Interstitial expression of heat-shock protein 47 correlates with capillary deposition of complement split product C4d in chronic allograft nephropathy

BACKGROUND: Chronic allograft nephropathy (CAN), associated with late-allograft dysfunction is caused by alloantigen-dependent and -independent mechanisms, and eventually leads to interstitial fibrosis (ci). Activation of complement cascade is considered to be a poor prognostic marker of graft survival. This study was designed to examine the relationship between the expression of C4d and heat-shock protein 47 (HSP47, a collagen-specific chaperone) in the development of interstitial fibroproliferative lesions in CAN. METHODS: Sixty-three renal allograft biopsy specimens, obtained from 48 patients, were examined for the expression of C4d, HSP47, CD68 and alpha-smooth muscle actin (alpha-SMA) by immunohistochemistry. Double-staining was performed to determine the colocalization of C4d and HSP47. The relationship of between the expression of C4d, HSP47, CD68 and alpha-SMA and the clinical and histopathological parameters were statistically analysed. RESULTS: No expression of C4d was noted in the tubulointerstitium including peritubular capillary (PTC) of the control kidney. C4d was expressed in PTC in one-third of allograft renal tissues with morphological evidences of CAN. The interstitial cells around the fibrotic areas of the PTC of CAN were positive for the expression of HSP47. The deposition of C4d in PTC correlated with interstitial expression of HSP47 around the PTC. Most HSP47 expressing cells were phenotypically altered myofibroblasts, as determined by the dual staining of alpha-SMA. CONCLUSIONS: The increased expression of HSP47 positively correlated with the expression of C4d in PTC, and might contribute to the progression of interstitial ci in CAN.     

Progressive renal fibrosis in murine polycystic kidney disease: an immunohistochemical observation

BACKGROUND: The appearance of interstitial fibrosis in polycystic kidneys is emblematic of progressive disease. Matrix forming this scar tissue is derived from local renal cells in response to cystogenesis. We investigated the phenotype of collagen-producing cells in the cystic kidneys of DBA/2-pcy mice to better characterize the spectrum of interstitial cells associated with renal fibrogenesis. METHODS: The extent of interstitial fibrosis and the number of fibroblasts in cystic kidneys were first quantitated over time using computer-assisted image analysis. Subsequently, antisera to four cell protein markers were studied by coexpression immunohistochemistry during progression of fibrosis using confocal microscopy. The antisera included fibroblast-specific protein 1 (FSP1) for fibroblast phenotype, alpha-smooth muscle actin (alpha-SMA) for contractile phenotype, vimentin (VIM) for mesenchymal phenotype, and heat shock protein 47 (HSP47) for interstitial collagen-producing phenotype. RESULTS: Interstitial fibrosis in cystic kidneys gradually increased throughout the 30-week observation period of our study. With progression of cystogenesis, most of the tubules in pcy mice either dilated or disappeared with time. FSP1+ fibroblasts were distributed sparsely throughout the renal interstitium of young pcy and wild-type mice. Their number increased in the widening fibrotic septa by 18 weeks of age and persisted through 30 weeks of the study interval. Some epithelia among remnant tubules trapped within fibrotic septa around adjacent cysts also acquired the phenotype of FSP1+, HSP47+ collagen-producing fibroblasts, suggesting a possible role for epithelial-mesenchymal transformation (EMT) in this process. Most FSP1+ fibroblasts were alpha-SMA-, but HSP47+, suggesting they were producing collagen proteins for the extracellular matrix. alpha-SMA+, FSP1-, HSP47+ or HSP47- cells were also observed, and the latter tended to distribute independently in a linear pattern, reminiscent of vasculature adjacent to forming cysts. VIM+ expression was not observed in alpha-SMA+ cells. CONCLUSIONS: Many nonoverlapping as well as fewer overlapping populations of FSP1+ and alpha-SMA+ cells shared in the collagen expression associated with progressive fibrogenesis in pcy mice undergoing cystogenesis. Some FSP1+ fibroblasts are likely derived from tubular epithelium undergoing EMT, while alphaSMA+, VIM- cells probably represent vascular smooth muscle cells or pericytes surviving vessel attenuation during the chaos of fibrogenesis. Importantly, not all interstitial cells producing collagens are alpha-SMA+.     

Vascular endothelial growth factor expression in human chronic renal allograft rejection

BACKGROUND: Chronic renal allograft rejection is characterized by interstitial fibrosis and vasculopathy. Vascular endothelial growth factor (VEGF) is an endothelial mitogen with increased expression in inflammation and vasculopathy. METHODS: Renal tissue from 17 patients with chronic rejection was examined for VEGF protein and the presence of CD 68-positive macrophages, and compared to biopsies from patients with temporary allograft dysfunction, acute rejection, and native kidneys with thin membrane disease. RESULTS: In the chronic rejection group, there was markedly increased expression of VEGF protein in the interstitium (P<0.0001). In serial sections, VEGF colocalized with the expression of CD 68-positive macrophages. Significantly more macrophages were in the tubulointerstitium in tissue with chronic rejection than in those with temporary allograft dysfunction (P<0.005). Additionally, VEGF protein expression in the glomeruli and the vascular compartment of patients with chronic rejection was increased. CONCLUSION: The up-regulation of VEGF in chronic renal allograft rejection may be important in inflammation and development of fibrosis.     

Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats

BACKGROUND: Peritoneal fibrosis is a serious complication in patients on continuous ambulatory peritoneal dialysis (CAPD), but the molecular mechanism of this process remains unclear. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for biosynthesis and secretion of collagen molecules, and is expressed in the tissue of human peritoneal fibrosis. In the present study, we examined the effect of HSP47 antisense oligonucleotides (ODNs) on the development of experimental peritoneal fibrosis induced by daily intraperitoneal injections of chlorhexidine gluconate (CG). METHODS: HSP47 antisense or sense ODNs were injected simultaneously with CG from day 14, after injections of CG alone. Peritoneal tissue was dissected out 28 days after CG injection. The expression patterns of HSP47, type I and type III collagen, alpha-smooth muscle actin (alpha-SMA), as a marker of myofibroblasts, ED-1 (as a marker of macrophages), and factor VIII were examined by immunohistochemistry. RESULTS: In rats treated with CG alone, the submesothelial collagenous compact zone was thickened, where the expression levels of HSP47, type I and type III collagen and alpha-SMA were increased. Marked macrophage infiltration was also noted and the number of vessels positively stained for factor VIII increased in the CG-treated group. Treatment with antisense ODNs, but not sense ODNs, abrogated CG-induced changes in the expression of HSP47, type I and III collagen, alpha-SMA, and the number of infiltrating macrophages and vessels. CONCLUSION: Our results indicate the involvement of HSP47 in the progression of peritoneal fibrosis and that inhibition of HSP47 expression might merit further clinical investigation for the treatment of peritoneal fibrosis in CAPD patients.     

Expression of heat shock proteins 47 and 70 in the peritoneum of patients on continuous ambulatory peritoneal dialysis

BACKGROUND: Peritoneal sclerosis, characterized by collagen accumulation, is a serious complication in continuous ambulatory peritoneal dialysis (CAPD) therapy. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperon and is closely associated with collagen synthesis. METHODS: We determined the expression of HSP47 and HSP70 (nonspecific for collagen synthesis) by immunohistochemistry in peritoneal tissues of patients on CAPD. The tissue for collagen III, alpha-smooth muscle actin (alpha-SMA), and CD68 (a marker for macrophages) were also stained. Thirty-two peritoneal samples were divided into three groups (group A1, 11 patients who had no ultrafiltration loss; group A2, 9 patients who had ultrafiltration loss; and group B, 12 specimens who had end-stage renal disease prior to induction of CAPD. RESULTS: In group B, staining for HSP47, HSP70, and collagen III in peritoneal tissues was faint, and only a few cells were positive for alpha-SMA and CD68. In contrast, HSP47, HSP70, and collagen III were expressed in areas of thickened connective tissues in fibrotic peritoneal specimens of CAPD patients. The expression level of HSP47, HSP70, collagen III, and alpha-SMA and the number of CD68-positive cells in group A2 were significantly higher than those in groups A1 and B. HSP47/HSP70-positive cells were mesothelial cells, adipocytes, and alpha-SMA-positive myofibroblasts. Furthermore, the expression level of HSP47 was significantly higher in peritoneal specimens from patients with refractory peritonitis than without it and was significantly higher in patients with more than 60 months of CAPD therapy than that in patients with less than 60 months of CAPD. CONCLUSION: Our results indicate that CAPD therapy may induce HSPs in the peritoneal tissue, and that peritonitis in CAPD patients may be associated with the progression of peritoneal sclerosis at least through HSP47 expression and chronic macrophage infiltration. Our data also suggest that the progression of peritoneal sclerosis in such patients is associated with deterioration of peritoneal ultrafiltration function.     

Epithelial to Mesenchymal Transition During Late Deterioration of Human Kidney Transplants: The Role of Tubular Cells in Fibrogenesis

The hallmark of failing renal transplants is tubular atrophy and interstitial fibrosis (TA/IF). Injury to tubular epithelial cells (TEC) could contribute to fibrogenesis via epithelial-mesenchymal transition (EMT). We examined the features of EMT in renal transplants that developed TA/IF. Biopsies from 10 allograft kidneys with impaired function and TA/IF and 10 biopsies from transplants with stable function were compared to their implantation biopsies. Relative to implantation biopsies, TEC in TA/IF kidneys showed loss of epithelial markers (E-cadherin, cytokeratin) with altered distribution. Some TEC also showed new cytoplasmic expression of mesenchymal markers vimentin, S100A4, and alpha smooth muscle actin (alpha-SMA) and collagen synthesis marker heat shock protein (HSP-47), both in deteriorating and atrophic tubules. Double immunostaining showed coexpression of cytokeratin and vimentin, S100A4 and HSP-47, indicating intermediate stages of EMT in TA/IF. These changes were absent or much less in transplants with stable function. EMT features in the TA/IF group correlated with serum creatinine (vimentin, S100A4, HSP-47), history of T-cell-mediated rejection (cytokeratin, S100A4) and proteinuria (cytokeratin). These findings support a model in which the TEC damage induces loss of epithelial features and expression of fibroblast features, as a common pathway of deterioration by either immunologic or nonimmunologic processes.     

Interstitial expression of alpha-SMA: an early marker of chronic renal allograft dysfunction.

BACKGROUND: Renal myofibroblast infiltration has been shown to be strongly associated with renal function decline in several chronic renal diseases. The purpose of the present study was to investigate whether early detection of myofibroblast infiltration using alpha-smooth-muscle actin (alpha-SMA) expression in time-zero biopsies predicts renal allograft dysfunction. METHODS: We studied renal tissue from 38 renal transplant patients from whom biopsies had been taken after vascular anastomosis during transplantation to ascertain whether myofibroblasts infiltration predicts renal graft survival. Immunohistochemistry was performed on time-zero biopsies to determine alpha-SMA expression, and this was compared to annual glomerular filtration rate (GFR) variation and other parameters including cold ischaemic time (CIT), donor and recipient age, number of acute rejections, and delayed graft function (DGF). GFR was measured by inulin clearance during of 3 years of follow-up after the transplantation. Progressors were defined as patients with an annual GFR decline >5 ml/min/year. RESULTS: We found a significant correlation between interstitial alpha-SMA expression in time-zero biopsies and GFR evolution during the post-transplantation course (r=0.60, P<0.001). Although progressors had greater interstitial alpha-SMA expression than non progressors (7.9+/-0.7 vs 4.3+/-0.4%), they showed only a tendency towards higher glomerular alpha-SMA expression. In addition, progressors had more interstitial fibrosis in time-zero biopsies than non-progressors. There was no relationship between alpha-SMA expression and CIT, donor and recipient ages, number of acute rejections, and occurrence of DGF. CONCLUSION: This study suggests that alpha-SMA evaluation in time-zero biopsies, especially the combination of alpha-SMA expression and interstitial fibrosis, can strongly predict chronic renal allograft dysfunctions.     

Role of atrophic changes in proximal tubular cells in the peritubular deposition of type IV collagen in a rat renal ablation model.

BACKGROUND: Tubular atrophy, dilation and interstitial fibrosis are common in tubulointerstitial lesions, but the precise roles and inter-relationships of these components in the development of interstitial lesions have not been determined. This study focused on the origin and roles of atrophic tubules in the peritubular deposition of type IV collagen in a rat renal ablation model. METHODS: Male Wistar rats underwent 5/6 nephrectomy or sham operation, and then were sacrificed at 4, 8 or 12 weeks, their remaining kidneys removed for histological and immuno-histochemical studies as well as in situ hybridization for type IV collagen mRNA. RESULTS: Immuno-histochemistry demonstrated the positive staining of atrophic tubules to vimentin, platelet-derived growth factor-B chain (PDGF) and heat shock protein 47 (HSP47). Cells positive to one or more of PDGF receptor beta, alpha-smooth muscle actin (alpha-SMA), and HSP47 accumulated around atrophic tubules. Type IV collagen was also increased in the proximity of the atrophic tubules. These intimate relationships were more clearly demonstrated in 'mosaic tubules', which are composed of both intact and atrophic proximal tubular epithelial cells, and which had a mixed pattern of staining with vimentin, PDGF and HSP47. The interstitial cells positive to alpha-SMA or HSP47, or both, were in close contact with atrophic but not with intact epithelial cells. Type IV collagen was exclusively deposited between atrophic tubules and HSP47-positive interstitial cells. In situ hybridization of type IV collagen mRNA demonstrated predominant expression in atrophic tubular epithelial cells, but not in surrounding interstitial cells. CONCLUSIONS: These findings suggest that atrophic proximal tubular cells are active in the development of collagen deposition in the peritubular space, i.e. in this model type IV collagen in the interstitial fibrotic area may be produced mainly by atrophic proximal tubules.     

Epidermal growth factor receptor expression in human renal allograft biopsies: an immunohistochemical study

OBJECTIVE: Epidermal growth factor receptor (EGFR) expression has been implicated in the progression of many tumors related to cell proliferation, differentiation, invasion and inhibition of apoptosis, and EGFR-targeted therapy options are under development. EGFR expression has been identified in normal and diseased native kidneys. However, its expression in renal allografts and its relation to rejection remains unclear. We aimed to investigate EGFR expression in renal allograft biopsies. MATERIAL AND METHODS: Sections from 52 renal allograft and 11 implantation biopsies were stained by EGFR antibody with streptavidin-biotin-immunoperoxidase method. Immunostaining of EGFR in renal tubules was scored semiquantitatively and the percentage of glomeruli expressing EGFR was determined. The results were correlated with Banff scores and serum creatinine values. RESULTS: Tubular EGFR expression was observed in 81.8% of implantation and 92.3% of allograft biopsies. Glomerular EGFR expression was identified in 18.2% of implantation and in 26.9% of allograft biopsies. The mean percentage (%) of glomeruli-expressing EGFR was 3.73+/-0.84 for implantation biopsies and 7.9+/-0.17 for allograft biopsies (p = 0.53, Mann Whitney U test). For implantation biopsies, tubular EGFR expression scores were 1.90+/-2.11 and for allograft biopsies, 2.89+/-2.01 (p = 0.08, Mann Whitney U test). Tubular EGFR expression was moderately correlated with interstitial fibrosis and tubular atrophy for allograft biopsies (p = 0.04, r = 0.3 and p = 0.04, r = 0.3, respectively, Spearman's rank correlation). CONCLUSIONS: These findings suggest a possible role of EGFR expression in renal scarring in the course of chronic allograft nephropathy, probably involved in a complex pathway.     

Vascular endothelial growth factor in chronic rat allograft nephropathy

BACKGROUND: Chronic allograft nephropathy (CAN) is a complex process of alloimmune responses and chronic inflammation leading to fibrosis and vasculopathy. We examined the biological role of proinflammatory vascular endothelial growth factor (VEGF) in a rat renal transplantation model of CAN. METHODS: Syngraft and allograft recipients were treated with a suboptimal dose of cyclosporine A which allows acute rejection and CAN to develop. Intragraft VEGF, VEGFR-1 and VEGFR-2 expressions were determined at 5, 14, 30 and 60 days. Protein tyrosine kinase inhibitor PTK787 was used to inhibit VEGFR activity. RESULTS: In nontransplanted kidneys and syngrafts, mild VEGF expression was observed in the glomeruli and tubuli. VEGFR-1 was detected in vascular structures and VEGFR-2 in glomeruli as well. In allografts, total intragraft VEGF expression and interstitial inflammatory cell VEGF expression were induced and correlated with the chronic allograft damage index (CADI) score. Total intragraft and interstitial inflammatory cell VEGFR-1 expression was induced and interstitial cell VEGFR-1 expression correlated with the CADI score. Blocking VEGF receptor signaling with PTK787 significantly reduced fibrosis and the CADI score, but did not affect early inflammation or VEGF, VEGFR-1, VEGFR-2 expressions compared to vehicle treated group. CONCLUSIONS: Interstitial inflammatory cell VEGF and VEGFR-1 expressions are induced during the development of CAN. Increased VEGF activity may enhance the alloimmune induced inflammatory responses leading to fibrosis and CAN.     

Localization of HSP47 in cisplatin-treated rat kidney: a possible role in tubulointerstitial damage

Background. A 47-kDa heat-shock protein (HSP47) is a major collagen-binding stress protein and is assumed to play an important role in the fibrotic process, but its role in cisplatin-induced tubulointerstitial fibrosis is not yet clear. To explore the possible role(s) of collagen-binding stress protein HSP47 in cisplatin-induced tubulointerstitial fibrosis, the expression of HSP47 was examined in cisplatin-treated rat kidneys. Methods. Eighteen male Wistar rats were divided into two groups; group I were age-matched controls and group II, animals were injected intraperitoneally with a single dose of cisplatin (6 mg/kg body weight). One hour after cisplatin injection, three rats from group II were killed along with control rats. The remaining rats in both groups were killed on the 7th, 14th, and 28th days of the experiment and the kidneys were collected for morphological and immunohistochemical study. The expression of collagen-binding HSP47 with various proteins implicated in phenotypic modulation (α-smooth muscle actin and vimentin) and fibrosis (type I and type III collagens) was examined in control and cisplatin-treated kidneys. Results. Cisplatin induced marked tubulointerstitial damage, including interstitial fibrosis, which was characterized by increased deposition of type I and type III collagens. Increased expression of HSP47 was also noted in and around the expanded interstitium in cisplatin-treated rats; by double-staining, HSP47-expressing cells were found to be α-smooth muscle actin-positive myofibroblasts and vimentin-positive tubular epithelial cells. In addition, colocalization of HSP47 and collagens was seen in and around the interstitial fibrosis in cisplatin-treated kidneys. Conclusion. From the results, we concluded that overexpression of HSP47 by phenotypically altered renal cells may play an important role in the excessive assembly of collagens and could thereby contribute significantly to the development of the tubulointerstitial fibrosis found in cisplatin-treated rat kidneys. Received: October 5, 1998 / Accepted: March 5, 1999     

Vascular Endothelial Growth Factor Expression and Cyclosporine Toxicity in Renal Allograft Rejection

The aim of this study was to evaluate the influence of vascular endothelial growth factor (VEGF) on renal function and on development of interstitial fibrosis (IF) in renal allografts. Tubular and interstitial expressions of VEGF and TNF-α, and density of macrophages in the interstitium were examined in 92 patients with nonrejected kidneys, acute rejection (AR), chronic allograft nephropathy (CAN), borderline changes (BC) and acute cyclosporin A (CsA) toxicity. Follow-up biopsy specimens from patients with AR and BC were evaluated for development of IF. A significant difference in tubular and interstitial VEGF expressions was found between patients with AR, BC, CAN and CsA toxicity (p < 0.001). Macrophage infiltration was positively correlated with VEGF and TNF-α expressions (p < 0.001). VEGF expression increased with increasing expression of TNF-α (p < 0.001). Renal function in first 6 months after initial biopsy was better in patients with marked tubular VEGF expression (p < 0.01); however, in follow-up, development of IF and graft loss was found earlier in these patients (p < 0.01 and p < 0.05, respectively). Increased renal VEGF expression has protective properties immediately following renal allograft but allows for increased risk of early IF, and therefore poor graft outcome in the long term.     

Increased expression of p21 (WAF1/CIP1) cyclin-dependent kinase (CDK) inhibitor gene in chronic allograft nephropathy correlates with the number of acute rejection episodes

The p21 (WAF1/CIP1) cyclin-dependent kinase (CDK) inhibitor gene is considered to be the senescence marker in some recent publications. Expression of the gene was evaluated in 14 normal human kidney tissues of different ages and in nine chronically rejected renal allografts. All normal kidneys were negative for p21 expression. Glomerular, tubular and interstitial expression of the marker was detected in 88.9% ( P<0.0001) and vascular expression in 66.7% of chronically rejected grafts ( P<0.001). No correlation was found between the intensity of p21 expression and recipient age, donor age or number of human leukocyte antigen (HLA) mismatches. The marker was expressed more in grade 3 of chronic allograft nephropathy (CAN) than in grade 2 ( P=0.059 for glomerular score). Tubular expression of p21 was correlated with the number of acute rejections: P<0.05 for three vs one and two, and P=0.0046 for three vs no previous acute rejection episodes.     

Unique changes in interstitial extracellular matrix composition are associated with rejection and cyclosporine toxicity in human renal allograft biopsies

Renal allograft loss from chronic rejection or cyclosporine toxicity (CsAT) is characterized by progressive interstitial fibrosis, yet the protein composition of these lesions is unknown. The normal tubular basement membrane (TBM) contains laminin (LM), collagen IV (containing collagen IV alpha chain 1 [COL4A1] and COL4A2), thrombospondin (TSP), and fibronectin (FN). Only TSP and FN extend beyond the TBM into the interstitial space. Very scanty amounts of interstitial collagens (I and III) are detected in the interstitium. In a pilot study of human renal allograft biopsy specimens, three patterns of extracellular matrix (ECM) composition were identified. Pattern 1 showed no change in ECM composition; pattern 2 showed generalized accumulation of collagens I and III in the interstitium; and pattern 3 showed new expression of COL4A3 and LM-beta2 in the proximal TBM. Criteria were established for the clinicopathological diagnosis of CsAT and rejection. These diagnoses were correlated with the ECM composition in 22 renal allograft biopsy specimens. Control groups were examined in a similar manner and included native kidney biopsy specimens from patients with other allografts (n = 7), renal biopsy specimens from patients with glomerular disease (n = 9), and renal allograft biopsy specimens from patients without clinicopathological evidence of renal disease. These data show that rejection is associated with pattern 3 and CsAT is associated with pattern 2. Thus, detection of ECM composition may be a useful adjunct to standard microscopy in distinguishing rejection from CsAT in renal allograft biopsy specimens. These data suggest that interstitial fibrosis associated with rejection and CsAT result from different pathogenic mechanisms.     

Fibroblast-specific protein 1 is a specific prognostic marker for renal survival in patients with IgAN

BACKGROUND: There is little direct evidence that fibroblasts are involved in the progression of the renal interstitial fibrosis in human glomerulonephritis. With the availability of a new specific marker for fibroblasts, we determined the presence of fibroblasts in kidneys with IgA nephropathy (IgAN) and correlated their numbers with various clinical parameters. In particular, we also prospectively asked if the number of fibroblasts in the renal interstitium correlates with prognosis. METHODS: Cells positive for fibroblast-specific protein 1 (FSP1) were localized in renal biopsy specimens using immunohistochemistry with anti-FSP1 antibody. Clinical features were analyzed by one-way analysis of variance (ANOVA) with the Bonferroni correction. To assess the prognostic impact of the number of FSP1+ fibroblasts on renal survival in 142 patients with normal serum creatinine, the relationship between covariates to renal survival were evaluated univariately using the log-rank test and multivariately using Cox proportional hazards. RESULTS: Fibroblasts identified by their expression of FSP1 accumulate in areas showing severe interstitial fibrosis. Some tubular epithelial cells undergoing epithelial-mesenchymal transition (EMT) in fibrotic areas also express FSP1. Numbers of FSP1+ fibroblasts directly correlate with serum creatinine (r = 0.74, P < 0.0001) and inversely correlate with estimated creatinine clearance (r = -0.54, P < 0.0001), and by multivariate analysis, the clinical factors influencing renal survival are urinary protein excretion [> or = 1.0 g/day, relative risk (RR) = 4.20, P= 0.032], hypertension (RR 5.85, P = 0.0027), and > or = 20 FSP1+ fibroblasts per high power field (HPF) (RR 7.39, P = 0.0015). Staining for FSP1+ fibroblasts is largely nonoverlapping with alpha-smooth muscle actin+ (alpha-SMA) cells in the interstitium. CONCLUSION: The target protein FSP1 identifies human fibroblasts and tubular epithelium undergoing EMT, and distinguishes them from the diaspora of alpha-SMA+ vascular smooth muscle cells. FSP1+ fibroblasts are critically related to the progression of IgAN; consequently, staining FSP1 in renal biopsy specimens provides a valuable histologic index of progression.     

The role of alpha-smooth muscle actin and platelet-derived growth factor-beta receptor in the progression of renal damage in human IgA nephropathy

BACKGROUND: The degree of tubulointerstitial damage can be considered a better indicator of renal function outcome in IgA nephropathy (N) than the extent of glomerular sclerosis. MATERIALS AND METHODS: To investigate the pathogenetic mechanisms of interstitial injury in IgAN, we used immunohistochemistry and in situ hybridization to evaluate the glomerular and tubolointerstitial expression of PDGF-beta receptor (R) and alpha-smooth muscle actin (SMA), two markers of mesenchymal cell activation, and correlated these findings with the histopathologic and clinical features of the disease. We studied 155 IgAN patients, divided into three groups based on the histological findings (mild, moderate and severe histological lesions). RESULTS: In normal kidneys and in patients with mild histological lesions, the interstitial areas showed scattered peritubular cells positive for PDGF-betaR and alpha-SMA, with a distribution resembling the capillary network. In the glomeruli several cells (mainly in the mesangial area) stained for PDGF-betaR, but only very few cells were positive for alpha-SMA. Alpha-SMA and PDGF-betaR staining, as expected, was also observed in vascular smooth muscle cells. Compared to patients with mild histological lesions, alpha-SMA expression was strikingly increased in patients with moderate to severe lesions, particularly in areas of tubulointerstitial fibrosis. In these patients, PDGF-betaR gene and protein expression, at the tubulointerstitial level, paralleled that in alpha-SMA. Both signals were significantly correlated with the interstitial damage (interstitial infiltrate and fibrosis). Interestingly, these patients showed a different pattern of distribution of alpha-SMA and PDGF-betaR in the glomeruli: PDGF-betaR expression was upregulated, whereas no changes were seen in alpha-SMA staining. In addition, glomerular PDGF-betaR staining was significantly correlated with mesangial cell proliferation, while alpha-SMA was not. Image analysis showed that 40.2+/-10.3/1,000 microm2 of interstitial cells were positive to both PDGF-betaR and alpha-SMA, but only 2.8+/-1.8/1,000 microm of glomerular cells expressed both signals. CONCLUSIONS: Our study supports the hypothesis that interstitial PDGF-betaR and alpha-SMA positive cells may play a key role in the pathogenesis of tubulointerstitial damage.     

Localization of SPARC in developing,mature, and chronically injured human allograft kidneys

BACKGROUND: The matricellular protein SPARC (secreted protein acidic and rich in cysteine) is expressed during development, tissue remodeling and repair. It functions as an endogenous inhibitor of cell proliferation, regulates angiogenesis, regulates cell adhesion to extracellular matrix, binds cytokines such as platelet derived growth factor and stimulates transforming growth factor-beta (TGF-beta) production. This study describes the expression of SPARC during human renal development, in normal kidneys and during renal allograft rejection. METHODS: A total of 60 renal specimens, including normal areas from tumor nephrectomies (N = 24), fetal kidneys (N = 27) and explanted renal allografts (N = 9), were included in the study. SPARC protein was localized by immunohistochemistry using two different antibodies. On consecutive sections SPARC mRNA was detected by in situ hybridization. RESULTS: In the normal adult kidney SPARC protein was expressed by visceral and parietal epithelial cells, collecting duct epithelium (CD), urothelium, smooth muscle cells of muscular arteries and focally in interstitial cells. During renal development immature glomeruli demonstrated a polarized SPARC expression in visceral epithelial cells at their surface abutting the capillary basement membranes. In the fully differentiated glomeruli the expression pattern mirrored that of the adult kidney. Furthermore, SPARC was abundantly expressed by derivatives of the ureteric bud, and smooth muscle cells of arterial walls. During chronic allograft rejection SPARC is expressed in neointimal arterial smooth muscle cells, infiltrating inflammatory cells as well as by interstitial myofibroblasts in areas of interstitial fibrosis. SPARC mRNA synthesis detected by in situ hybridization mirrored these protein expression patterns. CONCLUSION: These studies co-localize SPARC to several sites of renal injury previously shown to be sites of PDGF B-chain expression and/or activity. We speculate that SPARC could function as an accessory molecule in chronic PDGF-mediated sclerosing interstitial and vascular injury. SPARC localization to glomerular epithelial cells corresponds to similar findings in rodents, and may reflect its role in cell adhesion and /or regulation of cell shape.     

Longitudinal analysis of chronic allograft nephropathy: clinicopathologic correlations

BACKGROUND: Loss of the allograft from chronic allograft nephropathy and death of the patient from vascular, malignant, or infective disease are the major problems in renal transplantation today. Protocol biopsy of the long-term kidney has provided new data with which to develop strategies for prevention and treatment of chronic allograft nephropathy. METHODS: Two series of long-term protocol biopsies are reviewed. In the first, renal biopsies were obtained at time 0, and at 3 months and 12 months, and the recipients of the renal allografts were followed up for up to 15 years. In the second, the kidneys of recipients of simultaneous pancreas kidney transplants were biopsied annually for 10 years, and the results correlated with clinical events. RESULTS: Chronic allograft nephropathy is caused by acute and chronic immune-mediated damage, as well as by chronic calcineurin inhibitor nephrotoxicity. Both immune and nonimmune mechanisms exacerbate pre-existing donor disease and ischemia-reperfusion injury. Established interstitial fibrosis and arteriolar hyalinosis lead to progressive glomerular sclerosis and eventual loss of the graft. CONCLUSION: Protocol biopsies have shown that clinical parameters of renal function underestimate the severity of chronic graft damage. Strategies for preventing or treating chronic renal allograft dysfunction and subsequent graft loss must better control rejection and simultaneously avoid nephrotoxicity.