Gene therapy for arthritis

Arthritis is among the leading causes of disability in the developed world. There remains no cure for this disease and the current treatments are only modestly effective at slowing the disease's progression and providing symptomatic relief. The clinical effectiveness of current treatment regimens has been limited by short half-lives of the drugs and the requirement for repeated systemic administration. Utilizing gene transfer approaches for the treatment of arthritis may overcome some of the obstacles associated with current treatment strategies. The present review examines recent developments in gene therapy for arthritis. Delivery strategies, gene transfer vectors, candidate genes, and safety are also discussed.
Introduction
Rheumatoid arthritis (RA) is the most common inflammatory disorder, affecting approximately 0.5–1% of the North American adult population. It causes significant pathology and functional impairment in affected individuals. RA is a multifactorial disease whose main risk factors include genetic susceptibility, sex and age, smoking, infectious agents, hormones, diet, and socioeconomic and ethnic factors [1]. Those afflicted by RA report a decrease in quality of life measures such as persistent pain, functional disability, fatigue, depression, and an inability to perform daily tasks [2]. RA is also a significant burden on the health care system, averaging between $2,800–$28,500 per patient per year for direct and indirect costs in developed countries [3]. Although the pathogenesis of RA is not completely understood, specific HLA-DR genes, autoantibody and immune complex production, T cell antigen-specific responses, networks of cytokine production, and a hyperplastic synovium have all been shown to play a role.
RA primarily affects diarthrodial joints of the hands and feet. These joints are normally lined by a thin cell layer (1–3 cells) of both type I (macrophage-like) and type II (fibroblast-like) synoviocytes. In RA, the joint is inflamed and the synovium becomes hyperplastic, creating a pannus of synovial tissue comprised of CD4+ T cells, B cells, mast cells, dendritic cells, macrophages, and synoviocytes that invade and destroy nearby cartilage and bone. Neutrophils accumulate in the synovial fluid and also contribute to the destructive processes [4, 5]. Macrophages and fibroblast-like synoviocytes (FLS) secrete inflammatory cytokines, such as TNFα, IL-1β, and IL-6, all of which contribute to cartilage and bone destruction. They have been implicated as major contributors to many aspects of RA, including inflammatory cell infiltration, fibrosis, and T cell proliferative responses [6, 7]. These cytokines are thought to act primarily through MAP kinase and nuclear factor κB (NFκB) signaling pathways to activate transcription factors that turn on genes for chemokines and cell adhesion molecules, along with extracellular matrix degrading enzymes like matrix metalloproteinases (MMPs). Chemokines and chemokine receptors have also been linked to RA [6, 8, 9].
Initial treatment strategies for RA include drugs such as non-steroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids. While providing pain relief and decreased joint swelling, these drugs are unable to stop the progression of RA [10]. This led to the use of small molecules like methotrexate that were demonstrated to slow disease progression. For many decades these disease-modifying agents of rheumatic disease (DMARDs) were the best treatment option for RA. In recent years, a further understanding of the disease process has led to the development of biologic DMARDs primarily aimed at neutralizing the effects of pro-inflammatory cytokines. The most successful agents in this class are anti-TNF-α molecules and IL-1β blocking agents. Based on improvement criteria set forth by the American College of Rheumatology (ACR), these drugs are more effective than treatment with methotrexate alone. Even so, less than half of the patients show improvement of at least 50% in their ACR scores. In addition, the half-life of these drugs is relatively short and they require frequent systemic administration in order to be effective [11]. Gene transfer strategies have the potential to overcome some of these limitations, potentially leading to increased efficacy and decreased frequency of administration.
Gene delivery strategies
Although the majority of inflammation in RA is localized to the joints, there are systemic components to the disease. Therefore, when developing treatment strategies, one must consider whether the therapy should be delivered locally or systemically. Local administration is attractive because it has less potential for side effects and the treatment is delivered directly to the joint, the main site of inflammation. However, systemic features of the disease would seem to be left untreated. Interestingly, several researchers have observed a “contralateral effect” in animal models of local gene delivery where the delivered transgene is protective not only to the injected joint, but also to distal, untreated joints [12–14]. This effect appears to be independent of trafficking of modified immune cells to distal joints, because ex vivo modified fibroblasts alone are still able to confer a contralateral effect [15]. It is also independent of systemic circulating levels of transgene and non-specific immunosuppression and instead was found to depend on a complex antigen-specific mechanism [16]. Systemic delivery, typically by intravenous administration, would be expected to have a broader therapeutic effect, but is also associated with an increase in side effects and toxicity.
Another consideration for gene transfer is whether to employ in vivo or ex vivo delivery strategies. In vivo strategies have the advantage of being relatively easy and less expensive. In addition, many more studies have been performed in animal models looking at in vivo gene delivery. Ex vivo strategies, although expensive and time-consuming, have the advantage of being able to treat and select very specific cells, avoiding the possibility that the gene transfer vector could genetically modify a stem cell population and result in oncogene activation. A phase I clinical trial has been performed using a retroviral vector to deliver IL-1 receptor antagonist (IL-1ra) to cultured autologous synovial fibroblasts [17]. The cells were then injected into the RA patients' joints. After a scheduled arthroplasty 1 week later, significant expression of the transgene was seen in the injected joints. No adverse events were reported. This trial was initiated following previous experiments where antagonists of both IL-1β and TNF-α were delivered to autologous cultured rabbit fibroblasts ex vivo and injected into arthritic rabbit knee joints, with significant therapeutic benefit [15, 18].
Most ex vivo studies use fibroblast-like synoviocytes (FLS) as the target cell. These cells have been targeted specifically because they are thought to be directly responsible for cartilage destruction and drive and perpetuate the inflammatory response and autoimmunity [19]. The disadvantages of using this cell type are that FLS have a low proliferation rate, lack highly specific surface markers, and are a non-homogenous population. Future studies may examine other strategies, including gene delivery to T cells, dendritic cells, muscle cells, or mesenchymal stem cells [20].
Gene transfer vectors
Gene transfer vectors can be broadly categorized into two groups: viral and non-viral vectors. In general, viral vectors tend to provide for longer-term gene expression but often come with additional safety concerns, ranging from fears of generating replication competent virus during vector production, random insertion of the transgene into the genome following treatment, or development of a harmful immune response.
Plasmid DNA
The most common non-viral vector used in arthritis studies is plasmid DNA. Plasmid DNA can be delivered by liposomes, gene gun, or direct injection of the plasmid. The use of plasmid DNA tends to be less toxic and less immunogenic than the use of viral vectors and is also easy and relatively inexpensive to produce. However, plasmid DNA often leads to low transfection efficiency and short-term expression of the transgene, lasting only 1–2 weeks [21–23]. These limitations make it unlikely that local delivery of plasmid DNA in the joint will be successful.
The most success with the use of plasmid DNA in gene transfer for arthritis has been garnered through delivery of transgenes to skeletal muscle. Electrotransfer of soluble TNF-α receptor I variants to the tibial-cranial muscle at the onset of collagen-induced arthritis (CIA) led to a decrease in the clinical and histological signs of disease for up to 5 weeks [24]. Similarly, plasmids encoding cDNA for other anti-inflammatory molecules such as IL-1ra and a soluble TNFR-Fc fusion protein have been demonstrated to improve both macroscopic and microscopic scores of CIA when delivered intramuscularly [25, 26]. Plasmid encoding TGF-β delivered to skeletal muscle delayed progression of streptococcal cell wall induced arthritis when administered at the peak of the acute phase and virtually eliminated subsequent inflammation and arthritis when given at the beginning of the chronic phase of the disease [27]. Intramuscular injections of plasmids encoding immuno-modulatory molecules such as IL-4, IL-10, viral IL-10, and soluble complement receptor type I have given similar results [28–31]. Intramuscular delivery of a plasmid encoding TIMP-4, and inhibitor of matrix metalloproteinases, completely abolished the development of arthritis in a rat adjuvant-induced arthritis model [32]. Intravenous delivery of a plasmid encoding the heparin-binding domain of fibronectin inhibited leukocyte recruitment and decreased inflammation in CIA [33]. Intra-dermal injection of plasmid encoding IL-10 and intra-peritoneal injection of plasmid IL-10/liposome complexes have also been demonstrated to delay onset and progression of CIA [34, 35]. The liposome delivered DNA was able to maintain expression for only 10 days after injection, significantly less than the intramuscular studies mentioned above. Recently, the use of chitosan, a polycationic polysaccharide derived from crustacean shells, has been shown to act as an efficient gene carrier to rabbit knee joints both in vitro and in vivo [36].
Other non-viral vectors
Other non-viral gene delivery systems that have potential in the treatment of RA are the artificial chromosome expression (ACE) system and the sleeping beauty (SB) transposon system. The ACE system is attractive because it is non-integrating and can provide stable and long-term gene expression of one or multiple genes. A feasibility study was recently performed in a Mycobacterium tuberculosis rat arthritis model, which demonstrated that rat skin fibroblasts could be modified ex vivo to express a reporter gene from an artificial chromosome. These cells, when subsequently injected into rat joints, demonstrated engraftment into the synovial tissue microarchitecture and detectable transgene expression. The ACE system did not induce local inflammation at the injection site that is often associated with viral vector administration [37]. The sleeping beauty transposon system melds the advantages of both viral and non-viral vectors, allowing for both integration into the genome and long term expression. No studies have yet been performed in arthritis models using the sleeping beauty transposon system, but success has been found in both cancer and hemophilia models, suggesting that it might be have potential to successfully treat arthritis as well [38].
Viral vectors are by far the most widely used vectors for delivering transgenes in arthritic animal models [39]. There are several different viral vectors that have been examined for use in gene transfer for arthritis, including adenovirus, retrovirus, adeno-associated virus (AAV), and lentivirus, each with their respective advantages and disadvantages.
Adenovirus
Adenovirus is a non-encapsidated double-stranded DNA virus that can infect non-dividing cells and can be produced at high titers. Many gene-therapy studies have been performed with this vector but it has several limitations that may prevent it from being successful in the clinic. The high prevalence of neutralizing antibodies may prevent successful administration or re-administration. Injected adenovirus also causes a significant inflammatory immune response, which is a safety concern. In addition, adenovirus vectors typically only allow for 1–3 weeks of transgene expression, which would limit its long-term efficacy. Some improvements to adenoviral vectors have recently been made in an effort to improve delivery of transgenes to the synovium. FLS lack the coxsackie-adenovirus receptor (CAR) and are not efficiently transduced by adenovirus. By modifying the fiber knobs on the virus, adenoviral transgene delivery to synoviocytes and synovium was improved dramatically [40, 41]. Other recent improvements include the development of an adenoviral vector with an inflammation inducible promoter [42]. This would allow expression of the transgene during active disease, but expression would turn off once inflammation was brought under control.
Retrovirus
Retroviruses, mostly derived from the Moloney murine leukemia virus, have a relatively simple genome and structure. They are enveloped viruses and contain two identical copies of their RNA genome. The key feature of the retroviral life cycle is the ability of the RNA genome to be reverse transcribed into double-stranded DNA, which can then randomly integrate into the genome. They have been mostly used in ex vivo studies and are desirable vectors for several reasons. They can provide for long-term stable expression and their integration into the genome makes it possible to permanently correct a genetic defect [43]. For arthritis in particular, the inflamed synovium appears to be more susceptible to uptake of the virus [18]. The drawbacks to retroviral vectors are that they only infect non-dividing cells and are produced at low titers. The fact that these vectors integrate into the genome randomly is also a concern. In fact, in a recent clinical trial in France using a retrovirus to correct an X-linked SCID disorder, 3 out of 10 children developed leukemia after the vector inserted in or near a known oncogene. As a result, similar trials in the U.S. for this disorder have been halted until more information can be gathered [44, 45]. Future improvements to these vectors, including the development of self-inactivating vectors, which contain no retroviral promoter or enhancer elements, and use of vectors from non-oncogenic retroviruses will hopefully make them safer for clinical use [43].
Lentivirus
Lentivirus vectors are derived from retroviral vectors but have the advantage of infecting non-dividing cells. The most commonly studied lentiviral vectors are derived from either human immunodeficiency virus (HIV) or feline immunodeficiency virus (FIV), although equine anemia infectious virus and visna virus have also been examined [38]. The primary concern with HIV vectors is safety. In contrast, FIV is non-pathogenic in humans and does not cause serologic conversion [46]. Using an FIV vector, TNF-α was transduced into primary human FLS with high efficiency. When injected into knees of SCID mice, these cells induced cell proliferation and caused bone and joint destruction [47]. A replication-defective HIV vector encoding endostatin was injected into joints of TNF-α transgenic mice and was shown to decrease synovial blood vessel density and decrease the overall arthritis index [48]. Similarly, intra-articular expression of angiostatin inhibited the progression of CIA in mice [49]. A study examining a VSV-G pseudo-typed HIV vector, which has increased host range and stability, demonstrated that a transgene could be efficiently delivered to the synovium of rat knee joints, with transgene expression lasting up to 6 weeks in immunocompromised animals [50].
AAV
One of the most promising gene transfer vectors is AAV, which is a small, non-enveloped single-stranded DNA virus with broad tissue tropism. It belongs to the Parvoviridae family and has a 4.68kb genome. AAV normally requires adenovirus or herpesvirus to produce active infection. Several serotypes have been identified in primates, with AAV2 being the prototype for most gene transfer studies. Heparan sulfate proteoglycan has been identified as the primary attachment receptor for AAV, with fibroblast growth factor receptor 1 and integrin αvβ5 acting as co-receptors [51]. Although little is known about the details of AAV infection, some of the basic mechanisms have been described. The virus enters the cell by receptor-mediated endocytosis. Acidification of late endosomes leads to AAV release into the cytosol, with subsequent translocation of the virus to a perinuclear region [52–54]. The virus then enters the nucleus by an unknown mechanism that is independent of the nuclear pore complex [55]. Following uncoating, the single-stranded genome is converted to a double strand and the viral DNA integrates into chromosome 19 (AAVS1 locus) in a site-specific manner [56, 57].
AAV is an attractive vector for gene transfer studies for several reasons. It has been shown to deliver transgenes to a wide variety of tissues, has low immunogenicity, and mediates long-term gene expression [51]. Recombinant, replication incompetent AAV vectors have been designed that lack the Rep genes, which are required for integration, so long-term expression with these vectors is thought to be mediated by episomal viral DNA [58]. In addition, AAV vectors have been designed that are able to package double-stranded viral genomes, bypassing a rate-limiting step of viral transduction (second-strand synthesis) and allowing rapid and highly efficient transduction both in vitro and in vivo [59].
Several studies have demonstrated the efficacy of AAV vectors in arthritis models. Primary and recurrent arthritis were suppressed following a single injection of AAV encoding IL-1ra into knee joints of rats with LPS-induced arthritis. Surprisingly, disease-regulated expression of the transgene was observed [60]. Our laboratory has recently observed a similar phenomenon in in vitro cultured FLS infected with AAV in which inflammatory cytokines can increase transgene expression in these cells in a regulatable, PI3K-dependent manner. Protesosome inhibition has also been shown to enhance AAV transduction of human synoviocytes both in vitro and in vivo [61]. AAV encoding soluble TNF-α receptor type I decreased synovial cell hyperplasia and cartilage and bone destruction in human TNF-α transgenic mice injected intra-articularly with the virus [62]. Intra-articular or peri-articular delivery of AAV encoding IL-4 in CIA mice was also shown to decrease paw swelling, protect from cartilage destruction, and delay the onset of CIA [63, 64]. A vIL-10 transgene delivered by AAV under control of a tetracycline inducible promoter decreased the incidence and severity of CIA on a macroscopic, radiologic, and histologic level [65]. More recently, angiostatin, an anti-angiogenic molecule, was demonstrated to efficiently decrease development of CIA in the treated joint when delivered by AAV [66].
AAV vectors are in clinical trials for the treatment of cystic fibrosis and hemophilia B and preliminary results are promising [67]. Targeted Genetics Corporation (Seattle, WA) is currently conducting a phase I clinical trial (13G01; identifier NCT00126724) to assess the safety of using an AAV2 vector to deliver a soluble TNF receptor-Fc fusion gene in RA.
Gene transfer strategies
IL-1β inhibition
Several gene transfer strategies have been aimed at neutralizing the effects of IL-1β. IL-1β communicates with many different cell types in the joint. Its action on these cells leads to emigration of blood cells to the synovium, increased cartilage destruction, and increased production of other chemokines and pro-inflammatory mediators by macrophages, B cells, and T cells [7]. Neutralization of this key cytokine has proven beneficial in the treatment of RA. Several animal models support using gene transfer to block the effects of this cytokine. Primary and recurrent arthritis were suppressed following a single injection of AAV encoding IL-1ra into knee joints of rats with LPS-induced arthritis [60]. Adenoviral vectors encoding IL-1ra proved more effective than soluble type I TNF receptor-IgG fusion protein at reducing cartilage matrix degradation and decreasing leukocyte infiltration into the joint space of rabbits with antigen-induced arthritis [13]. Rabbit synovial fibroblasts modified ex vivo using a retrovirus encoding IL-1ra demonstrated a chondroprotective and mild anti-inflammatory effect when injected back into the joint after onset of antigen-induced arthritis [18]. This led to a phase I clinical trial using a retroviral vector to deliver IL-1ra to cultured human autologous synovial fibroblasts. The cells were then injected into RA patient joints. After a scheduled arthroplasty 1 week later, significant expression of the transgene was seen in the injected joints, and no adverse events were reported [17].
Intramuscular injection of plasmid DNA encoding IL-1ra has also been shown to decrease paw swelling and arthritis incidence in a CIA mouse model. Reduced synovitis and cartilage erosion were also seen [26, 68]. In a rat model of bacterial cell wall-induced arthritis, rat synoviocytes were modified ex vivo using a retroviral vector to express IL-1ra. When injected into ankle joints prior to reactivation of arthritis, a decreased severity of arthritis and attenuated destruction of cartilage and bone was observed [69]. In a SCID mouse model, human RA FLS transduced with retrovirus encoding IL-1ra co-implanted with normal human cartilage was able prevent progressive cartilage degradation compared to controls [70]. 3T3 mouse fibroblasts transfected with plasmid encoding IL-1ra were able to prevent the onset of CIA and cartilage destruction when injected into knee joints of CIA mice [12]. Similar effects on arthritis were seen in a rabbit model of antigen-induced arthritis in which a retroviral vector carrying the IL-1ra gene was used to transduce rabbit fibroblasts ex vivo, with subsequent injection of the cells into the knee joint [15]. More recently, the soluble form of interleukin-1 receptor accessory protein (sIL-1RAcP) was delivered to CIA mice either using an adenoviral vector or by injection of plasmid transfected 3T3 cells. In both instances, a profound prophylactic effect on the development of CIA was observed [71].
TNF-α inhibition
TNF-α is another pro-inflammatory cytokine that plays a key role in the pathogenesis of RA. Many of its effects overlap those of IL-1β listed above. Currently, therapies aimed at neutralizing this cytokine represent the most successful treatment strategies for RA. Current regimens involve receiving injections (etanercept) or infusions (infliximab) every 2 or 8 weeks, respectively. Gene transfer strategies have the potential to provide longer-term control and may also be delivered locally rather than systemically, potentially minimizing treatment side effects. Several studies in animal models have been performed. After one injection at onset of CIA, intramuscular electrotransfer of plasmids encoding soluble TNF receptor I variants led to a decrease in clinical and histological signs of CIA [24]. Expression lasted up to 5 weeks and was as least as efficient as repeated injections of the recombinant protein etanercept in controlling the disease. A similar study using a retroviral vector to deliver the transgene peri-articularly saw similar results and also observed a decrease in systemic levels of IgG2a antibodies to collagen type II [72].
Electrotransfer of a plasmid encoding a soluble p75 TNF receptor:Fc fusion protein was also beneficial in a CIA model and was associated with a decrease in the levels of IL-1β and IL-12 in the paw [25]. Other studies using electrotransfer or intramuscular injection of a plasmid with a doxycycline-regulated promoter to control expression of a dimeric soluble TNF receptor II molecule saw a therapeutic effect on CIA only when doxycycline was administered [73, 74]. In TNF-α transgenic mice, intra-articular delivery of soluble TNF receptor I by adeno-associated virus led to a decrease in synovial cell hyperplasia and cartilage and bone destruction [62]. Similarly, AAV5 encoding sTNFRI-Ig was also able to decrease paw swelling in a rat AIA model when expression was under control of an inflammation responsive promoter, but interestingly, not if expression was under control of the CMV promoter [75].
Splenocytes from arthritic DBA-1 mice can passively transfer collagen type II-induced arthritis when injected into SCID recipients. If these splenocytes were first modified ex vivo using retroviral vectors to express soluble p75 tumor necrosis factor receptor, the SCID recipients did not develop arthritis, bone erosion, or joint inflammation [76, 77]. Delivery of a rat TNF receptor:Fc fusion protein in a streptococcal cell wall-induced arthritis model by either plasmid or local or systemic administration of AAV vectors encoding the molecule led to decreased inflammation, pannus formation, bone and joint destruction, and mRNA expression of joint pro-inflammatory cytokines [21]. Adenoviral delivery of a soluble TNF receptor type I-IgG fusion protein directly to rabbit knees with antigen-induced arthritis reduced-cartilage matrix degradation and decreased leukocyte infiltration into the joint space, especially when administered in conjunction with a soluble IL-1 type I receptor-IgG fusion protein [13]. The above animal model data has led to the initiation of a phase I clinical trial using AAV vectors to deliver soluble TNF Receptor:Fc fusion protein (Targeted Genetics Corporation).
IL-18 inhibition
IL-18 is a pro-inflammatory cytokine that is overexpressed in the synovium of RA patients and correlates with inflammation. Elevated levels of this cytokine are also observed in serum and synovial fluid. Overexpression of an IL-18-binding protein using an adenoviral vector was able to ameliorate arthritis in a CIA model, indicating neutralization of IL-18 may be an effective target in the future treatment of RA [78].
Immune deviation
Previously, an imbalance between Th1 and Th2 cytokines was thought to play a role in the pathogenesis of several inflammatory diseases, including RA. Th1 cells secrete cytokines like IFN-γ that promote a pro-inflammatory environment, while Th2 cytokines secrete cytokines like IL-4, IL-10, and IL-13 that down-regulate Th1 activity. It was previously felt that overproduction of Th1 cytokines contributed significantly to the pathogenesis of RA. More recently, a new subset of T cells, termed Th17 cells, have been identified [79]. These cells produce, among other cytokines, IL-17, a pro-inflammatory cytokine previously implicated in the pathogenesis of CIA [80]. Dysregulation of Th17 cells and IL-17 overproduction has been implicated in the pathogenesis of inflammatory diseases and the development of severe autoimmunity. In fact, the pathogenesis of RA may be more directly related to an imbalance between Th17 cells and Foxp3-positive regulatory T cells than an imbalance between Th1 and Th2 cells [81]. Regardless of the exact mechanism, several strategies aimed at immune deviation have been successful in animal models of arthritis and are outlined below.
IL-13 inhibits activated monocytes/macrophages from secreting a variety of pro-inflammatory molecules. A possible role for this cytokine in the pathogenesis of RA was observed when adenoviral delivery of IL-13 to RA synovial tissues explants led to a decrease in IL-1β, TNF-α, IL-8, MCP-1, NAP-78, PGE2, and MIP-1α when compared to controls [82]. Subsequent studies demonstrated that adenoviral delivery of IL-13 directly to ankle joints in a rat antigen-induced arthritis model significantly decreased paw size, bony destruction, vascularization, inflammatory cell infiltration, and inflammatory cytokine production [83]. IL-13 overexpression during immune-complex-mediated arthritis significantly decreased chondrocyte death and MMP mediated cartilage destruction, despite the presence of enhanced inflammation [84].
Using IL-4 to skew the cytokine profile towards Th2, or perhaps by inhibiting Th17 cell production, has also proven successful. Adenoviral delivery of IL-4 to RA synovial tissue explants demonstrated decrease IL-1β, TNF-α, IL-8, MCP-1, and PGE2 in the cultured medium [85]. Intra-articular delivery of adenoviral vectors encoding IL-4 to CIA mice led to an enhanced onset of inflammation but less chondrocyte death and cartilage and bone erosion. Proteoglycan synthesis was enhanced and there was decreased MMP activity [86]. IL-17, IL-12, cathepsin K and osteoprotegrin ligand mRNA levels were also reduced [86, 87]. Kim et al. observed similar effects on CIA upon local and systemic administration of adenoviral vectors encoding IL-4 [88]. AAV-mediated delivery of IL-4 has also prove beneficial in CIA models [63, 64]. Electrotransfer of an IL-4 encoding plasmid prior to CIA onset decreased synovitis and cartilage destruction, with an associated decrease in IL-1β in the paw and an increased TIMP2:MMP2 ratio [29]. Similar results were also seen using either gene gun delivery or intra-dermal administration of plasmid encoding IL-4 [89]. Both retroviral and adenoviral delivery of IL-4 in a rat antigen-induced arthritis model had beneficial effects [90–92]. Cell-based therapies that deliver IL-4 in arthritis models have also been successful. Injection of fibroblast transfected with plasmid encoding IL-4 decreased histologic evidence of joint inflammation and destruction in a CIA model [93, 94]. Likewise, collagen type II pulsed antigen presenting cells engineered to secret IL-4 down-regulated CIA [95].
IL-10 is an anti-inflammatory cytokine that has demonstrated benefit in several animal models of RA. Either intramuscular administration via electrotransfer or intra-dermal injection of plasmid encoding IL-10 had beneficial effects on CIA [31, 35]. Similarly, systemic administration of a plasmid IL-10/liposome mixture decreased signs of CIA after a single intra-peritoneal injection [34]. Viral IL-10 (vIL-10) is homologous to human and mouse IL-10 but while retaining its immunosuppressive function, it lacks many of the immunostimulatory properties of IL-10, and therefore may be a superior treatment option. An adenoviral vector encoding vIL-10 was able to decrease CIA when delivered locally or systemically [14, 96, 97]. Electrotransfer of viral IL-10 decreased histologic evidence of arthritis in an arthrogen collagen-induced arthritis model and was associated with decreased TNF-α, IL-1β, and IL-6 transcripts in the joint [30]. A tet-inducible vIL-10 transgene delivered by AAV was able to decrease macroscopic, radiology, and histologic signs of CIA only when doxycycline was administered [65].
TGF-β is a pleiotropic cytokine with many different effects on many different cell types and has been suggested of playing a role in RA. While some of its effects, like immunosuppression, would appear to be beneficial for RA, it has also been associated with pro-inflammatory activity. Not surprisingly, data from animal models using gene transfer support both possibilities, making its true role in RA difficult to decipher. Splenocytes from CIA mice were isolated and infected ex vivo with a retroviral vector encoding TGF-β. These cells were then injected in the intra-peritoneal cavity 5 days after arthritis onset. Without TGF-β expression, an exacerbation of arthritis is normally observed. However, TGF-β expressing splenocytes were able to inhibit this exacerbation and also resulted in a decrease in MMP2 activity and a transient reduction in anti-collagen type II antibodies [98]. In a rat streptococcal cell wall-induced arthritis model, intramuscular delivery of plasmid encoding TGF-β showed significant decreases in inflammatory cell infiltration, pannus formation, bone and joint destruction, and inflammatory cytokine production [27]. In contrast to the above reports, another study found that injection of adenovirus encoding TGF-β into the knees of rabbits with antigen-induced arthritis resulted in significant pathology in the knee joint and surrounding tissue, suggesting that TGF-β therapy may not be suitable for treating arthritis in some models [99].
CTLA-4Ig fusion protein binds to the co-stimulatory molecules B7-1 and B7-2 present on antigen-presenting cells and blocks CD28/B7 interactions, resulting in decreased T cell activation. It has been shown to ameliorate several experimental autoimmune diseases, including CIA. A single intravenous injection of an adenovirus encoding CTLA-4Ig fusion protein suppressed established CIA as least as efficiently as repeated injections of monoclonal antibody to CTLA-4. Pathogenic cellular and humoral responses were also diminished in adenoviral vector treated group as compared to antibody treated and control groups [100]. CIA could also be inhibited both histologically and clinically by intra-articular administration of a low dose of adenovirus encoding CTLA-4Ig fusion protein [101].
Promoting apoptosis
One of the key features of RA is the increased cellularity of the synovial lining leading to pannus formation, which has been shown to contribute to cartilage invasiveness and bone destruction. Promoting synovial apoptosis has been suggested as a treatment strategy for RA. In a rabbit model of arthritis, intra-articular adenoviral delivery of TNF-related apoptosis-inducing ligand (TRAIL) was able to increase apoptosis in the synovial cell lining, decrease inflammatory cell infiltration, and promote new matrix deposition [102]. Mice injected with collagen type II-pulsed antigen presenting cells engineered to express TRAIL under control of a doxycycline inducible promoter decreased the incidence of CIA and infiltration of T cells in the joint in the presence of doxycycline. In situ TUNEL staining demonstrated TRAIL-induced apoptosis of activated T cells in the spleen [103]. Modulation of TRAIL receptor expression on RA synoviocytes has also been suggested as a gene therapy strategy for the treatment of RA [104].
Injection of adenovirus-expressing Fas ligand (FasL) into joints of CIA mice induced apoptosis and ameliorated CIA. IFN-γ production by collagen-specific T cells was also reduced [105]. Ex vivo modified T cells engineered to express FasL were injected into human RA synovial tissue that had been implanted in SCID mice. Analysis of the tissue following treatment demonstrated that synoviocytes and mononuclear cells present in the tissue had been eliminated by apoptosis through a Fas/FasL interaction [106]. Similar results were observed when adenovirus encoding FasL was injected directly into the implanted tissue [107]. Dendritic cells modified by adenoviral vectors to express FasL were able to suppress CIA when systemically injected and also demonstrated decreased IFN-γ production from spleen-derived lymphocytes and decreased T-cell proliferation in response to collagen stimulation [108]. Fas-associated death domain protein (FADD) also plays a key role in Fas-mediated apoptosis of synovial cells. It was found that adenoviral vectors expressing FADD could induce apoptosis in synoviocytes both in vitro and in vivo, suggesting that this strategy may be effective in the treatment of RA [109].
Anti-angiogenesis
Increased cellularity of the synovial lining is also associated with neo-vascularization in the local environment of the joint. This angiogenesis is necessary for the development and maintenance of the pannus and also provides nutrients required for the survival and proliferation of infiltrating inflammatory cells [110, 111]. A peptide targeted to the integrins present in the inflamed synovium and associated with angiogenesis was fused to an anti-apoptotic peptide. Systemic administration of this fusion peptide in a CIA model resulted in decreased clinical arthritis and increased apoptosis of synovial blood vessels [112]. 3T3 fibroblasts modified with retroviral vectors to express angiostatin were able to decrease pannus formation and cartilage erosion when injected into knee joints of mice with CIA. Arthritis-associated angiogenesis was also inhibited [113]. AAV and HIV vector-mediated delivery of angiostatin to CIA knee joints were similarly beneficial [49, 66]. The potent anti-angiogenic factor endostatin was able to decrease arthritis and reduce blood vessel density in a human TNF-transgenic mouse model of arthritis when delivered to knee joints using a lentiviral vector [48]. Tie2 has also been demonstrated to play a role in angiogenesis in arthritis. Adenoviral delivery of a soluble receptor for Tie2 resulted in a decreased incidence and severity of CIA, inhibition of angiogenesis, and was associated with decreased bone destruction that appeared to result from a decrease in RANKL [114]. VEGF is another angiogenic factor that promotes synovitis and bone destruction in arthritis. When soluble VEGF receptor I was administered via adenoviral vectors to CIA mice, disease activity was suppressed significantly [115]. Thrombospondins (TSP1 and 2) have also been shown to inhibit angiogenesis and also decrease pro-inflammatory cytokine production in animal models of arthritis [116, 117]. In addition, adenoviral gene transfer of a urokinase plasminogen inhibitor was also able to inhibit angiogenesis in a CIA model [118].
Targeting matrix degradation enzymes
Matrix metalloproteinases (MMPs) degrade extracellular matrix components and have been demonstrated to contribute to cartilage degradation in RA. Ribozymes and an antisense construct targeting the destruction of MMP-1 delivered to RA synovial fibroblasts via retroviral vectors decreased MMP-1 production and reduced the invasiveness of RA synovial fibroblasts in a SCID mouse model of RA, suggesting that this may be an effective approach to inhibiting cartilage destruction in RA [119, 120]. Another strategy that has proven successful is to increase the ratio of tissue inhibitors of matrix metalloproteinases (TIMPs) to MMPs. In a rat antigen-induced arthritis model, intramuscular injection of naked DNA encoding TIMP-4 completely abolished the development of the disease [32]. Likewise, adenoviral delivery of TIMP-1 and TIMP-3 to RA synovial fibroblasts significantly decreased their invasiveness both in vitro and in an in vivo SCID mouse model of RA. These molecules were found to act by both decreasing MMP production and reducing cell proliferation [121].
Targeting NFκB
The transcription factor NFκB plays a significant role in the activation of many cytokines that contribute to the pathogenesis of RA. Inhibition of this factor could lead to therapeutic benefit in RA. Injection of decoy oligodeoxynucleotides with high affinity for NFkB into ankle joints of CIA rats significantly decreased joint swelling and joint destruction. The levels of the pro-inflammatory cytokines TNF-α and IL-1β were also decreased in the treated joints [122]. Another group found that inhibiting NFκB in RA synovial fibroblasts using an adenovirus to deliver a dominant negative inhibitor of NFκB led to an increase in apoptosis upon stimulation with TNF-α. These cells are normally resistant to apoptosis when stimulated with TNF-α, suggesting that this strategy may be beneficial in the treatment of RA [123]. In adjuvant arthritis in rats, a dominant negative IkB kinase β (IKKβ) was used to inhibit the NFκB pathway. Delivering the molecule intra-articularly using an AAV5 vector resulted in significantly reduced paw swelling and decreased levels of IL-6 and TNF-α. Bone and cartilage destruction, as well as MMP-3 and TIMP-1 levels were unaffected. The same vector was also able to efficiently transduce ex vivo-cultured biopsies from joints of human RA patients. TNF-α-induced IL-6 production was significantly decreased in the ex vivo cultures receiving the vector encoding IKKβ [124].
Other strategies
Other molecules that have been demonstrated to play a role in arthritis using gene transfer in various in vitro or animal models are Csk, cathepsin L, fibronectin, galectin-1, p16INK4A, p21Cip1, SOCS3, soluble CR1, superoxide dismutase and catalase, Ras, and prothymosin α [28, 33, 125–136]. Vectors and genes used to successfully treat animal models of arthritis are summarized in Table 1.
Table 1Summary of vectors and genes used in animal models of arthritis AdenovirusAAVRetrovirusLentivirusPlasmid DNAGenes demonstrated to successfully treat arthritis in various animal modelsIL-1ra, sTNFRI-Ig fusion protein, sIL-1RacP IL-18-binding protein, IL-13, IL-4, vIL-10, CTLA4-Ig, TRAIL, Csk, IFN-β, p16INK4A, P21CIP1, prothymosin-α, VEGF receptor I, Tie2 soluble receptor, FADD, FasL, SOCS3, urokinase plasminogen inhibitor, TIMP-1, TIMP-3, Thromobospondin-1, dominant negative NFκB inhibitorIL-1ra, sTNFR-Ig, sTNFRI, sTNFR:Fc fusion protein, IL-4, IL-10, angiostatin, IKKβIL-1ra, sTNFRI variants, IL-4, TGF-β, angiostatin, soluble complement receptor I, superoxide dismutase, catalaseAngiostatin, endostatinIL-1ra, sTNFRI receptor variants, sTNFR:Fc fusion protein, sTNFRII, TGF-β, IL-4, IL-10, vIL-10, soluble complement receptor I, TIMP-4, fibronectin peptide, sIL-1RAcP
Safety concerns
As with most investigative therapies, one of the most important concerns with regards to gene transfer technologies is the issue of safety. While the FDA has outlined specific strict standards for clinical trial protocols for gene transfer studies, there have been several instances where the therapy has resulted in serious adverse events in patients enrolled in the studies. The most widely known is probably the September 1999 death of Jesse Gelsinger, who had a fatal systemic inflammatory response to adenoviral vector gene transfer [137]. Retroviral vectors have also resulted in insertional mutagenesis leading to a secondary malignancy [44]. Lentiviral vectors may have this potential as well as sharing a similar mechanism, although this has never been demonstrated in clinical studies. More recently, in July of 2007, Jolee Mohr, a patient enrolled in Targeted Genetics’ clinical trial for rheumatoid arthritis using AAV vectors, died several weeks following her second injection of the experimental treatment. The trial is currently suspended, and although the investigation into her death is still ongoing, initial results suggest she died of a systemic fungal infection that may be unrelated to the injected virus. This case and the final results of the investigation are likely to have a large impact on the future of gene therapy trials in the United States, as AAV has been heralded as one of the most promising vectors in the field, precisely because it has, until now, failed to reveal significant safety concerns in previous studies.
Future of gene therapy for arthritis
Much progress has been made in the past several years in the use of gene therapy for the treatment of arthritis. However, there are many obstacles that must be overcome in order for it to become a viable treatment option. Future studies will need to address improving targeted delivery of vectors, regulating transgene expression, obtaining long-term transgene expression, and improving the safety and efficacy of the vectors already in use before gene therapy becomes a viable clinical therapy for arthritis.
Even in light of the recent setback in clinical trials utilizing AAV vectors, the authors believe that the future of gene transfer for arthritis will rely heavily upon this vector. Once the safety issues have been clarified, these trials can hopefully move forward. Presently, AAV would seem to be the viral vector that has the best profile in terms of safety, efficacy, and level and length of transgene expression. Alternatively, strategies using siRNA technology or preventing the dysregulation of Th17 cells would appear to be emerging as treatment strategies that may one day outperform the current standards of care for rheumatoid arthritis.

gene therapy
gene transfer
rheumatoid arthritis
inflammation
cytokines


Mechanisms of progression of chronic kidney disease

Chronic kidney disease (CKD) occurs in all age groups, including children. Regardless of the underlying cause, CKD is characterized by progressive scarring that ultimately affects all structures of the kidney. The relentless progression of CKD is postulated to result from a self-perpetuating vicious cycle of fibrosis activated after initial injury. We will review possible mechanisms of progressive renal damage, including systemic and glomerular hypertension, various cytokines and growth factors, with special emphasis on the renin–angiotensin–aldosterone system (RAAS), podocyte loss, dyslipidemia and proteinuria. We will also discuss possible specific mechanisms of tubulointerstitial fibrosis that are not dependent on glomerulosclerosis, and possible underlying predispositions for CKD, such as genetic factors and low nephron number.
Introduction
Chronic kidney disease (CKD) occurs in all age groups, with an incidence in children between 1.5 per million and 3.0 per million. Renal developmental abnormalities (congenital abnormalities of the kidney and urinary tract, CAKUT) are the most common causes of CKD in children. Other diseases commonly underlying CKD in children include focal segmental glomerulosclerosis (FSGS), hemolytic uremic syndrome (HUS), immune complex diseases, and hereditary nephropathies, such as Alport’s disease [1]. The incidence of diabetes, especially type 2, is increasing in children. Although CKD secondary to diabetes usually does not develop until adulthood, early structural lesions of diabetic nephropathy start in childhood [2].
CKD shares a common appearance of glomerulosclerosis, vascular sclerosis and tubulointerstitial fibrosis, suggesting a common final pathway of progressive injury [3]. Adaptive changes in nephrons after initial injury are postulated ultimately to be maladaptive, eventually causing scarring and further nephron loss, thus perpetuating a vicious cycle that results in the end-stage kidney. We will review possible mechanisms of progressive renal damage, which include, but are not limited to, hemodynamic factors, the renin–angiotensin–aldosterone system (RAAS), various cytokines and growth factors, podocyte loss, dyslipidemia, proteinuria, specific mechanisms of tubulointerstitial fibrosis, and possible underlying predispositions for CKD, such as genetic factors and low nephron number.
Systemic and glomerular hypertension
Systemic hypertension often accompanies renal disease and may both result from, and contribute to, CKD. Progression of CKD is accelerated by hypertension, and control of blood pressure is key in the treatment of CKD. In addition, the glomerulus has a unique structure, with both an afferent and an efferent arteriole, which permits modulation of glomerular perfusion and pressure without corresponding systemic blood pressure change.
The remnant kidney model has been extensively studied to investigate CKD [4]. In this model, one kidney and infarction/removal of two-thirds of the remaining kidney (i.e. five-sixths nephrectomy) results in progressive hyperperfusion, hyperfiltration, hypertrophy and FSGS [4–6]. Additional models with initial podocyte injury, namely the puromycin aminonucleoside and adriamycin models of renal disease, show initial proteinuria and podocyte damage similar to human minimal-change disease, followed by progressive FSGS [7].
Direct micropuncture studies have demonstrated that single nephron function was increased after renal ablation, and led to the hypothesis that hyperfiltration caused sclerosis, setting in motion a vicious cycle of hyperfiltration and glomerulosclerosis [3, 8]. Maneuvers that decreased hyperfiltration, such as low-protein diet, angiotensin I converting enzyme inhibitors (ACEIs), lipid-lowering agents, or heparin, were, indeed, effective in ameliorating glomerular sclerosis. However, in some studies, glomerular sclerosis was decreased without altering glomerular hyperfiltration [9], and glomerular sclerosis occurred in some settings even in the absence of intervening hyperperfusion [10].
Thus, focus was shifted to glomerular hypertension as a key mediator of progressive sclerosis. Maneuvers that increase glomerular capillary pressure, such as therapy with erythropoietin, glucocorticoids, or high-protein diet, accelerated glomerulosclerosis, while decreasing glomerular pressure ameliorated sclerosis. These beneficial effects were particularly apparent in the comparison of agents such as ACEIs that preferentially decrease glomerular pressure even more than systemic BP to non-specific antihypertensive agents [11].
Renin–angiotensin–aldosterone system
The RAAS has been the focus of investigation of progression in CKD because of the efficacy of inhibition of its components in CKD. ACEIs decrease glomerular capillary pressure by preferential dilation of the efferent arteriole [1], likely mediated by both inhibition of angiotensin II (AngII) and especially by the effect of ACEIs in augmenting bradykinin, which is degraded by angiotensin I converting enzyme (ACE) [12]. Indeed, angiotensin type 1 receptor blockers (ARBs), which do not have this activity to increase bradykinin, do not preferentially dilate the efferent arteriole or decrease glomerular pressures to the extent of that seen with ACEIs in most experimental studies. However, both ACEIs and ARBs have shown superior efficacy in slowing progressive CKD in experimental models and in human CKD [13–16].
ARBs leave the angiotensin type 2 (AT2) receptor active, and may in theory even lead to augmented AT2 effects by allowing unbound AngII to bind to this receptor. The AT2 receptor counteracts some of the classic AT1 receptor actions and thus is mildly vasodilating and mediates growth inhibition and apoptosis [17–20]. Apoptosis often is associated with decreased injury, as injured cells are quickly removed without activation of profibrotic cytokines and chemokines. Absence of AT2 receptor actions, either by pharmacological inhibition or by genetic absence, indeed resulted in diminished apoptosis after injury, associated with increased fibrosis [21, 22].
Combined ACEI and AT1 receptor antagonist treatment could have a theoretic advantage, allowing further blockade of AngII actions while maintaining preferential local availability of the AT2 receptor [23]. In an experimental model, combined ACEI and ARB therapy did not result in added benefit on glomerulosclerosis when compared with single-drug therapy with similar blood pressure control [24, 25]. However, addition of AT2 receptor inhibition to ARB treatment prevented the beneficial effects of ARBs  [26]. A beneficial effect of the AT2 receptor in renal injury was also demonstrated in transgenic mice overexpressing the AT2 receptor. These mice developed less severe injury than did the wild type after subtotal nephrectomy [27]. Results from small clinical studies of human CKD suggest that the combination of ARBs and ACEIs has greater effect in the decrease of proteinuria, not attributable to effects on systemic blood pressure [28, 29]. In a large study of hypertensive patients with diabetic nephropathy and microalbuminuria, combined therapy resulted in greater reduction of  blood pressure and albuminuria than did therapy with either drug alone [30]. In a Japanese study, in addition to decreased proteinuria, the slope of decline of glomerular filtration rate (GFR) improved with combination ACEI and ARB versus monotherapy [31]. However, complete dose-range comparisons of combined therapy with monotherapy were not made in these clinical trials. A recent review of clinical trials with combination therapy with ACEI and ARB in CKD patients support that such combination therapy had increased effects to decrease proteinuria without significantly increasing adverse side effects [16].
Antifibrotic effects of combination therapy versus monotherapy could include augmented bradykinin and AT2 activity and also decreased urinary transforming growth factor (TGF)-β [32]. In addition, there may be greater suppression of the renin–angiotensin system (RAS) with combined therapy, decreasing both ligand generation by inhibition of ACE and binding of any remaining AngII to the AT1 receptor. However, even suprapharmacological doses of ACE inhibition did not achieve complete suppression of the local RAS in experimental models [33]. Similarly, patients receiving ACEIs long term still have measurable ACE in their plasma. These data support the notion that non-ACE-dependent AngII generation by chymotrypsin-sensitive generating enzyme occurs in humans. New directions under investigation include the development of renin antagonists that could obviate these obstacles to optimal inhibition of the RAAS. Renin itself may have direct effects, independent of activation of the RAAS, with renin receptor activity detected on mesangial cells [34].
Many profibrotic actions of the RAAS are mediated directly by AngII. AngII promotes migration of endothelial and vascular smooth muscle cells, and hypertrophy and hyperplasia of smooth muscle cells and mesangial cells [35, 36]. All components of the RAS are present in macrophages, which may thus serve as yet another source of AngII and also respond to ACEI and ARB. AngII also induces other growth factors, including basic fibroblast growth factor (basic FGF), platelet-derived growth factor (PDGF) and TGF-β, and plasminogen activator inhibitor-1 (PAI-1), all of which may impact on fibrosis (see below), [37–39].
Importantly, new data indicate that aldosterone has both genomic and non-genomic actions to promote fibrosis, independent of its actions to increase blood pressure by mediating salt retention [40, 41]. Aldosterone enhances angiotensin induction of PAI-1 (see below), and also has direct actions on fibrosis [40]. Conversely, aldosterone receptor antagonism with spironolactone decreased injury [40]. PAI-1 deficiency prevented aldosterone-induced glomerular injury, but interestingly did not alter cardiac or aortic injury in this mouse model, suggesting site-specific and perhaps species-specific mechanisms of aldosterone-PAI-1 mediated fibrosis [42]. In clinical trials, aldosterone antagonism has further decreased proteinuria when added to ACEI and ARB therapy [43, 44]. However, the potential risk of hyperkalemia may limit the ability to add aldosterone antagonism to angiotensin inhibition. Whether these approaches also apply to children with CKD has not been investigated.
Clearly, the RAAS has many non-hemodynamic actions and thus, doses beyond usual antihypertensive doses are potentially of additional benefit. Regression has even been achieved in experimental models with high-dose ACEI/ARB. A shift in the balance of synthesis/degradation of extracellular matrix (ECM) must occur to accomplish regression of sclerosis; endothelial cells must regenerate, mesangial cells must regrow, and finally, podocytes must be restored. New glomeruli cannot be generated after term birth in humans. However, remaining segments of non-sclerotic loops can give rise to more open capillary area by lengthening or branching of the remaining capillaries [45–48]. Recent experimental data show that regression can, indeed, be induced by high-dose ACEI or ARB or spironolactone, linked to decreased PAI-1, restored plasmin activity and capillary remodeling [25, 49–51]. Of note, regression was not associated with increased expression or activity of matrix metalloproteases-2 or -9 or decreased mRNA for TGF-β or local decreases in TGF-β expression as assessed by in situ hybridization. However, lack of changes in mRNA does not rule out that local changes in TGF-β actions could occur, and clearly, in many systems, TGF-β has been shown to impact on ECM accumulation. Regression is also possible in human CKD, demonstrated in principle by regression of early diabetic sclerosis and tubulointerstitial fibrosis in patients over a 10-year period when the underlying diabetes was cured by pancreas transplantation [52]. Regression of existing lesions also occurred in IgA nephropathy in response to high-dose corticosteroids and tonsillectomy [53].
Specific cytokines/growth factors and progression of CKD
Numerous cytokines/growth factors appear to modulate progression of glomerular and tubulointerstitial scarring. These factors and their roles may differ at the various stages of injury. Altered gene expressions and/or pharmacologic manipulations in pathophysiological settings have implicated e.g. PDGF, TGF-β, AngII, basic FGF, endothelin, various chemokines, peroxisome proliferator-activated receptor-γ (PPAR-γ) and PAI-1, among others, in progressive renal scarring [10, 54–56]. Current state-of-the-art approaches with proteomic and array analysis of renal tissue in human CKD and in animal models can identify novel targets and markers, and even mediators of progression [57, 58]. Of these many potential molecules of interest, we will discuss only a few that have been investigated in depth.
Increased PAI-1 is associated with increased cardiovascular disease and fibrotic kidney disease [59]. Conversely, PAI-1 could be decreased by inhibition of AngII and/or aldosterone, and linked to prevention of sclerosis or even regression of existing kidney fibrosis [25, 38, 51, 60]. AngII and aldosterone can also induce PAI-1 expression and subsequent fibrosis independent of TGF-β activation [61]. Some of the effects of PAI-1 in promoting fibrosis are independent of its effects on proteolysis. PAI-1 also modulates cell migration, perhaps by its effects on vitronectin interaction [59]. Thus, PAI-1 may in some inflammatory or interstitial disease settings increase fibrosis primarily by enhancing cell migration and epithelial-mesenchymal transition (EMT). In contrast, in the glomerulus, the effects of PAI-1 in the increase of sclerosis may predominantly be due to its ability to modulate ECM turnover [59]. These data support that mechanisms of fibrosis in the interstitium and glomerulus are not identical, and involve complex interactions of parenchymal and infiltrating cells and cytokines, with variable net effects on ECM accumulation.
TGF-β promotes ECM synthesis and is a key promoter of fibrosis. The biological actions of TGF-β are complex and depend not only on cell state, but also on the presence of decorin and latency-associated peptide (LAP), both of which can bind and modify its activity [37]. TGF-β also induces both PAI-1 and AngII [62]. Animals transgenic for TGF-β developed progressive renal disease [63]. Conversely, inhibition of either TGF-β or PDGF-B decreased mesangial matrix expansion in the anti-Thy1 model [64, 65]. Animals genetically deficient for TGF-β develop lymphoproliferative disease, thought to reflect a loss of TGF-β immune regulatory effect [66]. Interestingly, pharmacologic inhibition of TGF-β was more effective at lower dose, and with higher dose of anti-TGF-β associated with more fibrosis and greater macrophage influx, perhaps also reflecting effects on TGF-β immune modulation [67]. TGF-β may promote a more fibroblastic phenotype of the podocyte, with loss of differentiation markers and de novo expression of alpha-smooth muscle actin [68]. Although TGF-β promotes growth arrest and differentiation of podocytes at low doses, at higher doses, TGF-β causes podocyte apoptosis, mediated by Smad 7 signaling [69, 70]. Loss of podocytes (see below) is a key factor contributing to progressive kidney fibrosis.
PPAR-γ modifies numerous cytokines and growth factors, including PAI-1 and TGF-β. PPAR-γ is a transcription factor and a member of the steroid superfamily [71]. On activation, PPAR-γ binds the retinoic acid X receptor, translocates to the nucleus and binds to peroxisome proliferator activator response elements (PPREs) in selected target genes, modifying their expression. PPAR-γ agonists, such as the thiazolidinediones, are most commonly used to treat type 2 diabetes, due to their beneficial effects to increase insulin sensitivity and improve lipid metabolism, and they have been shown to decrease diabetic injury correspondingly in diabetic animal models [72]. Interestingly, PPAR-γ agonists also have antifibrotic effects in non-diabetic or non-hyperlipidemic experimental models of CKD. PPAR-γ agonist ameliorated the development of sclerosis in these non-diabetic models, linked to decreased PAI-1 and TGF-β and decreased infiltrating macrophages and protection of podocytes against injury [56, 73]. Further study is necessary to determine the specific role each of the above factors plays at varying stages of renal fibrosis.
Podocyte loss
Podocytes are the primary target in many glomerular diseases, including FSGS and the experimental models of adriamycin and puromycin aminonucleoside-induced nephropathies [74]. The podocytes are pivotal for maintenance of normal permselectivity, and are a source of matrix in both physiological and pathophysiological settings. The podocyte does not normally proliferate. Loss of podocytes after injury is postulated to be a key factor resulting in progressive sclerosis [74]. This principle was proven in experimental models in mice and rats, where podocyte-specific injury was produced by genetic manipulation of the podocytes to express toxin receptors only on this cell [75, 76]. Injection of toxin then resulted in podocyte loss, the degree of which depended on toxin dose. Animals subsequently developed progressive sclerosis. Of interest, even though only podocytes were initially injured, subsequent injury rapidly also developed in endothelial and mesangial cells, with resulting sclerosis. Even when chimeric mice were genetically engineered so that only a portion of their podocytes was susceptible to the toxin, all podocytes developed injury after toxin exposure [77]. These data show that injury can also spread from the initially injured podocyte to initially intact podocytes within a glomerulus, setting up a vicious cycle of progressive injury at the glomerular level [77].
The limited proliferation in the mature podocyte is accompanied by high expression of a cyclin-dependent kinase inhibitor, p27kip1, a rate-limiting step for the growth response of the podocyte [78]. Either too much or too little proliferation of the podocyte in response to genetic manipulation of p27kip1 is postulated be detrimental [79]. Inadequate growth of the podocyte is postulated to give rise to areas of dehiscence and insudation of plasma proteins, which progress to adhesions and sclerosis [80]. Another cyclin-dependent kinase inhibitor, p21, appears to be necessary for development of injury after five-sixths nephrectomy in mice, pointing to the crucial importance of cell growth responses in determining response to injury [81].
Podocytes normally produce an endogenous heparin-like substance, which inhibits mesangial cell growth; thus, injury may decrease this growth inhibitory effect and allow increased mesangial growth. Podocytes are also the main renal source of angiopoietin-1 and vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen that plays a key role in both physiologic and pathologic angiogenesis and vascular permeability [82]. Overexpression or partial loss of podocyte VEGF results in a collapsing lesion or pre-eclampsia-like endotheliosis lesion, respectively [82].
Podocyte genes and CKD
New studies of the molecular biology of the podocyte and identification of genes mutated in rare familial forms of FSGS and nephrotic syndrome, such as nephrin, WT-1, transient receptor potential cation channel-6 (TRPC-6), phospholipase C epsilon, α-actinin-4 and podocin, have given important new insights into mechanisms of progressive glomerulosclerosis. The gene mutated for congenital nephrotic syndrome, nephrin (NPHS1) is localized to the slit diaphragm of the podocyte and is tightly associated with CD2-associated protein (CD2AP) [83]. Nephrin functions as a zona occludens-type junction protein, and together with CD2AP, provides a crucial role in receptor patterning and cytoskeletal polarity and also provides signaling function of the slit diaphragm [84]. Mice with CD2AP knockout develop congenital nephrotic syndrome, similar to congenital nephrotic syndrome of Finnish type [85]. Autosomal dominant FSGS with adult onset is caused by mutation in α-actinin 4 (ACTN4) [86]. This is hypothesized to cause altered actin–cytoskeleton interaction, causing FSGS through a gain-of-function mechanism, in contrast to the loss-of-function mechanism implicated for disease caused by the nephrin mutation [85]. Patients with α-actinin 4 mutation progress to end-stage by age 30 years, with rare recurrence in a transplant. TRPC-6 encodes for a cation channel, which is present in several sites including podocytes. TRPC-6 is mutated in some kindreds with familial FSGS with adult onset in an autosomal dominant pattern [87]. Podocin, another podocyte-specific gene (NPHS2), is mutated in autosomal recessive FSGS with childhood onset with rapid progression to end-stage kidney disease [88]. Podocin interacts with the CD2AP-nephrin complex, indicating that podocin could serve in the structural organization of the slit diaphragm. In some series of steroid-resistant pediatric patients with non-familial forms of FSGS, a surprisingly high proportion, up to 25%, had podocin mutations [89, 90]. However, not all patients with nephrotic syndrome caused by mutation are steroid resistant. Diffuse mesangial sclerosis in a large kindred was recently linked to a truncating mutation of phospholipase C epsilon (PLCE1), and two of those patients responded to steroid therapy [91]. However, in two patients with missense mutation of this same gene, FSGS lesions developed, demonstrating that a spectrum of structural abnormalities may arise from varying mutations in the same gene. PLCE1 is expressed in the glomerulus, where it is postulated to play a key role in development, perhaps by interacting with other proteins that are crucial for the development and function of the slit diaphragm.
WT-1 mutation, which may occur sporadically with only FSGS or be associated with Denys–Drash syndrome, was found in only 5% of steroid-resistant patients [92]. Interestingly, mutations of podocin or WT-1 were not found in relapsing or steroid-dependent pediatric patients [93]. Acquired disruption or polymorphisms of some of these complexly interacting molecules have been demonstrated in experimental models and in human proteinuric diseases. Thus, in puromycin aminonucleoside nephropathy, a model of FSGS, nephrin localization and organization were altered [94]. Similar decreases in nephrin were observed in hypertensive diabetic rat models with significant proteinuria [95]. TRPC-6, a calcium channel, was induced in various non-genetic human proteinuric diseases [96]. Conversely, treatments that ameliorated these experimental models preserved e.g. glomerular nephrin expression, providing further support for a key causal role for slit diaphragm and key podocyte molecules in proteinuria [97]. Whether polymorphisms, compound heterozygosity for mutations or merely altered distribution and/or expression of any of these proteins contribute to proteinuria or progressive disease in various causes of CKD in humans has not been determined.
Dyslipidemia
Patients with CKD frequently have dyslipidemia and greatly increased cardiovascular disease risk, even beyond that predicted by lipid abnormalities [98]. Abnormal lipids are important in modulating glomerular sclerosis in rats; however, analogous studies in humans are still evolving [99–102]. Glomerular injury was increased in experimental CKD when excess cholesterol was added to the diets. Glomerular disease has been reported in the rare familial disease, lecithin cholesterol acyltransferase deficiency, and with excess apolipoprotein E. However, renal disease is not typical in the more common forms of primary hyperlipidemias. Patients with minimal-change disease or membranous glomerulonephritis, characterized by hyperlipidemia as part of their nephrotic syndrome, usually do not develop glomerular scarring. However, recently, post hoc and meta-analyses of clinical trial data support that abnormal lipids are associated with increased loss of GFR and that treatment with statins may not only benefit cardiovascular disease risk, but also be of benefit for progressive CKD. A post hoc analysis suggests that statins may even slow progression in patients with stage 3 CKD [102]. These beneficial effects of statins appear to extend beyond their lipid-lowering effects [98, 101].
Proteinuria
Proteinuria is a marker of renal injury, reflecting loss of normal permselecitvity. Further, proteinuria itself has been proposed to contribute to progressive renal injury inflammation [74, 103]. Increased proteinuria is associated with worse prognosis [104]. Whether proteinuria is merely a marker of injury or a contributor to progressive injury has been debated.
Albumin can in vitro in tubular cells increase AngII and in turn upregulate TGF-β receptor expression [105]. However, in most settings, pure albumin per se is not directly injurious. Other filtered components of the urine in proteinuric states, such as oxidized proteins, appear to be more potent in inducing direct injury of tubular epithelial cells and activating proinflammatory and fibrotic chemokines and cytokines. Complement and various lipoproteins are also present in the urine in proteinuric disease states and can activate reactive oxygen species [101, 106]. Proteinuria may thus alter tubule cell function directly, potentially contributing to a more profibrotic phenotype, and also augment interstitial inflammation, in particular by macrophages. Proteinuria may activate many profibrotic pathways through its ability to increase NF-kB, and also by other pathways. These include for instance complement synthesis from tubules [107].
Interventions that are particularly effective in decreasing proteinuria, such as the administration of ACEIs or ARBs, also decrease overall end organ injury. Whether these beneficial effects are dependent on the reduction of proteinuria has not been proven, in that these interventions have multiple parallel effects that may all contribute to the decrease of fibrosis [107].
Mechanisms of tubulointerstitial fibrosis
Tubulointerstitial fibrosis classically was thought merely to reflect glomerular injury and resulting whole nephron ischemia in most CKD. Interesting new data point to independent mechanisms of interstitial fibrosis and the importance of the tubulointerstitial lesion in progression. Decreased peritubular capillary density, possibly modulated by decreased VEGF or other angiogenic factors, has been proposed as a mechanism in various progressive renal diseases [108]. Future studies may demonstrate whether these interstitial microvascular lesions are causal or consequential in the development of interstitial injury.
Increased numbers of macrophages are closely correlated with both glomerulosclerosis and tubulointerstitial fibrosis and are usually decreased by interventions that decrease fibrosis. These cells are potential sources of numerous cytokines and eicosanoids that affect the glomerulus [109]. Support for this hypothesis is seen with the protective effects of maneuvers that decrease macrophage influx. In a rat model of unilateral ureteral obstruction (UUO), administration of ACEI ameliorated interstitial monocyte/macrophage infiltration and decreased fibrosis [110]. Studies in β6 integrin-deficient mice revealed that infiltrating macrophages do not inevitably transduce fibrotic effects; in these mice local activation of TGF-β is impaired, and they are protected from fibrosis despite abundant macrophage infiltration [61]. Macrophages may even play a beneficial role in scarring. The specific role of the macrophage AT1a receptor in renal fibrosis was examined in studies of bone marrow transplantation in wild type mice with UUO mice reconstituted with either wild type macrophages or macrophages devoid of the AT1a receptor. There was more severe interstitial fibrosis in mice with the AT1a deficient macrophages, even though fewer infiltrating macrophages were observed, suggesting that the macrophage AT1a receptor functions to protect the kidney from fibrogenesis [111].
In human diabetic nephropathy there is an early increase in total interstitial cell volume (which may represent increased cell size and/or number), preceding the accumulation of interstitial collagen [112]. This is in contrast to the diabetic glomerular lesion, where the expanded mesangial area is largely due to increased matrix accumulation rather than hypercellularity. These interstitial cells could possibly represent interstitial myofibroblasts, postulated to play a key role in interstitial fibrosis. These activated interstitial cells are a major source of collagen synthesis, and increased expression of α-smooth muscle actin (SMA), a marker of myofibroblasts, predicts progressive renal dysfunction both in human and experimental renal disease.
The source of interstitial myofibroblasts is a topic of controversy. Bone marrow-derived or potential renal stem cells may give rise not only to interstitial cells but also to regenerating parenchymal cells [113]. Epithelial–mesenchymal transformation (EMT) is another possible mechanism for generation of interstitial myofibroblasts [114]. This seamless plasticity of cells changing from epithelial to mesenchymal phenotypes exists during early development. EMT may also occur in the adult after injury, contributing approximately half of the interstitial fibroblasts in experimental models [114]. Injured tubular epithelial cells can change phenotype both in vivo and in vitro, with de novo expression of a fibroblast-specific protein (FSP1), and possibly migrate into the interstitium as myofibroblasts. The surrounding matrix and basement membrane underlying the tubular epithelium is disrupted by local proteolysis, modulated by an array of cytokines and growth factors, including insulin-like growth factors I and II, integrin-linked kinases, EGF, FGF-2 and TGF-β [114]. Several key factors inhibit EMT, including hepatocyte growth factor and bone morphogenetic factor-7, and thus inhibit fibrosis in experimental CKD [114].
Anatomic and genetic risks for CKD: nephron number and gene polymorphisms
Risk for development of CKD and its rate of progression varies in differing populations. CKD associated with hypertension and arterio-nephrosclerosis is particularly common in African Americans, and FSGS is more frequently the underlying cause of steroid-resistant FSGS in African Americans and Hispanics than in Caucasians [115, 116]. These varying disease trends in differing ethnic populations could represent both genetic and environmental factors. Low birth weight is epidemiologically linked to increased risk for cardiovascular disease, hypertension and CKD in adulthood. The link is postulated to be due to the decreased nephron number that accompanies low term birth weight, defined as less than 2,500 g [117, 118]. These fewer nephrons are postulated to be under greater hemodynamic stress, thus contributing to progressive sclerosis. Of interest, low birth weight is much more common in African Americans than in Caucasians and is not accounted for by socioeconomic status [119]. Further, glomerular size in normal African Americans is larger than in Caucasians and could possibly reflect smaller nephron number [120]. In Australian Aborigines, marked increase in incidence of CKD is associated with larger but fewer glomeruli and low birth weight [121, 122]. Mechanisms other than hemodynamic stress that could underlie these differences in normal glomerular populations and also relate to increased incidence of end-stage renal disease include functional polymorphisms of genes that are involved both in renal/glomerular development and contribute to amplified scarring mechanisms, such as the renin–angiotensin system [10].
African Americans also have increased severity of renal disease associated with several systemic conditions. The course of lupus nephritis in a prospective trial was more severe in African Americans than in Caucasians, with more extensive crescent formation and interstitial fibrosis and greater likelihood of end-stage renal disease [123]. Even the manifestations of HIV infection in the kidney differ markedly between African Americans and Caucasians: HIV-associated renal disease in African Americans is typically an aggressive collapsing type of FSGS, contrasting lower grade immune-complex-mediated glomerulonephritides in Caucasians with HIV infection and renal disease [124]. Genetic background also modulates susceptibility in experimental models, both to podocyte injury (e.g. only the balb/c mouse strain is susceptible to adriamycin) and to hypertension injury (e.g. in the five-sixths nephrectomy model, C57Bl mice are resistant, 129Sv/J mice are susceptible) and even to diabetic injury [125–127].
There is also accumulating evidence that specific genes in humans modulate the course and rate of organ damage. Polymorphisms in several genes within the RAAS system, including ACE, angiotensinogen and the angiotensin type 1 receptor, have been linked with cardiovascular and renal disorders, including diabetic nephropathy, IgA nephropathy and uropathies [128–133]. The ACE DD genotype, associated with increased RAS activity, was increased in patients with IgA nephropathy who ultimately experienced progressive decline in renal function during follow-up compared with those whose function remained stable over the same time [134].
Polymorphisms of TGF-β are also implicated in hypertension and progressive fibrosis. The Arg 25 polymorphism may be increased in African Americans, who may also have greater elevation of circulating TGF-β when they reach end-stage renal disease than do Caucasians [135].
These observations suggest that complex genetic traits can modulate the response of glomerular cells to pathogenic stimuli in experimental models. Whether ethnic differences in development of renal disease in humans reflect contributions of genetic and/or environmental influences remains to be definitively determined.
QUESTIONS (Answers appear following the reference list)
A 6-year-old African American boy presented with generalized edema, 24 h urine protein excretion of 1.5 g and normal complements, and serum creatinine of 0.7 mg/dl. His blood pressure was 110/70 mmHg. His nephrotic syndrome did not respond to an 8-week course of steroids, and a renal biopsy is planned. The most likely diagnosis in this patient is:
FSGS due to mutation of podocinMinimal-change diseaseFSGS, usual typeDiffuse mesangial sclerosisCollapsing glomerulopathyFor the same patient detailed in question 1, what additional treatment should be initiated at this time to decrease risk of CKD:
DiureticsSpironolactoneACEIsBeta blockersACEIs and ARBsIn the same patient detailed in the above questions, what parameters would be most important to follow and evaluate for adjustment of therapy:
EdemaWhite blood cell (WBC) countBlood pressureProteinuriaA 14-year-old Caucasian girl was diagnosed with IgA nephropathy, which on biopsy showed fibrocellular crescents, with focal proliferative and secondary sclerosing lesions of glomeruli. Her urine protein excretion was 1.0 g in 24 h. Urinalysis showed frequent red blood cell casts, serum creatinine was 1.2 mg/dl and her blood pressure was 120/93 mmHg. Which of the following mechanisms are likely to contribute to progression of her CKD:
Podocyte lossProteinuriaGlomerular hypertensionInfiltrating macrophagesAll of the aboveA 10-year-old Caucasian boy with a history of multiple episodes of steroid-dependent nephrotic syndrome since the age of 4 years now has proteinuria of 3.8 g in 24 h, with unremarkable urinalysis without red blood cell casts; his serum creatinine is 0.6 mg/dl, and his blood pressure is 98/64 mmHg. He has an increased cholesterol level of 480 mg/dl and triglyceride levels are 110 mg/dl. What mechanisms of renal injury are likely to be activated in this child:
podocyte lossproteinuriadyslipidemiaglomerular hypertension(b) and (c)

angiotensin i converting enzyme inhibitors (acei)