Hemodynamics and Vascular Regulation

Imatinib Attenuates Diabetic Nephropathy in Apolipoprotein E-Knockout Mice

Markus Lassila, Karin Jandeleit-Dahm, Kwee K. Seah, Craig M. Smith, Anna C. Calkin, Terri J. Allen and Mark E. Cooper
JASN February 2005, 16 (2) 363-373; DOI: https://doi.org/10.1681/ASN.2004050392

Abstract

In the diabetic kidney, clinical as well as experimental observations have shown an upregulation of growth factors such as PDGF. These studies, however, were not designed to address whether upregulation of PDGF is merely a manifestation of diabetic renal injury or whether PDGF plays an active role in the pathophysiology of diabetic nephropathy. The objectives of this study were first to assess whether PDGF-dependent pathways are involved in the development of diabetic nephropathy and second to determine the effects of PDGF receptor antagonism on this disorder and associated molecular and cellular processes. This study used the diabetic apolipoprotein E-knockout (apoE-KO) mouse, a recently described model of accelerated diabetic nephropathy. Diabetes was induced by injection of streptozotocin in 6-wk-old apoE-KO mice. Diabetic animals received treatment with a tyrosine kinase inhibitor that inhibits PDGF action, imatinib (STI-571, 10 mg/kg per d orally) or no treatment for 20 wk. Nondiabetic apoE-KO mice served as controls. This model of accelerated renal disease with albuminuria as well as glomerular and tubulointerstitial injury was associated with increased renal expression of PDGF-B, proliferating cells, and α-smooth muscle actin-positive cells. Furthermore, there was increased accumulation of type I and type IV collagen as well as macrophage infiltration. Imatinib treatment ameliorated both renal functional and structural parameters of diabetes as well as overexpression of a number of growth factors, collagens, proliferating cells, α-smooth muscle actin-positive cells, and macrophage infiltration within the kidney. Tyrosine kinase inhibition with imatinib seems to retard the development of experimental diabetic nephropathy.

There is increasing evidence to suggest that prosclerotic growth factors contribute to the development of diabetic nephropathy (1,2). During the past decade, most of the focus has been on TGF-β in the progression of this disease, whereas the role of PDGF remains less explored. As orally available inhibitors of various growth factors are becoming increasingly available, it is of considerable interest to determine whether specific antagonism of these pathways could be useful in the treatment of diabetic nephropathy.

Various PDGF isoforms bind to two structurally and functionally related receptors PDGF-Rα and PDGF-Rβ with different affinities. Ligand binding induces receptor dimerization and autophosphorylation, leading to activation of cytoplasmic SH2 domain-containing signal transduction molecules and initiation of different intracellular signaling pathways, including cellular tyrosine phosphorylation and c-fos mRNA induction. These, in turn, lead to cell growth, proliferation, chemotaxis, and differentiation (3).

PDGF-B, the PDGF isoform most closely linked with the development of various renal diseases (4), is produced by glomerular mesangial cells and tubular cells and released by infiltrating cells. PDGF potently increases proliferation and matrix synthesis (3,5,6).

PDGF has been suggested to play a role in the pathophysiology of various fibroproliferative diseases of the kidney (7–9). In the diabetic kidney, upregulation of the PDGF pathway has been shown in experimental diabetic nephropathy (10–12) and in the kidneys from patients with diabetes (13). Furthermore, amelioration of diabetic nephropathy by agents such as the inhibitor of advanced glycation, aminoguanidine, is associated with reduced renal PDGF expression (12). A nonspecific inhibitor of PDGF, trapidil, was able to ameliorate the early increase in glomerular volume (11), but no study has previously reported the effect of blockade of the PDGF pathway on the development of diabetic nephropathy. These previous studies had not been designed to address whether PDGF plays an active role in the pathophysiology of diabetic nephropathy or whether upregulation of PDGF is merely a manifestation of diabetic renal injury.

Orally active antagonists for the PDGF receptors such as imatinib (STI-571) have been developed. In the nondiabetic context, imatinib has been shown to reduce mesangial cell activation and proliferation as well as glomerular accumulation of extracellular matrix in anti-Thy 1.1 nephritis (9) and chronic allograft nephropathy (8). In the diabetic kidney, in vivo effects of PDGF inhibition remain to be determined. Recently, we showed that imatinib ameliorates atherosclerosis in diabetic apolipoprotein E-knockout (apoE-KO) mice (14). The objectives of this study were to assess whether PDGF-dependent pathways are involved in the development of diabetic nephropathy and specifically to determine the effects of tyrosine kinase inhibition of PDGF receptors with imatinib on the development of diabetic nephropathy and various cellular and molecular processes associated with this disorder using a recently described model of accelerated diabetic nephropathy, the diabetic apoE-KO mouse (15).

Materials and Methods

Six-week-old homozygous apoE-KO male mice (back-crossed 20 times from the C57BL/6 strain; Animal Resource Centre, Canning Vale, WA, Australia) were housed at the Biologic Research Laboratory at the Austin and Repatriation Medical Centre and were studied according to principles devised by the Animal Welfare Committee of the Austin and Repatriation Medical Centre. Thirty-four mice were rendered diabetic by five daily intraperitoneal injections of streptozotocin (Boehringer-Mannheim, Mannheim, Germany) at a dose of 55 mg/kg in citrate buffer (16). Only animals with blood glucose levels >15 mmol/L 2 d after the induction of diabetes were included in the study. Control mice (n = 20) received citrate buffer alone. The animals had unrestricted access to water and were maintained on a 12-h light-dark cycle in a non-pathogen-free environment on standard mouse chow (Barastoc, Pakenham, Victoria, Australia). After the induction of diabetes, the animals were randomized further to treatment with imatinib (Novartis Pharmaceuticals, Basel, Switzerland; n = 14) at a dose of 10 mg/kg body wt per d via gavage for 20 wk or no treatment (n = 20). Systolic BP was assessed by a noninvasive tail cuff system in conscious mice at the end of the study (17). Animals were habituated to the device before measuring the BP on five to six occasions over 15 min to enhance accurate measurements.

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). 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 analyses. Blood was collected from the left ventricle and centrifuged, and plasma and red blood cells were stored at −20°C and 4°C, respectively, for subsequent analyses. Glycated hemoglobin (HbA1c) was determined in lysates of red blood cells by high-pressure liquid chromatography (BioRad, Richmond, CA). Total cholesterol and triglyceride concentrations were measured by autoanalyzer (Hitachi 917, Tokyo, Japan).

Assessment of Renal Function

At week 20, the animals were housed in metabolic cages for 24 h to collect urine for subsequent measurements of albumin concentration by RIA. This assay is a modification of a previously established method for measuring rat albumin in our laboratory (18) with a rabbit anti-mouse albumin antibody (1:6000; Cappel, Aurora, OH) used. Plasma creatinine and urea concentrations were measured by autoanalyzer (Beckman Instruments, Fullerton, CA). Creatinine clearance was calculated using the formula creatinineurine × urine volume/creatinineserum × 1440 (ml/min). Albumin excretion was also expressed as albumin/creatinine ratio in urine.

Evaluation of Morphologic Changes

Two-micrometer kidney sections were prepared and stained with periodic acid-Schiff to evaluate renal pathology. Assessment of glomerular and tubulointerstitial injury was performed in a masked manner using a semiquantitative method. In brief, 40 glomeruli in each kidney were graded according to the severity of glomerular damage: grade 0, intact glomerulus; grade 1, glomerular damage or sclerosis involving <25% of the glomerulus; grade 2, 25 to 50% of the glomerulus; grade 3, 50 to 75% of the glomerulus; grade 4, severe damage or sclerosis involving >75% of the glomerulus. This index of glomerular injury was calculated using the following formula: Math where nx = number of glomeruli in each grade of glomerular injury. This index of glomerular injury was calculated by averaging the grades assigned to all glomeruli (19).

Tubulointerstitial area was assessed in the renal cortex by using a point-counting technique following a routine established method (20). In each field, 100 points were counted on a 1-cm2 eyepiece graticule with 10 equidistant grid lines. A total of 12 high-power fields (×400) per section were counted for each animal in all groups in the corticomedullary field. Each high-power field was 0.076 mm2, and 0.91 mm2 was the total area counted per slide. Tubulointerstitial area = number of tubulointerstitial grid intersections/total number of intersections. Glomerular size and mesangial area were quantified by a computerized image analysis system on periodic acid-Schiff-stained sections (21).

Immunohistochemistry

Frozen renal sections (6 μm) were stained for macrophages with a rat anti-mouse F4/80 antibody (Serotec, Oxford, UK; diluted 1:50) (22). 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% hydrogen peroxide (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., St. Louis, MO) as the chromogen. The slides were then counterstained with hematoxylin, dehydrated, and mounted.

Two-micrometer paraffin kidney sections were also used for immunohistochemistry. The primary antibodies used were a polyclonal rabbit anti-human PDGF-B antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:50), a monoclonal mouse anti-human α-smooth muscle actin (α-SMA) antibody (Dako A/S, Copenhagen, Denmark; diluted 1:50), a polyclonal goat anti-human type IV collagen antibody (1:1600; Southern Biotechnology, Birmingham, AL), a polyclonal goat anti-human type I collagen antibody (1:200; Southern Biotechnology), a polyclonal rabbit anti-TGF-β1 antibody (1:400; Santa Cruz Biotechnology), a polyclonal rabbit anti-mouse CTGF antibody (Abcam Ltd., Cambridge, UK; 1:800), and a monoclonal anti-mouse Ki-67 antibody (1:25; Dako A/S). In brief, sections to assess PDGF-B, α-SMA, Ki-67, and CTGF immunostaining 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 then were incubated with the primary antibody overnight at 4°C. Sections for TGF-β1 first were heated, dewaxed, and then underwent pressure cooker microwaving in 0.01 M citrate buffer at pH 6.0. This was followed by blockade with 3% H2O2 and Protein Blocking Agent and then incubation with the primary antibody for TGF-β1 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 predigested 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. Subsequently, sections were incubated with goat anti type I or type IV collagen antibodies overnight at 4°C in a humid atmosphere followed by avidin/biotin blocking. Thereafter, biotinylated horse anti-mouse Ig (1:250; Vector Laboratories) for α-SMA, biotinylated goat anti-rabbit Ig (1:250; Vector Laboratories) for PDGF-B, TGF-β1, and CTGF biotinylated horse anti-goat, 1:500 for type I and IV collagen, or biotinylated anti-rat (1:250; Vector Laboratories) for Ki-67 were applied, followed by the ABC Elite Kit (Vectastain ABC Elite, 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 of the sections were examined in a masked manner under light microscopy (Olympus BX-50; Olympus Optical, Tokyo, Japan) and digitized with a high-resolution camera. For the quantification of proportional area of staining, approximately 20 views (×400 magnification) were randomly located in the renal cortex and corticomedullary junction of each slide (Optimas 6.2-Video Pro-32; Bedford Park, SA, Australia). Some parameters were analyzed further with respect to glomerular, tubular, and interstitial expression by assessing expression within these individual renal compartments.

Reverse Transcription-PCR

Six micrograms of total RNA extracted from a whole kidney using Trizol method was used to synthesize cDNA with Superscript First Strand synthesis system for reverse transcription-PCR (RT-PCR; Life Technologies BRL, Grand Island, NY) (23). PDGF-B, PDGFR-β, TGF-β1, and CTGF gene expression were analyzed by real-time quantitative RT-PCR using the TaqMan system based on real-time detection of accumulated fluorescence (ABI Prism 7700; Perkin-Elmer, Foster City, CA) (23). Fluorescence for each cycle was quantitatively analyzed by an ABI Prism 7700 Sequence Detection System. To control 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; 18SrRNA, TaqMan Control Reagent kit; ABI Prism 7700). 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).

For amplification of the PDGF-B cDNA, the forward primer was 5′-TGTAATCGCCGAGTGCAAGA-3′ and the reverse primer was 5′-CATTGCACATTGCGGTTATTG-3′. The probe specific to PDGF-B was 5′-AGGTGTTCCAGATCTCGCGG-3′. For the PDGFR-β cDNA, the forward primer was TCACGGTCTGAGCCATTCG and the reverse primer was TCTGGCTGTCGATTTCAGCAT. The probe specific to PDGFR-β was CCACCATGAAAGTGG. For amplification of the TGF-β1 cDNA, the forward primer was GCAGTGGCTGAACCAAGGA and the reverse primer was GCAGTGAGCGCTGAATCGA. The probe specific to TGF-β1 was AAAGCCCTGTATTCCGT. For the CTGF cDNA, the forward primer was 5′-GAGGAAAACATTAAGAAGGGCAAA-3′ and the reverse primer was 5′-CGGCACAGGTCTTGATGA-3′. The probe specific to CTGF was FAM-5′-TTTGAGCTTTCTGGCTGCACCAGTGT-3′-TAMRA.

Every amplification was performed with the following time course: 50°C for 2 min and 95°C for 10 min and 40 cycles of 94°C for 20 s and 60°C for 1 min. Each sample was tested in triplicate. Results are expressed relative to values in the control apoE-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, correcting for multiple comparisons. Data are shown as mean ± SEM, unless otherwise specified. P < 0.05 was viewed as statistically significant.

Results

Metabolic Parameters and Systolic BP

Diabetic apoE-KO mice gained less weight than nondiabetic apoE-KO mice (Table 1). Diabetes was associated with an increase in HbA1c, plasma cholesterol, and triglyceride levels when compared with the nondiabetic apoE-KO mice. Systolic BP was not affected by diabetes. Imatinib treatment had no significant effect on body weight; HbA1c, cholesterol, or triglyceride levels; or systolic BP when compared with untreated diabetic apoE-KO mice (Table 1).

View this table:
Table 1.

Metabolic and physiologic parametersa

Renal Parameters

Diabetes was associated with increased serum urea and creatinine concentrations (Table 2). Induction of diabetes significantly increased urinary albumin excretion by week 10, and this increase was sustained at week 20 (Figure 1A). These findings with respect to albuminuria were observed, by expressing data either as 24-h urinary albumin excretion (Figure 1A) or as the urinary albumin/creatinine ratio (Table 2). Treatment with imatinib did not significantly affect plasma urea or creatinine concentrations when compared with untreated diabetic apoE-KO mice (Table 2). However, albuminuria was attenuated by treatment with imatinib at both the week 10 and week 20 time points (Figure 1A).

Figure 1.
Figure 1.

Albuminuria (μg/24 h) at weeks 10 and 20 (A), glomerular injury index (B), and tubulointerstitial area (C) in mice after the 20-wk treatment period. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 versus control apolipoprotein E-knockout (apoE-KO) mice; #P < 0.05, ##P < 0.01 versus diabetic apoE-KO mice.

View this table:
Table 2.

Renal functional, structural, and molecular parameters

Diabetes was associated with the development of glomerular injury and an increase in tubulointerstitial area (Figures 1, B and C, and 2). Glomerular injury in diabetic apoE-KO mice was characterized by glomerular hyperplasia and mesangial extracellular matrix expansion and glomerulosclerosis. Tubulointerstitial lesions in the diabetic mice were characterized by infiltration of mononuclear cells as well as tubulointerstitial fibrosis and tubular cell atrophy. Furthermore, dilation of both proximal and distal tubules was observed. Imatinib significantly reduced glomerular injury and tubulointerstitial area (Figures 1, B and C, and 2). Glomerular size and mesangial area were increased in diabetic apoE-KO mice compared with nondiabetic apoE-KO mice. Both morphologic parameters were attenuated but not normalized by imatinib treatment (Table 2).

Figure 2.
Figure 2.

Representative pictures of glomerulus (A through C) and tubulointerstitial area (D through F). (A and D) Control apoE-KO mice. (B and E) Diabetic apoE-KO mice. (C and F) Imatinib-treated diabetic apoE-KO mice. Magnification, ×400 in A through C, ×200 in D through F.

Macrophage Infiltration

In nondiabetic mice, macrophage infiltration, as assessed by F4/80-positive cells, was observed in the tubulointerstitium with few macrophages detected in the glomeruli (Figure 3). Diabetes was associated with increased interstitial and glomerular infiltration of macrophages (Figure 3). Treatment with imatinib was associated with a significant decrease in macrophage infiltration within both the glomeruli and the tubulointerstitium (Figure 3).

Figure 3.
Figure 3.

F4/80-positive macrophage staining in frozen section of the kidneys (quantification [A] and representative pictures [B through D]). (B) Control apoE-KO mice. (C) Diabetic apoE-KO mice. (D) Imatinib-treated diabetic apoE-KO mice. Data are shown as mean ± SEM. **P < 0.01 versus control apoE-KO mice; ##P < 0.01 versus diabetic apoE-KO mice. Magnification, ×200.

Expression of α-SMA-Positive Cells

In nondiabetic apoE-KO mice, immunostaining for α-SMA was found in the vasculature with only sparse α-SMA expression in the glomeruli and the tubulointerstitium (Figure 4). In kidneys from diabetic apoE-KO mice, α-SMA expression was increased within the glomeruli, particularly in mesangial cells and the interstitium and occasionally in proximal tubular epithelial cells (Figure 4). Treatment with imatinib prevented the increase in α-SMA-positive cell expression in the glomeruli and in the tubulointerstitium in diabetic apoE-KO mice (Figure 4).

Figure 4.
Figure 4.

Quantification (A) and representative pictures (B through D) of immunohistochemistry for α-smooth muscle actin (α-SMA) in the kidneys. (B) Control apoE-KO mice. (C) Diabetic apoE-KO mice. (D) Imatinib-treated diabetic apoE-KO mice. Data are shown as mean ± SEM. **P < 0.01 versus control apoE-KO mice; ##P < 0.01 versus diabetic apoE-KO mice. Magnification, ×400.

Collagen Expression

Diabetes was associated with an increase in type I and type IV collagen protein expression (Figure 5). The increase in collagen expression in diabetic kidneys was predominantly detected in the glomeruli as well as in the peritubular interstitium. Treatment with imatinib reduced accumulation of type I and type IV collagen in the glomeruli and the interstitium (Figure 5).

Figure 5.
Figure 5.

Immunohistochemistry for type I collagen (quantification [A] and representative pictures [B through D]) and type IV collagen (quantification [E] and representative pictures [F through H]) expression in the kidneys. (B and F) Control apoE-KO mice. (C and G) Diabetic apoE-KO mice. (D and H) Imatinib-treated diabetic apoE-KO mice. Data are shown as mean ± SEM. **P < 0.01 versus control apoE-KO mice; ##P < 0.01 versus diabetic apoE-KO mice. Magnification, ×400.

Renal Ki-67-Positive Cell Expression

Induction of diabetes was associated with increased expression of proliferative cells, as assessed by expression of Ki-67-positive cells, both in the glomeruli and in the tubules (Figure 6). Imatinib treatment reduced expression of Ki-67-positive cells at both of these sites (Figure 6).

Figure 6.
Figure 6.

Quantification (A) and representative pictures (B through D) of immunohistochemistry for Ki-67-positive proliferative cells in the kidneys. (B) Control apoE-KO mice. (C) Diabetic apoE-KO mice. (D) Imatinib-treated diabetic apoE-KO mice. Data are shown as mean ± SEM. **P < 0.01 versus control apoE-KO mice; ##P < 0.01 versus diabetic apoE-KO mice. Magnification, ×400 in A and C, ×200 in B and D.

Renal PDGF-B Expression

In the kidneys of nondiabetic apoE-KO mice, immunostaining for PDGF-B was found in the vasculature with only sparse expression in tubules. In the glomeruli, virtually no staining outside the vascular pole was detected (Figure 7). In the kidneys of diabetic apoE-KO mice, PDGF-B expression was increased within the glomeruli and the tubulointerstitium (Figure 7). Treatment with imatinib reduced PDGF-B expression at these sites when compared with the untreated diabetic apoE-KO mice (Figure 7). Gene expression of PDGF-B remained unchanged in the kidneys from diabetic apoE-KO mice (Table 2). Imatinib treatment was associated with reduced PDGF-B expression (Table 2).

Figure 7.
Figure 7.

Quantification (A) and representative pictures (B through D) of immunohistochemistry for PDGF-B in the kidneys. (B) Control apoE-KO mice. (C) Diabetic apoE-KO mice. (D) Imatinib-treated diabetic apoE-KO mice. Data are shown as mean ± SEM. **P < 0.01 versus control apoE-KO mice; ##P < 0.01 versus diabetic apoE-KO mice. Magnification, ×200.

Renal PDGFR-β Gene Expression

Renal PDGFR-β gene expression was increased in diabetic mice (Table 2). Imatinib treatment was associated with reduced PDGFR-β gene expression compared with nondiabetic as well as with diabetic apoE-KO mice (Table 2).

Renal TGF-β1 Expression

In the kidneys of nondiabetic apoE-KO mice, positive staining for TGF-β1 was localized to glomeruli as well to the tubulointerstitium (Figure 8). A prominent increase in TGF-β1 expression was detected in diabetic kidneys (Figure 8). This increase in renal TGF-β1 immunostaining in the diabetic kidney was seen in the glomerular epithelial and mesangial cells as well as in the tubulointerstitium. Treatment with imatinib reduced TGF-β1 expression in the glomeruli and the tubulointerstitium (Figure 8).

Figure 8.
Figure 8.

Quantification of TGF-β1 protein expression in the glomeruli (A) and the tubulointerstitium (B) as well as representative pictures of the kidneys (C through E) after TGF-β1 immunostaining. (C) Control apoE-KO mice. (D) Diabetic apoE-KO mice. (E) Imatinib-treated diabetic apoE-KO mice. Data are shown as mean ± SEM. **P < 0.01 versus control apoE-KO mice; ##P < 0.01 versus diabetic apoE-KO mice. Magnification, ×400.

TGF-β1 gene expression findings paralleled those seen with respect to TGF-β1 protein, albeit the changes were more modest. Specifically, there was an approximately 50% increase in renal TGF-β1 gene expression (Table 2), and there was a tendency for this to be decreased by imatinib (Table 2).

Renal CTGF Expression

Renal CTGF protein was primarily localized in the glomerulus with a marked increase in glomerular CTGF immunostaining in diabetic animals (4.30 ± 1.91 versus 0.14 ± 0.10% in apoE-KO control animals; P < 0.05). CTGF protein expression was reduced in imatinib-treated diabetic animals (0.34 ± 0.22; P < 0.05 versus untreated diabetic). Gene expression for CTGF, as assessed by RT-PCR, paralleled the findings for CTGF protein expression with a greater than twofold increase in CTGF mRNA levels in diabetic kidney, which was reduced in imatinib-treated animals (Table 2).

Discussion

The present study evaluated the novel therapeutic option of PDGF inhibition in the treatment of diabetic nephropathy. The results showed that treatment with the orally active tyrosine kinase inhibitor of PDGF receptors imatinib conferred renoprotection in a model of accelerated diabetic nephropathy, the diabetic apoE-KO mouse. Furthermore, amelioration of structural changes (glomerular and tubulointerstitial injury) as well as functional changes (albuminuria) by imatinib was associated with a reduction in a range of profibrotic pathways implicated in renal extracellular matrix accumulation.

Several growth factors, including PDGF, have been implicated in diabetic nephropathy on the basis of findings that these growth factor-dependent pathways are upregulated in diabetic renal injury and that treatment with various antihypertensive as well as antiglycation medications is associated with reduced growth factor expression (10–13). The protective effect of imatinib in the present study further extends our knowledge of the role of the PDGF pathway in diabetic nephropathy by demonstrating that renal PDGF expression is not merely a manifestation of renal injury in diabetes but that the PDGF pathway also plays a pivotal role in the pathophysiology of this disorder. The concomitant reduction in prosclerotic cytokines such as TGF-β and CTGF suggests that PDGF may be upstream of these other growth factors in diabetic nephropathy. However, one must be cautious in interpreting these findings with increasing evidence of significant cross-talk and other interactions among the various proliferative and fibrotic growth factors implicated in diabetic nephropathy (24).

Diabetic renal disease has been reported to be associated with increased mesangial cell (25) and interstitial (26) proliferation. Consistent with such findings, the present study observed increased expression of proliferating cells, as assessed by Ki-67-positive cells, in the kidneys from diabetic apoE-KO mice. Imatinib was able to reduce the number of proliferating cells, suggesting that cellular proliferation in diabetic renal disease is at least partly PDGF dependent. This finding is consistent with previous findings implicating PDGF in various forms of nondiabetic proliferative renal diseases (7–9). In vitro studies have described a pivotal role for PDGF-B in mesangial cell proliferation, whereas other growth factors, such as TGF-β1 and CTGF, had no effect on mesangial cell proliferation (27).

There is increasing evidence to suggest that α-SMA-positive myofibroblasts as well as activated mesangial cells play a major role in the pathogenesis of diabetic glomerulosclerosis and tubulointerstitial fibrosis (12,28,29). The increased glomerular and interstitial expression of α-SMA-positive cells in the kidneys of diabetic apoE-KO mice in the present study was completely abolished by imatinib treatment. This suggests that PDGF plays a major role in mesangial cell and fibroblast activation leading to glomerular and tubulointerstitial damage in diabetes. This result is consistent with earlier in vivo studies showing that exogenous PDGF-B administration led to tubulointerstitial proliferation in association with transdifferentiation of renal fibroblasts into α-SMA-positive myofibroblasts (6) as well as increased expression of α-SMA-positive mesangial cells (30). However, this has not been a universal finding, with PDGF-B reported to have no effect in vitro on mesangial cell activation as measured by α-SMA-positive cell expression, whereas TGF-β1 and CTGF induced mesangial cell activation (27). Therefore, it remains unclear whether reduction of α-SMA-positive cells by imatinib occurred as a result of a direct effect on the PDGF pathway or involved other growth factors that were activated through PDGF receptor-dependent pathways such as TGF-β1 and CTGF.

The renoprotective effects of imatinib were associated with amelioration of diabetes-induced increases in type I and IV collagen expression within the kidney. PDGF-B has been shown to stimulate accumulation of interstitial type III collagen (6) and glomerular type IV collagen (30). Amelioration of type IV collagen expression in imatinib-treated kidneys is consistent with earlier findings with blocking PDGF-dependent pathways either with imatinib (9) or with specific aptamers (31) in the anti-Thy 1.1 nephritis model.

Imatinib treatment reduced the prominent macrophage infiltration observed in the glomerular and tubulointerstitial compartments in the kidneys from these diabetic apoE-KO mice. This effect of imatinib on macrophage accumulation in the setting of diabetes is consistent with the effects of PDGF inhibition with specific aptamers in another model of renal disease, anti-Thy 1.1 nephritis (31), and in findings by our group in diabetes-associated atherosclerosis by imatinib (14).

The renoprotective effect of imatinib was associated with decreased expression of PDGF-B and TGF-β1, both growth factors being upregulated in the kidneys from these diabetic apoE-KO mice. Tyrosine kinase inhibition of PDGF receptors would not be predicted to reduce directly the level of a ligand for these receptors, PDGF-B or expression of the PDGF receptor per se. This suggests either that the reduction in PDGF-B and its receptor in response to imatinib is due to the renoprotection observed in the imatinib-treated animals or that there is an autocrine loop linking PDGF-B signal transduction to PDGF-B expression.

The mechanism of TGF-β1 reduction by imatinib in diabetic mice in the present study remains unknown but may be specific to the diabetic milieu. PDGF and glucose have been shown to upregulate TGF-β1 in proximal tubular cells in a synergistic manner involving an autocrine PDGF loop (32,33). This suggests that PDGF plays a crucial role in the regulation of TGF-β1, specifically in the context of diabetes. This hypothesis was supported in the present study by the prevention of upregulation of TGF-β1 in imatinib-treated diabetic mice. By contrast, in a nondiabetic model, anti-Thy 1.1 nephritis, PDGF-B antagonism with specific aptamers, despite beneficial effects on injury, did not affect renal TGF-β1 expression (34). Because neutralization of TGF-β1 by anti-TGF-β1 antibodies has been shown to attenuate kidney hypertrophy and enhanced extracellular matrix gene expression in streptozotocin-induced diabetic mice (2), this effect of imatinib in reducing TGF-β1 expression could partly explain the protective effects and in particular the reduction in extracellular matrix expansion observed in the present study. The reduction in α-SMA-positive myofibroblasts in response to imatinib could also be linked to the decrease in TGF-β1 expression because TGF-β1 has been demonstrated to be a potent stimulus for this phenomenon, including in the context of diabetes (29).

A number of in vitro studies have shown that imatinib not only is an inhibitor of PDGF receptors but also affects other tyrosine kinase receptors, including c-abl and c-kit with IC50 values of 0.025 to 0.1 μM. Therefore, it is possible that some of the renoprotection afforded by imatinib may involve effects linked to these other targets. Although there is no previous evidence of a role for c-abl or c-kit in progressive renal diseases including diabetic nephropathy, such a possibility warrants further investigation (35,36). Importantly, it should be appreciated that a wide range of other protein kinases are not inhibited by imatinib at concentrations of <100 μmol/L (35,36). These include the EGF receptor, HER-2/neu, insulin receptor, vascular endothelial growth factor receptor, IGF-1 receptor, c-FGR, c-LCK, c-LYN, c- and v-SRC, and a variety of protein serine/threonine kinases, including protein kinase A; phosphorylase kinase; protein kinase C types α, β1, β2, γ, ó, σ, ç, and æ; cdc2/cyclin; and casein kinases 1 and 2.

Although the present study was not designed to explore in detail the mechanisms by which PDGF inhibition decreased the expression of proinflammatory and profibrotic pathways, the results of this study suggest that there is a complex interplay among the different cytokines and growth factors that leads to a vicious circle in which PDGF plays a pivotal role. Blockade of this system is able to downregulate proinflammatory and proliferative pathways, including PDGF itself, that are induced by diabetes and this ultimately seems to translate to renoprotection.

Acknowledgments

This study was supported by grants from the Juvenile Diabetes Research Foundation. M.L. was supported by grants from the Finnish Academy, Einar and Karin Stroem's Foundation, The Helsingin Sanomat Centennial Foundation, and Paavo Nurmi Foundation. K.J.-D. is a National Heart Foundation of Australia postdoctoral clinical fellow. A.C.C. is a National Heart Foundation of Australia postgraduate biomedical scholar. T.J.A. is supported by a career development grant from the NHMRC/Diabetes Australia.

We thank Paula Aldersea, Gavin Langmaid, and MaryAnn Arnstein for excellent technical assistance. Imatinib was a gift from Dr. E Buchdunger, Novartis Pharmaceuticals (Basel, Switzerland).

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

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Imatinib Attenuates Diabetic Nephropathy in Apolipoprotein E-Knockout Mice
Markus Lassila, Karin Jandeleit-Dahm, Kwee K. Seah, Craig M. Smith, Anna C. Calkin, Terri J. Allen, Mark E. Cooper
JASN Feb 2005, 16 (2) 363-373; DOI: 10.1681/ASN.2004050392
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