Abstract
ABSTRACT. Renin-angiotensin system (RAS) inhibitors are effective in reducing renal disease progression in early diabetic nephropathy, but they provide imperfect protection at a later stage. Due to the pivotal role of transforming growth factor–β (TGF-β) in the pathogenesis of diabetic kidney disease, this study tested the effect of simultaneously interrupting TGF-β and angiotensin II on disease progression in diabetic rats with overt nephropathy. Diabetes was induced by streptozotocin injection in uninephrectomized rats. Diabetic rats received murine (1D11) or human (CAT-192) anti-TGF-β monoclonal antibodies alone or in combination with lisinopril, 13C4 irrelevant murine antibody, saline or lisinopril from month 4 (when animals had proteinuria) to month 8. Normal animals served as controls. Systolic BP increase was controlled by single treatments and even more by the combined therapies. 1D11 and lisinopril kept proteinuria at levels numerically lower than irrelevant antibody and saline, while CAT-192 was ineffective. The addition of either TGF-β antibody to lisinopril normalized proteinuria. Consistent results were obtained for glomerulosclerosis and tubular damage, which were abrogated by the combined therapy. Interstitial volume expansion and infiltration of lymphocytes/macrophages were limited by 1D11 and lisinopril and further reduced by their combination. The increase of type III collagen in the renal interstitium was partially attenuated by 1D11 and lisinopril while normalized by their combination. It is concluded that anti–TGF-β antibody when added to a background of chronic angiotensin-converting enzyme (ACE) inhibition fully arrests proteinuria and renal injury of overt diabetic nephropathy, providing a novel route to therapy and remission of disease for diabetic patients who do not respond to RAS inhibition. E-mail: zoja@marionegri.it
Received February 7, 2003. Accepted April 5, 2003.
Diabetic nephropathy, a major long-term complication of diabetes mellitus, is the most common cause of end-stage renal disease requiring dialysis worldwide (1,2⇓) and is becoming a staggering challenge to public healthcare systems due to the prohibitive costs of renal replacement therapy that could become unaffordable even for developed countries. Typical histologic features characterize diabetic nephropathy, including expansion of the extracellular matrix in the glomerular mesangium, thickening of glomerular and tubular membranes, and tubulointerstitial fibrosis, all of them contributing to the inexorable progressive deterioration of renal function (3).
Among treatment options for diabetes, agents that inhibit the renin-angiotensin system (RAS) are particularly effective in reducing renal disease progression (4). This is not simply a function of the effect on systemic and glomerular hypertension, but it can be attributed to the unique property of this class of drugs of limiting excess protein ultrafiltration and its deleterious consequences (5). Angiotensin-converting enzyme inhibitor (ACEi) effectiveness, however, depends on timing of treatment. Experimental data in rats with streptozotocin-induced diabetes indicate that ACEi given early after the induction of the disease significantly reduced systemic BP and normalized urinary protein excretion (6–8⇓⇓). By contrast, late administration of the drug was unable to affect proteinuria, despite effective control of BP (8). On the same line, RAS inhibition decreased albuminuria and markedly limited progression to overt nephropathy in diabetic humans with incipient nephropathy (9–12⇓⇓⇓). Conversely, when RAS inhibition was started later in type 2 diabetic patients with overt nephropathy, urinary protein excretion rate and the rate of progression to end-stage renal failure were decreased by only 25% and 20%, respectively (13,14⇓). It is therefore clear that agents that block angiotensin II (AngII) provide imperfect protection in diabetes, particularly in the advanced phase of the disease. On the other hand, a substantial proportion of those patients are referred to the nephrologist late, which implies the urgent need of better therapies.
Over the past several years, transforming growth factor–β (TGF-β) has been recognized as a central player in the fibrogenic process of diabetic nephropathy (15,16⇓), due to its activity of both stimulating matrix production and blocking matrix degradation (17,18⇓). In several animal models of diabetes, TGF-β mRNA and protein levels are significantly increased in the glomeruli and tubulointerstitium (19–21⇓⇓). Increased glomerular staining of TGF-β has also been described in patients with advanced diabetic nephropathy (22). Renal overexpression of the cytokine is attributable to hyperglycemia, as documented in vitro by data showing that high ambient glucose fosters mesangial and tubular cells to produce TGF-β (23,24⇓). Exaggerated tubular synthesis of TGF-β can be also induced by humoral factors, including AngII (25), or triggered by the toxic effect of excessive protein ultrafiltration. Data are indeed available showing that proximal tubular cells exposed to protein overload acquire an inflammatory and profibrogenic phenotype resulting in the increased generation of TGF-β (26).
In vivo the early administration of TGF-β neutralizing antibodies to mice with chemically induced diabetes prevented glomerular enlargement and suppressed the expression of genes encoding extracellular matrix components (20). Subsequent studies have shown that anti–TGF-β antibody given to db/db mice, which lack the hypothalamic leptin receptor and develop a type II diabetes-like disease, almost completely prevented the increase in collagen and fibronectin expression and mesangial matrix expansion (27).
The potential renoprotective properties of anti–TGF-β antibodies, along with the limited value of ACEi alone, prompted us to evaluate the effect of simultaneously interrupting TGF-β activity and AngII synthesis as novel strategy to afford renoprotection in overt diabetic nephropathy in the rat.
Materials and Methods
Experimental Design
Sixty-one male Sprague-Dawley rats (Charles River Italia s.p.a., Calco, Italy) with initial body weights of 260 to 300 g were used. Animal care and treatment were conducted in accordance with the institutional guidelines that are in compliance with national (Decreto Legislativo n.116, Gazzetta Ufficiale suppl 40, 18 febbraio 1992, Circolare n.8, Gazzetta Ufficiale 14 luglio 1994) and international laws and policies (EEC Council Directive 86/609, OJL358–1, December 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996). All animals were housed in a room in which the temperature was kept constant on a 12-h dark/12-h light cycle and allowed free access to standard diet containing 20% protein by weight and tap water. Animals were subjected to right nephrectomy under anesthesia 7 d before the induction of diabetes to hasten the development of the disease. Diabetes was induced by a single intravenous injection of streptozotocin (60 mg/kg body wt; Sigma Chemical Co., St Louis, MO). The presence of diabetes was confirmed 2 d later by the measurement of the tail blood glucose level with a reflectance meter (A. Menarini Diagnostics, Florence, Italy). Diabetic rats received daily evening injections of insulin (Ultratard HM or Protaphane HM; Nordisk Farmaceutici s.r.l., Roma, Italy) in doses individually adjusted to maintain a blood glucose level between 200 and 450 mg/dl. Blood glucose levels were monitored at least twice a week in all diabetic rats and occasionally in control animals for comparison. Four months after the induction of diabetes, animals were divided in seven groups and treated from month 4 (when animals had proteinuria) to month 8 as follows: group 1 (n = 8), 13C4 irrelevant murine antibody 0.5 mg/kg intraperitoneally (ip) three times/wk; group 2 (n = 8), 1D11 murine anti-TGF-β monoclonal antibody, 0.5 mg/kg ip three times/wk; group 3 (n = 8), 1D11 plus lisinopril; group 4 (n = 8), saline (CAT-192 vehicle) 0.5 ml ip twice/wk; group 5 (n = 8), CAT-192 anti-human TGF-β antibody, 5 mg/kg ip twice/wk; group 6 (n = 8), CAT-192 plus lisinopril; group 7 (n = 7), lisinopril (AstraZeneca, Basiglio, Milan, Italy) 12.5 mg/L in the drinking water. Six animals served as controls. The murine anti–TGF-β antibody neutralizes all three isoforms of TGF-β (28) and has a circulating half-life of 15.2 h in rats. The human anti-TGF-β antibody recognizes TGF-β1 and TGF-β2 and has a circulating half-life of 4 d in rats. 13C4 irrelevant murine antibody and 1D11 were obtained from Genzyme Corporation (Framingham, MA), and CAT-192 from Cambridge Antibody Technology (Melbourn Cambridgeshire, UK).
Systolic BP (SBP) was recorded by tail plethysmography in conscious rats. Twenty-four–hour urine samples were collected using metabolic cages, and proteinuria was determined by modified Coomassie blue G dye-binding assay for proteins with bovine serum albumin as standard (29). Blood was collected from the tail vein of anesthetized animals. Serum was obtained after whole blood clotting and kept frozen at −20°C until assayed. Serum creatinine was measured using an autoanalyzer (CX5, Beckman Instruments, Fullerton, CA).
Renal Histology
The removed kidneys were fixed overnight in Dubosq-Brazil, dehydrated in alcohol, and embedded in paraffin. Kidney samples were sectioned at 3-μm intervals, and the sections were stained with Masson’s trichrome, hematoxylin and eosin, or periodic-acid Schiff reagent (PAS stain). Tubular (atrophy, casts, and dilation) and interstitial changes (fibrosis and inflammation) were graded from 0 to 4+ (0, no changes; 1+, changes affecting <25% of the sample; 2+, changes affecting 25 to 50% of the sample; 3+, changes affecting 50 to 75% of the sample; 4+, changes affecting 75 to 100% of the sample). At least 100 glomeruli were examined for each animal, and the extent of glomerular damage was expressed as the percentage of glomeruli presenting sclerotic lesions. All renal biopsies were analyzed by the same pathologist who was unaware of the nature of the experimental groups.
Immunohistochemical Analyses
A mouse monoclonal antibody was used for the immunohistochemical detection of CD4+ cell surface glycoprotein, a 55-kD molecule expressed by helper T cells, thymocytes, and macrophages (W3/25; Serotec, Oxford, UK). CD4 antigen was analyzed by indirect immunofluorescence technique. Fragments of renal tissues were frozen in liquid nitrogen and cut at 3 μm using a Mikrom 500 O cryostat (Walldorf, Germany). The sections were blocked with 1% PBS/BSA, incubated overnight at 4°C with W3/25, 40 μg/ml, washed with PBS, and then incubated with Cy3-conjugated donkey anti-mouse IgG antibodies (5 μg/ml in PBS; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature. Positive cells were counted in at least ten randomly selected high power microscopic fields (×400) per each animal.
Evaluation of Type III Collagen Deposition
Type III collagen accumulation was detected by immunoperoxidase using a polyclonal rabbit anti-rat type III collagen antibody (Chemicon, Temecula, CA). Dubosq-Brazil–fixed, paraffin-embedded kidney sections were deparaffinized, rehydrated, and incubated for 30 min with 0.3% H2O2 in methanol to quench endogenous peroxidases. Tissues were treated with proteinase-K (20 μg/ml; Sigma-Aldrich, Milan, Italy) for 10 min at 37°C, followed by microwave and citrate buffer incubations to increase the reactivity of antibody to antigen. Primary antibody was then added overnight at 4°C (diluted 1:100) followed by the secondary antibody (biotinylated goat-anti-rabbit IgG diluted 1:200, Vector Laboratories), avidin-biotin peroxidase complex (ABC) solution, and finally developed with diaminobenzidine. The sections were then counterstained with Harris hematoxylin (Bio Optica, Milan, Italy). Negative control was obtained by omitting the primary antibody on an adjacent section on each slide. The signal intensity was graded on a scale of 0 to 3 (0, no staining; 1, weak staining, 2, staining of moderate intensity; 3, strong staining).
Fractional Interstitial Volume
Sections of paraffin-embedded renal tissue (3 μm) were stained with periodic-acid Schiff reagent (PAS stain). A videocamera (Panasonic, Matsushita Elect. Co., Osaka, Japan) connected to a light microscope and a computer was used. The sections under the microscope were simultaneously visualized on the computer display. The fractional volume of the cortex occupied by the interstitium was measured by point counting using a 10 × 10 orthogonal grid overlayed on the digital image on the computer monitor at original magnification of ×250. In each case, a minimum of 3000 points in each section were counted, and the fractional volumes of the interstitium were expressed as the percentages of points falling on these structural components.
Northern Blot Analyses
Total RNA was isolated from whole kidney tissue by the guanidium isothiocyanate method using TRIZOL (Life Technologies, San Giuliano Milanese, Italy) according to the manufacturer’s instruction. For mRNA preparation, total RNA of the kidneys from each experimental group was pooled. Poly(A)+ mRNA was selected by oligo (dT)-cellulose column chromatography (mRNA purification kit; Amersham Pharmacia Biotech, Buckinghamshire, UK). Six micrograms of mRNA was then fractionated on 1.2% agarose gel and blotted onto synthetic membranes (Zeta-probe, Bio-Rad, Richmond, CA). A 0.45-kb EcoRI/HindIII fragment of human TGF-β1 cDNA from plasmid pUC18 was used to detect 2.5-kb transcript. Plasmid containing murine JE/Monocyte Chemoattractant Protein-1 (MCP-1) probe was kindly provided by Dr. Charles D. Stiles (Harvard Medical School and Dana-Faber Cancer Institute, Boston, MA). MCP-1 mRNA was detected by using the 577 bp of a MCP-1 cDNA. The probes were labeled with α-32P dCTP by random-primed method. Hybridization was performed overnight in 0.25 mol/L Na2HPO4, pH 7.2, 7% SDS. Filters were washed twice for 30 min with 20 mmol/L Na2HPO4, pH 7.2, 5% SDS and two times for 10 min with 20 mmol/L Na2HPO4, pH 7.2, 1% SDS at 60°C. Membranes were subsequently probed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, taken as internal standard of equal loading of the samples on the membrane. TGF-β or MCP-1 mRNA optical density was normalized to that of the constituently released GAPDH gene expression.
Statistical Analyses
Data are expressed as mean ± SEM. Data were analyzed using the nonparametric Kruskal-Wallis test for multiple comparisons. The statistical significance level was defined as P < 0.05.
Results
Systemic Parameters
By the end of the study, one rat died in each of the groups treated with 1D11, 1D11 plus lisinopril, CAT-192 plus lisinopril. In the other groups all rats were alive.
Total food intake was comparable among diabetic groups for the entire study period. At the end of the 8-mo follow-up, the food intake averaged 31 ± 1 g for diabetic rats and 24 ± 1 for controls (P < 0.05). As shown in Table 1, diabetic rats gained weight along the study, although to a lesser extent than controls. Stable and comparable moderate hyperglycemia was maintained in all diabetic groups, whose values at the end of the study are reported in Table 1.
Table 1. Systemic parameters in diabetic rats given anti–TGF-β antibodies alone or in combination with angiotensin-converting enzyme inhibitor (ACEi)a
Rats with diabetes given the anti-TGF-β antibody vehicles, namely the irrelevant antibody 13C4 and saline, exhibited an increase in systolic BP in respect to controls (P < 0.05; Figure 1). Both murine (1D11) and human (CAT-192) TGF-β antibodies displayed an anti-hypertensive effect that was more remarkable for 1D11, which reduced BP to values lower than controls. Lisinopril limited BP increase to a similar extent as 1D11. Combination of either antibodies with lisinopril fully controlled systolic BP.
Figure 1. Anti-hypertensive effect of anti–TGF-β antibodies alone or in combination with lisinopril in diabetic rats. BP was measured at month 8 in control rats (n = 6) and in rats given the following from month 4 after diabetes induction: irrelevant murine antibody (13C4, n = 8); 1D11 (murine TGF-β antibody, n = 7); 1D11 plus lisinopril (n = 7); saline (n = 8); CAT-192 (human TGF-β antibody, n = 8); CAT-192 plus lisinopril (n = 7); or lisinopril (n = 7). Data are mean ± SE. ○P < 0.05 versus control; *P < 0.05 versus control and irrelevant antibody; ΔP < 0.01 versus saline; #P < 0.05 versus control, irrelevant antibody, saline and CAT-192; •P < 0.05 versus control, saline, and CAT-192.
Renal function, measured as serum creatinine concentration, was only mildly impaired in diabetic rats. None of the treatments affected serum creatinine values to any significant extent (Table 1).
Proteinuria
Diabetic rats developed proteinuria starting from month 3. Animals at month 4 had significantly higher values than controls (46.9 ± 1.25 mg/d; P < 0.05) and were randomized to receive treatments. Urinary protein excretion rates progressively increased with time in rats given irrelevant antibody or saline. Values measured at the end of the study are reported in Figure 2. Treatment with 1D11 reduced urinary protein excretion to values numerically, albeit not significantly, lower than diabetic animals receiving the irrelevant antibody. Conversely, CAT-192 did not substantially affect proteinuria. Lisinopril was as effective as 1D11. When 1D11 or CAT-192 were added to lisinopril, a marked and significant anti-proteinuric effect was achieved, leading to proteinuria values almost normalized.
Figure 2. Anti-TGF-β therapy combined with lisinopril arrests proteinuria in diabetic rats. Urinary protein excretion was measured at month 8 in control rats (n = 6) and in rats given the following from month 4 after diabetes induction: irrelevant murine antibody (13C4, n = 8); 1D11 (murine TGF-β antibody, n = 7); 1D11 plus lisinopril (n = 7); saline (n = 8); CAT-192 (human TGF-β antibody, n = 8); CAT-192 plus lisinopril (n = 7); or lisinopril (n = 7). Data are mean ± SE. *P < 0.01 versus control; •P < 0.05 versus irrelevant antibody; #P < 0.05 versus saline.
Renal Histology
Morphologic evaluations are reported in Table 2, and representative images are shown in Figure 3. Variable degree of glomerular sclerosis and hyalinosis with segmental collapse of the glomerular tuft was detected in diabetic rats given irrelevant antibody or saline, affecting on average 7.3 and 6.0% of glomeruli (P < 0.05 versus control). Administration of 1D11 led to a considerable reduction in the percentage of glomeruli with sclerotic changes. CAT-192 had no effect on this parameter. Lisinopril limited the percentage of glomeruli with sclerosis to a similar degree as 1D11. Remarkably, a complete renoprotection was achieved by the combined therapies so that glomerular morphology was comparable to age-matched controls.
Table 2. Effect of anti-TGF-β antibodies alone or in combination with ACEi on glomerular, tubular, and interstitial changes in diabetic ratsa
Figure 3. Light microscopy of periodic acid-Schiff–stained sections of renal cortex at month 8 representative of diabetic rats receiving irrelevant murine antibody (a), 1D11 (b), 1D11 plus lisinopril (c), CAT-192 (d), CAT-192 plus lisinopril (e), lisinopril (f), and control rats (g). Magnification, ×200.
In diabetic animals glomerular injury was associated with a mild tubular damage. Among single treatments, only lisinopril significantly reduced tubular damage scores to values similar to controls. The combined therapies normalized tubular changes.
Interstitial Accumulation of Mononuclear Cells and Type III Collagen
Diabetic animals given vehicles had higher numbers of CD4+ T cells/macrophages in the renal interstitium over controls, as evaluated by immunofluorescence at the end of the study (Table 2). 1D11 but not CAT-192 substantially, albeit not significantly, reduced the numbers of infiltrating cells. Similarly to murine TGF-β antibody, lisinopril tended to limit the interstitial accumulation of inflammatory cells. The degree of cell infiltration was instead reduced by the combination of 1D11 and lisinopril as compared with animals given vehicle, to a significant extent (P < 0.01), but not by the other add-on therapy.
Scores for immunoperoxidase staining of type III interstitial collagen are reported in Figure 4. Staining was found to be increased in diabetic rats given vehicles in respect to controls (P < 0.01). All diabetic animals but one showed consistently higher expression of the matrix protein. Neutralization of TGF-β with murine and human antibodies led to differential effects. 1D11 slightly but significantly (P < 0.05) reduced type III collagen deposition over diabetic rats given irrelevant antibody, while CAT-192 did not affect protein matrix accumulation. An effect similar to 1D11 was observed after lisinopril administration. Combination of 1D11 with lisinopril significantly (P < 0.01) limited type III collagen accumulation to levels comparable to controls and even numerically lower than those found with 1D11 alone, while CAT-192 plus ACEi slightly affected protein matrix deposition.
Figure 4. Deposition of type III collagen in the renal interstitium of diabetic rats was attenuated by 1D11 and lisinopril and further reduced by their combination. Type III collagen was measured at month 8 in control rats (n = 6) and in rats given the following from month 4 after diabetes induction: irrelevant murine antibody (13C4, n = 8); 1D11 (murine TGF-β antibody, n = 7); 1D11 plus lisinopril (n = 7); saline (n = 8); CAT-192 (human TGF-β antibody, n = 8); CAT-192 plus lisinopril (n = 7); or lisinopril (n = 7). Data are expressed as mean ± SE. ○P < 0.05, ∞P < 0.01 versus control; *P < 0.05, ** P < 0.01 versus irrelevant antibody; #P < 0.05 versus saline.
Fractional Interstitial Volume
Diabetes was characterized by a significant increase in fractional interstitial volume documented in rats given irrelevant antibody or saline with respect to controls (P < 0.05) (Table 2). Interstitial volume expansion was reduced significantly by 1D11 and only numerically affected by CAT-192. Lisinopril markedly limited the expansion of the interstitial volume, which was normalized in animals given 1D11 plus the ACEi. In contrast, combining CAT-192 to lisinopril did not potentiate the ACEi effect.
Renal Expression of TGF-β and MCP-1 mRNA
TGF-β mRNA was upregulated in the kidney cortex of diabetic rats given vehicles (Figure 5). Specifically, densitometric analysis of TGF-β mRNA factored for GAPDH demonstrated an average increase of threefold at the end of the study over controls. 1D11 did not affect TGF-β transcript levels, which were mildly reduced by CAT-192 (2.3-fold over control). TGF-β mRNA overexpression was partially reduced at almost the same extent by lisinopril either alone or combined with each antibody. Failure of antibodies to modulate renal TGF-β expression is at variance with previous studies of TGF-β block (20,30⇓), which suggested the existence of a positive feedback system for autostimulation of TGF-β.
Figure 5. Upregulation of TGF-β1 mRNA in renal cortex of diabetic rats was only partially modulated by lisinopril. Representative Northern blot of kidney mRNA probed with cDNA encoding human TGF-β1 and GAPDH, of diabetic rats treated with irrelevant murine antibody (13C4), 1D11 (murine TGF-β antibody), 1D11 plus lisinopril, saline, CAT-192 (human TGF-β antibody), CAT-192 plus lisinopril, or lisinopril, and control rats. For mRNA preparation, total RNA of the kidneys from each experimental group at month 8 was pooled together. Corresponding densitometry of the autoradiograph is shown. The optical density of the autoradiographic signals was quantitated and calculated as the ratio of TGF-β to GAPDH mRNA. The mRNA levels of diabetic rats were calculated by assuming the optical density of control as unit.
As shown in Figure 6, renal expression of MCP-1 was twofold increased in diabetic rats receiving irrelevant antibody or saline in respect to controls. 1D11 and CAT-192 slightly modified MCP-1 transcript levels, that were instead reduced by lisinopril alone or combined with CAT-192 and almost normalized by the association of 1D11 and lisinopril.
Figure 6. Representative Northern blot of kidney mRNA probed with cDNA encoding murine MCP-1 and GAPDH, of diabetic rats treated with irrelevant murine antibody, 1D11, 1D11 plus lisinopril, saline, CAT-192, CAT-192 plus lisinopril, or lisinopril, and control rats. For mRNA preparation, total RNA of the kidneys from each experimental group at month 8 was pooled together. Corresponding densitometry of the autoradiograph is shown. The optical density of the autoradiographic signals was quantitated and calculated as the ratio of MCP-1 to GAPDH mRNA. The mRNA levels of diabetic rats were calculated by assuming the optical density of control as unit.
Discussion
We previously showed a limited effect of ACEi on the progression of experimental diabetic nephropathy when the drug was given as delayed treatment (8). As a follow-up of such study, we here demonstrate that the remission from severe renal functional and structural injury in established diabetes can be achieved by a combined therapy that inhibits TGF-β and AngII. To counteract the actions of TGF-β, either a murine monoclonal antibody (1D11), which recognizes all three TGF-β (β1, β2, and β3) isoforms or a human monoclonal antibody (CAT-192) recognizing TGF-β1 and TGF-β2 were used (28).
Long-term administration of 1D11 alone to diabetic rats had a remarkable effect in limiting the progressive increase in urinary protein excretion. A similar result was obtained by lisinopril, which only partially ameliorated proteinuria and renal damage due to the fact that treatment was delayed to mimic events in clinical practice. Only the addition of either TGF-β antibody to lisinopril led to the normalization of urinary protein excretion values, however. The antiproteinuric potential of murine TGF-β antibody is in line with recent results in Dahl salt-sensitive rats fed a high-salt diet, showing that 1D11 given for 2 wk reduced proteinuria and BP without ameliorating renal injury (30). The mechanism by which TGF-β antibody lowered proteinuria in diabetic animals can be related to a direct action of the cytokine on the glomerular filtration barrier function. Similarly to AngII (31), TGF-β increases albumin permeability in isolated rat glomeruli early after the incubation, an effect independent of glomerular structural changes or upregulation of matrix protein genes — both requiring a more prolonged exposure — but attributed to the production of short-term mediators of damage including hydroxyl radicals (32).
Consistent with protein excretion data, the histologic findings of the present study also attest to partial renoprotection afforded by 1D11 therapy. Abrogation of glomerulosclerosis and tubular damage was accomplished by the addition of either TGF-β antibody to lisinopril. Accordingly, the combined therapy normalized interstitial volume expansion, a strong indicator of progressive renal injury in human diabetes (33). The profibrotic effect of excessive renal TGF-β (34) resulted in a higher interstitial accumulation of type III collagen. Neutralization of TGF-β by means of murine antibody substantially inhibited the matrix protein deposition, which was completely abolished by 1D11 plus ACEi. These data confirm previous observations of a favorable effect of long-term administration of anti–TGF-β antibody on renal structural injury and matrix accumulation in db/db diabetic mice (27). On the other hand, they indicate that in a severe model of type 1 diabetes, remission from overt disease can only be accomplished when anti–TGF-β antibodies are added to ACEi.
Streptozotocin-induced diabetes is accompanied by infiltration of inflammatory mononuclear cells into the kidney (35). It has been shown that macrophage infiltration precedes the development of glomerular injury and represents a key event in the pathogenesis of glomerulosclerosis in diabetes, to the extent that macrophage depletion prevented glomerular enlargement and damage in this model (35). Macrophage recruitment in experimental diabetes is largely driven by AngII-stimulated chemokine expression, as documented by a concomitant suppression of the time-dependent elevation of MCP-1 expression and glomerular macrophage number by ACEi or AngII receptor antagonist treatment (36). In the same study, inhibition of the RAS induced a transient decrease of TGF-β overexpression, which would have further contributed to the attenuation of inflammation. Among its various effects, TGF-β has been recognized to attract monocytes/macrophages and T lymphocytes to inflammatory sites (37,38⇓); however, a direct proof of the antiinflammatory potential of anti–TGF-β antibodies had not been provided so far. Here, we found accumulation of CD4+ lymphocytes/macrophages in the renal interstitium and periglomerular area of diabetic rats, which was significantly limited by the simultaneous interruption of pathogenetic pathways sustained by TGF-β and AngII. Finding that imposition of anti-TGF-β antibody to the ACEi significantly limited cell infiltrates in the kidney identifies a valuable antiinflammatory approach for diabetic nephropathy, also in view of the fact that ACEi alone did not consistently abrogate mononuclear cell accumulation in experimental diabetes (36). The antiinflammatory property of anti-TGF-β antibody combined with ACEi is also documented by normalization of the high levels of MCP-1 expressed in the kidney of diabetic rats. In the present study, the murine TGF-β antibody was more effective than the human one; because the time schedule of antibody administration was chosen to have comparable bioavailability, different effects could be attributed to the different spectrum of action toward TGF-β isoforms.
An interesting finding of the present study is that anti–TGF-β antibodies displayed an antihypertensive effect, which could be possibly ascribed to the preservation of medullary blood flow consequent to the reduction of matrix deposition in the kidney (30) or to the amelioration of glomerular hemodynamics. Actually, data are available showing that TGF-β2 caused a marked reduction in medullary perfusion and GFR (39). Moreover, it is well known that TGF-β alters the expression of a number of vasoactive factors, including endothelin-1 (40,41⇓), nitric oxide (42), and the RAS, which in turn regulate the vascular tone.
In conclusion, our data suggest that anti–TGF-β antibody added to a background of chronic ACE inhibition therapy fully protects from the development of proteinuria and renal injury of overt diabetic nephropathy. Combining TGF-β antibody and ACEi may represent a novel route to therapy and remission of disease for diabetic patients who do not fully benefit ACEi treatment.
Acknowledgments
This study was supported by a grant from Genzyme Corporation, Framingham, MA. Dr. Cristina Zatelli is recipient of a fellowship from the Associazione Ricerca Malattie Rare (ARMR), Bergamo, Italy. We thank Dr. Gustavo Coronel (Nordisk Farmaceutici s.r.l., Rome, Italy) for kindly providing insulin and Astra Zeneca (Basiglio, Milan, Italy) for lisinopril. We are indebted to Dr. Dario Cattaneo for helpful collaboration.
Footnotes
-
Ariela Benigni and Carla Zoja contributed equally to the paper.
- © 2003 American Society of Nephrology
References
Jump to section
More in this TOC Section
Cited By...
- Prevention of renal apoB retention is protective against diabetic nephropathy: role of TGF-{beta} inhibition
- TGF-{beta}/TGF-{beta} receptor system and its role in physiological and pathological conditions
- Mechanisms of Tubulointerstitial Fibrosis
- A Novel Transforming Growth Factor {beta} Receptor Kinase Inhibitor, A-77, Prevents the Peritoneal Dissemination of Scirrhous Gastric Carcinoma
- A PAI-1 Mutant, PAI-1R, Slows Progression of Diabetic Nephropathy
- Dual Roles of Immunoregulatory Cytokine TGF-{beta} in the Pathogenesis of Autoimmunity-Mediated Organ Damage
- Transforming Growth Factor-{beta} and the Immune Response: Implications for Anticancer Therapy
- Combination Therapy with an Angiotensin-Converting Enzyme Inhibitor and a Vitamin D Analog Suppresses the Progression of Renal Insufficiency in Uremic Rats
- Inhibition of Transforming Growth Factor-{beta} Signaling in Human Cancer: Targeting a Tumor Suppressor Network as a Therapeutic Strategy.
- Lipid Accumulation and Transforming Growth Factor-{beta} Upregulation in the Kidneys of Rats Administered Angiotensin II
- From the Periphery of the Glomerular Capillary Wall Toward the Center of Disease: Podocyte Injury Comes of Age in Diabetic Nephropathy
- N-Acetyl-Seryl-Aspartyl-Lysyl-Proline Prevents Renal Insufficiency and Mesangial Matrix Expansion in Diabetic db/db Mice
- Dual Blockade of the Renin-Angiotensin System: The Ultimate Treatment for Renal Protection?
- Diabetes Complications
- Epithelial to Mesenchymal Transition in Renal Fibrogenesis: Pathologic Significance, Molecular Mechanism, and Therapeutic Intervention