Original Paper
Nephron 1992;61:456-463
Second Department of Internal Medicine. Faculty of Medicine, Kyushu University, Fukuoka, Japan
Key Words Hypertension Glomerulosclerosis Extracellular matrix Fibronectin Subtotal nephrectomy Glomerular hypertrophy
Synthesis of Fibronectin by Isolated Glomeruli from IMephrectomized Hypertensive Rats
Abstract The accumulation of extracellular matrix (ECM) is an important feature of most forms of progressive glomerular diseases. In order to examine the relationship between ECM synthesis and glomerulosclerosis, we evaluated fibronectin syn thesis by glomeruli with the immunoprécipitation of conditioned media from isolated glomeruli in Yt>nephrectomized spontaneously hypertensive rats (5/*NSHR). There was no difference in blood pressure between VeN-SHR and control SHR throughout the experiment. Two weeks after the nephrectomy, most of the glomeruli were intact and no difference in the synthesis of fibronectin was observed between either groups. Twenty weeks after the nephrectomy, marked glomerulosclerosis associated with an increase in urinary protein was revealed in 5AN-SH R but no glomerular lesions in control SHR. The synthesis of fibronectin by isolated glomeruli increased in 5/«N-SHR compared to control SHR. The administration of enalapril or hydralazine-I-reserpine +hydrochlorothiazide markedly attenuated the glomerular sclerosis and urinary protein excretion to a comparable degree, although the later therapy reduced blood pressure more effectively. These antihypertensive therapies also suppressed fibronectin synthe sis in the %N-SHR group at week 20. In conclusion, increased synthesis of glomerular fibronectin appeared to contribute to the glomerulosclerosis caused by subtotal nephrectomy and hypertension.
Introduction Systemic hypertension either has a deteriorative effect on the development of renal disease or accelerate the loss of function in kidneys in which parenchymal disease is al ready established [1-7]. The glomerulosclerosis that occurs in rats with subtotal nephrectomy has been attributed, at least in part, to the effects of adaptive hemodynamic
Accepted: October 9.1991
changes and compensatory hypertrophy [8,9], The remnant nephrons undergo afferent arteriolar dilatation which re sults in a high glomerular flow and pressure accompanied by increased trapping of macromolecules in the mesangium [10, II], In such cases, systemic hypertension might augment intraglomerular hypertension with an increased glomerular blood flow and filtration rate, which results in further glomerular destruction.
Seiya O kuda. M D Second D epartm ent o f Internal Medicine Maidashi 3-1-1. Fukuoka 812 (Japan»
© 1992 S. K arger AG. Basel 0028 2 7 6 6 /9 2 / 06I4-0456S2.75/0
Downloaded by: University of Chicago Library 205.208.116.24 - 4/16/2018 3:49:50 AM
Seiya Okuda Hidetoshi Kanai Kiyoshi Tamaki Kaoru Onoyama Masatoshi Fujishima
Method Experimental Design Adult male SHR (Charles River Breeding, Tokyo, Japan) 8 weeks old, weighing about 250 g were used. These rats were fed the same regular diet containing 0.3% sodium and 24% protein. Half of the rats (n = 60) underwent %-nephrectomy (%N) while other rats (n = 60) were sham operated for control. The rats in the nephrectomized or sham operated groups were randomly assigned to the enalapril-treated group or hydrarazine +reserpine + hydrochlorothiazide-treated (tri ple therapy) group. Six groups were studied: (I) the %N group, n = 20; (2) the ViN-enalapril. n = 20; (3) the %N-triple therapy. n = 20; (4) the control SHR group, n = 20: (5) the control SHR-enalapril group n = 20; (6) the control SHR-triple therapy group, n = 20. Eight rats in each group were sacrificed at week 2 after the nephrectomy in order to study renal histology and the production of extracellular matrices by cultured glomeruli, and all other rats were sacrificed at week 20. A n tillypertensi ve Drugs Antihypertensive drugs were dissolved in drinking water. Treat ment with enalapril (Merk Sharp & Dohme Research Laboratories, Rathway. N.J., USA, 100 mg/l in drinking water) or hydralazine (Ciba-Geigy, Summit, N.J., USA, 100 mg/l in drinking water) + reserpine (Ciba-Geigy, 10 mg/i in drinking water) + hydrochlorothiazide (Ciba-Geigy, 50 mg/l in drinking water) was initiated after the subtotal nephrectomy and continued throughout the study. Methods for Subtotal Nephrectomy Subtotal nephrectomy was done in two stages according to the method of Morrison [18]. At the first stage, two-thirds of the left kidney (both upper and lower poles) was removed. At the second stage, 2 weeks after the first operation, the entire right kidney was removed, while a sham operation, was done in the control groups.
Measurement o f Blood Pressure and Proteinuria Systolic blood pressure and urinary protein were measured at week 2 and then every 4 weeks after the second operation. Systolic blood pressure was measured in conscious rats by the tail-cuff method. Rats were warmed for 10 min at 37°C before measurement. Twenty-fourhour urine collected in the metabolic cage was measured by the sulfosalicylic acid method [19]. Histologic Examination For light-microscopic study, kidneys were fixed with 10% neutral buffered formalin and embedded in paraffin. Sections 2 ,um thick were stained with hematoxylin and eosin (HE) and periodic acid-Schiff reagent (PAS). A semiquantitative score was used to evaluate the degree of glomerular sclerosis, according to the method of Raiji et al. [20]. Fifty glomeruli were examined independently, and the severity of the lesion was graded from 0 to 4 + according to the percentage of glomerular sclerosis. Thus, a I + lesion represented an involvement of 25% of the glomerulus, 2 + 25-50%, 3 + 50-75% and 4 -I- 75-100%. The number of glomeruli showing a lesion of 1+ was set at n,, 2 -F n :,3 + n? and 4 + n4, respectively. Then the sclerosis index was obtained by the following formula: [(lxn, + 2xn> + 3xn-,+ 4x110/50] x 100. The diame ter of 50 glomeruli which were randomly selected from each specimen was measured with a micrometer and averaged. A histological evalua tion was made independently by two investigators without prior knowledge of the experimental groups. The scores were averaged between two investigators per each specimen. For immunohistological studies, fresh kidney specimens were snap-frozen at -70°C in a dry ice-acetone mixture and sectioned with a cryostat microtome. Sections 4 pm thick were exposed to rabbit antiserum against rat fibronectin (Chemicon, Temecula, Calif., USA) for 30 min at room temperature. After washing with phosphate-buf fered saline ( PBS), they were exposed to fluorescein isothiocyanate conjugated anti-rat IgG (Cappel Lab., Cochranville, Pa.. USA) for 30 min at room temperature. The sections were examined under a fluores cence microscope (Olympus, Inc., Tokyo, Japan). Isolation o f Glomeruli Isolated glomeruli were prepared by use of a slight modification of the method of Schreiner et al. [21]. Briefly, the abdominal cavity was opened under pentobarbital sodium anesthesia. Kidneys were through ly perfused with 20-30 ml of PBS injected just above the bifurcation of the aorta. The kidneys were immediately removed and decapsulated. A part of the kidney removed was cut for histological studies. The remain ing cortices were dissected on ice and glomeruli were obtained by pressing slices of renal cortex through three consecutive sieves (mesh size 250.200 and 75 urn). Isolated glomeruli were suspended in Hank's balanced salt solution (HBSS) and then washed twice with HBSS. The isolated glomeruli were then treated with 60 U/ml collagénase type II with shaking for 10 min at 37 C to remove Bowman’s capsule. After the treatment, the preparations were washed five times with cold HBSS and then suspended in warm HBSS. The purity of the glomeruli and the removal of Bowman’s capsule were examined by light microscopy. Preparations were shown to contain more than 90% isolated glomeruli while most were free of Bowman’s capsule.The isolated glomeruli were resuspended at 5xl(F glomeruli/ml with serum-free and methioninefree Dulbecco’s modified Eagle's medium (Gibco Laboratories, Grand Island, N.Y., USA) and were biosynthetically labeled by the adition of 100 uCi/ml of "S-methionine (New England Nuclear, Boston, Mass., USA) for 24 h [22], The culture media were removed and stored at -20°C for immunoprécipitation study.
457
Downloaded by: University of Chicago Library 205.208.116.24 - 4/16/2018 3:49:50 AM
A common feature in the development of glomerulo sclerosis is a general increase in the amount of extracellular matrices (ECMs) [12]. Systemic hypertension has been re ported to increase the ECMs synthesis in vasculature, which contributes to pathogenesis of atherosclerosis [12-15], One of the common pathological characteristics between atherosclerosis and glomerulosclerosis is the increased ac cumulation of ECMs in tissue [16, 17]. Systemic hyperten sion may produce a harmful effect on the glomeruli by increasing the ECMs synthesis as well as glomerular hyper tension. We previously reported on extensive glomerulosclerosis in the 5/6-nephrectomized spontaneously hypertensive rats [5]. In this study, in order to examine the relationship between the ECMs synthesis and glomerulosclerosis in this model, isolated glomeruli from the rats were cultured and metabolically labeled, and then the synthesis of fibronectin was evaluated.
mm Hg
mm Hg
Time, weeks
Time, weeks
Fig. 1. Systolic blood pressure (mean ±SE). * p< 0.001 compared to %N or %N-triple therapy groups. **p< 0.001 compared to %N or %N-enalapriI groups.
Molecular Identification by Immunoprécipitation 500 pi antiserum was preincubated with the 500-ul pellets of protein A-Sepharose (Sigma, St. Louis, Mo., USA) diluted in immuno précipitation buffer(50 mMTris HC1 pH 7.5, 0.15 M NaCI, l%Triton X-100. 1% sodium deoxycholete, 0.1% SDS, I m.W EDTA) at room temperature for 120 min. Normal rabbit serum was used in a control experiment. 20 pi of preincubated pellets of protein A-Sepharose were added to 150 ul of conditioned medium and incubated in the buffer overnight at 4°C while mixing the microtubes. The samples were centrifuged for 10 min at 2,000 gand the supernatant was removed. The pellets were washed 5 times with immunoprécipitation buffer, and then were dissolved in 40 pi of SDS-PAGE sample buffer containing 3% SDS and 10% beta-mercaptoethanol (Sigma) and boiled for 5 min. Aliquots (20 pi) were equally applied to 4-20% gradient SDS-PAGE gel (Daiichi Pure Chemical Co., Tokyo, Japan) and run for 3 h at constant power. The gels were stained with Coomassie blue. Molecu lar size markers were from Bio-Rad (Richmond, Calif.. USA). Fluorography was performed by incubating gel in Enlightning (New England Nuclear). Statistical Method Statistical difference was calculated by analysis of variance among the six groups and unpaired t test with the Bonferroni correction.
influence on the growth (355 ± 3 4 g in the %-enalapril group, 362 ± 38 g in the %-triple therapy group). Blood Pressure and Proteinuria
Systolic blood pressure in the 5/«N group (213 ± 10 mm Hg at week 20) was not significantly different from that in thecontroISH Rgroup(210±12m m Hgat week 20) (fig. la, b). Both enalapril and triple therapy revealed a significant reduction in systolic blood pressure immediately after the beginning. Triple therapy reduced blood pressure more effectively than enalapril (135 ± 8 vs. 109±10 mm Hg at week 20). Urinary protein excretion began to increase at week 8 and continuously increased to 237 ±98 mg/day at week 20 in the 5AN group (fig. 2a). The increase was sup pressed by both enalapril and triple therapy. The reduction by enalapril was larger than that by triple therapy (67 ±22 vs. 112 ±37 m g/day at week 20) but the difference was not significant. No particular increase in proteinuria was ob served in the control groups (fig. 2b). Renal Histology
Body Weight
Body weight increased gradually in rats of all groups throughout the experiment. The body weight in the 5/«N group (364 ± 34 g) did not differ from that of the control SH R group (372 ± 24 g). The antihypertensive drugs had no
458
A light-microscopic study revealed no significant histo logical alterations in any of the groups at week 2. At week 20, polymorphous patterns of glomerular changes, such as proliferation, degeneration and sclerosis with fibrinoid or hyaline deposition were observed in the %N group. The increase in the mesangial matrix was prominent, while the increase in mesangial cells was moderate. Small vacuoles
O kuda/K anai/Tam aki/O noyam a/ Fujishima
Fibronectin Synthesis and Glomerulosclerosis
Downloaded by: University of Chicago Library 205.208.116.24 - 4/16/2018 3:49:50 AM
Results
Fig. 2. Urinary protein excretion (m ean±SE). * p< 0.001, **p
Nephron 1992;61:456-463
Second Department of Internal Medicine. Faculty of Medicine, Kyushu University, Fukuoka, Japan
Key Words Hypertension Glomerulosclerosis Extracellular matrix Fibronectin Subtotal nephrectomy Glomerular hypertrophy
Synthesis of Fibronectin by Isolated Glomeruli from IMephrectomized Hypertensive Rats
Abstract The accumulation of extracellular matrix (ECM) is an important feature of most forms of progressive glomerular diseases. In order to examine the relationship between ECM synthesis and glomerulosclerosis, we evaluated fibronectin syn thesis by glomeruli with the immunoprécipitation of conditioned media from isolated glomeruli in Yt>nephrectomized spontaneously hypertensive rats (5/*NSHR). There was no difference in blood pressure between VeN-SHR and control SHR throughout the experiment. Two weeks after the nephrectomy, most of the glomeruli were intact and no difference in the synthesis of fibronectin was observed between either groups. Twenty weeks after the nephrectomy, marked glomerulosclerosis associated with an increase in urinary protein was revealed in 5AN-SH R but no glomerular lesions in control SHR. The synthesis of fibronectin by isolated glomeruli increased in 5/«N-SHR compared to control SHR. The administration of enalapril or hydralazine-I-reserpine +hydrochlorothiazide markedly attenuated the glomerular sclerosis and urinary protein excretion to a comparable degree, although the later therapy reduced blood pressure more effectively. These antihypertensive therapies also suppressed fibronectin synthe sis in the %N-SHR group at week 20. In conclusion, increased synthesis of glomerular fibronectin appeared to contribute to the glomerulosclerosis caused by subtotal nephrectomy and hypertension.
Introduction Systemic hypertension either has a deteriorative effect on the development of renal disease or accelerate the loss of function in kidneys in which parenchymal disease is al ready established [1-7]. The glomerulosclerosis that occurs in rats with subtotal nephrectomy has been attributed, at least in part, to the effects of adaptive hemodynamic
Accepted: October 9.1991
changes and compensatory hypertrophy [8,9], The remnant nephrons undergo afferent arteriolar dilatation which re sults in a high glomerular flow and pressure accompanied by increased trapping of macromolecules in the mesangium [10, II], In such cases, systemic hypertension might augment intraglomerular hypertension with an increased glomerular blood flow and filtration rate, which results in further glomerular destruction.
Seiya O kuda. M D Second D epartm ent o f Internal Medicine Maidashi 3-1-1. Fukuoka 812 (Japan»
© 1992 S. K arger AG. Basel 0028 2 7 6 6 /9 2 / 06I4-0456S2.75/0
Downloaded by: University of Chicago Library 205.208.116.24 - 4/16/2018 3:49:50 AM
Seiya Okuda Hidetoshi Kanai Kiyoshi Tamaki Kaoru Onoyama Masatoshi Fujishima
Method Experimental Design Adult male SHR (Charles River Breeding, Tokyo, Japan) 8 weeks old, weighing about 250 g were used. These rats were fed the same regular diet containing 0.3% sodium and 24% protein. Half of the rats (n = 60) underwent %-nephrectomy (%N) while other rats (n = 60) were sham operated for control. The rats in the nephrectomized or sham operated groups were randomly assigned to the enalapril-treated group or hydrarazine +reserpine + hydrochlorothiazide-treated (tri ple therapy) group. Six groups were studied: (I) the %N group, n = 20; (2) the ViN-enalapril. n = 20; (3) the %N-triple therapy. n = 20; (4) the control SHR group, n = 20: (5) the control SHR-enalapril group n = 20; (6) the control SHR-triple therapy group, n = 20. Eight rats in each group were sacrificed at week 2 after the nephrectomy in order to study renal histology and the production of extracellular matrices by cultured glomeruli, and all other rats were sacrificed at week 20. A n tillypertensi ve Drugs Antihypertensive drugs were dissolved in drinking water. Treat ment with enalapril (Merk Sharp & Dohme Research Laboratories, Rathway. N.J., USA, 100 mg/l in drinking water) or hydralazine (Ciba-Geigy, Summit, N.J., USA, 100 mg/l in drinking water) + reserpine (Ciba-Geigy, 10 mg/i in drinking water) + hydrochlorothiazide (Ciba-Geigy, 50 mg/l in drinking water) was initiated after the subtotal nephrectomy and continued throughout the study. Methods for Subtotal Nephrectomy Subtotal nephrectomy was done in two stages according to the method of Morrison [18]. At the first stage, two-thirds of the left kidney (both upper and lower poles) was removed. At the second stage, 2 weeks after the first operation, the entire right kidney was removed, while a sham operation, was done in the control groups.
Measurement o f Blood Pressure and Proteinuria Systolic blood pressure and urinary protein were measured at week 2 and then every 4 weeks after the second operation. Systolic blood pressure was measured in conscious rats by the tail-cuff method. Rats were warmed for 10 min at 37°C before measurement. Twenty-fourhour urine collected in the metabolic cage was measured by the sulfosalicylic acid method [19]. Histologic Examination For light-microscopic study, kidneys were fixed with 10% neutral buffered formalin and embedded in paraffin. Sections 2 ,um thick were stained with hematoxylin and eosin (HE) and periodic acid-Schiff reagent (PAS). A semiquantitative score was used to evaluate the degree of glomerular sclerosis, according to the method of Raiji et al. [20]. Fifty glomeruli were examined independently, and the severity of the lesion was graded from 0 to 4 + according to the percentage of glomerular sclerosis. Thus, a I + lesion represented an involvement of 25% of the glomerulus, 2 + 25-50%, 3 + 50-75% and 4 -I- 75-100%. The number of glomeruli showing a lesion of 1+ was set at n,, 2 -F n :,3 + n? and 4 + n4, respectively. Then the sclerosis index was obtained by the following formula: [(lxn, + 2xn> + 3xn-,+ 4x110/50] x 100. The diame ter of 50 glomeruli which were randomly selected from each specimen was measured with a micrometer and averaged. A histological evalua tion was made independently by two investigators without prior knowledge of the experimental groups. The scores were averaged between two investigators per each specimen. For immunohistological studies, fresh kidney specimens were snap-frozen at -70°C in a dry ice-acetone mixture and sectioned with a cryostat microtome. Sections 4 pm thick were exposed to rabbit antiserum against rat fibronectin (Chemicon, Temecula, Calif., USA) for 30 min at room temperature. After washing with phosphate-buf fered saline ( PBS), they were exposed to fluorescein isothiocyanate conjugated anti-rat IgG (Cappel Lab., Cochranville, Pa.. USA) for 30 min at room temperature. The sections were examined under a fluores cence microscope (Olympus, Inc., Tokyo, Japan). Isolation o f Glomeruli Isolated glomeruli were prepared by use of a slight modification of the method of Schreiner et al. [21]. Briefly, the abdominal cavity was opened under pentobarbital sodium anesthesia. Kidneys were through ly perfused with 20-30 ml of PBS injected just above the bifurcation of the aorta. The kidneys were immediately removed and decapsulated. A part of the kidney removed was cut for histological studies. The remain ing cortices were dissected on ice and glomeruli were obtained by pressing slices of renal cortex through three consecutive sieves (mesh size 250.200 and 75 urn). Isolated glomeruli were suspended in Hank's balanced salt solution (HBSS) and then washed twice with HBSS. The isolated glomeruli were then treated with 60 U/ml collagénase type II with shaking for 10 min at 37 C to remove Bowman’s capsule. After the treatment, the preparations were washed five times with cold HBSS and then suspended in warm HBSS. The purity of the glomeruli and the removal of Bowman’s capsule were examined by light microscopy. Preparations were shown to contain more than 90% isolated glomeruli while most were free of Bowman’s capsule.The isolated glomeruli were resuspended at 5xl(F glomeruli/ml with serum-free and methioninefree Dulbecco’s modified Eagle's medium (Gibco Laboratories, Grand Island, N.Y., USA) and were biosynthetically labeled by the adition of 100 uCi/ml of "S-methionine (New England Nuclear, Boston, Mass., USA) for 24 h [22], The culture media were removed and stored at -20°C for immunoprécipitation study.
457
Downloaded by: University of Chicago Library 205.208.116.24 - 4/16/2018 3:49:50 AM
A common feature in the development of glomerulo sclerosis is a general increase in the amount of extracellular matrices (ECMs) [12]. Systemic hypertension has been re ported to increase the ECMs synthesis in vasculature, which contributes to pathogenesis of atherosclerosis [12-15], One of the common pathological characteristics between atherosclerosis and glomerulosclerosis is the increased ac cumulation of ECMs in tissue [16, 17]. Systemic hyperten sion may produce a harmful effect on the glomeruli by increasing the ECMs synthesis as well as glomerular hyper tension. We previously reported on extensive glomerulosclerosis in the 5/6-nephrectomized spontaneously hypertensive rats [5]. In this study, in order to examine the relationship between the ECMs synthesis and glomerulosclerosis in this model, isolated glomeruli from the rats were cultured and metabolically labeled, and then the synthesis of fibronectin was evaluated.
mm Hg
mm Hg
Time, weeks
Time, weeks
Fig. 1. Systolic blood pressure (mean ±SE). * p< 0.001 compared to %N or %N-triple therapy groups. **p< 0.001 compared to %N or %N-enalapriI groups.
Molecular Identification by Immunoprécipitation 500 pi antiserum was preincubated with the 500-ul pellets of protein A-Sepharose (Sigma, St. Louis, Mo., USA) diluted in immuno précipitation buffer(50 mMTris HC1 pH 7.5, 0.15 M NaCI, l%Triton X-100. 1% sodium deoxycholete, 0.1% SDS, I m.W EDTA) at room temperature for 120 min. Normal rabbit serum was used in a control experiment. 20 pi of preincubated pellets of protein A-Sepharose were added to 150 ul of conditioned medium and incubated in the buffer overnight at 4°C while mixing the microtubes. The samples were centrifuged for 10 min at 2,000 gand the supernatant was removed. The pellets were washed 5 times with immunoprécipitation buffer, and then were dissolved in 40 pi of SDS-PAGE sample buffer containing 3% SDS and 10% beta-mercaptoethanol (Sigma) and boiled for 5 min. Aliquots (20 pi) were equally applied to 4-20% gradient SDS-PAGE gel (Daiichi Pure Chemical Co., Tokyo, Japan) and run for 3 h at constant power. The gels were stained with Coomassie blue. Molecu lar size markers were from Bio-Rad (Richmond, Calif.. USA). Fluorography was performed by incubating gel in Enlightning (New England Nuclear). Statistical Method Statistical difference was calculated by analysis of variance among the six groups and unpaired t test with the Bonferroni correction.
influence on the growth (355 ± 3 4 g in the %-enalapril group, 362 ± 38 g in the %-triple therapy group). Blood Pressure and Proteinuria
Systolic blood pressure in the 5/«N group (213 ± 10 mm Hg at week 20) was not significantly different from that in thecontroISH Rgroup(210±12m m Hgat week 20) (fig. la, b). Both enalapril and triple therapy revealed a significant reduction in systolic blood pressure immediately after the beginning. Triple therapy reduced blood pressure more effectively than enalapril (135 ± 8 vs. 109±10 mm Hg at week 20). Urinary protein excretion began to increase at week 8 and continuously increased to 237 ±98 mg/day at week 20 in the 5AN group (fig. 2a). The increase was sup pressed by both enalapril and triple therapy. The reduction by enalapril was larger than that by triple therapy (67 ±22 vs. 112 ±37 m g/day at week 20) but the difference was not significant. No particular increase in proteinuria was ob served in the control groups (fig. 2b). Renal Histology
Body Weight
Body weight increased gradually in rats of all groups throughout the experiment. The body weight in the 5/«N group (364 ± 34 g) did not differ from that of the control SH R group (372 ± 24 g). The antihypertensive drugs had no
458
A light-microscopic study revealed no significant histo logical alterations in any of the groups at week 2. At week 20, polymorphous patterns of glomerular changes, such as proliferation, degeneration and sclerosis with fibrinoid or hyaline deposition were observed in the %N group. The increase in the mesangial matrix was prominent, while the increase in mesangial cells was moderate. Small vacuoles
O kuda/K anai/Tam aki/O noyam a/ Fujishima
Fibronectin Synthesis and Glomerulosclerosis
Downloaded by: University of Chicago Library 205.208.116.24 - 4/16/2018 3:49:50 AM
Results
Fig. 2. Urinary protein excretion (m ean±SE). * p< 0.001, **p
