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ACE inhibitors attenuate expression of renal transforming growth factor-β1 in humans
ORIGINAL INVESTIGATIONS
ACE Inhibitors Attenuate Expression of Renal Transforming Growth Factor-1 in Humans Gyu-Tae Shin, MD, Seung-Jung Kim, MD, Kyoung-Ai Ma, MD, Heung-Soo Kim, MD, and Dohun Kim, MD ● Progressive nephropathies are characterized by the enhanced accumulation of extracellular matrix in the kidney. Overproduction of transforming growth factor- (TGF-) was shown to result in pathological tissue fibrosis through the accumulation of extracellular matrix proteins. It has been proposed that angiotensin II stimulates TGF- production. Despite accumulating data supporting the effects of angiotensin-converting enzyme (ACE) inhibitors on the attenuation of TGF- in vitro and in rats, such studies in humans are lacking. The present study sought to determine the effects of ACE inhibitors on TGF-1 in patients with glomerulonephritis. Using competitive polymerase chain reaction and the sandwich enzyme-linked immunosorbent assay, TGF-1 messenger RNA (mRNA) abundance and TGF-1 protein levels were measured. Patients with immunoglobulin A nephropathy administered ACE inhibitors showed significantly lower renal TGF-1 gene expression than patients not administered these medications (mean ratios of TGF-1/-actin, 4.27 ⴞ 0.62 [SEM] versus 14.81 ⴞ 3.87; P < 0.05), whereas no difference was noted between patients administered ACE inhibitors and healthy controls (4.27 ⴞ 0.62 versus 2.78 ⴞ 0.71). ACE inhibitor therapy did not affect TGF-1 mRNA expression in freshly isolated mononuclear cells. Urine and serum TGF-1 protein levels were not affected by the administration of ACE inhibitors. However, possibly a longer duration of treatment would decrease TGF-1 levels in urine or blood. In conclusion, we observed a significant reduction in TGF-1 expression in the kidney by ACE inhibitors, and this suggests that the effects of ACE inhibitors observed in animals can be extrapolated to patients with chronic renal disease. © 2000 by the National Kidney Foundation, Inc. INDEX WORDS: Glomerulonephritis; transforming growth factor- (TGF-); angiotensin-converting enzyme (ACE) inhibitors.
A
CCUMULATING evidence shows the beneficial effects of angiotensin-converting enzyme (ACE) inhibitors in retarding the progression of chronic renal disease over the last decade.1,2 These effects of ACE inhibition may be derived from both hemodynamic and nonhemodynamic mechanisms.3 Chronic renal injury results in a reduction in renal mass, with a subsequent increase in intraglomerular pressure, that further aggravates the progression of renal disease. ACE inhibitors block angiotensin II (ANG II) formation, which increases efferent arteriolar resistance preferentially,4 and a subse-
From the Department of Nephrology, Ajou University School of Medicine, Suwon, South Korea. Received February 3, 2000; accepted in revised form May 19, 2000. Supported in part by a grant from Ajou University School of Medicine. Address reprint requests to Gyu-Tae Shin, MD, Department of Nephrology, Ajou University School of Medicine, San 5, Wonchon-dong, Paldal-gu, Suwon, 442-721, South Korea. E-mail: [email protected] © 2000 by the National Kidney Foundation, Inc. 0272-6386/00/3605-0002$3.00/0 doi:10.1053/ajkd.2000.19078 894
quent reduction in intraglomerular pressure is believed to have a role in mitigating renal injury. It also has been suggested that renoprotective effects of ACE inhibitors may be mediated through changes in transforming growth factor- (TGF-) expression. TGF- is a fibrogenic cytokine exerting its effects by inducing the synthesis of extracellular matrix, decreasing its degradation, and stimulating the synthesis of integrin matrix receptors.5,6 Progressive nephropathies are characterized by the enhanced accumulation of extracellular matrix in the kidney,7 and overproduction of TGF- can result in pathological tissue fibrosis through the accumulation of extracellular matrix proteins. Evidence shows that TGF- expression is increased in various types of human glomerulonephritis, including diabetic nephropathy,8,9 lupus nephritis,10,11 human immunodeficiency virus nephropathy,12 and immunoglobulin A (IgA) nephropathy.13-15 Recent findings suggested that ANG II is one of the main culprits in progressive renal injury, and such suggestion is supported by the suppression of renal fibrosis by treatment with ACE inhibitors.16 Despite increasing data in vitro and
American Journal of Kidney Diseases, Vol 36, No 5 (November), 2000: pp 894-902
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in animal models, published studies substantiating the effects of ACE inhibitors on the attenuation of TGF- in human glomerulonephritis are scarce. Given the differences of human renal diseases from experimentally induced renal diseases in animals, studies of humans are essential to determine whether similar effects can be observed in humans. In this regard, the present study was undertaken to determine the effects of ACE inhibitors on TGF- in patients with glomerulonephritis. MATERIALS AND METHODS
Patients The present study consisted of two different experimental protocols. First, we investigated whether ACE inhibitors attenuate the expression of TGF-1 in the kidney. Second, we studied the effect of ACE inhibitors on TGF-1 levels in peripheral blood and urine. Informed consent was obtained from all enrolled individuals, and the study was approved by the Institutional Review Board of the Ajou University Hospital (Suwon, South Korea). Experiment 1: gene expression of TGF-1 in the kidney. Thirty-nine patients diagnosed with IgA nephropathy by renal biopsy were included in this study. Until biopsy was performed, 16 of 39 patients were administered ACE inhibitors (fosinopril or ramipril) for a mean of 84.38 ⫾ 27.79 (SEM) days (range, 7 to 365 days; ACE-inhibitor patients), whereas 23 others were never administered ACE inhibitors (no-ACE-inhibitor patients). Other medications included calcium channel blockers (amlodipine or nifedipine) in 4 patients in the no-ACE-inhibitor group and furosemide in 5 and 3 patients in the no-ACE-inhibitor and ACE-inhibitor groups, respectively. A biopsy core of renal specimen, obtained by using an 18G automated biopsy gun, was snap frozen immediately after each biopsy and kept at ⫺70°C until further analysis. Experiment 2: gene expression and protein concentrations of TGF-1 in peripheral blood and urine. Venous blood samples for serum and peripheral-blood mononuclear cells (PBMCs) and urine samples were obtained from 16 patients with various kinds of glomerulonephritis before ACE-inhibitor therapy. Repeated samples were obtained from these patients after being administered ACE inhibitors for 15.6 ⫾ 2.5 days (range, 7 to 37 days). Thirteen patients were administered ramipril, 2.5 to 10 mg/d, and 3 other
Table 1. Primers
Sequences
-Actin
Sense Antisense Sense Antisense
TGF-1
patients were administered fosinopril, 10 to 20 mg/d. Disease distribution included 9 patients with IgA nephropathy, 4 patients with diabetic nephropathy, 1 patient with focal segmental glomerulosclerosis, 1 patient with chronic glomerulonephritis, and 1 patient with hypertensive nephrosclerosis. Blood and urine samples from healthy volunteers (n ⫽ 12, experiment 2) and the normal part of nephrectomized kidneys from tumor patients (n ⫽ 11, experiment 1) served as controls.
Methods RNA isolation. PBMCs isolated from fresh heparinized blood using Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation were immediately lysed in Trizol reagents (Life Technologies, Rockville, MD). Kidney specimens were homogenized in the same reagents. RNA was extracted with chloroform, precipitated with isopropanol, washed with 75% ethanol, and then redissolved in Tris-EDTA (TE) buffer. The isolated RNA was quantified by spectrophotometry. Reverse transcription. One microgram of total RNA was reverse transcribed into complementary DNA (cDNA) in a 20-L reaction mixture containing 50 mmol/L of Tris-HCl (pH 8.3), 75 mmol/L of KCl, 10 mmol/L of dithiothreitol (DTT), 3 mmol/L MgCl2, 200 U of Moloney murine leukemia virus reverse transcriptase, 100 ng/reaction of random hexanucleotide primers, and 0.5 mmol/L each of deoxynucleoside triphosphate (dNTP): deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate. The reaction mixtures were incubated at 37°C for 1 hour and then at 65°C for 10 minutes to deactivate the reverse transcriptase. The final volume was adjusted to 50 L with TE buffer (pH 8.0), and the cDNAs were stored at ⫺20°C until further analysis. Competitive polymerase chain reaction. Competitive polymerase chain reaction (PCR) for quantification of messenger RNA (mRNA) was performed using the competitor constructs of -actin and TGF-1 (PCR MIMIC construction kit; Clontech Lab, Hampshire, UK; Table 1). A constant amount of the cDNA was coamplified with known serial concentrations of aliquots (10 L) of each competitor DNA construct in 15 L of reaction mixture containing 83.5 mmol/L of KCl, 16.7 mmol/L of Tris-HCl (pH 8.3), 2.5 mmol/L of MgCl2, 0.01% gelatin, 0.33 mol/L of each primer (Table 1), 1 U Taq DNA polymerase, and 67.5 mol/L of each dNTP. PCR was performed for 33 cycles for TGF-1 and 29 cycles for -actin using a Perkin Elmer 9600
Sequences of Primers Used in PCR
5⬘-GGT CAC CCA CAC TGT GCC CAT-3⬘ 5⬘-GGA TGC CAC AGG ACT CCA TGC-3⬘ 5⬘-CTG CGG ATC TCT GTG TCA TT-3⬘ 5⬘-CTC AGA GTG TTG CTA TGG TG-3⬘
NOTE. Each set of primers recognizes both the cDNA and the competitor.
cDNA (bp)
Competitor (bp)
350
442
246
340
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thermocycler (Perkin Elmer, Norwalk, CT). The amplification profile consisted of denaturing at 94°C for 20 seconds, primer annealing at 57°C for 30 seconds, and primer extension at 72°C for 20 seconds. Before cycling, the samples were preheated at 94°C for 30 seconds, and after amplification, an extra 5 minutes was added for extension at 72°C. The PCR products were applied to a 2%/1% Nusieve (FMC BioProducts, Rockland, ME)/agarose gel, resolved by electrophoresis, visualized by ethidium bromide staining, and photographed (Fig 1). The negatives were analyzed by laser densitometry, and the absolute absorbance values of the PCR products were determined. The ratios of the relevant PCR product pairs (target cDNA to competitor DNA) were plotted against the concentrations of the competitor used. The abundance of TGF-1 mRNA was determined as ratios of TGF-1 (in femtograms) to -actin (in picograms). Sandwich enzyme-linked immunosorbent assay. Serum and urine samples were analyzed for TGF-1 using commercially available enzyme-linked immunosorbent assay kits (Promega Corp, Madison, WI) according to the manufacturer’s specifications. Urine samples were centrifuged at 1,500g for 5 minutes before the assay. To measure total TGF-1, urine and diluted serum (1:300 in sample buffer 1⫻) samples were activated by acidification using 1 N of HCl to convert latent into active TGF-1, followed by the addition of 1 N of NaOH for neutralization. Statistical analysis. All results are expressed as mean ⫾ SEM. Statistical significance between multiple groups was evaluated using analysis of variance with the Bonferroni correction. Unpaired t-test or paired t-test was applied to compare continuous variables between two groups. Chisquare test was used to compare proportions between groups. To test for associations between the results, Pearson’s correlation coefficients were calculated. P less than 0.05 is considered significant.
RESULTS
Experiment 1 Renal TGF-1 mRNA expression. Of 23 noACE-inhibitor patients, 4 patients were administered calcium channel blockers (amlodipine or nifedipine) for blood pressure control. There were no differences between no-ACE-inhibitor patients (n ⫽ 23) and ACE-inhibitor patients (n ⫽ 16) in age, sex, serum creatinine level, mean blood pressure, daily urinary protein excretion determined by urine protein-creatinine ratios, and pathological severity on the basis of World Health Organization (WHO) criteria17 (Tables 2 and 3). TGF-1 mRNA abundance examined by competitive PCR was determined as the ratio of TGF-1 to -actin. ACE-inhibitor patients showed significantly lower TGF-1 gene expression than no-ACE-inhibitor patients (4.27 ⫾ 0.62 versus 14.81 ⫾ 3.87; P ⬍ 0.05), whereas no difference was noted when ACE-
Fig 1. Effect of ACE inhibitors on renal TGF-1 expression. (A) Representative competitive PCR of TGF-1 in kidney specimens of a no-ACE-inhibitor patient (patient 6, Table 2). (B) The corresponding bands for -actin (patient 6, Table 2). (C) Representative competitive PCR of TGF-1 in kidney specimens of an ACE-inhibitor patient (patient 16, Table 3). (D) The corresponding bands for -actin (patient 16, Table 3).
TGF-1 IN GLOMERULONEPHRITIS Table 2.
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Clinical Characteristics and Renal TGF-1 Results of No-ACE-Inhibitor Patients in Experiment 1
Patient No.
Age (y)
Sex
Medications (mg/d)
WHO*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Total‡
20 36 20 17 31 21 38 56 21 22 41 21 17 66 58 51 32 34 22 20 23 30 31 31.7 ⫾ 3.0
M M F M M M M F F F M F M F M F M F F M M M M 61 (% men)
None Amlodipine (10) None None None None Fr (80) Fr (40), nifedipine (30) None Fr (80) None None Fr (80) None None None None Fr (80), amlodipine (10) None None None None Amlodipine (10)
4 4 4 4 3 3 4 4 3 4 4 5 4 4 3 4 4 4 3 2 3 4 4
Cr (mg/dL)
Proteinuria (g/d)
Mean BP (mm Hg)
TGF-1† (fg/ pg)
1.1 2.16 94.3 3.24 1.9 0.46 116.7 6.38 0.7 2.68 98.3 5.32 1.0 0.34 93.3 14.87 1.7 1.67 101.0 62.00 0.8 16.10 104.7 9.56 1.1 9.95 106.7 0.59 3.9 2.82 95.0 46.00 1.0 2.37 100.0 18.48 1.0 12.53 89.7 4.62 1.1 0.52 90.0 3.85 1.2 2.09 100.0 1.16 1.9 10.68 96.7 35.85 1.1 9.14 83.3 1.31 1.4 0.67 95.0 6.84 1.2 6.61 96.7 6.22 1.4 1.78 96.7 0.88 5.0 6.11 89.7 12.00 0.6 3.22 81.7 62.20 1.0 0.58 90.0 14.58 1.0 0.24 95.0 15.71 0.8 1.44 84.3 3.47 1.8 1.83 97.7 5.50 1.5 ⫾ 0.2 4.17 ⫾ 0.94 95.5 ⫾ 1.6 14.81 ⫾ 3.87
Abbreviations: Cr, creatinine; BP, blood pressure; Fr, furosemide. *WHO pathological criteria, from class 1 to class 5. †Ratios of TGF-1 (in femtograms) to -actin (in picograms). ‡Data expressed as mean ⫾ SEM.
inhibitor patients were compared with healthy controls (n ⫽ 11; 4.27 ⫾ 0.62 versus 2.78 ⫾ 0.71; Fig 2). TGF-1 mRNA abundance of the patients administered calcium channel blockers (n ⫽ 4) in the no-ACE-inhibitor group was similar to that of the rest of the patients in this group (n ⫽ 19; 17.47 ⫾ 9.62 versus 14.25 ⫾ 4.34; P ⫽ 0.76). Correlation of clinical parameters. TGF-1 gene expression in renal tissue had no relationship with serum creatinine level, urinary protein excretion, mean blood pressure, and WHO pathological criteria. Experiment 2 PBMC mRNA expression. Spontaneous gene expression from freshly isolated PBMCs was observed in this experiment. TGF-1 mRNA expression of patients with glomerulonephritis before ACE-inhibitor therapy (n ⫽ 16) was similar to that of controls (n ⫽ 12; TGF-1/-actin,
0.66 ⫾ 0.19 versus 0.44 ⫾ 0.07 fg/pg, P ⫽ 0.32). TGF-1 mRNA expression before and after ACE inhibitor therapy in 16 pairs of samples did not differ (TGF-1–-actin, 0.66 ⫾ 0.19 versus 0.77 ⫾ 0.29 fg/pg, P ⫽ 0.62; Table 4). Urinary TGF-1 excretion. Urine TGF-1 concentration in picograms per milliliter was corrected by concomitant urine creatinine concentration (in milligrams per deciliter) to minimize the effect of urine concentration. The urinary TGF-1 level in patients with glomerulonephritis before ACE-inhibitor therapy (n ⫽ 16) was significantly greater than that of controls (n ⫽ 12; 7.13 ⫾ 1.89 versus 0.24 ⫾ 0.05 pg/mg; P ⫽ 0.002); however, it did not differ from that after ACE-inhibitor therapy (n ⫽ 16 pairs; 7.13 ⫾ 1.89 versus 8.78 ⫾ 2.01 pg/mg; P ⫽ 0.16; Table 4). Serum TGF-1 levels. Serum TGF-1 concentrations were not different between patients with glomerulonephritis before ACE-inhibitor therapy (n ⫽ 16) and controls (n ⫽ 12; 106.72 ⫾
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SHIN ET AL Table 3.
Clinical Characteristics and Renal TGF-1 Results of ACE-Inhibitor Patients in Experiment 1
Patient No.
Age (y)
Sex
WHO*
Medications (mg/d)
Cr (mg/dL)
Proteinuria (g/d)
Mean BP (mm Hg)
TGF-1† (fg/ pg)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total‡
36 44 24 40 45 36 28 56 60 13 25 34 29 53 28 18 35.6 ⫾ 3.4
M F F M F M F M F F M F M F M M 50 (% men)
4 4 3 4 4 4 3 4 4 4 4 4 4 3 4 4
Ramipril (5) Ramipril (5) Ramipril (2.5) Fr (80), ramipril (10) Ramipril (5) Fosinopril (10) Ramipril (2.5) Ramipril (5) Fr (80), ramipril (10) Ramipril (5) Ramipril (10) Fosinopril (20) Ramipril (5) Fr (80), fosinopril (10) Fosinopril (10) Ramipril (2.5)
1.6 1.2 1.1 1.1 1.2 3.2 1.9 1.9 2.3 0.7 1.2 0.9 1.4 0.8 1.5 1.0 1.4 ⫾ 0.2
0.92 0.39 0.25 7.40 1.35 5.13 3.82 0.68 14.70 2.57 0.52 4.08 0.83 6.00 1.98 1.53 3.26 ⫾ 0.94
100.7 96.7 80.0 95.7 75.8 104.3 98.7 96.7 97.7 93.3 93.7 102.3 94.3 90.0 94.3 96.7 94.4 ⫾ 1.9
3.45 6.43 5.63 0.00 9.58 8.56 3.30 4.27 3.00 2.48 3.18 3.13 5.87 1.72 4.64 3.06 4.27 ⫾ 0.62
Abbreviations: Cr, creatinine; BP, blood pressure; Fr, furosemide. *WHO pathological criteria, from class 1 to class 5. †Ratios of TGF-1 (in femtograms) to -actin (in picograms). ‡Data expressed as mean ⫾ SEM.
18.93 versus 81.73 ⫾ 39.19 ng/mL; P ⫽ 0.54). There was no difference in serum TGF-1 levels before and after ACE-inhibitor therapy (n ⫽ 16 pairs; 106.72 ⫾ 18.93 versus 92.36 ⫾ 20.48 ng/mL; P ⫽ 0.55; Table 4).
Fig 2. The renal gene expression of TGF-1 expressed as TGF-1/-actin in patients with IgA nephropathy not administered ACE inhibitors (NoACEI) and administered ACE inhibitors (ACEI). Controls are the normal part of nephrectomized kidneys. Mean values ⴞ SEM are given. * P < 0.05 versus NoACEI (oneway analysis of variance).
DISCUSSION
In this study, heightened renal expression of TGF-1 was observed in patients with IgA nephropathy not administered ACE inhibitors compared with controls. This is in accordance with previous reports in which enhanced renal TGF- expression was shown in patients with IgA nephropathy.13-15 Conversely, the expression of TGF-1 in patients administered ACE inhibitors was similar to that in controls and significantly less than that in patients not administered ACE inhibitors. It has been proposed that ANG II stimulates the production of TGF-, and this pathogenic link is interrupted by ANG II blockade by either ACE inhibitors or ANG II-receptor antagonists, which in turn slows the progression of renal fibrosis.18 Increasing numbers of studies have investigated the effects of ANG II blockade on the attenuation of TGF- in various animal models. Wu et al19 observed that ANG II blockade significantly blunted the upregulation of renal TGF- gene transcription in the rat with subtotal nephrectomy. Similarly, ANG II antagonism reduced the elevation of TGF- expression in the remnant kidney in rats.20 In rat models with
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Table 4.
Group (no. of patients)
Control (12) Before ACE inhibitors (16) After ACE inhibitors (16)
Mean BP (mm Hg)
— 107.19 ⫾ 3.01 98.50 ⫾ 2.85§
Effect of ACE Inhibitors in Experiment 2 Serum Creatinine (mg/dL)
PBMC TGF1 (fg/pg)*
Serum TGF-1 (ng/mL)
Urinary TGF-1 (pg/mg)†
— 3.34 ⫾ 0.82
— 1.76 ⫾ 0.16
0.44 ⫾ 0.07 0.66 ⫾ 0.19
81.73 ⫾ 39.19 106.72 ⫾ 18.93
0.24 ⫾ 0.05 7.13 ⫾ 1.89‡
2.51 ⫾ 0.62㛳
1.84 ⫾ 0.18
0.77 ⫾ 0.29
92.36 ⫾ 20.48
8.78 ⫾ 2.01¶
Proteinuria (g/d)
NOTE. Data expressed as mean ⫾ SEM. Abbreviation: BP, blood pressure. *mRNA expression, ratios of TGF-1 (in femtograms) to -actin (in picograms). †Urine TGF-1 concentration (in picograms per milliliter) corrected by urinary creatinine concentration (in milligrams per deciliter). ‡P ⫽ 0.002 versus control. §P ⬍ 0.001 versus before ACE inhibitors. 㛳P ⫽ 0.009 versus before ACE inhibitors. ¶P ⫽ 0.001 versus control.
unilateral ureteral obstruction, ANG II blockade significantly blunted the increase in TGF- mRNA in the obstructed kidney.21,22 Again, ANG II blockade effectively reduced intrarenal TGF- expression and renal injury in experimentally induced glomerulonephritis.23,24 The ANG IIreceptor antagonist significantly prevented the increase in glomerular TGF- mRNA in twokidney, one-clip hypertensive rats25 and strokeprone spontaneously hypertensive rats.26 These all indicate that ANG II blockade can prevent the increase in TGF- production in the kidney, thus delaying the progression of renal disease. Despite such accumulating data in animal models, the publications showing the effects of ACE inhibitors on the attenuation of TGF- in humans are scarce. Because differences may exist between chronic renal diseases in humans and experimentally induced renal diseases in animals, it is possible that the former may respond to ANG II blockade in a way that is different from the latter. In this regard, our current study of patients with chronic glomerulonephritis is of particular value. We observed a significant reduction in TGF-1 expression in the kidney by ACE inhibitors, and this suggests that the effects of ANG II blockade observed in rats mentioned previously can be extrapolated to patients with chronic renal disease. Conflicting data exist on whether blood pressure per se may regulate the expression of TGF- in the kidney. In vitro studies have shown that
pressure-induced glomerular distention can stimulate TGF- synthesis and extracellular matrix production.27,28 Nifedipine,29 as well as triple therapy with hydralazine, reserpine, and hydrochlorothiazide,25 effectively prevented the increase in TGF- in the experimental animal models. These findings suggest that systemic hypertension generates glomerular stretch, which in turn stimulates the synthesis of TGF-. Conversely, some studies suggest that blood pressure is not a factor in the regulation of TGF- production. Junaid et al20 reported that triple therapy with hydralazine, reserpine, and hydrochlorothiazide did not induce a decrease in TGF- level in the rat remnant kidney, whereas the ANG II antagonist reduced the elevation of TGF- expression. Administration of a small dose, insufficient to reduce systemic blood pressure, of ANG II-receptor antagonist remarkably attenuated the increase in renal TGF- gene expression in stroke-prone spontaneously hypertensive rats.30 Similar findings have been reported with an ACE inhibitor in immune complex nephritis in the absence of an effect on blood pressure.31 Moreover, cell culture experiments, in which the effects on intraglomerular pressure are absent, favor the hypothesis that ANG II directly stimulates TGF- synthesis. For example, Kagami et al32 showed that ANG II stimulated matrix proteins through the induction of TGF- activity in mesangial cell cultures, and Wolf et al33 reported that ANG II stimulated TGF- synthesis in mu-
900
rine proximal tubular cell cultures. Because blood pressure was similar in both groups in the current study, differences in TGF- expression in these groups cannot be explained by the reduction in blood pressure with ACE inhibitors. Hence, it is likely that these differences may result from either a decrease in intraglomerular pressure through the inhibition of the intrarenal reninangiotensin system,16 blocking the direct stimulatory effect of ANG II on TGF-, or both. Hamaguchi et al29 reported that calcium channel blockers were effective in blunting TGF- upregulation in the kidney. Such effects of calcium channel blockers on TGF- could not be evaluated in the current study because only a small number of patients (n ⫽ 4) were administered these medications. However, at least there was no tendency toward downregulation of TGF- in the kidney by calcium channel blockers. The current study does not give information on which structure in renal tissue is responsible for the change in TGF-1 expression. Therefore, investigations in isolated glomeruli or localization of TGF-1 by in situ hybridization or immunohistochemistry remain to be performed to evaluate the source of renal TGF-1 expression. Recently, mononuclear cells have been implicated in the pathogenesis of glomerulonephritis. Arima et al34 observed renal trafficking of mononuclear cells in IgA nephropathy, and it was shown that PBMCs have the capacity to produce high levels of various cytokines in patients with glomerulonephritis.35,36 In addition, upregulation of TGF- by cultured PBMCs in patients with IgA nephropathy suggested the possibility of PBMCs as a source of TGF- in the kidney.37,38 PBMCs are known to harbor ANG II type 1 receptors,39,40 and ANG II stimulates the synthesis of cytokines in PBMCs through these receptors.41 Accordingly, heightened TGF- gene transcription was observed in monocytes infiltrating the renal interstitium, followed by its significant reduction by ANG II blockade in the rat kidney with subtotal nephrectomy.19 Based on these studies, we examined the status of TGF- expression and the effects of ACE inhibitors on the synthesis of TGF- in circulating PBMCs. In the current study, TGF-1 gene transcription was not enhanced in PBMCs from patients
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with glomerulonephritis compared with controls or attenuated by ACE inhibitors. Some possible reasons for these results follow. First, given the absence of hemodynamic effects in the environment in which PBMCs are circulating, ANG II may regulate TGF- synthesis through changes in intraglomerular pressure rather than directly regulate it. Second, systemic ANG II levels may not be elevated despite the increased ANG II in the kidney, which explains why the TGF- level is elevated in renal tissue but not in PBMCs. Previous studies have suggested that systemic ANG II may be dissociated from intrarenal ANG II.42 Last, the difference in TGF- expression between the groups may become evident if PBMCs are stimulated in vitro, as in previous studies.37,38 Additional studies are needed to confirm our findings and elucidate the underlying mechanism. In the current investigation, TGF-1 levels were significantly increased in urine of patients with glomerulonephritis. Data exist showing a correlation between urinary TGF- excretion and renal fibrosis in rats,43 as well as in patients with glomerulonephritis.44 However, in the present study, urinary TGF-1 levels were not decreased after ACE-inhibitor therapy. This is contradictory to our expectation, but it is possible that longer treatment is needed for the drug to accumulate in the organs and decrease TGF- production, although 4 to 8 days of ACE-inhibitor therapy was sufficient to see the effects on TGF- in the kidney and/or urine in certain animal models.20,22,24 Other possibilities are that TGF- mRNA expression can be dissociated from active protein secretion,45 or the urine TGF- level may be an insufficient indicator of the renal TGF- protein synthesis. In the present study, serum levels of TGF-1 were not increased in patients with glomerulonephritis or suppressed by ACE inhibitors. Again, this is contradictory to our expectation, but previous investigations showed that serum or plasma levels of TGF-1 were not increased in animal models of renal disease, including diabetic rats46; two-kidney, one-clip hypertensive rats25; and human immunodeficiency virus transgenic mice.47 Furthermore, plasma TGF-1 concentrations were not reduced by the administration of ACE
TGF-1 IN GLOMERULONEPHRITIS
inhibitors in their studies.25,47 However, possibly the duration of ACE inhibitor therapy in the current study was not long enough to see the effect of ACE inhibitors on serum TGF-1 levels. Previous reports showed a significant reduction of serum and/or plasma TGF-1 levels after 6 months of ACE-inhibitor treatment48 or 4 weeks of ANG II-receptor antagonist therapy.49 Although serum levels of TGF- reflect plateletderived TGF- contributed by platelet degranulation, as well as circulating TGF-,50 many studies have found that measurements of serum TGF- are informative in clinical diseases.51-53 It remains to be determined whether measurements of plasma TGF- may render different results from ours. In summary, our current study showed that ACE inhibitors attenuate the increase in renal TGF-1 expression in patients with IgA nephropathy. The downregulation of renal TGF-1 expression in patients with glomerulonephritis may be a central part of the beneficial effects of ACE inhibitors on chronic progressive nephropathies. REFERENCES 1. Anderson S, Rennke HG, Brenner BM: Therapeutic advantage of converting enzyme inhibitors in arresting progressive renal disease associated with systemic hypertension in the rat. J Clin Invest 77:1993-2000, 1986 2. Ruggenenti P, Perna A, Benini R, Bertani T, Zoccali C, Maggiore Q, Salvadori M, Remuzzi G: In chronic nephropathies, prolonged ACE inhibition can induce remission: Dynamics of time-dependent changes in GFR. J Am Soc Nephrol 10:997-1006, 1999 3. Hollenberg NK, Raij L: Angiotensin-converting enzyme inhibition and renal protection. Arch Intern Med 153:2426-2435, 1993 4. Denton KM, Fennessy PA, Alcorn D, Anderson WP: Morphometric analysis of the actions of angiotensin II on renal arterioles and glomeruli. Am J Physiol 262:367-372, 1992 5. Border WA, Noble NA: Transforming growth factor- in tissue fibrosis. N Engl J Med 331:1286-1292, 1994 6. Eddy AA: Molecular insights into renal interstitial fibrosis. J Am Soc Nephrol 7:2495-2508, 1996 7. Yamamoto T, Noble NA, Miller DE, Border WA: Sustained expression of TGF-1 underlies development of progressive kidney fibrosis. Kidney Int 45:916-927, 1994 8. Yamamoto T, Nakamura T, Noble NA, Rouslahti E, Border WA: Expression of TGF- in human and experimental diabetic nephropathy. Proc Natl Acad Sci U S A 90:18141818, 1993 9. Iwano M, Kubo A, Nishino T, Sato H, Nishioka H, Akai Y, Kurioka H, Fuji Y, Kanauchi M, Shiiki H, Dohi K: Quantification of glomerular TGF-1 mRNA in patients with diabetes mellitus. Kidney Int 49:1120-1126, 1996
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10. Yang CW, Hsueh S, Wu MS, Lai PC, Huang JY, Wu CH, Hu SA, Chen JF, Huang CC: Glomerular TGF-1 mRNA as a marker of glomerulosclerosis—Application in renal biopsies. Nephron 77:290-297, 1997 11. Yamamoto T, Noble NA, Cohen AH, Nast CC, Hishida A, Gold LI, Border WA: Expression of TGF- isoforms in human glomerular diseases. Kidney Int 49:461-469, 1996 12. Bodi I, Kimmel PL, Abraham AA, Svetkey LP, Klotman PE, Kopp JB: Renal TGF- in HIV-associated kidney diseases. Kidney Int 51:1568-1577, 1997 13. Niemir ZI, Stein H, Noronha IL, Kruger C, Andrassy K, Ritz E, Waldherr R: PDGF and TGF- contribute to the natural course of human IgA glomerulonephritis. Kidney Int 48:1530-1541, 1995 14. Yoshioka K, Takemura T, Murakami K, Okada M, Hino S, Miyamoto H, Maki S: TGF- protein and mRNA in glomeruli in normal and diseased human kidneys. Lab Invest 68:154-163, 1993 15. Taniguchi Y, Yorioka N, Masaki T, Yamashita K, Ito T, Ueda H, Yamakido M: Role of TGF-1 in glomerulonephritis. J Int Med Res 25:71-80, 1997 16. Matsusaka T, Hymes J, Ichikawa I: Angiotensin in progressive renal diseases: Theory and practice. J Am Soc Nephrol 7:2025-2043, 1996 17. D’Amico G: Immunoglobulin A nephropathy, in Jacobson HR, Striker GE, Klahr S (eds): The Principles and Practice of Nephrology. St Louis, MO, Mosby, 1995, pp 134-135 18. Border WA, Noble NA: Interactions of TGF- and angiotensin II in renal fibrosis. Hypertension 31:181-188, 1998 19. Wu LL, Cox A, Roe CJ, Dzidek M, Cooper ME, Gilbert RE: Transforming growth factor-1 and renal injury following nephrectomy in the rat: Role of the reninangiotensin system. Kidney Int 51:1553-1567, 1997 20. Junaid A, Hostetter TH, Rosenberg ME: Interaction of angiotensin II and TGF-1 in the rat remnant kidney. J Am Soc Nephrol 8:1732-1738, 1997 21. Pimentel JL Jr, Sundell CL, Wang S, Kopp JB, Montero A, Martı´nez-Maldonado M: Role of angiotensin II in the expression and regulation of TGF- in obstructive nephropathy. Kidney Int 48:1233-1246, 1995 22. Kaneto H, Morrissey J, Klahr S: Increased expression of TGF-1 mRNA in the obstructed kidney of rats with unilateral ureteral ligation. Kidney Int 44:313-321, 1993 23. Zoja C, Donadelli R, Corna D, Testa D, Facchinetti D, Maffi R, Luzzana E, Colosio V, Bertani T, Remuzzi G: The renoprotective properties of angiotensin-converting enzyme inhibitors in a chronic model of membranous nephropathy are solely due to the inhibition of angiotensin II: Evidence based on comparative studies with a receptor antagonist. Am J Kidney Dis 29:254-264, 1997 24. Zoja C, Abbate M, Corna D, Capitanio M, Donadelli R, Bruzzi I, Oldroyd S, Benigni A, Remuzzi G: Pharmacologic control of angiotensin II ameliorates renal disease while reducing renal TGF- in experimental mesangioproliferative glomerulonephritis. Am J Kidney Dis 31:453-463, 1998 25. Wolf G, Schneider A, Wenzel U, Helmchen U, Stahl RA: Regulation of glomerular TGF- expression in the
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contralateral kidney of two-kidney, one-clip hypertensive rats. J Am Soc Nephrol 9:763-772, 1998 26. Obata JE, Nakamura T, Kuroyanagi R, Yoshida Y, Guo DF, Inagami T: Candesartan prevents the progression of glomerulosclerosis in genetic hypertensive rats. Kidney Int 52:S229-S231, 1997 (suppl 63) 27. Riser BL, Cortes P, Heilig C, Grondin J, LadsonWofford S, Patterson D, Narins RG: Cyclic stretching force selectively upregulates TGF- isoforms in cultured mesangial cells. Am J Pathol 148:1915-1923, 1996 28. Yasuda T, Kondo S, Homma T, Harris RC: Regulation of extracellular matrix by mechanical stress in rat glomerular mesangial cells. J Clin Invest 98:1991-2000, 1996 29. Hamaguchi A, Kim S, Ohta K, Yagi K, Yukimura T, Miura K, Fukuda T, Iwano H: TGF-1 expression and phenotypic modulation in the kidney of hypertensive rats. Hypertension 26:199-207, 1995 30. Kim S, Ohta K, Hamaguchi A, Omura T, Yukimura T, Miura K, Inada Y, Wada T, Ishimura Y, Chatani F, Iwano H: Contribution of renal angiotensin II type I receptor to gene expressions in hypertension-induced renal injury. Kidney Int 46:1346-1358, 1994 31. Ruiz-Ortega M, Gonzalez S, Seron D, Condom E, Bustos C, Largo R, Gonzalez E, Ortiz E, Egido J: ACE inhibition reduces proteinuria, glomerular lesions and extracellular matrix production in a normotensive rat model of immune complex nephritis. Kidney Int 48:1778-1791, 1995 32. Kagami S, Border WA, Miller DE, Noble NA: Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor- expression in rat glomerular mesangial cells. J Clin Invest 93:24312437, 1994 33. Wolf G, Mueller E, Stahl RA, Ziyadeh FN: Angiotensin II-induced hypertrophy of cultured murine proximal tubular cells is mediated by endogenous TGF-. J Clin Invest 92:1366-1372, 1993 34. Arima S, Nakayama M, Naito M, Satu T, Takahashi K: Significance of mononuclear phagocytes in IgA nephropathy. Kidney Int 39:684-692, 1991 35. Scivittaro V, Gesualdo L, Ranieri E, Marfella C, Schewn SA, Emancipator SN, Schena FP: Profiles of immunoregulatory cytokine production in vitro in patients with IgA nephropathy and their kindred. Clin Exp Immunol 96:311-316, 1994 36. Ichinose H, Miyazaki M, Koji T, Furusu Y, Ozono T, Harada T, Shin M, Najkane PK, Hara K: Detection of cytokine mRNA-expressing cells in peripheral blood of patients with IgA nephropathy using non-radioactive in situ hybridization. Clin Exp Immunol 103:125-132, 1996 37. Lai KN, Ho RT, Leung JC, Lai FM, Li PK: Increased mRNA encoding for transforming growth factor-beta in CD4⫹ cells from patients with IgA nephropathy. Kidney Int 46:862-868, 1994 38. de Caestecker MP, Bottomley M, Telfer BA, Hutchinson IV, Vose BM, Ballardie FW: Detection of abnormal peripheral blood mononuclear cell cytokine networks in human IgA nephropathy. Kidney Int 44:1298-1308, 1993 39. Lijnen P, Fagard R, Petrov V: Cytosolic calcium changes induced by angiotensin II in human peripheral blood mononuclear cells are mediated via angiotensin II subtype 1 receptors. J Hypertens 15:871-876, 1997
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40. Shibata H, Suzuki H, Murakami M, Sato A, Saruta T: Angiotensin II type 1 receptor mRNA levels in human blood cells of patients with primary and secondary hypertension: Reference to renin profile. J Hypertens 12:1275-1284, 1994 41. Hahn AW, Jonas U, Buhler FR, Resink TJ: Activation of human peripheral monocytes by angiotensin II. FEBS Lett 347:178-180, 1994 42. Vallon V, Wead LM, Blantz RC: Renal hemodynamics and plasma and kidney angiotensin II in established diabetes mellitus in rats: Effect of sodium and salt restriction. J Am Soc Nephrol 5:1761-1767, 1995 43. Monteiro de Freitas AS, Coimbra TM, Costa RS, Baroni EA: Urinary TGF- excretion and renal production of TGF- in rats with subtotal renal ablation: Effect of enalapril and nifedipine. Nephron 78:302-309, 1998 44. Honkanen E, Teppo AM, Tornroth T, Groop PH, Gronhagen-Riska C: Urinary TGF-1 in membranous glomerulonephritis. Nephrol Dial Transplant 12:2562-2568, 1997 45. Assoian RK, Fleurdelys BE, Stevenson HC, Miller PJ, Madtes DK, Raines EW, Ross R, Sporn MB: Expression and secretion of type beta transforming growth factor by activated human macrophage. Proc Natl Acad Sci U S A 84:6020-6024, 1987 46. Sharma K, Jin Y, Guo J, Ziyadeh FN: Neutralization of TGF- by anti-TGF- antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice. Diabetes 45:522-530, 1996 47. Bird JE, Durham SK, Giancarli MR, Gitlitz PH, Pandya DG, Dambach DM, Mozes MM, Kopp JB: Captopril prevents nephropathy in HIV-transgenic mice. J Am Soc Nephrol 9:1441-1447, 1998 48. Sharma K, Eltayeb BO, McGowan TA, Dunn SR, Alzahabi B, Rohde R, Ziyadeh FN, Lewis EJ: Captoprilinduced reduction of serum levels of transforming growth factor-1 correlates with long-term renoprotection in insulindependent diabetic patients. Am J Kidney Dis 34:818-823, 1999 49. Campistol JM, Inigo P, Jimenez W, Lario S, Clesca PH, Oppenheimer F, Rivera F: Losartan decreases plasma levels of TGF-beta1 in transplant patients with chronic allograft nephropathy. Kidney Int 56:714-719, 1999 50. Wakefield LM, Letterio JJ, Chen T, Danielpour D, Allison RSH, Pai LH, Denicoff AM, Noone MH, Cowan KH, O’Shaughnessy JA, Sporn MB: TGF-1 circulates in normal plasma and is unchanged in advanced metastatic breast cancer. Clin Cancer Res 1:129-136, 1995 51. Grainger DJ, Kemp PR, Metcalfe JC, Liu AC, Lawn RM, William NR, Grace AA, Schofield PM, Chauhan A: The serum concentration of active TGF- is severely depressed in advanced atherosclerosis. Nat Med 1:74-79, 1995 52. Hirakata Y, Kitamura S: Elevated serum TGF-1 level in primary lung cancer patients with finger clubbing. Eur J Clin Invest 26:820-823, 1996 53. Benett A, Chao C, Hu S, Buchwald D, Fagioll L, Scur P, Peterson P, Komaroff A: Elevation of bioactive TGF- in serum from patients with chronic fatigue syndrome. J Clin Immunol 17:160-166, 1997
ACE Inhibitors Attenuate Expression of Renal Transforming Growth Factor-1 in Humans Gyu-Tae Shin, MD, Seung-Jung Kim, MD, Kyoung-Ai Ma, MD, Heung-Soo Kim, MD, and Dohun Kim, MD ● Progressive nephropathies are characterized by the enhanced accumulation of extracellular matrix in the kidney. Overproduction of transforming growth factor- (TGF-) was shown to result in pathological tissue fibrosis through the accumulation of extracellular matrix proteins. It has been proposed that angiotensin II stimulates TGF- production. Despite accumulating data supporting the effects of angiotensin-converting enzyme (ACE) inhibitors on the attenuation of TGF- in vitro and in rats, such studies in humans are lacking. The present study sought to determine the effects of ACE inhibitors on TGF-1 in patients with glomerulonephritis. Using competitive polymerase chain reaction and the sandwich enzyme-linked immunosorbent assay, TGF-1 messenger RNA (mRNA) abundance and TGF-1 protein levels were measured. Patients with immunoglobulin A nephropathy administered ACE inhibitors showed significantly lower renal TGF-1 gene expression than patients not administered these medications (mean ratios of TGF-1/-actin, 4.27 ⴞ 0.62 [SEM] versus 14.81 ⴞ 3.87; P < 0.05), whereas no difference was noted between patients administered ACE inhibitors and healthy controls (4.27 ⴞ 0.62 versus 2.78 ⴞ 0.71). ACE inhibitor therapy did not affect TGF-1 mRNA expression in freshly isolated mononuclear cells. Urine and serum TGF-1 protein levels were not affected by the administration of ACE inhibitors. However, possibly a longer duration of treatment would decrease TGF-1 levels in urine or blood. In conclusion, we observed a significant reduction in TGF-1 expression in the kidney by ACE inhibitors, and this suggests that the effects of ACE inhibitors observed in animals can be extrapolated to patients with chronic renal disease. © 2000 by the National Kidney Foundation, Inc. INDEX WORDS: Glomerulonephritis; transforming growth factor- (TGF-); angiotensin-converting enzyme (ACE) inhibitors.
A
CCUMULATING evidence shows the beneficial effects of angiotensin-converting enzyme (ACE) inhibitors in retarding the progression of chronic renal disease over the last decade.1,2 These effects of ACE inhibition may be derived from both hemodynamic and nonhemodynamic mechanisms.3 Chronic renal injury results in a reduction in renal mass, with a subsequent increase in intraglomerular pressure, that further aggravates the progression of renal disease. ACE inhibitors block angiotensin II (ANG II) formation, which increases efferent arteriolar resistance preferentially,4 and a subse-
From the Department of Nephrology, Ajou University School of Medicine, Suwon, South Korea. Received February 3, 2000; accepted in revised form May 19, 2000. Supported in part by a grant from Ajou University School of Medicine. Address reprint requests to Gyu-Tae Shin, MD, Department of Nephrology, Ajou University School of Medicine, San 5, Wonchon-dong, Paldal-gu, Suwon, 442-721, South Korea. E-mail: [email protected] © 2000 by the National Kidney Foundation, Inc. 0272-6386/00/3605-0002$3.00/0 doi:10.1053/ajkd.2000.19078 894
quent reduction in intraglomerular pressure is believed to have a role in mitigating renal injury. It also has been suggested that renoprotective effects of ACE inhibitors may be mediated through changes in transforming growth factor- (TGF-) expression. TGF- is a fibrogenic cytokine exerting its effects by inducing the synthesis of extracellular matrix, decreasing its degradation, and stimulating the synthesis of integrin matrix receptors.5,6 Progressive nephropathies are characterized by the enhanced accumulation of extracellular matrix in the kidney,7 and overproduction of TGF- can result in pathological tissue fibrosis through the accumulation of extracellular matrix proteins. Evidence shows that TGF- expression is increased in various types of human glomerulonephritis, including diabetic nephropathy,8,9 lupus nephritis,10,11 human immunodeficiency virus nephropathy,12 and immunoglobulin A (IgA) nephropathy.13-15 Recent findings suggested that ANG II is one of the main culprits in progressive renal injury, and such suggestion is supported by the suppression of renal fibrosis by treatment with ACE inhibitors.16 Despite increasing data in vitro and
American Journal of Kidney Diseases, Vol 36, No 5 (November), 2000: pp 894-902
TGF-1 IN GLOMERULONEPHRITIS
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in animal models, published studies substantiating the effects of ACE inhibitors on the attenuation of TGF- in human glomerulonephritis are scarce. Given the differences of human renal diseases from experimentally induced renal diseases in animals, studies of humans are essential to determine whether similar effects can be observed in humans. In this regard, the present study was undertaken to determine the effects of ACE inhibitors on TGF- in patients with glomerulonephritis. MATERIALS AND METHODS
Patients The present study consisted of two different experimental protocols. First, we investigated whether ACE inhibitors attenuate the expression of TGF-1 in the kidney. Second, we studied the effect of ACE inhibitors on TGF-1 levels in peripheral blood and urine. Informed consent was obtained from all enrolled individuals, and the study was approved by the Institutional Review Board of the Ajou University Hospital (Suwon, South Korea). Experiment 1: gene expression of TGF-1 in the kidney. Thirty-nine patients diagnosed with IgA nephropathy by renal biopsy were included in this study. Until biopsy was performed, 16 of 39 patients were administered ACE inhibitors (fosinopril or ramipril) for a mean of 84.38 ⫾ 27.79 (SEM) days (range, 7 to 365 days; ACE-inhibitor patients), whereas 23 others were never administered ACE inhibitors (no-ACE-inhibitor patients). Other medications included calcium channel blockers (amlodipine or nifedipine) in 4 patients in the no-ACE-inhibitor group and furosemide in 5 and 3 patients in the no-ACE-inhibitor and ACE-inhibitor groups, respectively. A biopsy core of renal specimen, obtained by using an 18G automated biopsy gun, was snap frozen immediately after each biopsy and kept at ⫺70°C until further analysis. Experiment 2: gene expression and protein concentrations of TGF-1 in peripheral blood and urine. Venous blood samples for serum and peripheral-blood mononuclear cells (PBMCs) and urine samples were obtained from 16 patients with various kinds of glomerulonephritis before ACE-inhibitor therapy. Repeated samples were obtained from these patients after being administered ACE inhibitors for 15.6 ⫾ 2.5 days (range, 7 to 37 days). Thirteen patients were administered ramipril, 2.5 to 10 mg/d, and 3 other
Table 1. Primers
Sequences
-Actin
Sense Antisense Sense Antisense
TGF-1
patients were administered fosinopril, 10 to 20 mg/d. Disease distribution included 9 patients with IgA nephropathy, 4 patients with diabetic nephropathy, 1 patient with focal segmental glomerulosclerosis, 1 patient with chronic glomerulonephritis, and 1 patient with hypertensive nephrosclerosis. Blood and urine samples from healthy volunteers (n ⫽ 12, experiment 2) and the normal part of nephrectomized kidneys from tumor patients (n ⫽ 11, experiment 1) served as controls.
Methods RNA isolation. PBMCs isolated from fresh heparinized blood using Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation were immediately lysed in Trizol reagents (Life Technologies, Rockville, MD). Kidney specimens were homogenized in the same reagents. RNA was extracted with chloroform, precipitated with isopropanol, washed with 75% ethanol, and then redissolved in Tris-EDTA (TE) buffer. The isolated RNA was quantified by spectrophotometry. Reverse transcription. One microgram of total RNA was reverse transcribed into complementary DNA (cDNA) in a 20-L reaction mixture containing 50 mmol/L of Tris-HCl (pH 8.3), 75 mmol/L of KCl, 10 mmol/L of dithiothreitol (DTT), 3 mmol/L MgCl2, 200 U of Moloney murine leukemia virus reverse transcriptase, 100 ng/reaction of random hexanucleotide primers, and 0.5 mmol/L each of deoxynucleoside triphosphate (dNTP): deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate. The reaction mixtures were incubated at 37°C for 1 hour and then at 65°C for 10 minutes to deactivate the reverse transcriptase. The final volume was adjusted to 50 L with TE buffer (pH 8.0), and the cDNAs were stored at ⫺20°C until further analysis. Competitive polymerase chain reaction. Competitive polymerase chain reaction (PCR) for quantification of messenger RNA (mRNA) was performed using the competitor constructs of -actin and TGF-1 (PCR MIMIC construction kit; Clontech Lab, Hampshire, UK; Table 1). A constant amount of the cDNA was coamplified with known serial concentrations of aliquots (10 L) of each competitor DNA construct in 15 L of reaction mixture containing 83.5 mmol/L of KCl, 16.7 mmol/L of Tris-HCl (pH 8.3), 2.5 mmol/L of MgCl2, 0.01% gelatin, 0.33 mol/L of each primer (Table 1), 1 U Taq DNA polymerase, and 67.5 mol/L of each dNTP. PCR was performed for 33 cycles for TGF-1 and 29 cycles for -actin using a Perkin Elmer 9600
Sequences of Primers Used in PCR
5⬘-GGT CAC CCA CAC TGT GCC CAT-3⬘ 5⬘-GGA TGC CAC AGG ACT CCA TGC-3⬘ 5⬘-CTG CGG ATC TCT GTG TCA TT-3⬘ 5⬘-CTC AGA GTG TTG CTA TGG TG-3⬘
NOTE. Each set of primers recognizes both the cDNA and the competitor.
cDNA (bp)
Competitor (bp)
350
442
246
340
896
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thermocycler (Perkin Elmer, Norwalk, CT). The amplification profile consisted of denaturing at 94°C for 20 seconds, primer annealing at 57°C for 30 seconds, and primer extension at 72°C for 20 seconds. Before cycling, the samples were preheated at 94°C for 30 seconds, and after amplification, an extra 5 minutes was added for extension at 72°C. The PCR products were applied to a 2%/1% Nusieve (FMC BioProducts, Rockland, ME)/agarose gel, resolved by electrophoresis, visualized by ethidium bromide staining, and photographed (Fig 1). The negatives were analyzed by laser densitometry, and the absolute absorbance values of the PCR products were determined. The ratios of the relevant PCR product pairs (target cDNA to competitor DNA) were plotted against the concentrations of the competitor used. The abundance of TGF-1 mRNA was determined as ratios of TGF-1 (in femtograms) to -actin (in picograms). Sandwich enzyme-linked immunosorbent assay. Serum and urine samples were analyzed for TGF-1 using commercially available enzyme-linked immunosorbent assay kits (Promega Corp, Madison, WI) according to the manufacturer’s specifications. Urine samples were centrifuged at 1,500g for 5 minutes before the assay. To measure total TGF-1, urine and diluted serum (1:300 in sample buffer 1⫻) samples were activated by acidification using 1 N of HCl to convert latent into active TGF-1, followed by the addition of 1 N of NaOH for neutralization. Statistical analysis. All results are expressed as mean ⫾ SEM. Statistical significance between multiple groups was evaluated using analysis of variance with the Bonferroni correction. Unpaired t-test or paired t-test was applied to compare continuous variables between two groups. Chisquare test was used to compare proportions between groups. To test for associations between the results, Pearson’s correlation coefficients were calculated. P less than 0.05 is considered significant.
RESULTS
Experiment 1 Renal TGF-1 mRNA expression. Of 23 noACE-inhibitor patients, 4 patients were administered calcium channel blockers (amlodipine or nifedipine) for blood pressure control. There were no differences between no-ACE-inhibitor patients (n ⫽ 23) and ACE-inhibitor patients (n ⫽ 16) in age, sex, serum creatinine level, mean blood pressure, daily urinary protein excretion determined by urine protein-creatinine ratios, and pathological severity on the basis of World Health Organization (WHO) criteria17 (Tables 2 and 3). TGF-1 mRNA abundance examined by competitive PCR was determined as the ratio of TGF-1 to -actin. ACE-inhibitor patients showed significantly lower TGF-1 gene expression than no-ACE-inhibitor patients (4.27 ⫾ 0.62 versus 14.81 ⫾ 3.87; P ⬍ 0.05), whereas no difference was noted when ACE-
Fig 1. Effect of ACE inhibitors on renal TGF-1 expression. (A) Representative competitive PCR of TGF-1 in kidney specimens of a no-ACE-inhibitor patient (patient 6, Table 2). (B) The corresponding bands for -actin (patient 6, Table 2). (C) Representative competitive PCR of TGF-1 in kidney specimens of an ACE-inhibitor patient (patient 16, Table 3). (D) The corresponding bands for -actin (patient 16, Table 3).
TGF-1 IN GLOMERULONEPHRITIS Table 2.
897
Clinical Characteristics and Renal TGF-1 Results of No-ACE-Inhibitor Patients in Experiment 1
Patient No.
Age (y)
Sex
Medications (mg/d)
WHO*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Total‡
20 36 20 17 31 21 38 56 21 22 41 21 17 66 58 51 32 34 22 20 23 30 31 31.7 ⫾ 3.0
M M F M M M M F F F M F M F M F M F F M M M M 61 (% men)
None Amlodipine (10) None None None None Fr (80) Fr (40), nifedipine (30) None Fr (80) None None Fr (80) None None None None Fr (80), amlodipine (10) None None None None Amlodipine (10)
4 4 4 4 3 3 4 4 3 4 4 5 4 4 3 4 4 4 3 2 3 4 4
Cr (mg/dL)
Proteinuria (g/d)
Mean BP (mm Hg)
TGF-1† (fg/ pg)
1.1 2.16 94.3 3.24 1.9 0.46 116.7 6.38 0.7 2.68 98.3 5.32 1.0 0.34 93.3 14.87 1.7 1.67 101.0 62.00 0.8 16.10 104.7 9.56 1.1 9.95 106.7 0.59 3.9 2.82 95.0 46.00 1.0 2.37 100.0 18.48 1.0 12.53 89.7 4.62 1.1 0.52 90.0 3.85 1.2 2.09 100.0 1.16 1.9 10.68 96.7 35.85 1.1 9.14 83.3 1.31 1.4 0.67 95.0 6.84 1.2 6.61 96.7 6.22 1.4 1.78 96.7 0.88 5.0 6.11 89.7 12.00 0.6 3.22 81.7 62.20 1.0 0.58 90.0 14.58 1.0 0.24 95.0 15.71 0.8 1.44 84.3 3.47 1.8 1.83 97.7 5.50 1.5 ⫾ 0.2 4.17 ⫾ 0.94 95.5 ⫾ 1.6 14.81 ⫾ 3.87
Abbreviations: Cr, creatinine; BP, blood pressure; Fr, furosemide. *WHO pathological criteria, from class 1 to class 5. †Ratios of TGF-1 (in femtograms) to -actin (in picograms). ‡Data expressed as mean ⫾ SEM.
inhibitor patients were compared with healthy controls (n ⫽ 11; 4.27 ⫾ 0.62 versus 2.78 ⫾ 0.71; Fig 2). TGF-1 mRNA abundance of the patients administered calcium channel blockers (n ⫽ 4) in the no-ACE-inhibitor group was similar to that of the rest of the patients in this group (n ⫽ 19; 17.47 ⫾ 9.62 versus 14.25 ⫾ 4.34; P ⫽ 0.76). Correlation of clinical parameters. TGF-1 gene expression in renal tissue had no relationship with serum creatinine level, urinary protein excretion, mean blood pressure, and WHO pathological criteria. Experiment 2 PBMC mRNA expression. Spontaneous gene expression from freshly isolated PBMCs was observed in this experiment. TGF-1 mRNA expression of patients with glomerulonephritis before ACE-inhibitor therapy (n ⫽ 16) was similar to that of controls (n ⫽ 12; TGF-1/-actin,
0.66 ⫾ 0.19 versus 0.44 ⫾ 0.07 fg/pg, P ⫽ 0.32). TGF-1 mRNA expression before and after ACE inhibitor therapy in 16 pairs of samples did not differ (TGF-1–-actin, 0.66 ⫾ 0.19 versus 0.77 ⫾ 0.29 fg/pg, P ⫽ 0.62; Table 4). Urinary TGF-1 excretion. Urine TGF-1 concentration in picograms per milliliter was corrected by concomitant urine creatinine concentration (in milligrams per deciliter) to minimize the effect of urine concentration. The urinary TGF-1 level in patients with glomerulonephritis before ACE-inhibitor therapy (n ⫽ 16) was significantly greater than that of controls (n ⫽ 12; 7.13 ⫾ 1.89 versus 0.24 ⫾ 0.05 pg/mg; P ⫽ 0.002); however, it did not differ from that after ACE-inhibitor therapy (n ⫽ 16 pairs; 7.13 ⫾ 1.89 versus 8.78 ⫾ 2.01 pg/mg; P ⫽ 0.16; Table 4). Serum TGF-1 levels. Serum TGF-1 concentrations were not different between patients with glomerulonephritis before ACE-inhibitor therapy (n ⫽ 16) and controls (n ⫽ 12; 106.72 ⫾
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SHIN ET AL Table 3.
Clinical Characteristics and Renal TGF-1 Results of ACE-Inhibitor Patients in Experiment 1
Patient No.
Age (y)
Sex
WHO*
Medications (mg/d)
Cr (mg/dL)
Proteinuria (g/d)
Mean BP (mm Hg)
TGF-1† (fg/ pg)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total‡
36 44 24 40 45 36 28 56 60 13 25 34 29 53 28 18 35.6 ⫾ 3.4
M F F M F M F M F F M F M F M M 50 (% men)
4 4 3 4 4 4 3 4 4 4 4 4 4 3 4 4
Ramipril (5) Ramipril (5) Ramipril (2.5) Fr (80), ramipril (10) Ramipril (5) Fosinopril (10) Ramipril (2.5) Ramipril (5) Fr (80), ramipril (10) Ramipril (5) Ramipril (10) Fosinopril (20) Ramipril (5) Fr (80), fosinopril (10) Fosinopril (10) Ramipril (2.5)
1.6 1.2 1.1 1.1 1.2 3.2 1.9 1.9 2.3 0.7 1.2 0.9 1.4 0.8 1.5 1.0 1.4 ⫾ 0.2
0.92 0.39 0.25 7.40 1.35 5.13 3.82 0.68 14.70 2.57 0.52 4.08 0.83 6.00 1.98 1.53 3.26 ⫾ 0.94
100.7 96.7 80.0 95.7 75.8 104.3 98.7 96.7 97.7 93.3 93.7 102.3 94.3 90.0 94.3 96.7 94.4 ⫾ 1.9
3.45 6.43 5.63 0.00 9.58 8.56 3.30 4.27 3.00 2.48 3.18 3.13 5.87 1.72 4.64 3.06 4.27 ⫾ 0.62
Abbreviations: Cr, creatinine; BP, blood pressure; Fr, furosemide. *WHO pathological criteria, from class 1 to class 5. †Ratios of TGF-1 (in femtograms) to -actin (in picograms). ‡Data expressed as mean ⫾ SEM.
18.93 versus 81.73 ⫾ 39.19 ng/mL; P ⫽ 0.54). There was no difference in serum TGF-1 levels before and after ACE-inhibitor therapy (n ⫽ 16 pairs; 106.72 ⫾ 18.93 versus 92.36 ⫾ 20.48 ng/mL; P ⫽ 0.55; Table 4).
Fig 2. The renal gene expression of TGF-1 expressed as TGF-1/-actin in patients with IgA nephropathy not administered ACE inhibitors (NoACEI) and administered ACE inhibitors (ACEI). Controls are the normal part of nephrectomized kidneys. Mean values ⴞ SEM are given. * P < 0.05 versus NoACEI (oneway analysis of variance).
DISCUSSION
In this study, heightened renal expression of TGF-1 was observed in patients with IgA nephropathy not administered ACE inhibitors compared with controls. This is in accordance with previous reports in which enhanced renal TGF- expression was shown in patients with IgA nephropathy.13-15 Conversely, the expression of TGF-1 in patients administered ACE inhibitors was similar to that in controls and significantly less than that in patients not administered ACE inhibitors. It has been proposed that ANG II stimulates the production of TGF-, and this pathogenic link is interrupted by ANG II blockade by either ACE inhibitors or ANG II-receptor antagonists, which in turn slows the progression of renal fibrosis.18 Increasing numbers of studies have investigated the effects of ANG II blockade on the attenuation of TGF- in various animal models. Wu et al19 observed that ANG II blockade significantly blunted the upregulation of renal TGF- gene transcription in the rat with subtotal nephrectomy. Similarly, ANG II antagonism reduced the elevation of TGF- expression in the remnant kidney in rats.20 In rat models with
TGF-1 IN GLOMERULONEPHRITIS
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Table 4.
Group (no. of patients)
Control (12) Before ACE inhibitors (16) After ACE inhibitors (16)
Mean BP (mm Hg)
— 107.19 ⫾ 3.01 98.50 ⫾ 2.85§
Effect of ACE Inhibitors in Experiment 2 Serum Creatinine (mg/dL)
PBMC TGF1 (fg/pg)*
Serum TGF-1 (ng/mL)
Urinary TGF-1 (pg/mg)†
— 3.34 ⫾ 0.82
— 1.76 ⫾ 0.16
0.44 ⫾ 0.07 0.66 ⫾ 0.19
81.73 ⫾ 39.19 106.72 ⫾ 18.93
0.24 ⫾ 0.05 7.13 ⫾ 1.89‡
2.51 ⫾ 0.62㛳
1.84 ⫾ 0.18
0.77 ⫾ 0.29
92.36 ⫾ 20.48
8.78 ⫾ 2.01¶
Proteinuria (g/d)
NOTE. Data expressed as mean ⫾ SEM. Abbreviation: BP, blood pressure. *mRNA expression, ratios of TGF-1 (in femtograms) to -actin (in picograms). †Urine TGF-1 concentration (in picograms per milliliter) corrected by urinary creatinine concentration (in milligrams per deciliter). ‡P ⫽ 0.002 versus control. §P ⬍ 0.001 versus before ACE inhibitors. 㛳P ⫽ 0.009 versus before ACE inhibitors. ¶P ⫽ 0.001 versus control.
unilateral ureteral obstruction, ANG II blockade significantly blunted the increase in TGF- mRNA in the obstructed kidney.21,22 Again, ANG II blockade effectively reduced intrarenal TGF- expression and renal injury in experimentally induced glomerulonephritis.23,24 The ANG IIreceptor antagonist significantly prevented the increase in glomerular TGF- mRNA in twokidney, one-clip hypertensive rats25 and strokeprone spontaneously hypertensive rats.26 These all indicate that ANG II blockade can prevent the increase in TGF- production in the kidney, thus delaying the progression of renal disease. Despite such accumulating data in animal models, the publications showing the effects of ACE inhibitors on the attenuation of TGF- in humans are scarce. Because differences may exist between chronic renal diseases in humans and experimentally induced renal diseases in animals, it is possible that the former may respond to ANG II blockade in a way that is different from the latter. In this regard, our current study of patients with chronic glomerulonephritis is of particular value. We observed a significant reduction in TGF-1 expression in the kidney by ACE inhibitors, and this suggests that the effects of ANG II blockade observed in rats mentioned previously can be extrapolated to patients with chronic renal disease. Conflicting data exist on whether blood pressure per se may regulate the expression of TGF- in the kidney. In vitro studies have shown that
pressure-induced glomerular distention can stimulate TGF- synthesis and extracellular matrix production.27,28 Nifedipine,29 as well as triple therapy with hydralazine, reserpine, and hydrochlorothiazide,25 effectively prevented the increase in TGF- in the experimental animal models. These findings suggest that systemic hypertension generates glomerular stretch, which in turn stimulates the synthesis of TGF-. Conversely, some studies suggest that blood pressure is not a factor in the regulation of TGF- production. Junaid et al20 reported that triple therapy with hydralazine, reserpine, and hydrochlorothiazide did not induce a decrease in TGF- level in the rat remnant kidney, whereas the ANG II antagonist reduced the elevation of TGF- expression. Administration of a small dose, insufficient to reduce systemic blood pressure, of ANG II-receptor antagonist remarkably attenuated the increase in renal TGF- gene expression in stroke-prone spontaneously hypertensive rats.30 Similar findings have been reported with an ACE inhibitor in immune complex nephritis in the absence of an effect on blood pressure.31 Moreover, cell culture experiments, in which the effects on intraglomerular pressure are absent, favor the hypothesis that ANG II directly stimulates TGF- synthesis. For example, Kagami et al32 showed that ANG II stimulated matrix proteins through the induction of TGF- activity in mesangial cell cultures, and Wolf et al33 reported that ANG II stimulated TGF- synthesis in mu-
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rine proximal tubular cell cultures. Because blood pressure was similar in both groups in the current study, differences in TGF- expression in these groups cannot be explained by the reduction in blood pressure with ACE inhibitors. Hence, it is likely that these differences may result from either a decrease in intraglomerular pressure through the inhibition of the intrarenal reninangiotensin system,16 blocking the direct stimulatory effect of ANG II on TGF-, or both. Hamaguchi et al29 reported that calcium channel blockers were effective in blunting TGF- upregulation in the kidney. Such effects of calcium channel blockers on TGF- could not be evaluated in the current study because only a small number of patients (n ⫽ 4) were administered these medications. However, at least there was no tendency toward downregulation of TGF- in the kidney by calcium channel blockers. The current study does not give information on which structure in renal tissue is responsible for the change in TGF-1 expression. Therefore, investigations in isolated glomeruli or localization of TGF-1 by in situ hybridization or immunohistochemistry remain to be performed to evaluate the source of renal TGF-1 expression. Recently, mononuclear cells have been implicated in the pathogenesis of glomerulonephritis. Arima et al34 observed renal trafficking of mononuclear cells in IgA nephropathy, and it was shown that PBMCs have the capacity to produce high levels of various cytokines in patients with glomerulonephritis.35,36 In addition, upregulation of TGF- by cultured PBMCs in patients with IgA nephropathy suggested the possibility of PBMCs as a source of TGF- in the kidney.37,38 PBMCs are known to harbor ANG II type 1 receptors,39,40 and ANG II stimulates the synthesis of cytokines in PBMCs through these receptors.41 Accordingly, heightened TGF- gene transcription was observed in monocytes infiltrating the renal interstitium, followed by its significant reduction by ANG II blockade in the rat kidney with subtotal nephrectomy.19 Based on these studies, we examined the status of TGF- expression and the effects of ACE inhibitors on the synthesis of TGF- in circulating PBMCs. In the current study, TGF-1 gene transcription was not enhanced in PBMCs from patients
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with glomerulonephritis compared with controls or attenuated by ACE inhibitors. Some possible reasons for these results follow. First, given the absence of hemodynamic effects in the environment in which PBMCs are circulating, ANG II may regulate TGF- synthesis through changes in intraglomerular pressure rather than directly regulate it. Second, systemic ANG II levels may not be elevated despite the increased ANG II in the kidney, which explains why the TGF- level is elevated in renal tissue but not in PBMCs. Previous studies have suggested that systemic ANG II may be dissociated from intrarenal ANG II.42 Last, the difference in TGF- expression between the groups may become evident if PBMCs are stimulated in vitro, as in previous studies.37,38 Additional studies are needed to confirm our findings and elucidate the underlying mechanism. In the current investigation, TGF-1 levels were significantly increased in urine of patients with glomerulonephritis. Data exist showing a correlation between urinary TGF- excretion and renal fibrosis in rats,43 as well as in patients with glomerulonephritis.44 However, in the present study, urinary TGF-1 levels were not decreased after ACE-inhibitor therapy. This is contradictory to our expectation, but it is possible that longer treatment is needed for the drug to accumulate in the organs and decrease TGF- production, although 4 to 8 days of ACE-inhibitor therapy was sufficient to see the effects on TGF- in the kidney and/or urine in certain animal models.20,22,24 Other possibilities are that TGF- mRNA expression can be dissociated from active protein secretion,45 or the urine TGF- level may be an insufficient indicator of the renal TGF- protein synthesis. In the present study, serum levels of TGF-1 were not increased in patients with glomerulonephritis or suppressed by ACE inhibitors. Again, this is contradictory to our expectation, but previous investigations showed that serum or plasma levels of TGF-1 were not increased in animal models of renal disease, including diabetic rats46; two-kidney, one-clip hypertensive rats25; and human immunodeficiency virus transgenic mice.47 Furthermore, plasma TGF-1 concentrations were not reduced by the administration of ACE
TGF-1 IN GLOMERULONEPHRITIS
inhibitors in their studies.25,47 However, possibly the duration of ACE inhibitor therapy in the current study was not long enough to see the effect of ACE inhibitors on serum TGF-1 levels. Previous reports showed a significant reduction of serum and/or plasma TGF-1 levels after 6 months of ACE-inhibitor treatment48 or 4 weeks of ANG II-receptor antagonist therapy.49 Although serum levels of TGF- reflect plateletderived TGF- contributed by platelet degranulation, as well as circulating TGF-,50 many studies have found that measurements of serum TGF- are informative in clinical diseases.51-53 It remains to be determined whether measurements of plasma TGF- may render different results from ours. In summary, our current study showed that ACE inhibitors attenuate the increase in renal TGF-1 expression in patients with IgA nephropathy. The downregulation of renal TGF-1 expression in patients with glomerulonephritis may be a central part of the beneficial effects of ACE inhibitors on chronic progressive nephropathies. REFERENCES 1. Anderson S, Rennke HG, Brenner BM: Therapeutic advantage of converting enzyme inhibitors in arresting progressive renal disease associated with systemic hypertension in the rat. J Clin Invest 77:1993-2000, 1986 2. Ruggenenti P, Perna A, Benini R, Bertani T, Zoccali C, Maggiore Q, Salvadori M, Remuzzi G: In chronic nephropathies, prolonged ACE inhibition can induce remission: Dynamics of time-dependent changes in GFR. J Am Soc Nephrol 10:997-1006, 1999 3. Hollenberg NK, Raij L: Angiotensin-converting enzyme inhibition and renal protection. Arch Intern Med 153:2426-2435, 1993 4. Denton KM, Fennessy PA, Alcorn D, Anderson WP: Morphometric analysis of the actions of angiotensin II on renal arterioles and glomeruli. Am J Physiol 262:367-372, 1992 5. Border WA, Noble NA: Transforming growth factor- in tissue fibrosis. N Engl J Med 331:1286-1292, 1994 6. Eddy AA: Molecular insights into renal interstitial fibrosis. J Am Soc Nephrol 7:2495-2508, 1996 7. Yamamoto T, Noble NA, Miller DE, Border WA: Sustained expression of TGF-1 underlies development of progressive kidney fibrosis. Kidney Int 45:916-927, 1994 8. Yamamoto T, Nakamura T, Noble NA, Rouslahti E, Border WA: Expression of TGF- in human and experimental diabetic nephropathy. Proc Natl Acad Sci U S A 90:18141818, 1993 9. Iwano M, Kubo A, Nishino T, Sato H, Nishioka H, Akai Y, Kurioka H, Fuji Y, Kanauchi M, Shiiki H, Dohi K: Quantification of glomerular TGF-1 mRNA in patients with diabetes mellitus. Kidney Int 49:1120-1126, 1996
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