Sotiris Tsakas

Transforming Growth Factor-β1 and Myofibroblasts: A Potential Pathway towards Renal Scarring in Human Glomerular Disease

Background/Aims: The cellular and humoral factors involved in the development and progression of renal scarring have not been fully investigated. Transforming growth factor-β (TGF-β1) is considered to be the main fibrogenic growth factor and it is implicated in the pathogenesis of renal fibrosis in experimental and clinical nephropathies. On the other hand, collagen III is an important component of the extracellular matrix. In this study we attempted to identify any possible links between TGF-β1 and collagen III synthesis and expression with the expression of myofibroblasts in the evolution of renal scarring in human glomerular diseases. Methods: We studied retrospectively 40 patients with various types of primary and secondary glomerulonephritis (GN), with either proliferative or nonproliferative pattern, with emphasis on the renal synthesis of TGF-β1 and collagen III (detected by in situ hybridization) and their expression (detected by immunohistochemistry) as well as myofibroblast expression. The possible links of TGF-β1 expression with myofibroblast distribution (α-smooth muscle actin, α-SMA(+) cells) and with conventional histopathology and renal function was also examined. Results: TGF-β1 protein and mRNA were detected in the renal tubular epithelial cells and interstitium and to a lesser extent within glomeruli of patients with GN. Collagen III was mainly detected in the interstitium (peritubular and periglomerular areas) and to a lesser extent in the glomeruli. Messenger RNA for collagen III followed a similar peritubular and periglomerular distribution to that of TGF-β1 and α-SMA(+) interstitial cells. The intensity of interstitial TGF-β1 protein expression was significantly related to the degree of interstitial fibrosis (r = 0.628, p < 0.01), tubular atrophy (r = 0.612, p < 0.01), interstitial collagen III expression (r = 0.478, p < 0.05), and serum creatinine values (r = 0.722, p < 0.001). Also there was a close positive correlation between the severity of interstitial myofibroblast expression and interstitial TGF-β1 (r = 0.412, p < 0.05), as well as collagen III (r = 0.409, p < 0.05). In addition, a significant correlation was found between glomerular TGF-β1 expression and severity of glomerulosclerosis (r = 0.620, p < 0.01). Conclusion: The results of this study suggest that TGF-β1 plays an important role in the pathogenesis of fibrosis developing in human kidney, during the evolution of glomerular disease. Interstitial myofibroblasts may contribute to interstitial fibrosis through the synthesis and release of both TGF-β1 and collagen III.

Innate immunity in insects: surface-associated dopa decarboxylase-dependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytes

Phagocytosis, melanization and nodulation in insects depend on phenoloxidase (PO) activity. In this report, we demonstrated that these three processes appear to be also dependent on dopa decarboxylase (Ddc) activity. Using flow cytometry, RNA interference, immunoprecipitation and immunofluorescence, we demonstrated the constitutive expression of Ddc and its strong association with the haemocyte surface, in the medfly Ceratitis capitata. In addition, we showed that Escherichia coli phagocytosis is markedly blocked by small interfering RNA (siRNA) for Ddc, antibodies against Ddc, as well as by inhibitors of Ddc activity, namely carbidopa and benzerazide, convincingly revealing the involvement of Ddc activity in phagocytosis. By contrast, latex beads and lipopolysaccharide (LPS) did not require Ddc activity for their uptake. It was also shown that nodulation and melanization processes depend on Ddc activation, because antibodies against Ddc and inhibitors of Ddc activity prevent haemocyte aggregation and melanization in the presence of excess E. coli. Therefore, phagocytosis, melanization and nodulation depend on haemocyte‐surface‐associated PO and Ddc. These three unrelated mechanisms are based on tyrosine metabolism and share a number of substrates and enzymes; however, they appear to be distinct. Phagocytosis and nodulation depend on dopamine‐derived metabolite(s), not including the eumelanin pathway, whereas melanization depends exclusively on the eumelanin pathway. It must also be underlined that melanization is not a prerequisite for phagocytosis or nodulation. To our knowledge, the involvement of Ddc, as well as dopa and its metabolites, are novel aspects in the phagocytosis of medfly haemocytes.

Accurate Measurement and Clinical Significance of Urinary Transforming Growth Factor-Beta1

Transforming growth factorβ1 (TGF-β1) is the main modulator of the healing process after tissue injury. In the kidney, if TGF-β1 release is not switched off, extracellular matrix components (ECM) are accumulated and tissue fibrosis occurs. Urinary TGF-β1 levels reflect its renal production and it has been determined in various types of glomerular disease. In this review, a critical analysis of the different immunoassays that have been used for the measurement of TGF-β1 in the urine is presented and the importance of the serial determination of urinary TGF-β1 levels in patients with various types of renal disease is discussed.

Apoptosis and Myofibroblast Expression in Human Glomerular Disease: A Possible Link with Transforming Growth Factor-Beta-1

Background/Aims: The pathophysiological pathways involved in the pathogenesis and evolution of renal fibrosis, have not been fully elucidated. Transforming growth factor-beta1 (TGF-β1) is involved in the development of renal scarring. Apoptosis is responsible for intrinsic cell deletion observed in end-stage kidney disease. Myofibroblasts are involved in the development of renal fibrosis. This study investigates whether there is a potential relationship between apoptosis, myofibroblast infiltration and TGF-β1 expression in the kidney of patients with glomerulonephritis (GN). Methods: Forty patients with various types of GN were included in the study. Myofibroblasts and TGF-β1 positive cells were detected in kidney biopsies by immunohistochemistry, while apoptotic cells were detected by the in situ end labelling of fragmented DNA. Results: Myofibroblasts were identified in the glomeruli of some patients with severe mesangioproliferative GN and glomerulosclerosis but a more intensive myofibroblast expression was found in the renal interstitium. TGF-β1 was expressed in the cytoplasm of tubular epithelial cells, in the renal interstitium and in the glomeruli of patients with GN. Apoptotic cells were mainly detected in the tubules and interstitium and were present in areas with intense myofibroblast infiltration. Positive correlations were observed between the intensity of myofibroblast expression in the interstitium and apoptosis in the tubulointerstitial area (r = 0.521, p < 0.01) as well as TGF-β1 expression (r = 0.462, p < 0.05) and degree of renal impairment (r = 0.430, p < 0.05). Conclusions: These observations suggest that myofibroblast infiltration and apoptosis along with TGF-β1 expression are associated with the development of interstitial fibrosis in patients with glomerular disease.

Temporally regulated protein synthesis in cultured haemocytes of the Mediterranean fruit fly Ceratitis capitata during larval and prepupal development: Internalization of larval serum proteins into the haemocytes

Detailed analysis of haemocyte proteins by one-dimensional polyacrylamide gel electrophoresis during the late larval stages and white pupae of the Mediterranean fruit fly Ceratitis capitata reveals more than 50 polypeptides. A numbering system is established based on molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detailed examination leads to the conclusion that each protein resolved by our methods has its own characteristic kinetics of accumulation. The most abundant protein is unequivocally the 36-kDa band with an isoelectric point at 5.07, followed by the 47- and 54-kDa proteins. The 36-kDa band constitutes approximately 9% of total haemocyte protein. Comparison of the haemocyte protein pattern to that of cell-free haemolymph clearly shows that the protein bands at 81, 82, 85, and 87 kDa are common in both sources. We demonstrated by immunoblotting and pulse-labeled experiments followed by 2D SDS-PAGE that these protein bands which constitute the larval serum proteins were internalized into the haemocytes from the serum. By pulse labeling cells with [35S]methionine, separating polypeptides by one-dimensional PAGE electrophoresis, and comparing the rates of synthesis of over 50 individual polypeptides by fluorography, the following results were obtained: Three protein groups with distinct patterns of synthesis were noted: (1) proteins that are synthesized throughout the developmental period studied, (2) proteins whose synthesis begins at the wandering stage, and (3) proteins that are synthesized only during the feeding period. The 47-kDa protein shows the highest relative rate of synthesis, but since it is not the most accumulated band it must have a high turnover rate. Most of the labeled proteins were secreted into the incubation medium. The intracellular transit time was estimated to be about 11 min. Patterns of protein synthesis in haemocytes are regulated in a precise temporal sequence during the transformation of larvae to pupae. Their study yields a useful system for the analysis of molecular events in gene control.

Urinary Transforming Growth Factor-beta 1 as a marker of response to immunosuppressive treatment, in patients with crescentic nephritis

BackgroundCrescentic nephritis is characterized by formation of cellular crescents that soon become fibrotic and result in irreversible damage, unless an effective immunosuppressive therapy is rapidly commenced. TGF-β1 is involved in the development of crescents through various pathways. The aim of this study was to identify whether the determination of urinary TGF-β1 levels in patients with crescentic nephritis could be used as a marker of response to treatment.MethodsFifteen patients with crescentic nephritis were included in the study. The renal expression of TGF-β1 was estimated in biopsy sections by immunohistochemistry and urinary TGF-β1 levels were determined by quantitative sandwich enzyme immunoassay (EIA). TGF-β1 levels were determined at the time of renal biopsy, before the initiation of immunosuppressive treatment (corticosteroids, cyclophosphamide and plasma exchange). Twelve patients with other types of proliferative glomerulonephritis and ten healthy subjects were used as controls.ResultsImprovement of renal function with immunosuppressive therapy was observed in 6 and stabilization in 4 patients (serum creatinine from 3.2 ± 1.5 to 1.4 ± 0.1 mg/dl and from 4.4 ± 1.2 to 4.1 ± 0.6 mg/dl, respectively). In 5 patients, with severe impairment of renal function who started on dialysis, no improvement was noted. The main histological feature differentiating these 5 patients from others with improved or stabilized renal function was the percentage patients with poor response to treatment were the percentage of glomeruli with crescents and the presence of ruptured Bowmans capsule and glomerular necrosis. Urinary TGF-β1 levels were significantly higher in patients who showed no improvement of renal function with immunosuppressive therapy (930 ± 126 ng/24 h vs. 376 ± 84 ng/24 h, p < 0.01). TGF-β1 was identified in crescents and tubular epithelial cells, whereas a significant correlation of TGF-β1 immunostaining with the presence of fibrocellular cresents was observed (r = 0.531, p < 0,05).ConclusionIncreased TGF-β1 renal expression and urinary excretion that is related to the response to immunosuppressive therapy was observed in patients with crescentic nephritis. Evaluation of urinary TGF-β1 levels may be proved a useful marker of clinical outcome in patients with crescentic nephritis.

Detection of haemocyte proteins in the integument of the developing Mediterranean fruit flyCeratitis capitata

SummaryStudies of the synthesis of integumental proteins during the feeding and non-feeding stages ofCeratitis capitata demonstrated stage specificity. The synthetic profile changed dramatically, showing a maximum of protein synthesis just before the larval wandering stage, followed by an abrupt decline. The comparison between synthetic and accumulation profiles indicated that some polypeptides must be internalized into the integument from the haemolymph. The major haemolymph proteins or arylphorins have already been documented to be incorporated into the integument. In the present work, we demonstrated the interalization of some haemocyte proteins into the integument. For that purpose, polyclonal antibodies were raised against total haemocyte proteins. Immunoblot analysis of haemocyte salt extractable proteins revealed that the protein bands at 36, 54, 58, 84, 110 and 130 kDa were immunoreactive with the total haemocyte antibodies. Cell-free protein synthesis, organ culture experiments and immunoblot analysis indicated that the 36-, 54- and 58-kDa polypeptides were synthesized only in the haemocytes and were probably internalized into the integument from the serum. The 36-kDa polypeptide was also demonstrated to be internalized into the fat body of white puparia. The immunofluorescence experiments suggested that the internalization of haemocyte proteins first occurs into the epidermal cells and then into the cuticle. The presence of haemocyte proteins in the integument was also demonstrated by immunofluorescence experiments in twoC. capitata mutants. These mutations affect the darkening and stiffening of the cuticle. The demonstration of 36-, 54- and 58-kDa haemocyte polypeptides in the integument reveals a hitherto unknown function of this cell type. Moreover, the demonstration of tyrosine binding to the 54- and 58-kDa polypeptides points to their potential involvement in the sclerotization process in the cuticle.

Endothelin Receptors in the Kidney of Patients with Proteinuric and Non-Proteinuric Nephropathies

Background. Endothelin-1 (ET-1), which acts via the specific receptors ET-A and ET-B, has been implicated in the development of renal scarring. The activation of the endothelin system was observed in experimental models of glomerular diseases and was attributed to the toxic action of proteinuria on the tubular epithelial cells. The aim of this study was to investigate whether the endothelin system in the kidney is altered in glomerular diseases and possibly related to proteinuria. Methods. Thirty-seven patients with different types of glomerulonephritis and 14 controls were included. Patients presented either nephrotic syndrome (n=25) or mild proteinuria (<1g/24h, n=12). The expression of ET-A and ET-B receptors in the renal tissue was examined immunohistochemically. At the time of biopsy, urinary ET-1 was determined. Results. Both receptors were mainly localized within tubular epithelial cells, and their expression was significantly higher in patients with glomerulonephritis compared to controls. The expression of ET-B was higher in nephrotic compared to non-nephrotic patients, while no difference was observed in the expression of ET-A receptors. A significant positive correlation of the degree of proteinuria with the excreted ET-1 (r= 0.487, p<0.05) and the extent of immunostaining for ET-B receptors (r=0.420, p<0.05) was observed. The expression of ET-B receptors and the excretion of ET-1 decreased significantly in patients with remission of nephrotic syndrome after therapy. Conclusion. This study provides evidence that the endothelin system is activated in human glomerular disease, confirming data from experimental studies. Proteinuria seems to be related to the activation of endothelin system, though further investigation is necessary to clarify this issue.

Expression of Apoptosis‐Related Proteins Bcl‐2 and Bax Along with Transforming Growth Factor (TGF‐β1) in the Kidney of Patients with Glomerulonephritides

Background. Apoptosis, a gene‐directed form of cell death, has been involved in the resolution of renal injury but also in the development of scarring. Bcl‐2 and bax are proteins related to apoptotic process that either provides a survival advantage to rapidly proliferating cells (bcl‐2) or promote cell death by apoptosis (bax). Various cytokines and growth factors are involved in this process. This study investigates the expression of bcl‐2 and bax and the presence of apoptotic bodies in relation to the TGF‐β1 expression at the time of diagnosis in the renal biopsies of patients with glomerulonephritis (GN). Methods. Fifty patients with various types of GN and ten controls were included in the study. Bcl‐2, bax and Transforming Growth Factor (TGF‐β1) positive cells were detected in kidney biopsies by immunohistochemistry, while apoptotic cells were detected by in situ end labeling of fragmented DNA (ISEL). Morphometric analysis was used for quantitation of immunostaining. Results. The intensity of bcl‐2, bax and TGF‐β1 immunostaining in the renal tissue of patients with GN was significantly more to the observed in the control biopsies. Bcl‐2 and bax were expressed within the epithelial cells of proximal, distal and collecting tubules and in the renal interstitium. Bax and bcl‐2 proteins were also identified within the glomeruli in a few patients but their distribution was not related to the type of GN. TGF‐β1 was expressed in the cytoplasm of tubular epithelial cells and to a lesser extent in the renal interstitium and glomeruli. A positive correlation of TGF‐β1 with the extent of bax immunostaining (r = 0.498, p < 0.05) and an inverse correlation with that of bcl‐2 (r = − 0.490, p < 0.05) were identified. Apoptotic bodies were identified only in the renal tissue of patients with GN and were mainly localized among tubular epithelial and interstitial cells. Conclusion. The intensity of bcl‐2 and bax proteins expression and the presence of apoptotic bodies in the renal tissue of patients with GN suggest that apoptotic process is ongoing during the evolution of renal disease. The correlation of TGF‐β1 expression with that of apoptosis‐related proteins might represent an implication of this growth factor with apoptotic process in the human diseased kidney.

Incorporation of arylphorins (LSP-1) and LSP-2 like protein into the integument of Ceratitis capitata during pupariation

The arylphorins and LSP-2 like polypeptide were detected by immunoblotting analysis during development in the integument of C. capitata. In vitro translation of total RNA from fat body and integument during pupariation, clearly revealed that the polypeptides under consideration were exclusively synthesized in the fat body. Furthermore, in vitro experiments demonstrated that radiolabeled arylphorins and LSP-2 like polypeptide were taken up by the integument, in an undegraded state. Immunofluorescence experiments in cross sections of wandering stage larvae and white pupae revealed that the LSP-2 like polypeptide was mainly localized in the epidermal cells, and a very weak signal was also given by the cuticle. Furthermore, the presented results indicated that a small portion of the extracted proteins exist in high molecular weight aggregate(s).