Wayne A. Border

Transforming Growth Factor β in Tissue Fibrosis

Progressive fibrosis in the kidney, liver, lung, heart, bone marrow, and skin is both a major cause of suffering and death and an important contributor to the cost of health care. All of this is li...

Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerular mesangial cells.

Angiotensin II (Ang II) has been implicated in the development of progressive glomerulosclerosis, but the precise mechanism of this effect remains unclear. In an experimental model, we have shown previously that TGF-beta plays a key role in glomerulosclerosis by stimulating extracellular matrix protein synthesis, increasing matrix protein receptors, and altering protease/protease-inhibitor balance, thereby inhibiting matrix degradation. We hypothesized that Ang II contributes to glomerulosclerosis through induction of TGF-beta. Ang II treatment of rat mesangial cells in culture increased TGF-beta and matrix components biglycan, fibronectin, and collagen type I at both the mRNA and protein levels in a time- and dose-dependent manner. Saralasin, a competitive inhibitor of Ang II, prevented the stimulation. Ang II also promoted conversion of latent TGF-beta to the biologically active form. Coincubation of mesangial cells with Ang II and neutralizing antibody to TGF-beta blocked the Ang II-induced increases in matrix protein expression. Continuous in vivo administration of Ang II to normal rats for 7 d resulted in 70% increases in glomerular mRNA for both TGF-beta and collagen type I. These results indicate that Ang II induces mesangial cell synthesis of matrix proteins and show that these effects are mediated by Ang II induction of TGF-beta expression. This mechanism may well contribute to glomerulosclerosis in vivo.

Elevated expression of transforming growth factor-beta and proteoglycan production in experimental glomerulonephritis. Possible role in expansion of the mesangial extracellular matrix.

Glomerular accumulation of extracellular matrix is a prominent feature of progressive glomerulonephritis. Previously, we have shown that transforming growth factor-beta (TGF-beta) is unique among growth factors in regulating the production of the proteoglycans biglycan and decorin by glomerular mesangial cells in vitro. We now provide evidence of an elevated expression of TGF-beta, proteoglycans, and fibronectin in glomerulonephritis induced in rats by injection of anti-thymocyte serum (ATS). Glomeruli were cultured from rat kidneys at 1, 4, 7, 14, and 28 d after ATS administration. Increased proteoglycan synthesis was detected beginning on day 4, which peaked at a 4,900% increase compared with control on day 7, and returned toward control levels by day 28. The increased proteoglycan synthesis by cultured nephritic glomeruli, as well as that of fibronectin, were greatly reduced by addition of antiserum raised against a synthetic peptide from TGF-beta. Conditioned media from ATS glomerular cultures, when added to normal cultured mesangial cells, induced elevated proteoglycan synthesis that also peaked on day 7 and that mimicked the response to added exogenous TGF-beta. The stimulatory activity of the conditioned media was blocked by addition of TGF-beta antiserum. Prior addition of the immunizing peptide to the antiserum abolished the blocking effect. The main induced proteoglycans were identified as biglycan and decorin by immunoprecipitation with antiserum made against synthetic peptides from the proteoglycan core proteins. Glomerular histology showed mesangial matrix expansion in a time course that roughly paralleled both the elevated proteoglycan synthesis by the ATS glomeruli and the ability of the conditioned media from these glomeruli to induce proteoglycan synthesis. At the same time there was an increased expression of TGF-beta mRNA and TGF-beta protein in the glomeruli. These results suggest a central role for TGF-beta in the accumulation of pathological extracellular matrix in glomerulonephritis.

Interactions of Transforming Growth Factor-β and Angiotensin II in Renal Fibrosis

Overproduction of transforming growth factor-beta clearly underlies tissue fibrosis in numerous experimental and human diseases. Transforming growth factor-betas powerful fibrogenic action results from simultaneous stimulation of matrix protein synthesis, inhibition of matrix degradation, and enhanced integrin expression that facilitates matrix assembly. In animals, overexpression of transforming growth factor-beta by intravenous injection, transient gene transfer, or transgene insertion has shown that the kidney is highly susceptible to rapid fibrosis. The same seems true in human disease, where excessive transforming growth factor-beta has been demonstrated in glomerulonephritis, diabetic nephropathy, and hypertensive glomerular injury. A possible explanation for the kidneys particular susceptibility to fibrosis may be the recent discovery of biologically complex interactions between the renin-angiotensin system and transforming growth factor-beta. Alterations in glomerular hemodynamics can activate both the renin-angiotensin system and transforming growth factor-beta. Components of the renin-angiotensin system act to further stimulate production of transforming growth factor-beta and plasminogen activator inhibitor leading to rapid matrix accumulation. In volume depletion, transforming growth factor-beta is released from juxtaglomerular cells and may act synergistically with angiotensin II to accentuate vasoconstriction and acute renal failure. Interaction of the renin-angiotensin system and transforming growth factor-beta has important clinical implications. The protective effect of inhibition of the renin-angiotensin system in experimental and human kidney diseases correlates closely with the suppression of transforming growth factor-beta production. This suggests that transforming growth factor-beta, in addition to blood pressure, should be a therapeutic target. Higher doses or different combinations of drugs that block the renin-angiotensin system or entirely new drug strategies may be needed to achieve a greater antifibrotic effect.

Induction of Membranous Nephropathy in Rabbits by Administration of an Exogenous Cationic Antigen: DEMONSTRATION OF A PATHOGENIC ROLE FOR ELECTRICAL CHARGE

We examined the role of antigenic electrical charge as a determinant of glomerular immune complex localization in the rabbit. Serum sickness nephritis was induced in groups of New Zealand white rabbits by daily 25-mg intravenous injections of bovine serum albumin (BSA) chemically modified to be cationic (pI > 9.5) or more anionic (pI, 3.5-4.6); an additional group received unmodified native BSA (pI, 4.5-5.1). Factors known to influence immune complex localization, e.g., molecular size of the administered antigen and resulting circulating immune complexes, immunogenicity, and disappearance time from the circulation were examined and found to be similar for both anionic and cationic BSA. Charge modification did increase the nonimmune clearance of cationic and anionic BSA compared with native BSA. Injected cationic BSA was shown in paired label experiments to bind directly to glomeruli compared with native BSA. The renal lesion produced by cationic BSA was markedly different from that found in rabbits given anionic or native BSA. Animals receiving cationic BSA uniformly developed generalized diffuse granular capillary wall deposits of IgG, C3, and BSA detected after 2 wk of injections and increasing until death at 6 wk. Qualitatively similar deposits were produced by the administration of low doses of cationic BSA of only 1 or 10 mg/d. In contrast, the injection of both anionic and native BSA resulted in mesangial deposits at 2 and 4 wk with capillary wall deposits appearing by 6 wk. Ultrastructural examination of animals receiving cationic BSA revealed pure, extensive formation of dense deposits along the lamina rara externa of the glomerular basement membrane whereas such deposits were absent or rare in animals injected with the anionic or native BSA. Albuminuria was significantly greater at 6 wk in the groups receiving cationic BSA with a mean of 280 mg/24 h compared with 53 mg/24 h in the combined groups injected with anionic or native BSA. Blood urea nitrogen values were similar in all groups at 2 and 4 wk but higher in the animals receiving cationic BSA at 6 wk. These experiments describe the reproducible induction of epimembranous immune deposits by administration of an exogenous cationic antigen. They suggest that antigenic charge can play an important role in the pathogenesis of membranous nephropathy by permitting direct glomerular binding of an antigen and predisposing to in situ immune complex formation.

Cytokines in Kidney Disease: The Role of Transforming Growth Factor-β

Cytokines such as transforming growth factor-beta (TGF-beta) are peptide factors that regulate embryogenesis, development, inflammation, tissue repair, and carcinogenesis. Growing evidence indicates that dysregulation of cytokine actions may underlie the pathogenesis of serious autoimmune, degenerative, and fibrotic diseases. Studies in a model of acute mesangial proliferative glomerulonephritis show that overproduction of TGF-beta is the cause of pathologic accumulation of extracellular matrix in the nephritic glomeruli. Transforming growth factor-beta acts to increase matrix production, inhibit matrix degradation, and modulate matrix receptors in the glomerulonephritic rats. It may also play a role in the glomerular matrix build-up that is a central feature of diabetic nephropathy. Elevated expression of TGF-beta mRNA and TGF-beta protein were found in the glomeruli of diabetic rats along with increased levels of proteoglycans and other matrix components that are known to be induced by TGF-beta. The study of human diabetic glomeruli has also showed markedly elevated levels of TGF-beta protein. Glomeruli from normal kidneys and nonprogressive kidney disorders were negative. The striking ability of TGF-beta to cause exuberant matrix formation may be due to the fact that TGF-beta can induce its own production by resident cells at a site of injury. Thus, the potential for TGF-beta to do harm may be due to this autoinduction mechanism whereby TGF-beta expression can become chronic, creating a vicious circle. As the role that TGF-beta plays in chronic fibrotic diseases becomes better understood, it is likely that TGF-beta inhibitors will become important future drugs for treating these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

A mutant, noninhibitory plasminogen activator inhibitor type 1 decreases matrix accumulation in experimental glomerulonephritis

In fibrotic renal disease, elevated TGF-beta and angiotensin II lead to increased plasminogen activator inhibitor type 1 (PAI-1). PAI-1 appears to reduce glomerular mesangial matrix turnover by inhibiting plasminogen activators, thereby decreasing plasmin generation and plasmin-mediated matrix degradation. We hypothesized that therapy with a mutant human PAI-1 (PAI-1R) that binds to matrix vitronectin but does not inhibit plasminogen activators, would enhance plasmin generation, increase matrix turnover, and decrease matrix accumulation in experimental glomerulonephritis. Three experimental groups included normal, untreated disease control, and PAI-1R-treated nephritic rats. Plasmin generation by isolated day 3 glomeruli was dramatically decreased by 69%, a decrease that was reversed 43% (P < 0.02) by in vivo PAI-1R treatment. At day 6, animals treated with PAI-1R showed significant reductions in proteinuria (48%, P < 0.02), glomerular staining for periodic acid-Schiff positive material (33%, P < 0.02), collagen I (28%, P < 0.01), collagen III (34%, P < 0.01), fibronectin (48%, P < 0.01), and laminin (41%, P < 0.01), and in collagen I (P < 0.01) and fibronectin mRNA levels (P < 0.02). Treatment did not alter overexpression of TGF-beta1 and PAI-1 mRNAs, although TGF-beta1 protein was significantly reduced. These observations strongly support our hypothesis that PAI-1R reduces glomerulosclerosis by competing with endogenous PAI-1, restoring plasmin generation, inhibiting inflammatory cell infiltration, decreasing local TGF-beta1 concentration, and reducing matrix accumulation.

Clinical and antibody responses after influenza immunization in systemic lupus erythematosus.

After immunization with A/New Jersey/76 and A/Victoria/75 influenza vaccines, 11 patients with systemic lupud erythematosus were serially evaluated for changes in disease activity, serologic abnormalities, and their capability to generate specific antibodies. One patient, with active disease, developed a diffuse, proliferative glomerulonephritis. None of the other patients or control subjects had significant local or systemic side effects. Significant levels of antibodies were generated to A/New Jersey/76 in eight of the 11 patients and in seven of eight control subjects and to A/Victoria/75 in seven of 11 patients and five of eight control subjects. The geometric mean responses of both total and IgG antibodies to each viral antigen were no different in patients with systemic lupus erythematosus than in control subjects. In patients with stable systemic lupus erythematosus, immunization with killed influenza viral vaccine appears to be safe and effective.

In vivo interactions of TGF-β and extracellular matrix

TGF-beta, a multifunctional cytokine, plays an important role in embryogenesis and in regulating repair and remodeling following tissue injury. Many of the biological actions of TGF-beta are mediated by widespread effects on deposition of extracellular matrix. TGF-beta stimulates the synthesis of individual matrix components including proteoglycans, collagens and glycoproteins. TGF-beta also blocks matrix degradation by decreasing the synthesis of proteases and increasing the synthesis of protease inhibitors. Finally, TGF-beta increases the synthesis of matrix receptors and alters their relative proportions on the surface of cells in a manner that could facilitate adhesion to matrix. All of these events have largely been demonstrated in vitro in cultured cells. In an experimental model of glomerulonephritis we have shown that TGF-beta is responsible for the accumulation of pathological matrix in the glomeruli following immunological injury. Furthermore, all three of TGF-betas actions on extracellular matrix--increased synthesis, decreased degradation and modulation of receptors--have now been documented to be involved in matrix deposition in vivo in this model. Administration of the proteoglycan decorin suppressed TGF-beta-induced matrix deposition in the nephritic glomeruli, thus confirming a physiological role for decorin as a regulator of TGF-beta. Inhibitors of TGF-beta may be important future drugs in treating fibrotic diseases caused by overproduction of TGF-beta.

Electrical Charge. ITS ROLE IN THE PATHOGENESIS AND PREVENTION OF EXPERIMENTAL MEMBRANOUS NEPHROPATHY IN THE RABBIT

Intravenous cationic bovine serum albumin (BSA, pI > 9.5) induces membranous nephropathy in immunized rabbits. In this study, unimmunized rabbits received intravenous injections of cationic (n = 3) or native (n = 3) or native (n = 3) BSA, followed by ex vivo isolated left renal perfusions with sheep anti-BSA antibody. Capillary wall deposits of IgG and C3 were seen exclusively in the group receiving cationic BSA, confirming an in situ pathogenesis for cationic, BSA-induced membranous nephropathy, and demonstrating the importance of a cationic antigen for its production. We then explored whether membranous nephropathy in this model is prevented by the concomitant injection of protamine sulfate, a filterable, relatively non-immunogenic polycation. An in vitro study demonstrated that protamine sulfate incubated with glomerular basement membrane (GBM) decreased the subsequent binding of radiolabeled cationic BSA (P < 0.05). In vivo, protamine sulfate was shown to bind to anionic sites in the glomerular capillary wall after intravenous injection.Groups of rabbits received 3 wk of daily intravenous injections of cationic BSA alone (n = 15) or cationic BSA and protamine (n = 18). After 2 wk of injection of cationic BSA alone, typical membranous nephropathy developed. Granular deposits of IgG and C3 were present along the GBM associated with subepithelial dense deposits, foot process effacement, and marked albuminuria. Protamine significantly reduced or prevented the formation of deposits (P < 0.001) and in6 of 18 protamine-treated animals, existing deposits decreased or disappeared between 2 and 3 wk of injection. Albuminuria was significantly reduced in protamine-treated animals with a mean of 124+/-55 mg/24 h compared to 632+/-150 mg/24 h in the control group receiving cationic BSA alone. No significant differences between the groups were noted in serum lev9lsof IgG, C3, anti-BSA antibody, or circulating immune complex size. Studies in additional animals (n = 5) given radiolabeled cationic BSA showed that protamine did not alter the clearance of cationic BSA from serum. Control experiments showed that protamines beneficial effects were not related to its weak anticoagulant property or toits theoretical ability to deplete tissue histamine. The administration of heparin (n = 6) or diphenhydramine (n = 6) had no effect on the development of the epimembranous lesion compared to the group receiving cationic BSA alone. In addition, homogenized whole kidney histamine content was not significantly different in the group receiving cationic BSA alone compared to the group receiving cationic BSA and protamine. This work shows that a cationic BSA-induced glomerular lesion can be produced by a renal perfusion technique involving in situ complex formation and that this process requires a cationic antigen for its development. We believe that the demonstrated beneficial effects of protamine are due to its ability to bind to glomerular anionic sites, and that this electrostatic interaction results in inhibition for the further binding of the cationic antigen, thereby limiting the severity of glomerulonephritis in this model.