Effects of substrate stiffness and actin velocity on in silico fibronectin fibril morphometry and mechanics.

Assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils is a critical step during embryonic development and wound healing; misregulation of FN fibril assembly has been implicated in many diseases, including fibrotic diseases and cancer. We have previously developed a computational model of FN fibril assembly that recapitulates the morphometry and mechanics of cell-derived FN fibrils. Here we use this model to probe two important questions: how is FN fibril formation affected by the contractile phenotype of the cell, and how is FN fibril formation affected by the stiffness of the surrounding tissue? We show that FN fibril formation depends strongly on the contractile phenotype of the cell, but only weakly on in vitro substrate stiffness, which is an analog for in vivo tissue stiffness. These results are consistent with previous experimental data and provide a better insight into conditions that promote FN fibril assembly. We have also investigated two distinct phenotypes of FN fibrils that we have previously identified; we show that the ratio of the two phenotypes depends on both substrate stiffness and contractile phenotype, with intermediate contractility and high substrate stiffness creating an optimal condition for stably stretched fibrils. Finally, we have investigated how re-stretch of a fibril affects cellular response. We probed how the contractile phenotype of the re-stretching cell affects the mechanics of the fibril; results indicate that the number of myosin motors only weakly affects the cellular response, but increasing actin velocity results in a decrease in the apparent stiffness of the fibril and a decrease in the stably-applied force to the fibril. Taken together, these results give novel insights into the combinatorial effects of substrate stiffness and cell contractility on FN fibril assembly.

Protein-crystal interface mediates cell adhesion and proangiogenic secretion.

The nanoscale materials properties of bone apatite crystals have been implicated in breast cancer bone metastasis and their interactions with extracellular matrix proteins are likely involved. In this study, we used geologic hydroxyapatite (HAP, Ca10(PO4)6(OH)2), closely related to bone apatite, to investigate how HAP surface chemistry and nano/microscale topography individually influence the crystal-protein interface, and how the altered protein deposition impacts subsequent breast cancer cell activities. We first utilized Förster resonance energy transfer (FRET) to assess the molecular conformation of fibronectin (Fn), a major extracellular matrix protein upregulated in cancer, when it adsorbed onto HAP facets. Our analysis reveals that both low surface charge density and nanoscale roughness of HAP facets individually contributed to molecular unfolding of Fn. We next quantified cell adhesion and secretion on Fn-coated HAP facets using MDA-MB-231 breast cancer cells. Our data show elevated proangiogenic and proinflammatory secretions associated with more unfolded Fn adsorbed onto nano-rough HAP facets with low surface charge density. These findings not only deconvolute the roles of crystal surface chemistry and topography in interfacial protein deposition but also enhance our knowledge of protein-mediated breast cancer cell interactions with apatite, which may be implicated in tumor growth and bone metastasis.

Fibronectin remodelling: cell-mediated regulation of the microenvironment.

The biophysical, mechanical and chemical characteristics of extracellular matrixes influence many cellular functions to control tissue homoeostasis and drive progression of cancer and inflammatory diseases. To maintain normal tissue function, fibronectin-rich matrixes are subject to dynamic cell-mediated structural and chemical modification. In this article, we discuss how localized application of mechanical force, heterodimer-specific integrin engagement and matrix proteolysis regulate fibronectin assembly and turnover. We also speculate that recently identified integrin trafficking, syndecan signalling and adhesion receptor-growth factor receptor cross-talk mechanisms might dynamically control the function, assembly and mechanical properties of a viable, and mechanoresponsive, fibronectin network.

Ultrastructurally confirmed myofibrosarcoma: a series of 10 new cases, with a discussion on diagnostic criteria.

Some view ultrastructure as key to myofibrosarcoma diagnosis, whereas others argue that electron microscopy is too little used in contemporary practice to be considered an important diagnostic tool. These views are discussed in the context of 10 ultrastructurally confirmed cases of myofibrosarcoma, some occurring at rare sites such as skin and penis. Patient age ranged from 21 to 83 years, with a 6:4 male to female ratio. Size ranged from 2 to 7.5 cm and all had infiltrative margins. Histologically, all consisted of variably cellular fascicles of spindle cells with mild to moderately pleomorphic nuclei, small punctate nucleoli, and eosinophilic cytoplasm. All cases showed α-smooth muscle actin positivity and 2 showed very focal weak positivity for desmin. Ultrastructurally, the tumor cells contained rough endoplasmic reticulum, mainly peripheral smooth-muscle myofilaments, and fibronectin fibrils or fibronexus junctions at the cell surface. The most confident diagnosis of myofibrosarcoma is provided by ultrastructural examination. However, given the right histological appearance, use of a panel of antibodies that includes α-smooth muscle actin, desmin, and h-caldesmon, serves as an acceptable practical way of diagnosing myofibrosarcoma.

Multiscale grooved titanium processed with femtosecond laser influences mesenchymal stem cell morphology, adhesion, and matrix organization.

The femtosecond laser processing enabled the structuring of six types of surfaces on titanium-6aluminium-4vanadium (Ti-6Al-4V) plates. The obtained hierarchical features consisted of a combination of microgrooves and oriented nanostructures. By adjusting beam properties such as laser polarization, the width of the microgrooves (20 or 60 µm) and the orientation of the nanostructures (parallel or perpendicular to the microgrooves) can be precisely controlled. Mesenchymal stem cells (MSCs) grown on these structured surfaces produced cytoplasmic extensions with focal contacts, while on the smooth titanium, the cells were found to be well spread and without any focal contact 12 h postseeding. The 600-nm wide nanostructures on their own were sufficient to orient the MSCs. For the multiscale structured areas, when the orientation of the nanostructures was orthogonal in relation to the microgrooves, there was an important decrease in or even a loss of cell alignment signifying that cells were sensitive to the directional nanostructures in the microgrooves. At 7 days, cell proliferation was not affected but the direction of nanostructures controlled the matrix organization. The ultrafast laser, as a new method for producing micro-nanohybrid surfaces, is a promising approach to promote desired tissue organization for tissue engineering.

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells.

BACKGROUND: Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. METHODS: We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. RESULTS: We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is ß1-integrin/FAK mediated. CONCLUSION: Cancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and ß1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking ß1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-induced effects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

Adipose progenitor cells increase fibronectin matrix strain and unfolding in breast tumors.

Increased stiffness represents a hallmark of breast cancer that has been attributed to the altered physicochemical properties of the extracellular matrix (ECM). However, the role of fibronectin (Fn) in modulating the composition and mechanical properties of the tumor-associated ECM remains unclear. We have utilized a combination of biochemical and physical science tools to evaluate whether paracrine signaling between breast cancer cells and adipose progenitor cells regulates Fn matrix assembly and stiffness enhancement in the tumor stroma. In particular, we utilized fluorescence resonance energy transfer imaging to map the molecular conformation and stiffness of Fn that has been assembled by 3T3-L1 preadipocytes in response to conditioned media from MDA-MB231 breast cancer cells. Our results reveal that soluble factors secreted by tumor cells promote Fn expression, unfolding, and stiffening by adipose progenitor cells and that transforming growth factor-ß serves as a soluble cue underlying these changes. In vivo experiments using orthotopic co-transplantation of primary human adipose-derived stem cells and MDA-MB231 into SCID mice support the pathological relevance of our results. Insights gained by these studies advance our understanding of the role of Fn in mammary tumorigenesis and may ultimately lead to improved anti-cancer therapies.

A fibrillar form of fibronectin induces apoptosis by activating SHP-2 and stress fiber formation.

Fibronectin (FN) is an endogenous ligand of integrins, which plays a critical role in cell adhesion and growth. Here, we converted globular FN (G-FN) into a fibrillar form (F-FN) and found that, even though both G-FN and F-FN interacted with integrin alpha5beta1, G-FN induced cellular proliferation, whereas F-FN resulted in apoptosis that was associated with deactivation of Akt/GSK-3beta and phosphorylation of SHP-2. SHP-2 inhibitor and anti-sense oligodeoxynucleotide decreased SHP-2 level and reversed the F-FN mediated apoptosis. F-FN also induced stress fiber formation associated with activation of RhoA, Rho kinase (ROCK), and filamin. Inhibition of ROCK by ROCK inhibitor or dominant negative plasmid treatment modulated F-FN mediated apoptosis. Pharmacological studies revealed that F-FN was effective in inhibiting the survival of SKOV-3 and MCF-7 cancer cells. These findings thus demonstrate that unlike G-FN, F-FN exhibits fibrillar structure to induce cell apoptosis that is associated with phosphorylation of SHP-2, activation of RhoA/ROCK and formation of stress fibers as well as deactivation of Akt/GSK-3beta.

Atomic force microscopy evidence for conformational changes of fibronectin adsorbed on unmodified and sulfonated polystyrene surfaces.

The effect of polystyrene surface polarity on the conformation of adsorbed fibronectin (FN) has been studied with atomic force microscopy. We demonstrated that bare sulfonated and nonsulfonated polystyrene surfaces featured similar topographies. After the FN adsorption, direct comparison of both types of substrata revealed drastically different topographies, roughness values, and also cell-adhesive properties. This was interpreted in terms of FN conformational changes induced by the surface polarity. At high-solute FN concentrations the multilayer FN adsorption took place resulting, for the sulfonated substratum, in an increase of surface roughness, whereas for the nonsulfonated one the roughness was approximately stable. Conversely, the FN conformation characteristic for the first saturative layer tended to be conserved in the consecutive layers, as evidenced by height histograms. The height of individual FN molecules indicated, consonantly with the derived thickness of the adsorbed protein layer (the latter value being 1.4 nm and 0.6 nm, respectively, for an unmodified and sulfonated polystyrene surface), that molecules are flattened on polar surfaces and more compact on nonsulfonated ones. It was also demonstrated that the FN adsorption and conformation on polymeric substrata, and hence the resultant cell-adhesive properties, depended on the chemistry of the original surface rather than on its topography. Our results also demonstrated the ability of surface polarity to influence the protein conformation and its associated biological activity.

Epithelioid sarcoma: a case report with ultrastructural confirmation of myofibroblastic differentiation based on fibronexus junctions.

Epithelioid sarcoma is an uncommon but well-described malignancy, which is found predominantly in the soft tissues of the young and middle-aged, and which pursues an indolent to aggressive course. It shows a degree of both mesenchymal and epithelial differentiation. Myofibroblastic differentiation has been recorded in epithelioid sarcoma for some time, the evidence being based mainly on the presence of smooth-muscle-type myofilaments and, more recently, on alpha-smooth-muscle actin and muscle-specific actin immunostaining. Myofibroblastic differentiation based on the stricter criterion of the fibronectin fibril/fibronexus junction has not so far been demonstrated except for a single atypical case with spindle-cell morphology and a cytokeratin-negative immunophenotype. The authors describe a conventional epithelioid sarcoma showing myofibroblastic differentiation based on the presence of fibronectin fibrils and fibronexus junctions. The patient was a 35-year-old Chinese male with a tumor that initially developed on his left forefinger: it recurred, then metastasised first to the left arm and eventually to the scalp. Histologically, the tumors had the typical features of epithelioid sarcoma: by immunohistochemistry, cytokeratin, epithelial membrane antigen, and vimentin were positive. By electron microscopy, rough endoplasmic reticulum, intermediate filaments, peripheral myofilaments, and fibronexus junctions were observed. This is the first epithelioid sarcoma of conventional histological type to show myofibroblastic differentiation on the basis of the more stringent criterion of the fibronexus. The findings are discussed in relation to the unusually varied differentiation reported for this tumor.

Fibronexus in low-grade myofibrosarcoma: a case report.

Fibronexus (fibronexus junction) has been thought to be a characteristic ultrastructural feature of myofibroblasts, but it is controversial as to whether fibronexus is a characteristic of various myofibroblastic tumors. We report here a case of low-grade myofibrosarcoma with fibronexus arising in the right arm of an 80-year-old man. Histologically, the tumor was composed of relatively uniform and slender spindle cells arranged in fascicles. The nuclei with fusiform and tapered shapes were mildly hyperchromatic, but never exhibited pleomorphism. Mitotic figures were common, but no atypical mitosis was identified. At the tumor periphery, tumor cells had invaded into the surrounding skeletal muscle tissue. Tumor cells were positive diffusely for alpha-smooth muscle actin and less intensely for desmin, but were negative for h-caldesmon and S-100 protein. Ultrastructurally, tumor cells had well developed cytoplasmic organelles and varying amounts of peripheral or subplasmalemmal bundles of thin myofilaments with focal density. In addition, well formed, long fibronectin fibrils adjacent to the cell surface and fibronexus contacting intracellular myofilaments were easily identified. We believe that fibronexus is a useful ultrastructural feature for differentiating myofibrosarcoma from other myogenic sarcomas.

Structure and reactivity of adsorbed fibronectin films on mica.

Understanding the interactions of adsorbed fibronectin (Fn) with other biomolecules is important for many biomedical applications. Fn is found in almost all body fluids, in the extracellular matrix, and plays a fundamental role in many biological processes. This study found that the structure (conformation, orientation) and reactivity of Fn adsorbed onto mica is dependent on the Fn surface concentration. Atomic force microscopy and x-ray photoelectron spectroscopy were used to determine the surface coverage of adsorbed Fn from isolated molecules at low surface coverage to full monolayers at high surface coverage. Both methods showed that the thickness of Fn film continued to increase after the mica surface was completely covered, consistent with Fn adsorbed in a more upright conformation at the highest surface-Fn concentrations. Time-of-flight secondary ion mass spectrometry showed that relative intensities of both sulfur-containing (cystine, methionine) and hydrophobic (glycine, leucine/isoleucine) amino acids varied with changing Fn surface coverage, indicating that the conformation of adsorbed Fn depended on surface coverage. Single-molecule force spectroscopy with collagen-related peptides immobilized onto the atomic force microscope tip showed that the specific interaction force between the peptide and Fn increases with increasing Fn surface coverage.

Effects of sterilization on an extracellular matrix scaffold: part I. Composition and matrix architecture.

The impact of peracetic acid (PAA), lyophilization, and ethylene oxide (EO) sterilization on the composition and three dimensional matrix structure of small intestinal submucosa (SIS), a biologic scaffold used to stimulate the repair of damaged tissues and organs, was examined. Fibronectin and glycosaminoglycans are retained in SIS following oxidation by peracetic acid and alkylation using ethylene oxide gas. Significant amounts of FGF-2 are also retained, but VEGF is susceptible to the effects of PAA and is dramatically reduced following processing. Further, matrix oxidation, lyophilization, and sterilization with EO can be performed without irreversibly collapsing the three dimensional structure of the native SIS. These structural features and growth promoting extracellular matrix constituents are likely to be important variables underlying cellular attachment, infiltration and eventual incorporation of SIS into healing host tissues.

Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity: impact of vascular endothelial growth factor.

Endothelial cells play a crucial role in the pathogenesis of many diseases and are highly sensitive to low gravity conditions. Using a three-dimensional random positioning machine (clinostat) we investigated effects of simulated weightlessness on the human EA.hy926 cell line (4, 12, 24, 48 and 72 h) and addressed the impact of exposure to VEGF (10 ng/ml). Simulated microgravity resulted in an increase in extracellular matrix proteins (ECMP) and altered cytoskeletal components such as microtubules (alpha-tubulin) and intermediate filaments (cytokeratin). Within the initial 4 h, both simulated microgravity and VEGF, alone, enhanced the expression of ECMP (collagen type I, fibronectin, osteopontin, laminin) and flk-1 protein. Synergistic effects between microgravity and VEGF were not seen. After 12 h, microgravity further enhanced all proteins mentioned above. Moreover, clinorotated endothelial cells showed morphological and biochemical signs of apoptosis after 4 h, which were further increased after 72 h. VEGF significantly attenuated apoptosis as demonstrated by DAPI staining, TUNEL flow cytometry and electron microscopy. Caspase-3, Bax, Fas, and 85-kDa apoptosis-related cleavage fragments were clearly reduced by VEGF. After 72 h, most surviving endothelial cells had assembled to three-dimensional tubular structures. Simulated weightlessness induced apoptosis and increased the amount of ECMP. VEGF develops a cell-protective influence on endothelial cells exposed to simulated microgravity.

Rhizopus oryzae fungus cells producing L(+)-lactic acid: kinetic and metabolic parameters of free and PVA-cryogel-entrapped mycelium.

Spores of the filamentous fungus Rhizopus oryzae were entrapped in macroporous poly(vinyl alcohol) cryogel (PVA-cryogel). To prepare immobilised biocatalyst capable of producing L(+)-lactic acid (LA), the fungus cells were cultivated inside the carrier beads. The growth parameters and metabolic activity of the suspended (free) and immobilised cells producing LA in a batch process were comparatively investigated. The immobilised cells possessed increased resistance to high concentrations of accumulated product and gave much higher yields of LA in the iterative working cycle than the free cells did. Detailed kinetic analysis of the changes in the intracellular adenosine triphosphate concentration, specific rate of growth, substrate consumption and LA production showed that the fungus cells entrapped in PVA-cryogel are more attractive for biotechnological applications compared to the free cells.

Monitoring mesenchymal stromal cell developmental stage to apply on-time mechanical stimulation for ligament tissue engineering.

To evaluate the appropriate time frame for applying mechanical stimuli to induce mesenchymal stromal cell (MSC) differentiation for ligament tissue engineering, developmental cell phenotypes were monitored during a period of in vitro culture. MSCs were seeded onto surface-modified silk fibroin fiber matrices and cultured in Petri dishes for 15 days. Cell metabolic activity, morphology, and gene expression of extracellular matrix (ECM) proteins (collagen type I and III and fibronectin), ECM receptors (integrins alpha-2, alpha-5, and beta-1), and heat-shock protein 70 (HSP-70) were monitored during the culture of MSC. MSCs showed fluctuations in cell metabolic activity, ECM, integrin, and HSP-70 transcription potentially correlating to innate developmental processes. Cellular response to mechanical stimulation was dependent on the stage of cell development. At day 9, when levels of cell metabolic activity, ECM, integrin, and HSP-70 transcription peaked, mechanical stimulation increased MSC metabolic activity, alignment, and collagen production. Mechanical stimulation applied at day 1 and 3 showed detrimental effects on MSCs seeded on silk matrices. The results presented in this study identify a unique correlation between innate MSC development processes on a surface-modified silk matrix and dynamic environmental signaling.

Expression of alpha5beta1 integrin and fibronectin during early pregnancy in pigs.

The implantation in pig is superficial and non-invasive, involving phases of apposition, adhesion and attachment of conceptuses to endometrial surface epithelium. The role of integrins and ECM proteins is suggested. In the study, the expression of beta5beta1 integrin and FN on conceptus trophectoderm and endometrium during implantation and early pregnancy was investigated. The immunohistochemical localization of alpha5beta1 integrin and FN was performed on formalin-fixed, paraffin-embedded tissue sections using the ABC method. The results indicate that both conceptus and uterus expressed alpha5beta1 integrin and FN during early porcine pregnancy. The most intensive staining for alpha5beta1 integrin and FN was found in conceptus trophectoderm and endometrial surface epithelium in all investigated periods. During placentation the immunohistochemical staining for both alpha5beta1 integrin and FN was increased in trophectoderm and all endometrial structures. Since placenta in pigs is non-invasive, it can be suggested that both alpha5beta1 integrin and FN participate in molecular events leading to successful implantation and placentation in species with true epitheliochorial placenta.

Chronic renal failure in male and female rats.

Gender influences the progression of chronic renal failure (CRF). We studied male (M) and female (F) Wistar rats for 90 days: castrated (CMc,n=7;CFc,n=6) and non castrated controls (CM,n=9;CF,n=6); castrated (CRFMc,n=8; CRFFc,n=6) and non castrated animals submitted to 5/6 nephrectomy (CRFM,n=13;CRFF,n=6). Data are expressed as mean +/-SEM. Proteinuria (PTN) was higher in CRFM (554+/-69 mg/24h) compared to CRFMc (277+/-85 mg/24h), but not in females (CRFF=193+/-20mg/24h, CRFFc= 164+/-71 mg/24h). Mesangial fractional volume increased in all CRF animals. CRF animals showed an increase of glomerular sclerosis index (GSI) and tubulointerstitial damage (TID) but in a smaller proportion in male castrated animals; the opposite occurred with females: castration induced an increase of these parameters. CRF animals showed increased cortical and glomerular fibronectin (FN) rates. Castration decreased glomerular and cortical FN rates in CRFM but not in females. In conclusion, proteinuria was higher in CRFM and probably led to glomerular and interstitial damage, as well as to FN accumulation, castration seems to protect against development of PTN, TID and FN accumulation in males. Castrated female rats presented mesangial expansion, with no changes in PTN, TID and FN rates. It seems that female sex hormones do not protect against renal disease progression, instead, we suggest that male sex hormones lead to acceleration of CRF.