The renal expression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats

Glomerulosclerosis and tubulointerstitial fibrosis are the main structural changes found in the later stages of diabetic nephropathy, which is clinically characterized by proteinuria, and progressive renal insufficiency. Heat shock protein (HSP) 47, a collagen-binding stress protein, has a specific role in the intracellular processing of procollagen molecules during collagen synthesis. It is implicated in the pathogenesis of various fibrotic diseases. However, the expression and significance of HSP47 in acute and chronic phases of diabetic nephropathy is not yet known. In this study, we studied the expression of HSP47 in the kidneys obtained from streptozotocin-induced diabetic rats, in both short- and long-term diabetes. To determine the renal expression of HSP47, and collagens (type III and IV) in acute (days 1, 3 and 14) and chronic (weeks 4, 12 and 24) diabetes, we have performed a time-course study using streptozotocin-induced diabetic rats. The expression pattern of alpha-smooth muscle actin (to identify mesangial cell damage), vimentin (to identify tubular epithelial cell damage), and desmin (to identify glomerular epithelial cell damage) was also determined in kidneys of these diabetic rats. Antibodies specific for HSP47, type III and type IV collagens, alpha-smooth muscle actin, vimentin, and desmin were used to assess the relative expression of their proteins in paraffin-embedded kidney sections by immunohistochemistry. Compared to control rat kidneys, no significant changes in the expression of HSP47 was found in the kidneys of acute diabetic rats. However a significant increase in the expression of HSP47 was noted in the kidneys of chronic diabetic rats; increased expression of HSP47 correlated with an increased renal deposition of types III and IV collagens. Similarly, compared to kidneys of control and acute diabetic rats, an increased expression of alpha-smooth muscle actin (in mesangial cells), vimentin (in tubular epithelial cells), and desmin (in glomerular epithelial cells) was detected in the kidneys of chronic diabetic rats; by dual immunostaining, these phenotypically-altered renal cells in kidneys of chronic diabetic rats were found to be HSP47-producing cells. Importantly, HSP47 up-regulation coincided with the initiation and progression of renal fibrosis, as determined by the expression and deposition of collagens. Our results strongly support a pathological role for HSP47 in the later stages (sclerotic phase) of streptozotocin-induced diabetic nephropathy, which is associated with glomerulosclerosis and tubulointerstitial fibrosis.     

Collagens,collagen-binding heat shock protein 47 and transforming growth factor-beta 1 are induced in cicatricial pemphigoid: possible role(s) in dermal fibrosis

Cicatricial pemphigoid (CP) is an autoimmune mucocutaneous blistering disease associated with scarring. Heat shock protein 47 (HSP47) is thought to play an important role in fibrogenesis, but its role in skin lesions of cicatricial pemphigoid is not yet known. In the present study, we examined the role of HSP47 in dermal fibrosis in cutaneous lesions of a CP patient. Skin biopsies from a patient with CP, and from normal subjects were studied for the expression of HSP47, and interstitial collagens (type I and type III collagens) by immunohistochemistry. Dermal fibroblasts isolated from skin of normal individuals and from fibrotic skin of a CP patient were used to study the expression of HSP47, transforming growth factor beta 1 (TGF-beta 1), type I and type III collagens. Compared to the control skin sections, an increased expression of HSP47 was associated with an increased deposition of interstitial collagens in the fibrotic skin section of the CP patient. Similarly, in contrast to control dermal fibroblasts, the fibroblasts isolated and cultured from fibrotic skin of the CP patient, and grown in vitro, exhibited increased expression of HSP47, type I and type III collagens. Furthermore, compared to the normal control fibroblasts, an increased expression of TGF-beta 1 was detected in the dermal fibroblasts isolated from fibrotic skin of the CP patient. When dermal fibroblasts were treated with various concentrations of TGF-beta 1 (6.25, 12.5, 25, 50 and 100 ng/ml for 24 h), it induced the expression of both type I collagen and HSP47, as determined by quantitative real-time PCR. In conclusion, the expression of TGF-beta 1, HSP47, type I collagen and type III collagen was up-regulated in the fibrotic skin of CP patient, and a complex interaction of these molecules may initiate and propagate the fibrotic cascade in the skin of CP patients.     

Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47

To explore the possible role of heat shock protein (HSP) 47 in the age-related renal changes in Fischer 344 (F 344) rats, the expression of collagen-binding HSP47 with various proteins implicated in phenotypic modulation (α-smooth muscle actin, desmin, and vimentin) and fibrosis (type I, type III, and type IV collagens) was examined in young and old F 344 rat kidneys. Male F 344 rats often develop spontaneous nephropathy in old age. Kidneys obtained from 24-month-old F 344 rats showed glomerulosclerosis with marked tubulointerstitial damage including interstitial fibrosis, while no significant histological alteration was found in the kidneys of 6-month-old rats. Immunohistochemical analysis showed an increased accumulation of type I, type III, and type IV collagens in areas of glomerulosclerosis and interstitial fibrosis in old rat kidneys. In kidneys of young rats, collagen-binding HSP47 expression was weak in the glomeruli and occasionally seen in the interstitial cells. In contrast, strong immunostaining for HSP47 was noted in the glomeruli, tubular epithelial cells, and interstitial cells in kidneys of old rats. In addition, phenotypic alterations of mesangial cells and interstitial cells (immunopositive for α-smooth muscle actin), glomerular epithelial cells (immunopositive for desmin), and tubular epithelial cells (immunopositive for vimentin) were found in the kidneys of old F 344 rats. Double immunostaining showed that all these phenotypically altered renal cells express HSP47 and that increased expression of HSP47 was always associated with increased expression of collagens in the old rat kidneys. From the above observations, it is concluded that overexpression of HSP47 by phenotypically altered renal cells might play an important role in the excessive assembly of collagens and could thereby contribute to the glomerulosclerosis and interstitial fibrosis found in kidneys of aged F 344 rats.     

Life-long Dietary Restriction Modulates the Expression of Collagens and Collagen-binding Heat Shock Protein 47 in Aged Fischer 344 Rat Kidney

It has been shown that the expression of HSP47 and collagens is substantially increased in the sclerotic/fibrotic process in various organs, including kidney. However, the factors regulating the increased expression of HSP47 are not yet clear. In this study, we examined the effect of dietary restriction for the expression of collagens and collagen-binding HSP47 in the kidneys of 6- and 24-month-old male Fischer 344 (F 344) rats fed ad libitum or 30% diet-restricted. No significant histological alteration was found in the kidneys of 6-month-old fed or diet-restricted rats. Kidneys obtained from 24-month-old freely fed rats showed glomerulosclerosis with marked tubulointerstitial damage including interstitial fibrosis, while in the kidneys of 24-month-old diet-restricted rats, renal damage was remarkably less than those noted in 24-month-old freely fed rat kidneys. Immunohistochemical analysis showed an increased accumulation of type I, type III and type IV collagens in areas of glomerulosclerosis and interstitial fibrosis in old rat kidneys. Dietary restriction significantly reduces renal accumulation of collagens in old age. Aging enhanced expression of HSP47 in 24-month-old freely fed rat kidneys whereas dietary restriction suppressed its expression in 24-month-old diet-restricted rat kidneys. Also, phenotypic alterations of mesangial cells and interstitial cells (immunopositive for -smooth muscle actin), glomerular epithelial cells (immunopositive for desmin) and tubular epithelial cells (immunopositive for vimentin) were seen in 24-month-old freely fed rat kidneys and found to express HSP47. Dietary restriction significantly diminished phenotypically altered renal cells in 24-month-old rat kidneys. Our results suggest that increased expression of HSP47 is associated with age-related renal damage and that diet-restricted alteration of its expression is associated with the modulation of age-associated renal sclerosis/fibrosis.     

Expression profiles of collagens,HSP47, TGF-beta1, MMPs and TIMPs in epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is a chronic, uncommon, sub-epidermal blistering disease involving the skin and mucous membranes that heals with scar formation and milia. Collagens, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are important components that play an essential role(s) in matrix remodeling during scar formation. However, the possible involvement of these components in EBA-induced scarring is not yet known. In the present study, we examined the expression profile of collagens, collagen-binding heat shock protein 47 (HSP47), MMPs and their inhibitory enzymes, TIMPs, in matrix remodeling during conjunctival scarring. The involvement of TGF-beta1, a fibrogenic factor, was also studied. Compared to the controls, an increased expression of type I collagen, type III collagen and HSP47 was detected in conjunctival biopsy sections of patient with EBA using immunohistochemistry. Similar increase in the expression of type I collagen, type III collagen and HSP47 was noted in conjunctival fibroblasts obtained from the patient with EBA. Up-regulation in the expression of MMP-1 and MMP-14 was also noted in conjunctival fibroblasts isolated from the patient with EBA, while no significant changes in the expression of MMP-3, MMP-8, MMP-9 and MMP-13 were seen. As for TIMPs, conjunctival fibroblasts isolated from the patient with EBA, grown in vitro, exhibited increased expression of TIMP-1, TIMP-2 and TIMP-3, when compared with fibroblasts grown from control conjunctival tissues, although the expression level varies with different molecules of the same family. Additionally, compared to the control conjunctival fibroblasts, an increased expression of TGF-beta1 was detected in fibroblasts isolated from the conjunctival tissues of patient with EBA.This study suggests that there is up-regulation in the production of collagens (type I and III), collagen-binding protein (HSP47), matrix degrading collagenases (MMP-1 and 14), and their inhibitory enzymes (TIMP-1, 2 and 3) during the process of conjunctival matrix remodeling in the patient with EBA. The presented data is preliminary and could serve as a basis for further studies to enhance our understanding about the molecular mechanisms of conjunctival scarring in patients with EBA.     

Inhibition of MCP-1/CCR2 pathway ameliorates the development of diabetic nephropathy

Monocyte chemoattractant protein (MCP-1) is an important mediator for macrophage recruitment in atherosclerosis and various glomerulonephritis. However, the role of MCP-1 and its receptor CCR2 in the progression of diabetic nephropathy remains unknown. Using a type 1 diabetic nephropathy model that shows noticeable glomerulosclerosis, we examined the role of MCP-1/CCR2 by propagermanium (Pro; CCR2 antagonist) treatment, and confirmed it by transfection of plasmids carrying the 7ND (a mutant of MCP-1) gene. We measured the mesangial matrix expansion, type IV collagen (Col4), transforming growth factor (TGF)-beta1 positive area, and macrophage infiltration in glomeruli after 12 weeks. Mesangial matrix expansion and macrophage infiltration were increased in diabetic mice and inhibited by Pro or 7ND-treatment. Increased glomerular expression of Col4 and TGF-beta1 in diabetic mice was also ameliorated. Thus blocking the MCP-1/CCR2 pathway ameliorated glomerulosclerosis, indicating that the MCP-1/CCR2 pathway plays a crucial role in the progression of diabetic nephropathy.     

Pirfenidone inhibits the expression of HSP47 in TGF-beta1-stimulated human lung fibroblasts

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and patients with idiopathic pulmonary fibrosis (IPF). Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen and plays an important role in the pathogenesis of IPF. The present study evaluated the in vitro effects of pirfenidone on expression of HSP47 and collagen type I in cultured normal human lung fibroblasts (NHLF). Expression levels of HSP47 and collagen type I in NHLF stimulated by transforming growth factor (TGF)-beta1 were evaluated genetically, immunologically and immunocytochemically. Treatment with TGF-beta1 stimulated both mRNA and protein expressions of both HSP47 and collagen type I in NHLF, and pirfenidone significantly inhibited this TGF-beta1-enhanced expression in a dose-dependent manner. We concluded that the anti-fibrotic effect of pirfenidone may be mediated not only through direct inhibition of collagen type I expression but also at least partly through inhibition of HSP47 expression in lung fibroblasts, with a resultant reduction of collagen synthesis in lung fibrosis.     

Unilateral nephrectomy leads to up-regulation of syndecan-2- and TGF-beta-mediated glomerulosclerosis in syndecan-4 deficient male mice.

Syndecan-4 is an ubiquitous, plasma membrane-spanning heparan sulfate proteoglycan involved in proliferation, differentiation, adhesion and migration of cells in vitro. Syndecan-4 knockout (KO) mice show no obvious defects but respond abnormally to experimental stress conditions. In the adult, syndecan-4 is the most abundant syndecan of renal tissue. We therefore investigated the consequences of syndecan-4 deficiency during progression of kidney disease using unilaterally nephrectomized mice, a model of glomerular hyperfiltration and renal hypertrophy. 60 days after unilateral nephrectomy (UNX), mesangial expansion, enhanced matrix production (collagens I and IV, fibronectin) and focal segmental glomerulosclerosis, resembling early stages of diabetic nephropathy, was apparent in male but not female syndecan-4 KO mice. No defect was detected in wild type UNX males. Syndecan-2 mRNA and protein were not detectable in renal glomeruli of wild type mice, but were induced specifically in the glomeruli of the syndecan-4 deficient kidneys after unilateral nephrectomy. Due to the structural similarities of syndecans-2 and -4 we hypothesize that de novo-production of syndecan-2 in kidneys after unilateral nephrectomy reflects a compensatory response. However, this response is counterproductive since syndecan-2 supports the pro-sclerotic activity of TGF-beta1 which is increased in parallel with syndecan-2 synthesis. By contrast, signaling through syndecan-4 negatively controls the production of pro-sclerotic TGF-beta1.     

HSP47 as a collagen-specific molecular chaperone: function and expression in normal mouse development

A large family of molecular chaperones can be divided into two major groups: general chaperone and substrate-specific chaperone. HSP47 is a collagen-specific molecular chaperone residing in the endoplasmic reticulum (ER). Recent studies revealed that HSP47 is essential molecular chaperone for mouse development and is essential for collagen molecular maturation in the ER. In the absence of HSP47, collagen microfibril formation and basement membrane formation are impaired in mouse embryos because the failure in the molecular maturation of types I and IV collagens, respectively. The tissue-specific expression of HSP47 is always correlated with that of various types of collagens and closely related with the collagen-related diseases including fibrosis in various organs. The importance of HSP47 in the therapeutic strategy for fibrotic diseases as well as for a marker of collagen-related autoimmune diseases will also be discussed.     

The collagen-specific molecular chaperone HSP47: is there a role in fibrosis?

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that is required for molecular maturation of various types of collagens. Recent studies have shown a close association between increased expression of HSP47 and excessive accumulation of collagens in scar tissues of various human and experimental fibrotic diseases. It is presumed that the increased levels of HSP47 in fibrotic diseases assist in excessive assembly and intracellular processing of procollagen molecules and, thereby, contribute to the formation of fibrotic lesions. Studies have also shown that suppression of HSP47 expression can reduce accumulation of collagens to delay the progression of fibrotic diseases in experimental animal models. Because HSP47 is a specific chaperone for collagen synthesis, it provides a selective target to manipulate collagen production, a phenomenon that might have enormous clinical impact in controlling a wide range of fibrotic diseases. Here, we outline the fibrogenic role of HSP47 and discuss the potential usefulness of HSP47 as an anti-fibrotic therapeutic target.     

Role of Smad1 in diabetic nephropathy: Molecular mechanisms and implications as a diagnostic marker

Diabetic nephropathy (DN) is the leading cause of chronic kidney failure. Moreover, DN is associated with elevated cardiovascular morbidity and mortality. DN is characterized by progressive expansion of the mesangial matrix and thickening of the glomerular basement membrane, resulting in the obliteration of glomerular capillaries. Advanced glycation endproducts (AGEs) produced as the result of hyperglycemia are known to stimulate the production of extracellular matrix (ECM) proteins, resulting in glomerulosclerosis. Exposure of cultured mesangial cells to AGEs results in a receptor-mediated upregulation of mRNA and protein secretion of type IV collagen (Col4), which is a major component of ECM. Here we review recent novel insights into the pathogenesis and diagnosis of DN, with a special emphasis on the emerging concept that diabetic glomerulosclerosis can result from activation of the signaling cascade leading to irreversible ECM overproduction. Finally, we describe signaling pathways involved in the initial change of DN and how these pathways can be manipulated for therapeutic benefit.     

The molecular basis of increased glomerulosclerosis after blockade of the renin angiotensin system in growth hormone transgenic mice.

BACKGROUND: Angiotensin converting enzyme inhibitor (ACEi) therapy delays the onset of renal failure in diabetic nephropathy and inhibits or delays the onset of proteinuria in several animal models. MATERIALS AND METHODS: We examined this question using a transgenic model of chronic glomerulosclerosis caused by an excess production of growth hormone (GH) in which there is progressive glomerular scarring leading to uremia. In addition, since GH mice do not have systemic hypertension or an elevated glomerular filtration rate, we could address the question of whether ACEi or angiotensin II receptor antagonists (AII RA) had an effect on the development of glomerulosclerosis under these conditions. Since excess matrix accumulates in glomerulosclerosis because of alterations in the balance between its synthesis and degradation, we examined the effect of ACEi and AII RA on these parameters. RESULTS: Systemic blood pressure was unaffected by ACEi treatment, but the glomerular filtration rate decreased 85%. ACEi-treated mice had increased mesangial deposition of type I collagen and decreased 105 kD complex collagenase activity. In addition, ACEi-treated GH mice had increased glomerular alpha 1 type I collagen, alpha 1 type IV collagen, and alpha-smooth muscle cell actin mRNAs. No changes were noted in beta actin, or 72 kD metalloproteinase mRNAs. The result of these changes was a net increase in sclerosis. Surprisingly, GH mice treated with ACEi or AngII RA developed marked renal arteriolar lesions. CONCLUSIONS: In some forms of glomerulosclerosis, the lesions develop independently of angiotensin II. Pharmacological inhibition of angiotensin II, in this circumstance, may aggravate the lesions through disregulation of the levels and the balance between glomerular matrix synthesis and degradation.     

The plasmid encoding HSP47 enhances collagen expression and promotes skin wound healing in an alloxan-induced diabetic model

The objective of this work was initially to investigate the effects on skin wound healing process by local injection of HSP47 recombinant plasmid in an alloxan-induced diabetic rat model and assess the possibility and utility of gene therapy based on HSP47 plasmid to improve the diabetic skin wound healing. Rats were injected intraperitoneally with alloxan (120 mg/kg) to induce diabetes. The fragment containing the rat 47 kDa heat shock protein (HSP47) gene lacking its own promoter was cloned into plasmids containing a promoter and green fluorescent protein (GFP). The resulting gene constructs were first tested in vitro using 3T3 fibroblast cell line and subsequently in vivo after inducing wounds with alloxan in diabetic rats. Immunohistochemistry, quantitative fluorescent RT-PCR, and Western blotting 3-5 days after plasmid injection were performed to measure the expression changes of HSP47 and collagen I. The results demonstrate an increase of HSP47 levels in vitro in 3T3 fibroblast cells and in vivo in diabetic rat after treatment with plasmids expressing HSP47. The level of collagen I around the wound during the repair process was higher in the treated group than that in the control group, indicating that the constructs may have use in human gene therapy in cases of impaired skin wound healing in diabetes.     

Nitric oxide in the pathogenesis of diffuse pulmonary fibrosis

By studying the responses of nitric oxide in pulmonary fibrosis, the role of inducible nitric oxide synthase in diffuse pulmonary fibrosis as caused by lipopolysaccharide (LPS) treatment was investigated. When compared to rats treated with LPS only, the rats pretreated with 1400W (an iNOS-specific inhibitor) were found to exhibit a reduced level in: (i) NOx (nitrate/nitrite) production, (ii) collagen type I protein expression, (iv) soluble collagen production, and (iv) the loss of body weight and carotid artery PO2. In the pulmonary fibroblast culture, exogenous NO from LPS-stimulated secretion by macrophages or from a NO donor, such as DETA NONOate, was observed to induce the expression of TIMP-1, HSP47, TGF-beta1, and collagen type I as well as the phosphorylation of SMAD-2. After inhalation of NO for 24 h, an up-regulation of collagen type I protein was also noted to occur in rat pulmonary tissue. The results suggest that the NO signal pathway enhanced the expression of TGF-beta1, TIMP-1, and HSP47 in pulmonary fibroblasts, which collectively demonstrate that the NO signal pathway could activate the SMAD-signal cascade, by initiating a rapid increase in TGF-beta1, thereby increasing the expression of TIMP-1 and HSP47 in pulmonary fibroblasts, and play an important role in pulmonary fibrosis.     

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.     

Metal-catalyzed oxidation of extracellular matrix proteins promotes human mesangial cell apoptosis and is associated with enhanced expression of Bax and caspase activation

Oxidative injury in glomerular disease may oxidize extracellular matrix proteins which might modulate their interaction with mesangial cells and thereby account for the hypocellularity seen in advanced glomerulosclerosis. In this study we evaluated whether oxidation of extracellular matrix could modulate human mesangial cell apoptosis. Human mesangial cells were seeded onto plates coated with unmodified (control) or oxidized Matrigel, laminin, or type IV collagen. Mesangial cell apoptosis was increased on oxidized Matrigel as well as on oxidized laminin and type IV collagen. Mesangial cells behaved similarly on plates coated with control and oxidized forms of the integrin ligand-containing peptide GRGDSP. Cells on oxidized matrix demonstrated enhanced expression of Bax, increased fragmentation of PARP, and diminished apoptosis in the presence of the interleukin-1 beta converting enzyme inhibitor Ac-Tyr-Val-Ala-Asp-aldehyde. These data suggest that oxidation of extracellular matrix proteins may enhance human mesangial cell apoptosis via a mechanism that appears to involve enhanced expression of Bax and caspase activation. This may account for irreversible mesangial hypocellularity in glomerulosclerosis.