Frank Strutz

Renal Fibrosis: Collagen Composition and Assembly Regulates Epithelial-Mesenchymal Transdifferentiation

Type IV collagen is a major component of basement membranes and it provides structural and functional support to various cell types. Type IV collagen exists in a highly complex suprastructure form and recent studies implicate that protomer (the trimeric building unit of type IV collagen) assembly is mediated by the NC1 domain present in the C-terminus of each collagen alpha-chain polypeptide. Here we show that type IV collagen contributes to the maintenance of the epithelial phenotype of proximal tubular epithelial cells, whereas type I collagen promotes epithelial-to-mesenchymal transdifferentiation (EMT). In addition, the recombinant human alpha1NC1 domain inhibits assembly of type IV collagen NC1 hexamers and potentially disrupts the deposition of type IV collagen, facilitating EMT in vitro. Inhibition of type IV collagen assembly by the alpha1NC1 domain up-regulates the production of transforming growth factor-beta1 in proximal tubular epithelial cells, an inducer of EMT. These results strongly suggest that basement membrane architecture is pivotal for the maintenance of epithelial phenotype and that changes in basement membrane architecture potentially lead to up-regulation of transforming growth factor-beta1, which contributes to EMT during renal fibrosis.

Renal Fibroblasts and Myofibroblasts in Chronic Kidney Disease

Fibroblasts and myofibroblasts are believed to be the key effector cells in renal fibrogenesis responsible for the synthesis and deposition of extracellular matrix components. In an editorial written 15 yr ago, Gown claimed to have the mysteries of the fibroblast (partially) unmasked ([1][1]).

Activation of toll-like receptor-9 induces progression of renal disease in MRL-Fas(lpr) mice

How bacterial or viral infections trigger flares of autoimmunity is poorly understood. As toll‐like receptor (TLR)‐9 activation by exogenous or endogenous CpG‐DNA may contribute to disease activity of systemic lupus erythematosus, we examined the effects of CpG‐ oligodeoxynucleotides (ODN) or DNA derived from Escherichia coli (E. coli) on the course of nephritis in MRLlpr/lpr mice. In kidneys of these mice, TLR9 localized to glomerular, tubulointerstitial, and perivascular infiltrates. After intraperitoneal injection labeled CpG‐ODN localized to glomerular and interstitial macrophages and dendritic cells in nephritic kidneys of MRLlpr/lpr mice but not in healthy MRL controls. Furthermore, murine J774 macrophages and splenocytes from MRLlpr/lpr mice, but not tubular epithelial cells, renal fibroblasts, or mesangial cells, expressed TLR9 and up‐regulated CCL5/RANTES mRNA upon stimulation with CpG‐ ODN in vitro. In vivo both E. coli DNA and CpG‐ODN increased serum DNA autoantibodies of the IgG2a isotype in MRLlpr/lpr mice. This was associated with progression of mild to crescentic glomerulonephritis, interstitial fibrosis, and heavy proteinuria. CpG‐ODN increased renal CCL2/MCP‐1 and CCL5/RANTES expression associated with increased glomerular and interstitial leukocyte recruitment. In contrast control GpC‐ODN had no effect. We conclude that TLR9 activation triggers disease activity of systemic autoimmunity, for example, lupus nephritis, and that adaptive and innate immune mechanisms contribute to the CpG‐DNA‐induced progression of lupus nephritis.

Renal fibrosis: an update.

Tubulointerstitial fibrosis invariably accompanies the course of chronic renal failure towards end-stage renal disease. Tubular epithelial cells, the predominant cell type in the tubulointerstitium, are increasingly being recognized for playing a dominant role as mediators of renal fibrogenesis. Tubular epithelial cells become activated either by the glomerular ultrafiltrate from their apical side or by mononuclear cells from their basolateral side. They initiate the scarring process by secreting chemokines, which in return attract mononuclear cells as well as growth factors that stimulate interstitial fibroblasts. In later phases of renal fibrogenesis, cellular changes of tubular epithelial cells contribute to the chronic impairment of renal function. Whereas tubular epithelial cells react by proliferation or hypertrophy to initial stimuli, they may undergo apoptosis or transdifferentiate into fibroblasts, and thus contribute to tubular atrophy in later stages of progressive renal disease. Resident interstitial fibroblasts are also important in renal fibrogenesis, and recent research has demonstrated that these cells are much more heterogeneous than expected. Cytokines such as fibroblast growth factor type 2 and epithelial growth factor have been shown to be pro-fibrogenic, whereas hepatocyte growth factor and bone morphogenic protein type 7 may inhibit fibrogenesis. Despite recent progress, further research is mandatory for a better understanding and the development of novel therapeutic approaches.

Safety and clinical outcomes of rituximab therapy in patients with different autoimmune diseases: experience from a national registry (GRAID)

IntroductionEvidence from a number of open-label, uncontrolled studies has suggested that rituximab may benefit patients with autoimmune diseases who are refractory to standard-of-care. The objective of this study was to evaluate the safety and clinical outcomes of rituximab in several standard-of-care-refractory autoimmune diseases (within rheumatology, nephrology, dermatology and neurology) other than rheumatoid arthritis or non-Hodgkins lymphoma in a real-life clinical setting.MethodsPatients who received rituximab having shown an inadequate response to standard-of-care had their safety and clinical outcomes data retrospectively analysed as part of the German Registry of Autoimmune Diseases. The main outcome measures were safety and clinical response, as judged at the discretion of the investigators.ResultsA total of 370 patients (299 patient-years) with various autoimmune diseases (23.0% with systemic lupus erythematosus, 15.7% antineutrophil cytoplasmic antibody-associated granulomatous vasculitides, 15.1% multiple sclerosis and 10.0% pemphigus) from 42 centres received a mean dose of 2,440 mg of rituximab over a median (range) of 194 (180 to 1,407) days. The overall rate of serious infections was 5.3 per 100 patient-years during rituximab therapy. Opportunistic infections were infrequent across the whole study population, and mostly occurred in patients with systemic lupus erythematosus. There were 11 deaths (3.0% of patients) after rituximab treatment (mean 11.6 months after first infusion, range 0.8 to 31.3 months), with most of the deaths caused by infections. Overall (n = 293), 13.3% of patients showed no response, 45.1% showed a partial response and 41.6% showed a complete response. Responses were also reflected by reduced use of glucocorticoids and various immunosuppressives during rituximab therapy and follow-up compared with before rituximab. Rituximab generally had a positive effect on patient well-being (physicians visual analogue scale; mean improvement from baseline of 12.1 mm).ConclusionsData from this registry indicate that rituximab is a commonly employed, well-tolerated therapy with potential beneficial effects in standard of care-refractory autoimmune diseases, and support the results from other open-label, uncontrolled studies.

Renal fibrosis is attenuated by targeted disruption of KCa3.1 potassium channels

Proliferation of interstitial fibroblasts is a hallmark of progressive renal fibrosis commonly resulting in chronic kidney failure. The intermediate-conductance Ca2+-activated K+ channel (KCa3.1) has been proposed to promote mitogenesis in several cell types and contribute to disease states characterized by excessive proliferation. Here, we hypothesized that KCa3.1 activity is pivotal for renal fibroblast proliferation and that deficiency or pharmacological blockade of KCa3.1 suppresses development of renal fibrosis. We found that mitogenic stimulation up-regulated KCa3.1 in murine renal fibroblasts via a MEK-dependent mechanism and that selective blockade of KCa3.1 functions potently inhibited fibroblast proliferation by G0/G1 arrest. Renal fibrosis induced by unilateral ureteral obstruction (UUO) in mice was paralleled by a robust up-regulation of KCa3.1 in affected kidneys. Mice lacking KCa3.1 (KCa3.1−/−) showed a significant reduction in fibrotic marker expression, chronic tubulointerstitial damage, collagen deposition and αSMA+ cells in kidneys after UUO, whereas functional renal parenchyma was better preserved. Pharmacological treatment with the selective KCa3.1 blocker TRAM-34 similarly attenuated progression of UUO-induced renal fibrosis in wild-type mice and rats. In conclusion, our data demonstrate that KCa3.1 is involved in renal fibroblast proliferation and fibrogenesis and suggest that KCa3.1 may represent a therapeutic target for the treatment of fibrotic kidney disease.

New insights into mechanisms of fibrosis in immune renal injury

Abstract. Renal fibrosis is the final common pathway for many kidney lesions that lead to chronic progressive organ failure. The tubulointerstitial space occupies up to 90% of kidney volume, indicating that pathological changes in that space can not be without functional significance. By analogy to wound healing, renal fibrogenesis can be divided arbitrarily into three phases: induction, inflammatory, and post-inflammatory phases. The latter phase is of particular importance, since its length often exceeds what would be required for healing. The induction phase is characterized by the infiltration of the tubulointerstitial space by mononuclear inflammatory cells. This influx is mediated by proinflammatory cytokines and chemokines often secreted by activated tubular epithelial cells. Subsequently, these infiltrating mononuclear cells stimulate a heterogeneous group of resident fibroblasts and tubular epithelial cells to undergo phenotypic conversion into activated fibroblasts that secrete abundant extracellular matrix. Tubular epithelial cells contribute to this process through epithelial-mesenchymal transition. During the inflammatory phase these activated fibroblasts are stimulated to produce collagenous matrix mainly by cytokines, such as TGF-β1, EGF, ET-1, and FGF-2, which are secreted by inflammatory and injured somatic cells. Occasionally however, when inflammation subsides, the matrix synthesis in the post-inflammatory phase of renal fibrogenesis continues and may be more dependent on autocrine stimulation from resident renal cells such as remaining tubular epithelium. Eventually, the collagenous matrix of fibrogenesis destroys blood supply and the perimeter of viability for fibroblasts regresses to the point where scars become acellular.

EMT and proteinuria as progression factors

Tubulointerstitial fibrosis is an integral part of the structural changes of the kidney in chronic progressive renal failure. The accumulation of the extracellular matrix in the tubulointerstitial space is mediated mainly by myofibroblasts. These are derived from resident interstitial fibroblasts, tubular epithelial cells, periadventitial cells, and possibly also mesenchymal stem cells and endothelial cells. Fibrosis is usually preceded by tubulointerstitial infiltration of mononuclear inflammatory cells. Proteinuria is one of several mechanisms of primary glomerular or vascular disease to transmit the disease process to the interstitial space. Increased protein filtration may have direct toxic effects on tubular epithelial cells, induce chemokine and cytokine secretion and result in increased expression of adhesion molecules, all contributing to the influx of mononuclear cells. Inflammatory cells in return secrete cytokines, which stimulate resident fibroblasts and tubular epithelial cells to differentiate into matrix-producing cells. The phenotypic conversion of primary epithelial cells into mesenchymal cells, termed epithelial-mesenchymal transition (EMT), has been studied in great detail in recent years. Several signal transduction pathways of this process have been clarified and may eventually result in novel therapeutic approaches. The severity of proteinuria and the extent of EMT have both been associated with the decline in renal function in clinical studies. Limiting proteinuria results in a slower decline of renal function deterioration, whereas reducing EMT has had beneficial effects in a number of animal studies, including those indicating reversal of fibrotic lesions. However, the association between proteinuria and EMT and vice versa is far from clear and has not been carefully studied.

Decrease in expression of bone morphogenetic proteins 4 and 5 in synovial tissue of patients with osteoarthritis and rheumatoid arthritis

Bone morphogenetic proteins (BMPs) have been identified as important morphogens with pleiotropic functions in regulating the development, homeostasis and repair of various tissues. The aim of this study was to characterize the expression of BMPs in synovial tissues under normal and arthritic conditions. Synovial tissue from normal donors (ND) and from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) were analyzed for BMP expression by using microarray hybridization. Differential expression of BMP-4 and BMP-5 was validated by semiquantitative RT-PCR, in situ hybridization and immunohistochemistry. Activity of arthritis was determined by routine parameters for systemic inflammation, by histological scoring of synovitis and by semiquantitative RT-PCR of IL-1β, TNF-α, stromelysin and collagenase I in synovial tissue. Expression of BMP-4 and BMP-5 mRNA was found to be significantly decreased in synovial tissue of patients with RA in comparison with ND by microarray analysis (p < 0.0083 and p < 0.0091). Validation by PCR confirmed these data in RA (p < 0.002) and also revealed a significant decrease in BMP-4 and BMP-5 expression in OA compared with ND (p < 0.015). Furthermore, histomorphological distribution of both morphogens as determined by in situ hybridization and immunohistochemistry showed a dominance in the lining layer of normal tissues, whereas chronically inflamed tissue from patients with RA revealed BMP expression mainly scattered across deeper layers. In OA, these changes were less pronounced with variable distribution of BMPs in the lining and sublining layer. BMP-4 and BMP-5 are expressed in normal synovial tissue and were found decreased in OA and RA. This may suggest a role of distinct BMPs in joint homeostasis that is disturbed in inflammatory and degenerative joint diseases. In comparison with previous reports, these data underline the complex impact of these factors on homeostasis and remodeling in joint physiology and pathology.

INTERSTITIAL PATHOMECHANISMS UNDERLYING PROGRESSIVE TUBULOINTERSTITIAL DAMAGE

Progressive renal disease poses an increasing problem for the medical community. Though the causes of end–stage renal failure are multiple, the histologic pictures of chronic renal disease are remarkably similar being characterized by interstitial infiltration, fibrosis, tubular atrophy and dilatation. This similarity points to a final common pathway. In addition, renal disease often progresses despite elimination or amelioration of the inciting stimulus. This review deals with the pathomechanisms of progressive renal failure proposing a three–step model of fibrogenesis with an induction phase, a phase of inflammatory, and, lastly, a phase of postinflammatory matrix synthesis. The central role of the tubular epithelial cell as a mediator of interstitial inflammation and its participation in matrix synthesis will be discussed particularly. Finally, a brief overview is listed on new therapeutic approaches to limit the progressive nature of fibrogenesis.