Katsushige Abe

Predominant role for C5b-9 in renal ischemia/reperfusion injury

Previous work has indicated that complement is a mediator of ischemia/reperfusion (I/R) injury. To investigate the components of complement responsible for this effect, we examined a model of renal I/R injury in C3-, C4-, C5-, and C6-deficient mice. We occluded the renal arteries and veins (40-58 minutes) and, after reperfusion (0-72 hours), assessed renal structural and functional injury. C3-, C5-, and C6-deficient mice were protected from renal I/R injury, whereas C4-deficient mice were not protected. C6-deficient mice treated with antibody to block C5a generation showed no additional protection from I/R injury. Reconstitution with C6 alone restored the I/R injury in C6-deficient mice. Tubular epithelial cells were the main structures damaged by complement-mediated attack, and, in contrast, the renal vessels were spared. Neutrophil infiltration and myeloperoxidase activity were reduced in C-deficient mouse kidney, but by a similar extent in C3-deficient and C6-deficient mice. We conclude that the membrane attack complex of complement (in which C5 and C6 participate) may account for the effect of complement on mouse renal I/R injury. Neither C5a-mediated neutrophil infiltration nor the classic pathway, in which C4 participates, appears to contribute to I/R injury in this model. By contrast with other organs, such as the heart, the primary effect of complement in the ischemic area is on the parenchymal cell rather than the vascular endothelial cell. The membrane attack complex of complement is a potential target for prevention of I/R injury in this model.

Albumin stimulates interleukin-8 expression in proximal tubular epithelial cells in vitro and in vivo.

Renal tubulointerstitial injury is characterized by inflammatory cell infiltrate; however, the stimuli for leukocyte recruitment are not fully understood. IL-8 is a potent chemokine produced by proximal tubular epithelial cells (PTECs). Whether nephrotic proteins stimulate tubular IL-8 expression remains unknown. Acute exposure of human PTECs to albumin induced IL-8 gene and protein expression time- and dose-dependently. Apical albumin predominantly stimulated basolateral IL-8 secretion. Electrophoretic mobility shift assay demonstrated nuclear translocation of NF-kappaB, and the p65/p50 subunits were activated. NF-kappaB activation and IL-8 secretion were attenuated by the NF-kappaB inhibitors pyrrolidine dithiocarbamate and cell-permeable peptide. Albumin upregulated intracellular reactive oxygen species (ROS) generation, while exogenous H2O2 stimulated NF-kappaB translocation and IL-8 secretion. Albumin-induced ROS generation, NF-kappaB activation, and IL-8 secretion were endocytosis- and PKC-dependent as these downstream events were abrogated by the PI3K inhibitors LY294002 and wortmannin, and the PKC inhibitors GF109203X and staurosporin, respectively. In vivo, IL-8 mRNA expression was localized by in situ hybridization to the proximal tubules in nephrotic kidney tissues. The intensity of IL-8 immunostaining was higher in nephrotic than non-nephrotic subjects. In conclusion, albumin is a strong stimulus for tubular IL-8 expression, which occurs via NF-kappaB-dependent pathways through PKC activation and ROS generation.

Pirfenidone attenuates expression of HSP47 in murine bleomycin-induced pulmonary fibrosis

Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen. The present study was undertaken to investigate whether treatment with the antifibrotic drug pirfenidone attenuates the bleomycin (BL)‐induced overexpression of HSP47 in the lungs. Male ICR mice were intravenously injected with BL or saline (SA). Pirfenidone or control drug (CD) was administered 14 days after commencement of BL or SA, and continued throughout the course of the experiment. The mice were randomly divided into three experimental groups: 1) SA‐treated with CD (SA group); 2) BL‐treated with CD (BL group); and 3) BL‐treated with pirfenidone (pirfenidone group). Lungs of the pirfenidone group showed a marked reduction of fibrotic lesions compared with the corresponding BL group. Immunohistochemical studies showed that BL treatment significantly increased the number of macrophages, myofibroblasts, HSP47‐positive type II pneumocytes and HSP47‐positive interstitial spindle-shaped cells. Treatment with pirfenidone significantly reduced the number of these cells compared with the corresponding BL group. Furthermore, treatment with pirfenidone significantly suppressed the BL‐induced increase of the positive ratio of HSP47 and α‐smooth muscle actin to interstitial spindle-shaped cells. The present study results showed that pirfenidone inhibited heat shock protein 47‐positive cells and myofibroblasts, the principal cells responsible for the accumulation and deposition of extracellular matrix seen in pulmonary fibrosis.

Epithelial secretion of C3 promotes colonization of the upper urinary tract by Escherichia coli

To assess the role of complement in renal infection, we studied a model of Escherichia coli-induced pyelonephritis in mice deficient in complement components C3 and C4. Renal infection occurred less frequently in C3- and C4-deficient mice compared with wild-type mice. In vitro, renal epithelial cells internalized fewer bacteria in the absence of C3 or in the presence of blockade of C3 bound to the bacteria. Moreover, upregulation of epithelial C3 production by stimulation with lipopolysaccharide enhanced bacterial internalization. Here we provide evidence that uropathogenic E. coli might use host C3 to invade the renal epithelium and that local complement production is sufficient for the bacteria to achieve this effect.

In Situ Localization of C3 Synthesis in Experimental Acute Renal Allograft Rejection

Recent evidence has implicated complement in renal transplant injury and identified the kidney as a source of complement components. We therefore investigated the local gene expression of complement component C3, pivotal to complement activation pathways and a mediator of inflammatory injury, in a rat renal transplant model. By reverse transcriptase-polymerase chain reaction, the expression of C3 mRNA increased in two phases. The first phase coincided with post-ischemic injury over 2 days post-transplantation and was localized by in situ hybridization to vessels and glomerular mesangial cells in allogeneic and syngeneic (control) kidney transplants. In allografts only, a second phase was found in tubular epithelial cells, glomerular parietal cells, vessel walls and some infiltrating cells, which peaked on day 4 together with rapid influx of leukocytes, tubule cell damage, the induction of interleukin-2 and interferon-gamma mRNA, and the up-regulation of tumor necrosis factor-alpha and interleukin-1beta mRNA in the graft. In vitro studies showed that interleukin-2 and interferon-gamma up-regulate C3 production in renal tubule cells. We conclude that post-ischemic injury led to transient up-regulation of glomerular expression of C3 mRNA. Subsequent cellular rejection was associated with tubulointerstitial/glomerular parietal cell expression of C3 mRNA. This differential expression of local C3, immediately post-transplant or associated with acute rejection, may have implications for putative therapeutic complement inhibition in clinical transplantation.

Detection of nuclear factor-κB in IgA nephropathy using Southwestern histochemistry

BACKGROUND The transcription factor nuclear factor-kappaB (NF-kappaB) is involved in inflammatory and immune responses through induction of various cytokines and growth factors. The aim of this study is to examine the correlation between NF-kappaB expression and severity of tissue injury in immunoglobulin A (IgA) nephropathy and the mechanism of such correlation. METHODS The study included 43 renal tissue samples from 28 patients, including 28 samples of IgA nephropathy, 5 samples of non-IgA mesangial proliferative glomerulonephritis (non-IgA nephropathy), and 10 samples with nonproliferative glomerulonephritis (membranous nephropathy [MN] n = 5; minimal change nephrotic syndrome [MCNS]; n = 5). Tissue sections were examined by Southwestern histochemistry and immunohistochemistry for monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and intercellular cell adhesion molecule-1 (ICAM-1), which are regulated by NF-kappaB. Normal portions of surgically resected kidney with adenocarcinoma served as controls. RESULTS In normal kidney, MCNS, and MN sections, NF-kappaB expression was detected in a few mesangial cells and tubular epithelial cells. In IgA nephropathy and non-IgA nephropathy samples, NF-kappaB was expressed in mesangial, glomerular endothelial and epithelial cells, tubular epithelial cells, and infiltrating cells. Expression in both glomeruli and interstitium correlated with progression of tissue injury. In IgA nephropathy samples, MCP-1 and GM-CSF expression was increased in both glomeruli and interstitium and correlated with progression of tissue injury. Glomerular ICAM-1 expression was weaker in severe lesions, whereas interstitial expression correlated with progression of tissue injury. CONCLUSION Our results indicate that NF-kappaB is involved in the progression of tissue injury in IgA nephropathy through the induction of transcriptionally regulated genes.

Suppression of Renal Tubulointerstitial Fibrosis by Small Interfering RNA Targeting Heat Shock Protein 47

Background/Aim: Unilateral ureteral obstruction (UUO) is a well-established model for tubulointerstitial fibrosis. During the progression of tubulointerstitial fibrosis, upregulation of collagen synthesis and subsequent accumulation of collagen were observed in the tubulointerstitial area. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone and plays an essential role in regulating collagen synthesis. We designed small interfering RNA (siRNA) sequences for HSP47 mRNA to examine whether HSP47 is involved in the progression of renal tubulointerstitial fibrosis in a mouse UUO model. Methods: The HSP47 siRNA was injected once via the ureter at the time of UUO preparation. We also applied a new gene delivery system for siRNA using cationized gelatin microspheres. The kidneys were harvested 7 and 14 days after UUO. The HSP47 and type I, III, and IV collagen expression levels were analyzed by immunohistochemistry and Western blotting. Results: Seven days after UUO, the expression levels of HSP47 and type I, III, and IV collagens were markedly upregulated in obstructed kidneys or green fluorescent protein siRNA treated obstructed kidneys. HSP47 siRNA injection significantly reduced the protein expression levels and significantly diminished the accompanying interstitial fibrosis. Moreover, cationized gelatin microspheres as a delivery system enhanced and lengthened the antifibrotic effect of HSP47 siRNA. Conclusions: Our results indicate that HSP47 is a candidate target for the prevention of tubulointerstitial fibrosis and that selective blockade of the HSP47 expression by using siRNA could be a potentially useful therapeutic approach for patients with renal disease.

Significance of IL‐1β and IL‐1 receptor antagonist (IL‐1Ra) in bronchoalveolar lavage fluid (BALF) in patients with diffuse panbronchiolitis (DPB)

We evaluated the effect of erythromycin therapy on pulmonary function tests and the airway inflammatory response of patients with DPB. The number of neutrophils in BALF obtained from DPB patients was significantly higher than that of healthy volunteers. Treatment with erythromycin (600 mg/day for 12.9 γδ+9.5 months (mean γδ+s.d.)) significantly reduced the total number of cells and neutrophils in the airway, and significantly improved pulmonary function tests. The levels of IL‐1β and IL‐8 were significantly higher in DPB compared with healthy volunteers (P < 0.05, P < 0.05, respectively). IL‐1 Ra in patients is considered to have a weak inhibitory activity for IL‐1β, with approximately five‐fold concentration of IL‐1β compared with that in healthy volunteers (approx. nine‐fold concentration of IL‐1β). Erythromycin therapy significantly reduced these cytokines to levels comparable to those of healthy volunteers, and produced a trend toward reduction in the level of IL‐1Ra in BALF. The level of IL‐1β correlated significantly with the concentration of neutrophils in BALF (r= 0.72, P < 0.01), as well as with the level of IL‐1Ra (r= 0.688, P < 0.05) and IL‐8 (r= 0.653, P < 0.05). A nearly significant or significant correlation was observed between the concentration of neutrophils and levels of IL‐1Ra or IL‐8 in BALF (r= 0.526, P= 0.053 or r= 0.776, P < 0.01, respectively). There was also a significant relationship between FEV, and the concentration of neutrophils in BALF (r= 0.524, P < 0.05). Our results suggest that the relative amounts of IL‐1β and IL‐1Ra or IL‐8 may contribute, at least in part, to the neutrophil‐mediated chronic airway inflammation in patients with chronic airway disease, and long‐term erythromycin therapy may down‐regulate the vigorous cycle between the cytokine network and neutrophil accumulation, with resultant reduction of neutrophil‐mediated inflammatory response.

Protection and injury: the differing roles of complement in the development of glomerular injury

The role of complement in autoimmune glomerulonephritis (as in other autoimmune diseases) is paradoxical, in that complement activation mediates acute inflammatory injury, yet inherited deficiency of complement may predispose to immune complex disease in particular immune complex glomerulonephritis. We have investigated the role of complement in experimentally induced glomerulonephritis in C3‐deficient mice, using antibodies against the mouse glomerular basement membrane (GBM). In the acute phase of the disease, which is initiated by binding of heterologous antibody to the GBM, we confirmed that the inflammatory injury was positively complement dependent, with C3‐deficient mice developing less severe injury. In contrast, in the autologous phase of the disease, mediated by theimmune response against the heterologous antibody fixed in the GBM, the disease was negatively complement dependent. That is, by 14 days after disease induction the C3‐deficient mice had heavier proteinuria and more severe uremia (p < 0.001) compared to the complement sufficient mice. The C3‐deficient mice also showed a greater accumulation of electron‐dense deposits in the GBM. Thesefindings were reproduced in an accelerated model of this disease in which C3‐deficient mice also develop more severe functional disturbance and demonstrate a higher rate of immune complex deposition. These data illustrate the potential for the net effect of complement to switch from a detrimental to a protective mode at different stages of autoimmune injury.

The membrane attack complex, C5b-9, up regulates collagen gene expression in renal tubular epithelial cells

Evidence suggesting a direct role for proteinuria in the pathogenesis of renal tubulointerstitial fibrosis is accumulating. However the mechanism by which proteinuria leads to injury is unknown. In proteinuric states complement proteins are filtered through the glomerulus and could contribute to the tubular damage. The aim of this study was to investigate the role of complement activation in the progression of interstitial fibrosis. To determine whether complement activation may be responsible for the pro‐fibrotic response that occurs in the tubulointerstitial compartment we stimulated primary cultures of proximal tubular epithelial cells with membrane attack complex, C5b‐9. This led to increased mRNA concentrations of both collagen type IV and its intracellular chaperone, Heat Shock Protein 47 (HSP47). To determine whether this occurred in vivo Adriamycin was used to induce proteinuria in female Balb/c mice. The expression of collagen type IV and HSP47 was increased in proteinuric mice compared to control mice. In proteinuric mouse kidney, C3 was deposited at sites of tubulointerstitial injury and there was a relationship between C3 deposition and immunochemical staining for collagen type IV and HSP47. In situ hybridization suggested that the renal tubular epithelium was actively expressing HSP47 mRNA and, by implication, excess collagen. These observations support the hypothesis that complement activation on tubular epithelial cells can directly increase the pro‐fibrotic process associated with tubulointerstitial damage.