Mechanistic connection between inflammation and fibrosis

Abstract

Fibrosis of the kidney is caused by the prolonged injury and deregulation of normal wound healing and repair processes, and by an excess deposition of extracellular matrices. Despite intensive research, our current understanding of the precise mechanism of fibrosis is limited. There is a connection between fibrotic events involving inflammatory and noninflammatory glomerulonephritis, inflammatory cell infiltration, and podocyte loss. The current review will discuss the inflammatory response after renal injury that leads to fibrosis in relation to non-inflammatory mechanisms.

Introduction

Regardless of the underlying etiology, most forms of chronic kidney diseases are characterized by progressive fibrosis as a final common pathway that eventually affects all substructures of the kidney with the final consequence of end-stage renal disease. Although there has been a great deal of research, comprehensive understanding of the pathogenetic mechanisms of kidney fibrosis remains uncertain and that hampers the development of effective therapeutic strategies.

Fibrosis is a process of normal wound healing and repair that is activated in response to injury to maintain the original tissue architecture and functional integrity. However, prolonged chronic injurious stimuli may cause deregulation of normal processes and result in an excess deposition of extracellular matrix and fibrosis. It involves a complex multistage inflammatory process with inflammatory cell infiltration, mesangial and fibroblast activation, tubular epithelial to mesenchymal transition, endothelial to mesenchymal transition, cell apoptosis, and extracellular matrix expansion that is orchestrated by a network of cytokines/chemokines, growth factors, adhesion molecules, and signaling processes.^1,2 These events consist of (1) injury to the tissue, (2) recruitment of inflammatory cells, (3) release of fibrogenic cytokines, and finally (4) activation of collagenproducing cells.

There are various kinds of injuries, such as immunological (immunoglobulin A nephropathy, lupus nephritis, Goodpasture’s disease), metabolic (diabetic nephropathy), emodynamic (hypertension), ischemic (shock), and toxic (drugs, microbials) assaults. Irrespective of the initial injury to the tissue, an inflammatory response will follow.^3-6 Only in the embryo can a loss of tissue be repaired without inflammation, scarring, or fibrosis.^3,4 After birth, repair is always associated with an inflammatory process, irrespective of the eventual outcome, such as healing or limited or progressive fibrosis. That is, inflammation is closely related to tissue repairs with a regeneration of parenchymal cells and the filling of tissue defects with fibrous tissue, namely, scar formation. The inflammatory response therefore represents a two-sided sword: beneficial in terms of the repair process to injury; detrimental when proceeding in an uncontrolled manner, which then leads to progressive fibrosis with a loss of function.⁷ Thus, controlling excessive inflammation would be of great potential therapeutic benefit for inhibiting progressive fibrosis of kidney.

This review discusses inflammatory responses after renal injury, as well as the process after inflammation that leads to renal fibrosis in relatively earlier stages of fibrosis pathways, to investigate the connection between inflammation, reaction, and fibrosis in the kidney.

Inflammatory responses after renal injury and its connection to fibrosis

There is little doubt that inflammation has an important role in the development and progression of most chronic kidney diseases. At end stage, the kidney is characterized histologically by chronic inflammation, including infiltration by leukocytes and fibrosis. Markers of inflammation, including C-reactive protein, interleukins (IL)-1 and 6, and tumor necrosis factor-α, are elevated in plasma of patients with chronic kidney disease.⁸ Kidney fibrosis is almost always preceded by and closely associated with chronic interstitial inflammation.^9-12 The overall aim of the inflammatory process is to eliminate the original insult by removing cell and matrix debris, and to repair the lost tissue components. Ideally, this results in a reconstitution of the original tissue architecture and function.⁷

The pathogenesis of inflammation is complex and multifactorial, involving the interaction of cytokines, chemokines, and adhesion molecules. Regardless of the initial insult, renal inflammation is characterized by glomerular and tubulointerstitial infiltration by inflammatory cells, including neutrophils, macrophages, lymphocytes, and so forth. Such cellular infiltrates are evident in experimental models of renal disease and human renal biopsy specimens.¹³ Vigorous cellular response is generally observed in renal diseases in which immune deposits form in the glomerular basement membrane (GBM) (anti-GBM disease), on the inner surface of the capillary wall (type-4 lupus nephritis), or in the mesangium (immunoglobulin A nephropathy). Unlike the products resulting from subepithelial immune complex formation in membranous nephropathy, these chemoattractants complement activation products, and cytokines directly access the vascular space, thereby resulting in the infiltration of circulating inflammatory cells (neutrophils, macrophages, and lymphocytes) and in the upregulation of leukocyte adhesion molecules. Resident glomerular cells also proliferate, particularly mesangial cells.

Initial inflammation is caused by cytokine-mediated endocytosis/phagocytosis. Neutrophils are the first cells recruited, as they uptake cell debris and phagocytose apoptotic bodies. Activated neutrophils degranulate, release inflammatory and profibrogenic cytokines, and apoptose. Following neutrophils, macrophages infiltrate damaged tissues, phagocytose, and secrete fibrogenic cytokines. Macrophages are a major source of transforming growth factor-β1 (TGF-β1) in fibrosing organs. T and B lymphocytes are also recruited to the site of injury and further facilitate secretion of fibrogenic cytokines.^1,14 At the same time, TGF-β1 is a potent chemoattractant for cells of macrophage-monocytic lineage. In addition to TGF-β1, monocyte chemoattractant protein-1, macrophage inflammatory protein-1, and macrophage inflammatory protein-2 are also involved in recruitment of inflammatory cells.¹⁵

In renal fibrosis, the activation of the renin-angiotensin-aldosterone system and its main effector angiotensin II stimulates inflammation, including the expression of cytokines, chemokines, growth factors, and reactive oxygen species.¹⁶ Angiotensin II induces vascular inflammation, endothelial dysfunction, upregulation of adhesion molecules, and recruitment of infiltrating cells into the kidney.¹⁷

Cellular mediators of renal inflammation

Neutrophils

Recently, evidence has clearly shown that neutrophils also have roles in activating the immune response beyond their conventional role of microbial clearance. In glomerulonephritis (GN), neutrophil participation is evident in the most aggressive forms of GN. A significant neutrophil presence is prominent in acute poststreptococcal GN,¹⁸ immunoglobulin A nephropathy,¹⁹ membranoproliferative GN,²⁰ lupus nephritis,²¹ vasculitis-associated GN,²² and is associated with both anti-proteinase-^3,23 anti-myeloperoxidase anti-neutrophil autoantibodies, and with other forms of rapidly progressive GN.²⁴ Except the usually self-limiting acute poststreptococcal GN, most cases of GN that have prominent neutrophil infiltration show progressive fibrosis.

Evidence from renal biopsy and experimental animal models suggests that neutrophil localization in glomerular capillaries is dependent on the generation of chemotactic factors within and around an inflammatory area by antibody and complement deposition. The most prominent chemoattractants for neutrophils in glomerular disease are C5a derived by activation of the complement and several chemokines such as IL-8, which can be bound to endothelial cells via heparin sulfate proteoglycans.¹¹

Once localized to the site of immune deposit formation, neutrophils attempt to ingest the immune complexes and they are subsequently activated, undergoing a respiratory burst that generates reactive oxygen species.²⁵ Neutrophils also store cationic serine proteases, such as elastase and cathepsin G, within azurophilic granules. The activation of neutrophils within glomeruli causes the extracellular release of these cytotoxic proteins, thereby resulting in the degradation of elements of the GBM. The importance of leukocytemediated immune glomerular injury is supported by many observations, including the beneficial effect of both neutrophil depletion and interference with adhesion molecule function and studies using animals genetically deficient in neutrophil enzymes.^26,27

Macrophages

Macrophages are key cellular mediators of inflammation and also prominent constituents of several proliferative GN cases, including all forms of rapidly progressive GN, lupus nephritis, and cryoglobulinemic nephropathy.^20,28,29 Studies of the functional contribution of macrophages to GN have generally highlighted the proinflammatory and profibrogenic role of macrophages.^30-33 Macrophages localize to glomeruli via interactions with both deposited immunoglobulins and several chemokines, such as monocyte chemoattractant protein-1, macrophage inflammatory protein-1, and regulated upon activation, normal T-cell expressed and secreted.^28,34,35

Members of the TGF-β superfamily are the most extensively studied macrophage-derived growth factors that have been linked to renal fibrosis.³⁶ Macrophages, tubular epithelial cells, and myofibroblasts are all capable of synthesizing TGF-β at different stages during the development of renal fibrotic lesions.³⁷ However, the observation that macrophage ablation markedly attenuates fibrosis in various conditions suggests that these cells are among the main producers of this growth factor.^38,39

A renewed appreciation of the heterogeneity of macrophages⁴⁰ and the identification of subsets with distinct phenotypic markers⁴¹ have led to an appreciation of their more diverse functional capabilities, including important roles in tissue repair and remodeling.⁴²

The animal model of unilateral ureteral obstruction in the rodent generates progressive renal fibrosis, as well as obstructive nephropathy,^43-45 and has proved to be valuable for the study of the pathogenesis of inflammation and fibrosis within the tubulointerstitium. Recent studies have revealed major pathways leading to the development of renal interstitial fibrosis following unilateral ureteral obstruction: (1) interstitial infiltration by macrophages that produce cytokine responsible for tubular apoptosis and fibroblast proliferation and activation; (2) tubular cell death by apoptosis and necrosis leading to the formation of atubular glomeruli and tubular atrophy; and (3) phenotypic transition of resident renal cells. Chronic unilateral ureteral obstruction activates the renin-angiotensin-aldosterone system with production of nuclear factor-κB and reactive oxygen species, which promotes more macrophage infiltration⁴⁶ and renal tubular apoptosis and interstitial fibrosis in rats.⁴⁷ Classically activated macrophages (M1) can generate tumor necrosis factor-α, which mediates proapoptotic signaling and renal tubular cell apoptosis following unilateral ureteral obstruction.⁴⁸ By contrast, alternatively activated macrophages (M2) generate anti-inflammatory cytokines, and induce cell survival and proliferation.⁴⁹

Modulating macrophage phenotype and function has been reported to reduce renal injury in models of renal disease, including GN,^50,51 allograft injury,⁵² and interstitial fibrosis.⁵³ More recently, an experiment provided direct evidence that ex vivo manipulation of macrophages can reduce renal injury and facilitate repair by using adoptive transfer of IL-4/IL-13-induced M2 macrophages injected into SCID mice with Adriamycin nephropathy in an in vivo model of chronic inflammatory renal disease analogous to human focal segmental glomerulosclerosis (FSGS).⁵⁴ This demonstrates that macrophages not only function as effectors of immune injury but can also be induced to provide protection against immune injury.

Although more recent studies have begun to explore the role of macrophages in repair of inflammatory renal injury, the heterogeneity of macrophages, their diverse roles in inflammation and tissue remodeling, and the coordinated activation and programming by other inflammatory cells are not fully understood.^13,55

Lymphocytes and dendritic cells

T cells, B cells, and dendritic cells are key components of adaptive immune system and have important roles in renal inflammation driven by aberrant immunological responses. Although T cells may be directly cytotoxic and nephritogenic, they primarily facilitate the activation of other cells, which subsequently function as effector cells, including macrophages, via interferon-γ production and B lymphocytes, leading to antibody production.^56,57 B cells are key factors in the humoral component of acute renal inflammation, whether it is in the setting of acute GN or a humoral rejection of a renal transplant. B-cell-deficient mice are protected from renal ischemia-reperfusion injury and this ischemia-reperfusion injury appears to be the result of a serum factor generated by B cells rather than by the B-cell itself.⁵⁸

Dendritic cells have an important role in the maintenance of tolerance and mounting robust immune responses to pathogens. They are highly efficient antigen-presenting cells and present antigen to T cells in the context of various costimulatory molecules. T-cell activation is critically dependent on dendritic cells, the role of which in renal disease appears to be protective, but the underlying mechanisms of which are largely unknown yet.

Mast cells

Mast cells are tissue cells of hematopoietic origin that secrete a variety of mediators such as histamine, cytokines, and chemokines. Recent studies suggest that mast cells contribute to renal disease with tubulointerstitial mast cell infiltration, which is evident in various human diseases such as lupus nephritis, FSGS, Ig A nephropathy, antineutrophil autoantibodies-associated GN, as well as diabetes, in which mast cell numbers correlated with interstitial fibrosis.^59,60 However, experimental studies in recent years using mast cell-deficient animals suggested the beneficial effects of mast cells, therefore revealing a more complex picture of mast cell actions. Mast cell-deficient mice showed an increased mortality and worse histopathological disease in a anti-GBM model of GN.⁶¹ The area of interstitial fibrosis was greater in mast cell-deficient rats compared with normal rats in a puromycin aminonucleoside-nephrosis model.⁶² Although it is likely that it is the physiological context involving the interaction with other cells and inflammatory mediators that determines the final action of mast cells in the development of kidney fibrosis,² the role of mast cells in both renal fibrosis and repair needs further study.

Non-inflammatory mechanisms of renal injury and fibrosis

Minimal change disease/FSGS and membranous nephropathy are initially characterized by a dramatic increase in glomerular permeability, causing nephrotic range proteinuria in association with little or no cellular infiltration or proliferation, in comparison with inflammatory GN such as acute poststreptococcal GN, membranoproliferative GN, lupus nephritis, and crescentic GN.

Podocytes seem to be the major target in this group. Podocytes, also called glomerular visceral epithelial cells, are highly specialized, terminally differentiated epithelial cells, with a quiescent phenotype.⁶³ There is an increasing body of experimental and clinical literature showing a decrease in podocyte number in diabetic and non-diabetic glomerular diseases.^64-67 The consequences of reduced podocyte number include proteinuria and glomerulosclerosis. The mechanism underlying proteinuria is simply owing to a lack of charge and size selectivity in areas of podocyte loss, as these barriers are now devoid.⁶⁸ Studies have shown that proteinuria increases as podocyte number decreases.^65,66,69 Although there is no direct evidence that podocyte loss is sufficient to induce glomerulosclerosis, several data support this notion. As soon as damage to podocytes exceeds a certain threshold (approximately 30%), glomerulosclerosis ensues.⁷⁰ In patients with a surgical reduction of ≥75% of renal mass, a relative lack of podocytes and subsequent FSGS in the originally healthy remnant kidney can lead to renal failure.⁷¹ In a rat model of puromycin aminonucleoside nephrosis, the region of the glomerulus devoid of podocytes developed glomerulosclerosis, and this area progressively increased as podocytes were progressively depleted, suggesting a role of podocyte injury and depletion in the development of glomerulosclerosis and disease progression.⁷² In patients with both type 1 and 2 diabetic glomerulosclerosis or immunoglobulin A nephropathy, the number of podocytes was reduced in proportion to the severity of injury and degree of proteinuria.^65,69,73 An explanation as to how a reduced podocyte number leads to glomerulosclerosis has been proposed.⁶⁴ In brief, podocyte loss leads to areas of ‘bare or denuded’ GBM, in which podocytes are reduced, and then from this area, outward bulging of the GBM occurs as the loss of opposing power provided by podocytes against the outward forces of glomerular pressures. Attachment of parietal epithelial cells to bare GBM invariably occurs when bare GBM coexists with architectural lesions, leading to the formation of a tuft adhesion to Bowman’s capsule, and the first ‘committed’ lesion progressing to segmental sclerosis. Therapeutic strategies targeted toward altering podocyte function will likely be of benefit in glomerular diseases, especially those that are dominantly nephrotic non-inflammatory GN, such as minimal change disease, early FSGS, and membranous nephropathy.

Conclusion

Renal fibrosis involves a complex multistage inflammatory process that is orchestrated by a network of cytokines/chemokines, growth factors, adhesion molecules, and signaling processes. Recent studies are more focused on the later stages of the fibrosis pathway, such as TGF-β, the major fibrogenic cytokine, and myofibroblasts transformation via epithelial to mesenchymal transition or endothelial to mesenchymal transition. However, the precise mechanism of renal inflammation that leads to fibrosis has yet to be elucidated. Still, our current therapies for inflammatory GN mainly focus on reducing the proinflammatory behavior of cellular mediators or inhibiting cell division using nonspecific immunosupressants or antimetabolic agents. Therefore, more significant advances in our understanding of renal inflammation can help develop earlier therapeutic intervention in inflammatory GN and renal fibrosis.