Shuji Tatsukawa

Transformation of interstitial fibroblasts and tubulointerstitial fibrosis in diabetic nephropathy

 The developmental mechanism of tubulointerstitial fibrosis in diabetic nephropathy (DN) has not been elucidated. Tubulointerstitial fibrosis, as well as glomerulosclerosis, occurs in DN. Myofibroblasts which overproduce extracellular matrix are present in the renal interstitium in diabetics, although they are almost never seen in normal kidneys. The myofibroblasts appear to originate from interstitial fibroblasts. In addition, transforming growth factor-β1 (TGF-β1), which can evoke myofibroblast transformation, is detected in interstitial cells in the diabetic kidney, but not in the normal kidney. Taken together, these findings led us to speculate that TGF-β1 induces the transformation of interstitial fibroblasts into myofibroblasts, followed by tubulointerstitial fibrosis. Based on this speculation, we discuss the developmental mechanism of tubulointerstitial fibrosis in this review.

Epiplakin accelerates the lateral organization of keratin filaments during wound healing

BACKGROUND Epiplakin (EPPK) belongs to the plakin family of cytolinker proteins and, resembling other members of the plakin family such as BPAG1 (an autoantigen of bullous pemphigoid) and plectin, EPPK has plakin repeat domains (PRDs) that bind to intermediate filaments. Elimination of EPPK by gene targeting in mice resulted in the acceleration of keratinocyte migration during wound healing. EPPK is expressed in proliferating keratinocytes at wound edges and, in view of its putative function in binding to keratin, we postulated that the keratin network in EPPK-null (EPPK(-/-)) mice might be disrupted during wound healing. OBJECTIVE To examine this hypothesis and to determine the precise localization of EPPK in relation to keratin filaments, we compared the non-wounded and wounded epidermis of wild-type and EPPK(-/-) mice. METHODS Non-wounded epidermis and wounded epidermis from wild-type and EPPK(-/-) mice were examined by immunofluorescence staining and electron microscopy before and after double immunostaining. RESULTS EPPK was colocalized with keratin 17 (K17) more extensively than with other keratins examined in wounded epidermis. The expression of K5, K10, K6, and K17 was the same in EPPK(-/-) mice after wounding as in normal mice, but diameters of keratin filaments were reduced in EPPK(-/-) keratinocytes. Electron microscopy after immunostaining revealed that EPPK colocalized with K5, K10 and K6 after wounding in wild-type mice. CONCLUSION Our data indicate that EPPK accelerates keratin bundling in proliferating keratinocytes during wound healing and suggest that EPPK might contribute to reinforcement of keratin networks under mechanical stress.

Transient expression of mouse pro-α3(V) collagen gene (Col5a3) in wound healing.

The α3(V) chain is poorly characterized among type V collagen chains. Pro-α3(V) collagen is expressed in newly synthesized bone as well as in the superficial fascia of developing muscle. Present study examined the expression in a mouse model of wound healing. Real-time reverse transcriptase polymerase chain reaction and in situ hybridization revealed transient expression of pro-α3(V) chain at a lower level than other fibrillar collagen genes after injury. Immunohistochemistry showed a similar expression pattern in the injured skin. In addition, electron microscopy showed that pro-α3(V) chain was localized in the amorphous nonfibrillar region, but not in fine or dense fibrils. The pro-α3(V) chain co-localized with heparan sulfate, which appeared in the skin after injury and might bind via an acidic segment of the pro-α3(V) chain. The matrix containing the pro-α3(V) chain may therefore be needed for the initiation of wound healing.

Contraction of tubulointerstitial fibrosis tissue in diabetic nephropathy, as demonstrated in an in vitro fibrosis model

Tubulointerstitial fibrosis in diabetic nephropathy (DN) was investigated using an in vitro tissue model of remodeling, to determine the pathogenic mechanism of fibrosis that leads to renal atrophy, i.e., renal failure. The remodeling model consisted of a renal fibroblast-populated collagen lattice (FPCL). The overexpression of transforming growth factor (TGF)-β1 in the diabetic kidney gave rise to FPCL contraction. FPCL relaxation was induced by the subsequent addition of cytochalasin D. The FPCL failed to contract when exposed to TGF-β1 plus Y27632, a Rho kinase inhibitor. TGF-β1 induced the phosphorylation of myosin light chains, and Y27632 blocked this activity. TGF-β1-induced FPCL contraction was suppressed by the addition of 2,3-butanedione monoxime, a myosin ATPase inhibitor. As shown in the video, the contraction rate of the projections of the cells in the FPCL was significantly greater in the TGF-β1 group than in the control group. Collectively, these results indicate that TGF-β1-induced FPCL contraction is attributable to actin–myosin interactions in the fibroblasts through the activation of Rho kinase, the phosphorylation of myosin light chains, and the subsequent activation of myosin ATPase. We propose that via these mechanisms, tubulointerstitial fibrosis generates tissue contraction that leads to renal atrophy and renal failure in DN.

Morphological and functional changes of the rat parotid glandular cells by clipping and reopening the parotid duct, using HAM8 antibody

The purpose of this experiment is to examine the proliferative process of rat acinar cells after parotid duct ligation and reopening. Two experimental groups were observed. The first group was killed from 0 to 14 days after the duct ligation. In the second group, the duct was clipped for 14 days, and it was reopened. Following a period of from 2 to 28 days after removal of the clip, the glands were removed to perform a histological analysis, including hematoxylin-eosin (HE), immunofluorescent staining using HAM8 antibody, which recognizes connexin 32, and transmission electron microscopy (TEM). In the experimental gland from the 1st group at 6 days after ligation (I-6D), the acinar cells disappeared. In the tissue from the 2nd group 8 days after reopening (II-8D), newly formed acinar cells were found again. Lobular structure of the parotid glands recovered in the II-21D. HAM8 signals were observed between normal acinar cells, while they declined in the tissue from I-1D, and they were not observed in the I-2D. HAM8 signals were first observed in the II-25D and then subsequently returned to normal levels in the II-28D. These results suggest that the intercellular communication and functional recovery was not complete 25 days after reopening of the duct. In conclusion, the recovery of the acinar structure was recognized during an extended period of duct ligation, however, a time lag between the morphological and functional recovery was found to exist.

Pore alterations of the endothelial lining of rat fenestrated intestinal capillaries exposed to acute stress.

Stress-induced inflammatory responses in the portal system are characterized by elevations in serum concentrations of interleukin-6 (IL-6) and endotoxins such as lipopolysaccharides (LPS). LPS translocation from the intestinal to the capillary lumen occurs via LPS endocytosis by the capillary endothelium. Because the capillary endothelium of the small intestinal submucosa is fenestrated, we determined the role of pore modifications within the fenestrated endothelium in relaying inflammatory stress responses in the portal vein. We evaluated changes in the diameter and density of endothelial pores of the lamina propria of intestinal villi induced by continuous light (CL) exposure for 48 h and the correlation between these changes and serum IL-6 concentration in the portal vein in a rat model. We found significant increases in both the pore diameter and density, accompanied by a significant increase in portal IL-6 concentration; these changes were significantly attenuated by pretreatment with propranolol, a beta adrenergic receptor antagonist. In contrast, intravenous noradrenaline administration mimicked CL-induced modifications of the diameter and density of pores and the elevation of portal vein IL-6 concentration. These findings suggested that stress-induced inflammatory responses in the portal system may be a part of the modifications of the endothelial pores triggered by sympathetic activation.

The Contribution of Fibronectin ED-A Expression to Myofibroblast Transdifferentiation in Diabetic Renal Fibrosis

It is well known that glomerulosclerosis and tubulointerstitial fibrosis occur during the early stages of diabetic nephropathy. It has been demonstrated that the development of tubulointerstitial lesions is more closely correlated with a progressive decline in renal function compared to glomerular lesions (Bohle et al., 1991). Tubulointerstitial fibrosis ultimately leads to renal failure as a result of renal atrophy. The myofibroblasts emerging in tubulointerstitial fibrosis tissue have been indicated to play a crucial role in the development and progression of fibrosis (Simonson, 2007). They overproduce extracellular matrix (ECM) molecules, including type I collagen and fibronectin, and repress ECM degradation through the production of tissue inhibitor of metalloproteinase1 (TIMP-1) (Edwards et al., 1987) and plasminogen activator inhibitor-1 (PAI-1) in response to transforming growth factor-┚1 (TGF-┚1), which is increased in DN (Laiho et al., 1987), followed by ECM accumulation (i.e. fibrosis). Furthermore, the myofibroblasts may induce fibrosis tissue contraction via increased cell contraction promoted by ┙smooth muscle actin (┙-SMA) expression, leading to renal atrophy and failure. Myofibroblasts are considered to be derived from various kinds of cells, e.g. fibroblasts (Strutz and Zeisberg, 2006), epithelial cells (Iwano et al., 2002), endothelial cells (Zeisberg et al., 2008), pericytes (Humphreys et al., 2010), and bone marrow-originated cells (Keeley et al., 2010), via stimulation by TGF-┚1. Although ┙-SMA expression, which is the most pronounced characteristic of myofibroblasts, has been considered to cause tissue contraction, the detailed mechanism has not yet been determined. Myofiboblasts were first described in wound-healing granulation tissue