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BASIC SCIENCE |
*Danielle Alberti Memorial Centre for Diabetes Complications, Vascular Division, Wynn Domain, Baker Heart Research Institute, Melbourne, Victoria, Australia; and Department of Medicine, Central and Eastern Clinical School, Monash University, Alfred Hospital, Melbourne, Australia
Correspondence to Dr. Karin A.M. Jandeleit-Dahm, Baker Heart Research Institute, PO Box 6492, Commercial Road, Melbourne 8008, VIC 3004, Australia. Phone: +61-3-9276-2983; Fax: +61-3-8532-1288; E-mail: karin.jandeleit-dahm{at}baker.edu.au
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Abstract |
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1 (TGF-1), increased 
-smooth muscle actin immunostaining and macrophage infiltration, and increased serum and renal AGE. The two treatments, which attenuated renal AGE accumulation in a disparate manner, were associated with less albuminuria, structural injury, macrophage infiltration, TGF-1, and collagen expression. The accelerated renal injury that was observed in diabetic apo E-KO mice was attenuated by approaches that inhibit renal AGE accumulation. |
Introduction |
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Diabetes is characterized by chronic hyperglycemia, which accelerates the reaction between glucose and proteins, lipids, and nucleic acids and leads to the formation of advanced glycation end products (AGE). These chemically heterogeneous compounds have a wide range of chemical, cellular, and tissue effects implicated in the development and progression of diabetic nephropathy (8–10). Renal AGE accumulation can be blocked by inhibiting AGE formation with agents such as aminoguanidine or by cleavage of preformed AGE with a cross-link breaker such as ALT-711 [4,5-dimethyl-3-(2-oxo2-phenylethyl)-thiazolium chloride]. Such interventions that reduce renal AGE accumulation have been shown to confer renoprotection in the context of diabetes (8,11).
AGE mediate their effects through a number of different pathways (8). The formation of inter- and intramolecular cross-links after AGE modification leads to functional and structural alterations of renal proteins in the kidney. AGE also directly interact with a variety of receptors in the kidneys, which may lead to activation of several inflammatory pathways and growth factors such as TGF-1 (12,13).
An earlier study using diabetic apo E-KO mice reported that relatively short-term diabetes (6 wk) resulted in increased renal expression of the receptor for AGE (RAGE) and infiltration of inflammatory cells (14). Soluble RAGE administration reduced infiltration of inflammatory cell into the kidneys from diabetic animals, although detailed functional and structural effects were not described (14).
The aim of the present study was to characterize renal injury in a model combining long-term diabetes and hyperlipidemia, the diabetic apo E-KO mouse. It was hypothesized that this model would demonstrate accelerated renal injury and that AGE would play a major role in promoting renal injury in this model. The present study used two disparate approaches to inhibit renal AGE accumulation in this model: an inhibitor of AGE formation, aminoguanidine, and the putative cross-link breaker ALT-711, which cleaves preformed AGE (15).
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Materials and Methods |
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After 20 wk, the animals were anesthetized by an intraperitoneal injection of pentobarbitone sodium (55 mg/kg body wt; Nembutal;, Boehringer Ingelheim, Artarmon, NSW, Australia). Blood was collected from the left ventricle and centrifuged, and plasma and red blood cells were stored at –20°C and at 4°C, respectively, for subsequent analyses. Glycated hemoglobin (HbA1c) was determined in lysates of red blood cells by HPLC (Bio-Rad, Richmond, CA). Total cholesterol and triglyceride concentrations were measured by autoanalyzer (Hitachi 917, Tokyo, Japan). The mouse kidneys were rapidly dissected and snap-frozen in liquid nitrogen and stored at –80°C or stored in buffered formalin (10%) for subsequent immunohistochemical studies.
Assessment of Renal Function
At week 20, the animals were housed in metabolic cages for 24 h for collection of urine samples for the later measurements of albumin concentration by RIA. This assay is a modification of a previously established method for measuring rat albumin in our laboratory (18). In brief, a rabbit anti-mouse albumin antibody (Cappel, Aurora, OH) was used followed by a second antibody, a sheep anti-rabbit antibody (ProSearch, Melbourne, Australia). Plasma urea and creatinine concentrations at the end of the study were measured by autoanalyzer (Beckman Instruments, Fullerton, CA).
Evaluation of Morphologic Changes
Two-micrometer-thick cross-sections of the kidneys were prepared and stained with periodic acid-Schiff to evaluate renal pathology. Assessment of the amount of glomerular and tubulointerstitial injury was performed in a blinded manner using a semiquantitative method (19). Glomerular injury included mesangial matrix expansion and/or hyalinosis with focal adhesions, capillary dilation, and true glomerular tuft occlusion and sclerosis. The assessment of tubulointerstitial injury included tubular dilation, interstitial fibrosis, and tubular cell atrophy. In brief, 40 glomeruli in each kidney were graded according to the severity and amount of the glomerular injury: grade 0, intact glomerulus; grade 1, glomerular injury involving <25% of the glomerulus; grade 2, glomerular injury affecting 25 to 50% of the glomerulus; grade 3, glomerular injury affecting 
50 to 75% of the glomerulus; grade 4, severe damage or sclerosis involving >75% of the glomerulus. For tubulointerstitial injury, 40 tubulointerstitial fields were graded according to the severity and amount of the tubular and interstitial damage: grade 0, no tubular injury, no interstitial fibrosis; grade 1, <25% of the field involved; grade 2, 25 to 50% of the field involved; grade 3, 50 to 75% of the field involved; grade 4, >75% of the tubules and interstitium per field involved. The indices for glomerular and tubulointerstitial injury were calculated using the following formula:
GSI/TI = (1 x n1) + (2 x n2) + (3 x n3) + (4 x n4)/(n0 + n1+ n2 + n3 + n4)
where nx = number of glomeruli/tubulointerstitial fields in each grade of glomerular/tubulointerstitial injury. These indices of glomerular/tubulointerstitial injury were calculated by averaging the grades assigned to all glomeruli/tubulointerstitial fields (20). For assessing renal lipid accumulation, frozen renal sections (6 µm) were stained for lipids with Sudan IV-Herxheimer’s solution (Sigma Chemical Co., St. Louis, MO) (16).
Immunohistochemistry
Frozen renal sections (6 µm) were used to immunostain for macrophages with a rat anti-mouse F4/80 antibody (Serotec, Oxford, UK; diluted 1:50) (21). In brief, frozen sections were fixed with cold acetone and blocked with normal rabbit serum (1/5 in Tris-buffered saline [TBS]). The sections were incubated with the primary antibody overnight at 4°C. Endogenous nonspecific binding for biotin was blocked using an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA), and endogenous peroxidase was inactivated using 0.6% H2O2 in TBS. Biotinylated rabbit anti-rat Ig (Vector Laboratories), diluted 1:200 in TBS, was used as the secondary antibody for 60 min, followed by Vectastain ABC reagent (Vector Laboratories) for 30 min. Peroxidase activity was identified by reaction with 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co) as the chromogen. The slides were then counterstained with hematoxylin, dehydrated, and mounted.
Two-micrometer paraffin sections of kidneys were used to stain for -smooth muscle actin [-SMA], collagen type I and IV, TGF-1, AGE, and RAGE. The primary antibodies that were used included a monoclonal mouse anti-human -SMA antibody (Dako A/S, Copenhagen, Denmark; diluted 1:50), a polyclonal goat anti-collagen type IV antibody (diluted 1:1600; Southern Biotechnology, Birmingham, AL), a polyclonal goat anti-collagen type I antibody (diluted 1:200; Southern Biotechnology), a polyclonal rabbit anti–TGF-1 antibody (diluted 1:400; Santa Cruz Biotechnology, Santa Cruz, CA), a monoclonal mouse anti-mouse AGE antibody (AGE 4G9, which recognizes the AGE carboxymethyllysine [CML]; diluted 1:100; a gift from Dr. H. Founds, Alteon, Ramsey, NJ), and a polyclonal goat anti-RAGE antibody (diluted 1:250; Chemicon, Temecula, CA). In brief, sections for -SMA and AGE were dewaxed, hydrated, and quenched with 3% H2O2 in PBS (pH 7.6) to inhibit endogenous peroxidase activity. This was followed by incubation in Protein Blocking Agent (Lipshaw-Immunon, Pittsburgh, PA) for 30 min at room temperature. The sections were then incubated with anti–-SMA, anti-AGE, or goat anti-RAGE antibodies overnight at 4°C. Sections for TGF-1 were first heated, dewaxed, and then underwent pressure-cooker microwaving in 0.01 M citrate buffer at pH 6.0. This was followed by incubation with 3% H2O2 in PBS (pH 7.6) and Protein Blocking Agent and incubation with the primary antibody for TGF-1 for 1 h at room temperature. For type I and type IV collagen, dewaxed and hydrated sections were quenched with 3% H2O2 in PBS. In addition, sections were digested with 0.4% pepsin (Sigma Chemical Co.) in 0.01 M HCl at 37°C. This was followed by incubation with 1% normal horse serum diluted in TBS to block nonspecific binding. Subsequently, sections were incubated with the primary antibody for either type I or type IV collagen overnight at 4°C in a humid atmosphere followed by avidin/biotin blocking. Thereafter, biotinylated anti-mouse Ig (Vector Laboratories, Burlingame, CA), diluted 1:250 for -SMA and AGE, biotinylated anti-rabbit Ig (Vector Laboratories), diluted 1:500 for TGF1, or anti-goat, diluted 1:500 for collagen type I and IV and RAGE, was applied as the secondary antibody, followed by horseradish peroxidase–conjugated streptavidin (VECTASTAIN Elite ABC Staining Kit, Vector Laboratories). Peroxidase conjugates were subsequently visualized using 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) in 0.08% H2O2/PBS as the chromogen. Finally, sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted.
All sections were examined under light microscopy (Olympus BX-50, Olympus Optical) and digitized with a high-resolution camera. For the quantification of proportional area of staining, 20 views were randomly located in the renal cortex and corticomedullary junction of each slide (Optimas 6.2-Video Pro-32; Bedford Park, SA, Australia). For some parameters with obvious differences detected with respect to glomerular, tubular, and interstitial expression, separate analyses were performed for these individual renal compartments. All assessments were performed in a blinded manner.
AGE Peptides in Serum
Fluorescence AGE peptides in serum were assayed using HPLC with on-line spectrofluorometric detection (22,23). Spectrofluorometric detection of AGE peptides was measured at 440 nm with excitation at 247 nm. Samples were run in triplicate, and the area under the curve was used for signal measurements. The assay was calibrated against AGE peptide obtained from enzymatic hydrolysis of AGE-BSA (10 g/L). Total peptide content was estimated using an on-line spectrophotometric detector set to a wavelength of 280 nm.
Reverse Transcription–PCR
Six micrograms of total RNA extracted from a whole kidney using Trizol method were used to synthesize cDNA with Superscript First Strand synthesis system for reverse transcription–PCR (Life Technologies BRL, Grand Island, NY) (24). RAGE, AGE receptor-2 (AGE R-2), and AGE R-3 gene expression were analyzed by real-time quantitative reverse transcription–PCR using the TaqMan system based on real-time detection of accumulated fluorescence (ABI Prism 7700, Perkin-Elmer, Foster City, CA) (24). Fluorescence for each cycle was quantitatively analyzed by an ABI Prism 7700 Sequence Detection System (Perkin-Elmer, PE Biosystems). For controlling for variation in the amount of DNA available for PCR in the different samples, gene expression of the target sequence was normalized in relation to the expression of an endogenous control, 18S ribosomal RNA (rRNA) (18S rRNA TaqMan Control Reagent kit; ABI Prism 7700, Perkin-Elmer). Primers and Taqman probes for the target sequences and the endogenous reference 18S rRNA were constructed with the help of Primer Express (ABI Prism 7700, Perkin-Elmer).
For amplification of the RAGE cDNA, the forward primer was 5'-GGGAAGGAGGTCAAGTCCAACTA-3' and the reverse primer was 5'-TGAGTTCAGAGGCAGGATCCA-3'. The probe specific to RAGE was 5'-TCTACCAGATTCCTGGGAAGCCAGAAATTG-3'. For the AGE R-2 cDNA, the forward primer was 5'-ACGAGCTCACCACCAATGAGT-3' and the reverse primer was 5'-TTTGGGTTTCTGGGAGACCA-3'. The probe specific to AGE R-2 was 5'-CGTCTACCGGCTTTGCCCCTTCA-3'. For the AGE R-3 cDNA, the forward primer was 5'-CAGACAGCTTTTCGCTTAACGA-3' and the reverse primer was 5'-CCATGCACCCGGATATCC-3'. The probe specific to AGE R-3 was FAM-5'-TTAGCTGGCTCTGGAAACCCAAACCCT-3'-TAMRA. Every amplification was performed with the following time course: 50°C, 2 min and 10 min at 95°C and 40 cycles of 94°C, 20 s, 60°C, 1 min. Each sample was tested in triplicate. Results are expressed relative to values in the control apo E-KO group, which were arbitrarily assigned a value of 1.
Statistical Analyses
Data were analyzed by ANOVA using Statview V (Brainpower, Calabasas, CA). Comparisons of group means were performed by Fisher least significant difference method. Data are shown as mean ± SEM, unless otherwise specified. P < 0.05 was viewed as statistically significant.
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Renal Parameters
Nondiabetic apo E-KO mice had higher kidney/body weight ratios than C57BL/6 mice. Diabetes in both strains was associated with an increase in the kidney/body weight ratio, but no difference in this parameter was detected between diabetic apo E-KO and diabetic C57BL/6 mice. Plasma urea and creatinine concentrations were similar in nondiabetic mice of either strain (Table 1). Diabetes was associated with increased serum urea concentrations in both strains, but serum creatinine concentrations were increased by diabetes in apo E-KO mice only. There was no difference in urinary albumin excretion rate between nondiabetic apo E-KO mice and C57BL/6 mice (Figure 1A). In both strains, induction of diabetes significantly increased urinary albumin excretion. The highest albumin excretion rate was seen in diabetic apo E-KO mice, which showed increased albuminuria when compared with both diabetic C57BL/6 mice and nondiabetic apo E-KO mice (Figure 1A).
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In nondiabetic C57BL/6 mice, renal structure was preserved. In nondiabetic apo E-KO mice, there was a slight but significant increase in the glomerular injury index as well as in the tubulointerstitial injury index (Figure 1, B and C). Glomerular changes were characterized by deposits of lipids in the mesangium and in the capillaries, slight glomerular hyperplasia, deposition of extracellular matrix (ECM) in the mesangium associated with mesangial expansion, and mesangial hypercellularity. Tubulointerstitial changes included macrophage infiltration and a modest increase in interstitial space as a result of deposition of ECM. The induction of diabetes was associated with an increase in the degree and severity of the glomerular and tubular changes compared with nondiabetic apo E-KO mice. These changes included glomerular capillary dilation, increased capillary and mesangial lipid deposits, and increased mesangial expansion with increased infiltration by inflammatory cells and true glomerulosclerosis with glomerular tuft occlusion (Figure 1D). There was not only an increase in tubulointerstitial lesions but also an increase in their severity. These changes included increased infiltration of mononuclear cells, tubulointerstitial ECM deposition, and dilation of proximal and distal tubules and occasional tubular cell atrophy (Figure 1E). The indices for glomerular and tubulointerstitial injury were greater in diabetic apo E-KO mice when compared with diabetic C57BL/6 mice (Figure 1, B and C). Treatment with aminoguanidine or ALT-711 was associated with amelioration of these renal pathologic changes with reduced glomerular and tubulointerstitial injury indices in both treatment groups (Figure 1, B and C).
Glomerular Lipid Accumulation
In nondiabetic or diabetic C57BL/6 mice, no staining for Sudan IV was observed. In contrast, nondiabetic apo E-KO mouse kidneys showed clear lipid accumulation (0.74 ± 0.20%). In diabetic apo E-KO mice, there was a significant increase in glomerular lipid accumulation (1.72 ± 0.46%; P < 0.05 versus nondiabetic apo E-KO mice). Aminoguanidine (1.63 ± 0.40%) or ALT-711 (1.53 ± 0.46%) had no effect on glomerular lipid accumulation.
Macrophage Infiltration
In the nondiabetic animals, macrophage infiltration, as assessed by F4/80-positive cells, was observed in the interstitium with virtually no macrophages detected in glomeruli (Figure 2). Diabetes was associated with increased interstitial and glomerular infiltration of macrophages in both mouse strains (Figure 2). Treatment with aminoguanidine or ALT–711 was associated with a significant decrease in macrophage infiltration in both the glomerulus and the tubulointerstitium (Figure 2).
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-SMA–Positive Cells-SMA was essentially confined to renal arterioles and arteries, with only a little staining observed in the peritubular interstitium (Figure 3). Nondiabetic apo E-KO mice showed a slight increase in -SMA expression in glomeruli and the tubulointerstitium when compared with C57BL/6 mice (Figure 3). In kidneys of diabetic mice, -SMA expression was increased in glomeruli and in the tubulointerstitium when compared with nondiabetic mice. There was no significant difference between diabetic apo E-KO mice and diabetic C57BL/6 mice. In the diabetic kidneys, -SMA–positive cells were detected in virtually all glomeruli, particularly in the mesangial cells as well as in the interstitium and occasionally in tubular cells. Treatment with ALT-711 prevented the increase in -SMA–positive cell expression in diabetic apo E-KO mice (Figure 3). By contrast, aminoguanidine treatment had no effect on this parameter.
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Renal AGE Receptor Expression
Gene expression of the AGE receptors RAGE and AGE R-3 was increased in diabetic kidneys in both mouse strains to a similar degree (Table 2). No changes in renal AGE R-2 gene expression in diabetic kidneys were detected (Table 2). Immunohistochemical analysis showed that RAGE protein was increased in glomerular and tubular cells of nondiabetic apo E-KO mice. Induction of diabetes caused a further increase in RAGE immunostaining, but there was no difference between the diabetic groups of both strains (Table 2).
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Renal TGF-1 Expression
In the kidneys of nondiabetic C57BL/6 mice, there was minimal staining for TGF-1 (Figure 6). In nondiabetic apo E-KO mice, positive staining for TGF-1 was seen in the glomeruli as well as in the tubulointerstitial areas (Figure 6). Diabetes was associated with a prominent increase in renal TGF-1 immunostaining, which was seen in the glomerular epithelial and mesangial cells as well as in the tubulointerstitium. Tubulointerstitial TGF-1 staining was significantly higher in diabetic apo E-KO mice than in diabetic C57BL/6 mice, whereas no difference in glomerular TGF-1 staining was observed between these two diabetic groups (Figure 6). Treatment with ALT-711 or aminoguanidine reduced TGF-1 expression in the glomeruli as well as in the tubulointerstitium (Figure 6) to levels similar to that observed in nondiabetic apo E-KO mice.
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Discussion |
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AGE accumulation in both the serum and the kidney was increased in diabetic apo E-KO mice when compared with mice with diabetes or hyperlipidemia alone. In the serum, fluorescence AGE peptides, and in the kidney, increased deposition of the specific nonfluorescence AGE, CML, using an immunohistochemical approach were observed. CML has been reported to be the major AGE in mesangium, glomerular basement membrane, and tubular basement membrane (25) and has been shown to correlate with the severity of nephropathy in diabetic patients (25,26). CML is considered by some investigators to be a highly relevant and pathogenic advanced glycation/lipoxidation end product and is formed by both glucose- and lipid-dependent mechanisms (27). It is interesting that renal as well as circulating AGE were increased not only in diabetic animals but also in apo E-KO mice when compared with C57BL/6 mice both with and without diabetes. This suggests not only that the AGE in this model are derived from glucose-dependent pathways but also that lipids may contribute to the accumulation of these AGE. Indeed, recent evidence has emphasized the importance of lipids as an important source of chemical modification of tissue proteins, even in the absence of hyperglycemia, leading to production of advanced lipoxidation products and consequently to renal injury (28). These chemically modified proteins include CML, which is also a major AGE. It is hypothesized that these AGE/advanced lipoxidation products, which accumulate in settings such as hyperlipidemia and chronic hyperglycemia, can induce renal injury by cross-link formation with collagens in the ECM, glomerular basement membrane, and tubular basement membrane or by inducing activation of a range of cellular processes via receptor-dependent pathways (28,29).
The renal injury in diabetic apo E-KO mice was associated with functional changes, including increased albuminuria and elevated serum urea and creatinine concentrations as well as various structural changes in both the glomerulus and the tubulointerstitium. Although diabetes per se in both mouse strains was associated with a range of abnormalities, including increased collagen accumulation and -SMA expression, as well as functional and structural injury, not all of these parameters were increased in diabetic apo E-KO versus diabetic C57BL/6 mice. The major differences between the two strains with the induction of diabetes included increased albuminuria, glomerular injury index, tubulointerstitial injury, and tubular TGF-1 immunostaining in the setting of a marked increase in renal AGE staining in these diabetic apo E-KO mice. The other major differences between the two diabetic strains, which had similar levels of glycemic control, were an increase in lipids and AGE in the diabetic apo E-KO mice. This suggests that AGE and lipids may play important roles in amplifying injury in the context of diabetes.
In the clinical context, the link between diabetic hyperlipidemia and renal damage has been postulated on the basis of a number of observations. For example, in subjects with diabetes with microalbuminuria or with overt proteinuria, plasma triglyceride levels are elevated compared with those without nephropathy (2). Furthermore, elevated serum cholesterol levels have been reported to be a predictor of rapid loss of renal function in patients with type 1 diabetes (3). The importance of hyperlipidemia in the development of diabetic nephropathy is further supported, albeit indirectly, by recent findings of a correlation between apo E gene polymorphisms and nephropathy in individuals with diabetes (30,31). However, the role of apo E polymorphism in diabetic nephropathy still remains controversial (32,33).
In addition to its effects on lipoprotein clearance, it has been suggested that apo E may confer protective renal effects independent of its effect on lipids (34). Mesangial cells are the major source of apo E in the kidneys, and apo E inhibits mesangial cell proliferation in an autocrine manner (34). Although the aggravated renal injury observed in diabetic apo E-KO mice in the present study most likely occurred as a result of the combination of diabetes and hyperlipidemia, these lipid-independent effects of apo E deficiency cannot be excluded in the progression of the disease.
The synergistic effects of hyperlipidemia and diabetes on the development of renal injury observed in the present study further extends our knowledge of the role of hyperlipidemia as an aggravating factor for diabetic nephropathy. Although diabetes was associated with renal damage in both C57BL/6 and apo E-KO mice, the hyperlipidemic animals with diabetes displayed a more extensive and severe degree of renal glomerular and tubulointerstitial injury as well as higher levels of urinary albumin excretion when compared with the normolipidemic diabetic animals. An earlier renal study using diabetic apo E-KO mice reported that relatively short-term diabetes (6 wk) resulted in increased renal expression of RAGE in association with infiltration of inflammatory cells (14). That study suggested that long-term diabetes might lead to accelerated renal disease in diabetic apo E-KO mice (14). Indeed, the present study has demonstrated that long-term diabetes leads to accelerated renal injury in diabetic apo E-KO mice.
The development of diabetic renal disease in hyperlipidemic animals in the current study is in agreement with studies using obese Zucker rats with diabetes (35). Because diabetic apo E-KO mice and Zucker rats are models of insulin deficiency and resistance, respectively, it is likely that the renal injury in these models is related to hyperglycemia per se.
The apo E-KO mouse is the most common experimental model currently used in the study of atherosclerosis. Renal pathology in this model has recently been characterized in detail, albeit in the absence of concomitant diabetes (4). As these mice get older, they develop mild progressive renal injury with spontaneous glomerular lesions with foam cells and widening of the mesangial area resembling the renal changes seen in human type III hyperlipoproteinemia (4). The present study has confirmed that hyperlipidemia alone does not cause significant renal impairment or major pathologic alterations in these animals, with only mild glomerular changes and negligible tubulointerstitial injury detected in the nondiabetic apo E-KO mice.
The interventional arm of the current study demonstrated that inhibitors of AGE accumulation conferred renoprotective effects, as assessed by a range of functional and structural parameters. These findings support the view that AGE play a pivotal role in the development and progression of renal injury in the diabetic apo E-KO mouse. In earlier studies, it was shown that soluble RAGE is able to confer renal protection in apo E-KO mice with short-term diabetes (14) as well as in genetically diabetic db/db mice (36). It needs to be appreciated that RAGE cannot be considered a highly specific AGE receptor because not only do AGE interact with other receptors, but also RAGE itself has multiple ligands, including S-100, which is itself a proinflammatory molecule (37). The present study provides further evidence to emphasize the role of advanced glycation and is consistent with the view that at least some of the renoprotective effects of approaches such as soluble RAGE are via effects on inhibiting AGE-dependent pathways.
The renoprotective effects of inhibition of AGE in experimental diabetic nephropathy have been previously described, albeit not in the context of hyperlipidemia. The prototype inhibitor of AGE formation, aminoguanidine, was shown to attenuate albuminuria and mesangial expansion in diabetic rats (9). Because aminoguanidine is also an inhibitor of the inducible form of NO synthase (iNOS), subsequent studies using agents that are either iNOS inhibitors alone or more selective inhibitors of AGE formation (ALT-946) (38) that do not significantly affect NOS have also been examined. These studies have shown that the renoprotective effects of aminoguanidine relate to its AGE inhibitory action and not its ability to inhibit NOS (38). More recently, ALT-711 was shown to confer renoprotection in experimental type 1 diabetes (8) and in preliminary reports in the db/db mouse, a model of type 2 diabetes (39,40).
Increased renal accumulation of AGE in diabetic apo E-KO mice compared with nondiabetic apo E-KO mice was associated with overexpression of the prosclerotic growth factor TGF-1. Furthermore, expression of TGF-1 was ameliorated by treatment with either aminoguanidine or ALT-711. It is likely that the elaboration of TGF-1 occurs partly as a result of AGE-induced expression of these proteins via receptor-dependent pathways (29). Indeed, both mesangial (12) and proximal tubular epithelial cells (13,41) have been demonstrated to express TGF-1 upon exposure to AGE, which in turn induces rapid accumulation of ECM. Production of TGF-1 may at least partly be in response to binding of AGE to their receptor RAGE (13). The effects of other AGE receptors have not been investigated as extensively, although the changes observed with respect to these receptors in response to both the diabetic milieu and the antiglycation therapies is consistent with these receptors’ also playing an important role in the development of renal injury. The increase in renal AGE R3 expression observed in the present study in the diabetic kidney is in agreement with similar observations by our group in the diabetic rat heart (42).
Induction of diabetes in apo E-KO mice was associated with increased renal type I and IV collagen expression compared with nondiabetic apo E-KO mice. Blockade of the glycation pathway with aminoguanidine or ALT-711 was associated with a significant reduction in collagen I and IV immunostaining with a possible superiority of ALT-711 over aminoguanidine treatment. Previous studies by our group in a nonhyperlipidemic model of diabetic nephropathy have shown reduced expression of both type I and IV collagen with aminoguanidine or ALT-711 treatment (8,43). It is possible that the reduction in type I collagen expression is an important manifestation of the reduced tubulointerstitial injury seen with the antiglycation therapies. However, there was no difference in type I or IV collagen expression between diabetic C57BL/6 and diabetic apo E-KO mice despite the differences in renal structural and functional parameters, suggesting that the change in the expression of collagens is more closely linked with the diabetic milieu than to concomitant dyslipidemia.
Increased renal expression of -SMA–positive cells in diabetic kidneys of C57BL/6 and apo E-KO mice observed in our study is in agreement with increasing evidence of the role of -SMA–positive myofibroblasts, or activated fibroblasts, as well as activated mesangial cells in the pathogenesis of diabetic glomerulosclerosis and tubulointerstitial fibrosis (44,45). It has been shown that myofibroblast expression is markedly increased in areas of interstitial fibrosis (44), and this increased expression has been specifically demonstrated in human diabetic nephropathy (13).
ALT-711 treatment reduced expression of -SMA–positive cells in both the glomeruli and the interstitium, whereas aminoguanidine had no effect on this parameter. This was an unexpected finding because both drugs had similar effect on AGE accumulation in both the kidney and the serum in the setting of renoprotection and reduced collagen deposition as well as attenuated TGF-1 overexpression. The underlying explanation for this difference is unknown but may be related to the different mechanisms of action of these drugs. As mentioned previously, in addition to its principal action as an inhibitor of AGE formation, aminoguanidine has inhibitory action on NOS, particularly the inducible form (iNOS) (46). The effects of ALT-711 on -SMA expression have been previously demonstrated by our group (13). Indeed, in those studies, it was shown that AGE per se promote tubulointerstitial transition as assessed by -SMA immunostaining and that this phenomenon seems to act via RAGE- and TGF-1–dependent pathways. The present study further extends these findings to the in vivo context linking AGE accumulation, RAGE, and TGF-1 expression to the presence of -SMA-positive cells, not only in the tubulointerstitium but also in the glomerulus.
It should be noted that not all molecular and cellular parameters assessed in this study were increased in diabetic apo E-KO mice when compared with diabetic C57BL/6 mice. This lack of effect of the addition of dyslipidemia to hyperglycemia on various parameters including macrophage infiltration and glomerular TGF-1 and collagen expression is consistent with some of these pathways being more closely linked to glucose- rather than lipid-dependent pathways. Indeed, glucose promotes end-organ injury via a number of additional pathways to advanced glycation, such as activation of the renin-angiotensin system, induction of certain protein kinase C isoforms, promotion of mitochondrial oxidative stress, and increased flux via the hexosamine pathway (47,48). Although beyond the scope of this study, it is likely that some of these putative pathogenic mechanisms were induced by the diabetic milieu and amplify renal injury induced by AGE-dependent pathways.
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Acknowledgments |
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The authors wish to thank Paula Aldersea, Gavin Langmaid, Maryann Arnstein, and Josefa Pete for excellent technical assistance. Reprinting of the pictures was supported by Alteon Inc.
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References |
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-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. J Biol Chem 271: 9982–9986, 19961 and type IV collagen in the renal tubulointerstitium in experimental diabetes: Effects of ACE inhibition. Diabetes 47: 414–422, 1998[Abstract]
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 E. E. Tchekneva, E. M. Rinchik, D. Polosukhina, L. S. Davis, V. Kadkina, Y. Mohamed, S. R. Dunn, K. Sharma, Z. Qi, A. B. Fogo, et al. A Sensitized Screen of N-ethyl-N-nitrosourea-Mutagenized Mice Identifies Dominant Mutants Predisposed to Diabetic Nephropathy J. Am. Soc. Nephrol., January 1, 2007; 18(1): 103 - 112. [Abstract] [Full Text] [PDF]
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A. C. Calkin, S. Giunti, K. A. Jandeleit-Dahm, T. J. Allen, M. E. Cooper, and M. C. Thomas PPAR-{alpha} and -{gamma} agonists attenuate diabetic kidney disease in the apolipoprotein E knockout mouse Nephrol. Dial. Transplant., September 1, 2006; 21(9): 2399 - 2405. [Abstract] [Full Text] [PDF]
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M. B. Marrero, A. K. Banes-Berceli, D. M. Stern, and D. C. Eaton Role of the JAK/STAT signaling pathway in diabetic nephropathy Am J Physiol Renal Physiol, April 1, 2006; 290(4): F762 - F768. [Abstract] [Full Text] [PDF]
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E. Galkina and K. Ley Leukocyte Recruitment and Vascular Injury in Diabetic Nephropathy J. Am. Soc. Nephrol., February 1, 2006; 17(2): 368 - 377. [Abstract] [Full Text] [PDF]
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S. Genuth, W. Sun, P. Cleary, D. R. Sell, W. Dahms, J. Malone, W. Sivitz, V. M. Monnier, and for the DCCT Skin Collagen Ancillary Study Group Glycation and Carboxymethyllysine Levels in Skin Collagen Predict the Risk of Future 10-Year Progression of Diabetic Retinopathy and Nephropathy in the Diabetes Control and Complications Trial and Epidemiology of Diabetes Interventions and Complications Participants With Type 1 Diabetes Diabetes, November 1, 2005; 54(11): 3103 - 3111. [Abstract] [Full Text] [PDF]
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J. M. Bohlender, S. Franke, G. Stein, and G. Wolf Advanced glycation end products and the kidney Am J Physiol Renal Physiol, October 1, 2005; 289(4): F645 - F659. [Abstract] [Full Text] [PDF]
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Z. Wang, T. Jiang, J. Li, G. Proctor, J. L. McManaman, S. Lucia, S. Chua, and M. Levi Regulation of Renal Lipid Metabolism, Lipid Accumulation, and Glomerulosclerosis in FVBdb/db Mice With Type 2 Diabetes Diabetes, August 1, 2005; 54(8): 2328 - 2335. [Abstract] [Full Text] [PDF]
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M. Lassila, K. Jandeleit-Dahm, K. K. Seah, C. M. Smith, A. C. Calkin, T. J. Allen, and M. E. Cooper Imatinib Attenuates Diabetic Nephropathy in Apolipoprotein E-Knockout Mice J. Am. Soc. Nephrol., February 1, 2005; 16(2): 363 - 373. [Abstract] [Full Text] [PDF]
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M. D. Breyer, E. Bottinger, F. C. Brosius III, T. M. Coffman, R. C. Harris, C. W. Heilig, K. Sharma, and for the AMDCC Mouse Models of Diabetic Nephropathy J. Am. Soc. Nephrol., January 1, 2005; 16(1): 27 - 45. [Abstract] [Full Text] [PDF]
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P < 0.01 versus appropriate C57BL/6 group; #P < 0.05, ##P < 0.01 versus diabetic apo E-KO mice. Magnifications: x400 in D, x200 in E.
