then performed and normalized to the intensity of the actin band. Increased GRP78 or ORP150 expression in glomeruli of rats with anti-Thy1 nephritis at day 7 was confirmed. **P < 0.001 versus untreated control rats.
Figure 3.
Figure 3.
Preconditioning with ER stress ameliorated the severity of the manifestations of anti-Thy1 nephritis in rats. Periodic acid-Schiff staining (A) followed by computer-assisted morphometry was performed for evaluation of glomerular tuft area (B) and number of glomerular cells (C) in untreated control rats and rats with anti-Thy1 nephritis (at day 3 or day 7) with or without ER stress preconditioning. Preconditioning was induced by tunicamycin or thapsigargin injection 4 d before the induction of anti-Thy1 nephritis. The pathologic features of anti-Thy1 nephritis, including an increase in glomerular size and glomerular hypercellularity, were dramatically improved by preconditioning with ER stress, which had no histologic effect in untreated control rats. TUN, tunicamycin pretreatment; THG, thapsigargin pretreatment. *P < 0.05 versus control rats without pretreatment; #P < 0.05 versus preconditioned anti-Thy1 nephritis rats at day 3; &P < 0.05 versus preconditioned rats with anti-Thy1 nephritis at day 7. Magnification, ×400.
Figure 4.
Figure 4.
Improvement in anti-Thy1 nephritis by preconditioning with ER stress was associated with a decrease in proteinuria. Urine samples were collected from control rats and rats with anti-Thy-1 nephritis (day 7) pretreated with or without tunicamycin or thapsigargin (n = 5 for each). The histologic improvement in anti-Thy1 nephritis by this pretreatment was associated with a significant decrease in proteinuria. *P < 0.05 versus control rats without pretreatment; #P < 0.05 versus rats with anti-Thy1 nephritis without pretreatment.
Figure 5.
Figure 5.
Preconditioning with ER stress modulated GRP78 expression induced by anti-Thy1 nephritis. Immunohistochemical analysis for detection of GRP78 (A) followed by quantitative analysis using computer-assisted morphometry (B) was performed in the kidney of nondisease control rats and rats with anti-Thy1 nephritis (at day 3 or day 7) with or without ER stress preconditioning. Preconditioning was induced by tunicamycin or thapsigargin injection 4 d before the induction of anti-Thy1 nephritis. Each renal paraffin-embedded section of five rats derived from each group was examined, and averages were expressed as means ± SD (B). Preconditioning with ER stress significantly enhanced basal GRP78 expression in the kidney of nondisease control rats and suppressed the remarkable increase in GRP78 expression observed in rats with anti-Thy1 nephritis. *P < 0.05 versus control rats without pretreatment; #P < 0.05 versus tunicamycin-pretreated rats with anti-Thy1 nephritis at day 3. Magnification, ×400.
Figure 6.
Figure 6.
Preconditioning with ER stress modulated ORP150 expression induced by anti-Thy1 nephritis. Immunohistochemical analysis for detection of ORP150 (A) followed by quantitative analysis using computer-assisted morphometry (B) was performed as described in Figure 5. Temporal expression profiles of ORP150 were similar to those of GRP78. *P < 0.05 versus control rats without pretreatment; #P < 0.05 versus tunicamycin-pretreated rats with anti-Thy1 nephritis at day 3. Magnification, ×400.
Figure 7.
Figure 7.
Comparison of binding levels of anti-Thy1 antibody with mesangial cells in rats with anti-Thy1 nephritis with or without preconditioning with ER stress. Renal frozen tissues of nondisease control rats and rats with anti-Thy1 nephritis with or without ER stress preconditioning (n = 3 for each) were examined by immunofluorescence microscopy for evaluation of binding level of anti-Thy1 antibody with mesangial cells. Anti-Thy1 antibody binding with mesangial cells was detected both in the rats pretreated or not pretreated with tunicamycin, and its degree did not change between them. Magnification, ×400.
Figure 8.
Figure 8.
ER stress–inducible signal transduction pathway in rats with anti-Thy1 nephritis was modulated by preconditioning with ER stress. Induction of the ER stress–inducible signal transduction pathway for shutdown of protein translation was assessed by Western blot analysis for the detection of phosphorylated PERK or eIF2α (A) followed by quantitative densitometry (B). Proteins from isolated glomeruli of control rats and rats with anti-Thy1 nephritis with or without ER stress preconditioning (n = 5 for each) were examined. Whereas phosphorylation of PERK or eIF2α was barely observed in control rats, it was markedly augmented in rats with anti-Thy1 nephritis. Tunicamycin or thapsigargin pretreatment enhanced basal levels of phosphorylation of PERK or eIF2α, which in turn led to the amelioration of further phosphorylation induced by anti-Thy1 nephritis. *P < 0.05 versus control rats without pretreatment; #P < 0.05 versus rats with anti-Thy1 nephritis without pretreatment. p-PERK, phosphorylated PERK; p-eIF2α, phosphorylated eIF2α.
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