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Immunoglobulin A (IgA) nephropathy is recognized worldwide as the most common primary glomerulopathy. Although the mechanisms underlying the development of IgA nephropathy are gradually being clarified, their details remain unclear, and a radical cure for this condition has not yet been established. It has been clinically demonstrated that the immunoreactivities of intrarenal heme oxygenase-1 (HO-1) and 4-hydroxy-2-nonenal (4-HNE) — markers of reactive oxygen species (ROS) — and those of intrarenal angiotensinogen (AGT) and angiotensin II (Ang II) — markers of renin angiotensin system (RAS) — in IgA nephropathy patients were significantly increased as compared to those of control subjects. In an animal study, high IgA of ddY (HIGA) mice were used as an IgA nephropathy model and compared with BALB/c mice, which served as the control. The levels of markers for ROS (urinary 8-isoprostane and intrarenal 4-HNE), RAS (intrarenal AGT and Ang II), and renal damage in the HIGA mice were significantly increased as compared to those in the BALB/c mice. Moreover, an interventional study using HIGA mice demonstrated that the expressions of 2 lines of intrarenal ROS markers (4-HNE and HO-1), 2 lines of intrarenal RAS markers (AGT and Ang II) and renal damage decreased significantly in HIGA mice receiving treatment with the Ang II receptor blocker olmesartan but not in HIGA mice receiving treatment with RAS-independent antihypertensive drugs (hydralazine, reserpine, and hydrochlorothiazide) when compared with HIGA mice that were not treated.
These data suggest that intrarenal ROS and RAS activation plays a pivotal role in the development of IgA nephropathy.
Immunoglobulin A (IgA) nephropathy is characterized by the predominant deposition of IgA in the glomerular mesangium.1
IgA nephropathy is the most common form of primary glomerulopathy among races in Europe,2–4 Asia,5, 6 and Australia.7 Nair et al.8 have demonstrated that the incidence of IgA nephropathy has increased and that in the USA, individuals with this condition account for a large proportion of young adults (20–39 years old) with primary glomerulopathy.
The clinical course of IgA nephropathy is well established. Floege et al.9 demonstrated that approximately 40% to 50% of patients with IgA nephropathy present with recurrent macroscopic hematuria, which usually coincides with mucosal infection or exercise. Nephrotic syndrome is unusual, and is observed in only 5% of all IgA nephropathy cases. Acute renal insufficiency is uncommon in IgA nephropathy; it occurs in only 5% of all IgA nephropathy cases. IgA nephropathy has the potential for causing slow progressive chronic renal impairment and, eventually, end-stage renal disease (ESRD). In any published cohort study, it has been found that renal replacement therapy is required in approximately 25% to 30% of all cases within 20–25 years of presentation with IgA nephropathy. On the basis of initial symptoms, it has been estimated that 1.5% of patients with IgA nephropathy develop ESRD per year.1
The mechanisms underlying the pathogenesis and development of IgA nephropathy are gradually being clarified.
It has been reported that functional abnormalities in B and/or T cells and the aberrantly glycosylated IgA1 underlie the pathogenesis of IgA nephropathy.10, 11 Allen et al.12 reported that B-cell-restricted reduction of β-1.3 galactosyltransferase activity (responsible for the aberrant O-glycosylated serum IgA1) may cause IgA nephropathy, while Hurtado et al.13 reported that Th1/Th2 imbalance, especially for a predominant Th2 immune response, may play an important role in IgA nephropathy. When IgA immune complexes and polymeric IgA are deposited in mesangial cells through nonspecific size-dependent mesangial trapping by the O-glycosylation defect, mesangial cells are capable of local complement activation using endogenously generated C3.1 As a result, a proinflammatory and profibrotic phenotypic transformation is induced in mesangial cells.14 The transformed mesangial cells upregulate the secretion of some factors that induce the production of extracellular matrix components and cell proliferation; transforming growth factor-(TGF)-β, platelet-derived growth factor (PDGF), interleukin-1-β, interleukin-6, tumor necrosis factor (TNF)-α, and monocyte chemotactic protein-1 (MCP-1).1 Macrophages are recruited by MCP-1, and patients with IgA nephropathy manifest an abnormal increase in the number of macrophages infiltrating the glomeruli.15, 16 Macrophages are also capable of producing several growth factors, such as PDGF, interlukin-1, and epidermal growth factor,17 and they play a major role in the pathogenesis and progression of IgA nephropathy.
The detailed mechanism underlying IgA nephropathy remains unclear, and a radical cure for this condition has not yet been established. However, recently, there has been progress in clarifying its etiology and establishing a radical cure on the basis of the levels of markers of intrarenal reactive oxygen species (ROS) and renin angiotensin system (RAS).
There are some clinical evidences indicating that intrarenal ROS and RAS are associated with renal damage. In this section, we discuss the evidences obtained for chronic kidney diseases (CKDs), including IgA nephropathy.
In diabetic nephropathy, hyperglycemia is the primary determinant of the initiation and progression of renal injury, and it not only generates more reactive oxygen metabolites but also attenuates antioxidative mechanisms via non-enzymatic glycation of oxidant enzymes.18 Hyperglycemia also results in the production of advanced glycation end products (AGEs). AGEs are heterogeneous cross-linked sugar-derived proteins that may accumulate in the glomerular basement membrane, mesangial cells, endothelial cells, and podocytes in diabetic and/or ESRD patients. AGEs are thought to be involved in the pathogenesis of diabetic nephropathy via multi-factorial mechanisms such as oxidative stress generation and overproduction of various growth factors and cytokines.19 It has been reported that TNF-α-stimulated superoxide production and N-formyl-methionyl-leucylphenylalanine-stimulated aggregation were significantly higher in diabetic patients with triopathy than in those without triopathy; further, the greater the number of diabetic complications, the higher the amount of TNF-α-stimulated superoxide produced by polymorphonuclear leukocytes.20 RENAAL Study was a double-blind, placebo-controlled trial in which 1 513 patients with type 2 diabetic nephropathy were examined to evaluate the renal protective effects of losartan, which is an Ang II type 1 receptor blocker (ARB). In this study, it was found that losartan conferred significant renal benefits in patients with type 2 diabetic nephropathy, suggesting that the activated RAS plays a role in the development and progression of diabetic nephropathy.21
These results were obtained by the examination of samples from diabetic nephropathy patients and the clinical study for diabetic nephropathy patients, and they indicated that intrarenal ROS and RAS play an important role in the pathogenesis and development of diabetic nephropathy.
The activation of intrarenal ROS and RAS is also associated with histological damage in hemodialysis patients.
It has been reported that the combination of hypertension and oxidative stress induced by the stimulation of the RAS results in the accelerated progression of atherosclerosis in hemodialysis patients.22 Kadowaki et al.23 reported that when 6 hypertensive hemodialysis patients were treated with olmesartan, an ARB, the ratio of oxidized albumin to unoxidized albumin decreased markedly after 4 weeks, and these low levels were maintained at 8 weeks. These data suggest that intrarenal ROS and RAS are activated and that their upregulation plays a role in the development of disease status in hemodialysis patients.
It has also been reported that renal damage in IgA nephropathy patients is associated with the increase in the expression levels of intrarenal ROS and RAS markers.
Nakamura et al.24 indicated that the urinary levels of 8-hydroxydeoxyguanosine (8-OHdG), a sensitive biomarker of oxidative DNA damage and oxidative stress,25 are higher in patients with IgA nephropathy than in age- and gender-matched healthy controls. They also indicated that these levels were correlated with the urinary levels of liver-type fatty acid-binding protein, which increase with deteriorating kidney function, and are useful clinical biomarkers.26 These data suggest that the expression of activated ROS is associated with renal dysfunction in IgA nephropathy. ROS is thought to be activated in IgA nephropathy patients because in renal biopsy specimens obtained from patients with IgA nephropathy and non-IgA mesangial proliferative glomerulonephritis, both the protein and mRNA expressions of the copper and zinc forms of superoxide dismutase (SOD) are lower in moderately or severely damaged tissues than in normal or mildly damaged tissues. Further, these reduced levels of SOD activity may suppress the superoxide-scavenging reaction, thus rendering the tissue more vulnerable to oxidative stress.27
Takamatsu et al.28 reported that activated RAS is associated with histological damage in IgA nephropathy. The expression of AGT protein by glomerular endothelial cells and mesangial cells is higher in nephritic glomeruli in IgA nephropathy than in glomeruli in minor glomerular abnormalities. Additionally, a study of renal biopsy samples showed that the levels of glomerular AGT protein are well correlated with those of glomerular Ang II, TGF-β, α-smooth-muscle actin, glomerular cell number, and glomerulosclerosis.
In clinical studies, the blockade of the RAS was performed in IgA nephropathy patients. For example, a multicenter, randomized, placebo-controlled study was performed on 39 IgA nephropathy patients with the aim of comparing the effects of the angiotensin-converting enzyme (ACE) inhibitor fosinopril and placebo on proteinuria.29 The mean basal values of proteinuria remained unchanged during the placebo sequence (1.79±1.20 g/24 h) and reduced to 1.37±0.98 g/24 h after 4 months of fosinopril treatment. In various clinical studies and Maschio’s study, the symptoms of IgA nephropathy were successfully alleviated, suggesting that the activated RAS plays a role in the development and progression of IgA nephropathy.29–31
More recently, 39 IgA nephropathy patients were recruited in a study and compared with the 5 control patients.32 The levels of hemeoxygenase (HO)-1 and 4-hydroxy-2-nonenal (4-HNE) were examined as ROS markers in the study. HO-1 is an inducible form of HO, which is a rate-limiting enzyme involved in heme catabolism. Activated HO-1 is considered to be involved in antioxidant and anti-inflammatory defense mechanisms via the degradation of cellular heme (pro-oxidant) and elevation of concentrations of the antioxidant biliverdin.33 4-HNE is the main aldehydic product of lipid peroxidation, and it has been suggested to play an important role in tissue toxicity associated with lipid peroxidation.34 It has been shown that the immunoreactivity of intrarenal HO-1 in tubules (Figure 1; A to C) and intrarenal 4-HNE accumulation (Figure 1; D to F) in IgA nephropathy patients were significantly increased as compared with that in the control patients.32 The immunoreactivity of intrarenal AGT and Ang II — RAS markers — in the IgA nephropathy patients was compared with that in the control patients.32 AGT was predominantly localized in the proximal tubular cells in the kidneys of both the control patients and IgA nephropathy patients, and it was also expressed in the glomerulus in IgA nephropathy patients. Ang II was localized in tubules. The immunoreactivity of intrarenal AGT (Figure 1; G to I) and Ang II (Figure 1; J to L) in the kidneys of IgA nephropathy patients was significantly increased as compared with that in the normal kidneys. Moreover, the correlation between the immunoreactivity of AGT and individual clinical data was examined.32 It was observed that the immunoreactivity of AGT was significantly correlated positively with urinary occult blood (r=0.81), urinary protein-to-creatinine ratio (r=0.72), urinary protein excretion (r=0.69), and serum creatinine (r=0.54), and that it was also significantly correlated negatively with creatinine clearance (r=−0.57).
These data indicate that intrarenal ROS and RAS expression levels increased and that they are associated with the pathogenesis and development of some CKDs, including IgA nephropathy.
Some reports discuss not only human diabetic nephropathy but also indicate the relationships between intrarenal ROS and RAS activation and renal damage in some diabetic nephropathy models in vivo and the contribution of hyperglycemia to ROS and RAS activation in vitro.
Namikoshi et al.35 performed an experiment using Zucker diabetic fatty (ZDF) obese rats, type II diabetic nephropathy rats. They observed that 4-HNE accumulated in the glomeruli and that the expression of the mRNA of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components (p22 phox and p47 phox) was upregulated in the renal cortex. They also found that these events were inhibited by olmesartan, an ARB. Miyata et al.36 indicated that the intrarenal ROS-RAS axis plays a pivotal role in the progression of diabetic nephropathy model as follows: after an increase in the urinary levels of 8-isoprostane — the major urinary metabolite of F2-isoprostanes, which is formed non-enzymatically by the action of superoxide radicals on arachidonic acid and used as a marker of oxidative stress 37 — the intrarenal levels of AGT and Ang II were increased in ZDF obese rats as compared with the controls; this increase in the intrarenal levels of ROS and RAS markers caused glomerular and tubulointerstitial damages in ZDF obese rats.
The spontaneous hypertensive/NIH-corpulent rat (SHR/NDmcr-cp) is a genetic model of obesity and diabetes and the strain exhibits both metabolic and histopathological characteristics associated with non-insulin-dependent diabetes mellitus in humans. It has been reported that when olmesartan was administered to the SHR/NDmcr-cp rats, the blood pressure and kidney pentosidine content, which is a marker of AGE, were significantly reduced, and the histological renal damage and proteinuria were improved.38 It has also been demonstrated that when olmesartan was administered to SHR/NDmcr-cp rats for 20 weeks, it significantly reduced proteinuria and prevented glomerular and tubulointerstitial damage (mesangial activation, podocyte injury, tubulointerstitial injury, and inflammatory cell infiltration).39 In addition, abnormal iron deposition in the interstitium, expressions of HO and p47phox (a subunit of NADPH oxidase), and pentosidine formation were increased in SHR/NDmcr-cp rats as compared to those in the control, and they were suppressed by olmesartan treatment.
These observations suggest that ROS and RAS may contribute to the pathogenesis and progression of diabetic nephropathy.
In in vitro studies, Vidotti et al.40 found that in primary rat mesangial cells, prorenin secretion was decreased and intracellular renin activity was significantly increased due to high glucose exposure. They also demonstrated that the mRNA levels of AGT and ACE were significantly elevated by exposure to high levels of glucose for 72 h, and that the mRNA levels of prorenin and cathepsin B as well as ACE were significantly increased by exposure to high levels of glucose for 24 h. As a result of these changes, mesangial Ang II generation is increased by high levels of glucose. Singh et al.41 reported that in primary rat mesangial cells, incubation of cells for 24 h at high glucose concentrations increased AGT levels by 1.5-fold, as revealed by ELISA, and increased AGT mRNA expression, as seen in a Northern blot analysis. They also reported that a significant increase in the formation of Ang II from angiotensin I (Ang I) and angiotensin 1–9 ([Ang (1–9)]) was stimulated by high glucose concentrations. These findings demonstrate that glucose increases mesangial Ang II levels via an increase in AGT, Ang I, and Ang (1–9). Hsieh et al.42 demonstrated that the stimulatory action of high glucose levels on the AGT gene expression in immortalized renal proximal tubular cells is mediated, at least in part via ROS generation and subsequent p38 mitogen-activated protein kinase (MAPK) activation. It has been shown that in vitro, the overexpression of catalase prevents the stimulation of ROS and AGT mRNA in proximal tubular cells of mice owing to elevated glucose or Ang II.43
These in vitro data indicate that high glucose concentrations increase ROS and the RAS components, including AGT in some component cells in the kidney.
Experiments using some hypertensive animal models indicate the relationships between intrarenal ROS and/or RAS activation are associated with renal damage.
It has been revealed that urinary excretion of thiobarbituric acid reactive substances, a lipid peroxidation marker, and kidney AGT were significantly increased in the Dahl salt-sensitive hypertensive rats (DS) and high salt group as compared to those in the DS and low salt group. Further, tempol (superoxide dismutase mimetic) treatment prevented these upregulations.44 The evidence suggests that the ROS-dependent enhancement of AGT may be involved in the development and progression of renal injury observed in DS.
It has been shown that intrarenal AGT mRNA and protein levels and 6 parameters of renal damage (1. urinary excretion rate of total protein; 2. glomerular sclerosis; 3. interstitial expansion; 4 and 5 numbers of monocyte/macrophages in interstitium or glomeruli, and 6 arterial proliferation) changed in parallel, and that treatment with ARB, but not hydralazine, reserpine, and hydrochlorothiazide (HRH), prevented these increases in spontaneous hypertensive rats (SHR).45 These results indicate that SHR have enhanced intrarenal AGT production that contributes to increased Ang II levels, leading to the development of hypertension and renal injury.
Ingelfinger et al.48 reported that in in vitro studies, Ang II upregulates the expression of AGT and Ang II type 1 and type 2 receptors in a line of origin-defective SV40 plasmid transformed immortalized rat proximal tubular cells, and that this upregulation is prevented by losartan, an ARB. These data demonstrate that increase in AGT levels by Ang II infusion is AT1-dependent and that the Ang II-AGT positive-feedback loop was observed in both in vivo and in vitro studies. In addition, it is well known that Ang II stimulates NADPH oxidase O2-production by neutrophils,49 vascular smooth muscle cells,50 and adventitial fibroblasts 51, 52 and increases vascular mRNA levels of p22phox and p67phox,52, 53 which in turn contributes to endothelial dysfunction and vascular inflammation.54
Anti-thymocyte serum (ATS) nephritis rat is the model of mesangial proliferative glomerulonephritis that IgA nephropathy represents. It is known that acute self-limiting ATS nephritis is induced by a single ATS injection, and chronic progressive ATS nephritis by multiple ATS injections.55, 56 Using this acute self-limiting ATS nephritis model, it was shown that the levels of glomerular AGT mRNA and intrarenal Ang II and mesangial proliferation with transient proteinuria in the ATS-treated rats (ATS group) were significantly increased as compared to those in the control rats. In addition, the levels of renal lesions, proteinuria, and intrarenal RAS activity in ATS rats treated by olmesartan were significantly decreased as compared to those in the ATS group.57 These data suggest that an increase in kidney-specific RAS activity plays an important role in the development of ATS nephritis.
High IgA of ddY (HIGA) mice are an inbred strain with the high serum IgA level of ddY mice, which are an outbred strain with IgA nephropathy-like characters.58 HIGA mice are used as an IgA nephropathy model, because HIGA mice exhibit high serum IgA levels from a young age and IgA deposition in mesangial areas, proliferation of mesangial cells, and high urinary protein excretion, as is also observed in IgA nephropathy patients, although these mice do not develop hematuria, which is one of the characteristics of IgA nephropathy.59
The association between renal damage and alterations in ROS and the RAS were examined using HIGA mice, with BALB/c mice as the control mice.60
The urinary levels of 8-isoprostane and intrarenal 4-HNE, the markers for ROS were examined. From 21 to 25 weeks, a gradual increase was observed in the urinary excretion of 8-isoprostane in the HIGA mice as compared with the observations in the control mice, and the difference became significant at week 25 (Figure 2). The accumulation of renal 4-HNE was significantly greater in the HIGA mice as compared to that in the control mice at week 25 (Figure 3).
Intrarenal AGT and Ang II levels were evaluated at week 25. AGT immunoreactivity (Figure 4A, B) and intrarenal AGT protein expression levels determined by western blot (Figure 4C, D) were significantly greater in the HIGA mice than those in the control mice. Ang II immunoreactivity was detected mainly in some distal tubular cells, and some proximal tubular cells demonstrated weak Ang II immunoreactivity in the control mice. In the HIGA mice, the Ang II staining significantly increased in the proximal and distal tubular cells (Figure 4; E, F).
Desmin is a well known marker that indicates the increase in immunoreactivity in the mesangial cells and podocytes of mice with increasing renal damage.61 At week 25, weak immunoreactivity for desmin was noted in some podocytes and mesangial cells in the control mice, while in the HIGA mice, desmin staining was significantly increased in the podocytes and mesangial cells (Figure 4; G, H). Persistently increasing proteinuria was noted in the HIGA mice from 21 to 25 weeks, and the differences observed from week 24 were significant as compared with the proteinuria in the control mice (Figure 5).
Moreover, HIGA mice were subdivided into 3 groups for the purpose of interventional study: those that were not treated (HIGA and null group), those that received long-term treatment with olmesartan (which has been reported to exhibit antioxidant activity as well as to exert inhibitory effects on Ang II) (HIGA and OLM group),23, 35, 39, 62 and those that received long-term treatment with RAS-independent antihypertensive drugs (hydralazine, reserpine, and hydrochlorothiazide; [HIGA and HRH group]).63
The accumulation of renal 4-HNE, as determined by western blot, was significantly lower in the HIGA and OLM group but not in the HIGA and HRH group, when compared with the HIGA and null group at week 25. In addition, immunohistochemistry for HO-1, which was performed on the tissues of the HIGA and null group mice, revealed that this marker was distributed in both the proximal and distal tubules of the kidneys, and that the intensity of HO-1 staining in the tubular cells was significantly lower in the HIGA and OLM group but not in the HIGA and HRH group when compared to the intensity in the HIGA and null group.
The AGT protein expression levels determined by performing western blot were significantly lower in the HIGA and OLM group but not in the HIGA and HRH group when compared to the levels in the HIGA and null group at week 25. Although the Ang II immunoreactivity signals were distributed in the proximal and distal tubular cells in the HIGA and null group at week 25, olmesartan treatment significantly decreased the immunoreactivity signals that were largely limited to the distal tubular cells. Significant differences were noted between the HIGA and null and HIGA and OLM groups, while no significant differences were observed between the HIGA and null and HIGA and HRH groups.
In addition, it has been demonstrated that the immunoreactivity for desmin decreased significantly in the HIGA+OLM group but not in the HIGA+HRH group when compared with the immunoreactivity in the HIGA+null group.
These data indicate that increases in intrarenal ROS and intrarenal RAS expression levels are associated with the pathogenesis and development of nephropathy in HIGA mice.
The mechanisms underlying the pathogenesis and development of IgA nephropathy, which is the most common primary glomerulopathy, remain unclear. However, it is gradually being clarified that intrarenal ROS and RAS activation plays a pivotal role in the pathogenesis and development of IgA nephropathy.