Original Paper
Nephron 1992:61:415-421
Third Department of Internal Medicine, Okayama University Medical School, Okayama, Japan
Heparan Sulfate Proteoglycans Are Lost in Patients with Diabetic Nephropathy
Key Words
Abstract
Diabetes mellitus Heparan sulfate proteoglycan Glomerular basement membrane Laminin Type IV collagen Fibronectin Proteinuria IgA nephropathy Glomerulosclerosis
The pathogenesis of diabetic nephropathy relative to the changes in the glomeru lar extracellular matrices was investigated. Renal tissues from 10 diabetic pa tients were immunostained with antibodies directed against heparan sulfate proteoglycans (HS-PGs), laminin, type IV collagen and fibronectin. Seven patients were nephrotic and had advanced glomerulosclerosis with nodular lesion, while the other 3 had no renal manifestations or minor glomerular tissue alterations. Controls included kidneys removed from patients with renal tumors and specimens obtained by renal biopsy from patients with IgA nephropathy. Relationships among proteinuria, intensity of fluorescence and glomerular changes were studied. In diabetes 3 patients with minor glomerular lesions were found to have no changes in various components of extracellular matrices. A marked reduction in the intensity of staining with anti-HS-PG antibodies was observed in renal specimens from patients with nodular glomerulosclerosis and proteinuria, while a mild decrease in the intensity of fluorescence was observed in tissues stained with antilaminin antibodies. An increase compared to normal control sample findings in type IV collagen and fibronectin was observed in the mesangium of sclerosing glomeruli. No loss of HS-PG was observed in patients with IgA nephropathy. These results indicate that glomerular extracellular matrix HS-PG is lost in association with diabetic nephropathy; this loss results in alteration of the charge-selective properties of glomerular capillaries. This altera tion may, in part, be the cause of the proteinuria associated with diabetic nephropathy.
Introduction
The glomerular basement membrane (GBM) and mes angial matrix are together referred to as the glomerular extracellular matrices. These extracellular matrices are formed by a group of proteins that include type IV collagen [1], laminin [2], proteoglycans [3], fibronectin [4], entactin [5] and nidogen [6].
Accepted : October 9.199!
The changes most characteristic of diabetes are observed in the glomerular extracellular matrices and include thick ening of the GBM and increase in the mesangial matrix. Changes in the glomerular extracellular matrix components also occur. Among these, the loss of heparan sulfate prote oglycan (HS-PG) is believed to play an important role in the etiology of diabetic nephropathy [7-15]. Rohrbach et al. [7, 8] reported a reduced level of synthesis of extracellular
Hirofumi Makino. MI) Third Department of Medicine Okayama University Medical School 2-5-1 Shikata-cho. Okayama 700 (Japan)
©1992S.Kargcr AG.Basel (K)28-2766/92/ 0614-0415S2.75/0
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 1:04:55 PM
Hirofumi Makino Shuji Ikecla Toshinori Haramoto Zensuke Ota
Table 1. Clinico pathological find ings in patients with diabetes mellitus
Patient No
Age years
Sex
Type of disease
Duration years
Proteinuria g/day
Q.r ml/min
Sclerosis grade
Nodule (glomeruli)
I 2 3 4 5 6 7 8 9 I0
59 54 5! 39 66 53 32 45 53 45
F F F M M M F M M M
NIDDM NIDDM NIDDM IDDM NIDDM NIDDM IDDM NIDDM NIDDM NIDDM
0.2 0.3 12.0 24.0 10.0 5.0 15.0 n.k. 16.0 n.k.
0.I 0.3 LI 4.0 3.5 6.0 4.0 4.9 7.5 5.0
68 102 II4 50 20 54 56 45 63 28
0 0 I 2 3 3 3 3 4 4
0 0 0 0 4/9 7 /2 1 7 /I7 4/12 6/18 8/15
IDDM = Insulin-dependent diabetes mellitus; NIDDM = non-insulin-dependent diabetes mellitus; n.k. = not known: Ccr= creatinine clearance.
Patients and Methods Ten patients with diabetic nephropathy participated in the study. Daily urinary protein was measured using Esbach methods. Creati nine clearances were also measured. Percutaneous renal biopsy was performed for each patient. For light-microscopic study, specimens were stained with hematoxylin and eosin, periodic acid-Schiff and periodic acid-silver methenamine. The degree of glomerulosclerosis observed was scored between 0 and 4 using the classification system of Gellman et al. [17], The presence or absence of diabetic nodules was determined for each glomerulus. As controls, normal-appearing parts of the kidney removed from patients with kidney tumor at surgery as well as specimens obtained by renal biopsy from 10 patients with IgA nephropathy were used. The degree of glomerulosclerosis observed under the light microscope was scored between 0 and 3 in patients with IgA nephropathy. The distribution of various glomerular components was studied using indirect immunofluorescence techniques as described previous ly [18]. In brief, the kidney biopsy specimen was immediately snapfrozen, and 4-um-thick unfixed cryostat sections were prepared. Antibasement-membrane-HS-PG antibody was prepared by immunizing
416
rabbits with rat renal basement membrane HS-PG. The authenticity of specificity has been described in our previous publication [16). Anti laminin antibody was purchased from EY Laboratories (San Mateo, Calif., USA), and anti-type-I V-collagen and antifibronectin antibodies were purchased from Advance Co. (Tokyo, Japan). The second anti body. antirabbit IgG conjugated with fluorescein isothiocyanate (FITC), was obtained from Cappel Co. (West Chester. Pa., USA). The cryostat sections were first stained for various GBM components, washed in phosphate-buffered saline (PBS) and then stained with antirabbit IgG conjugated with FITC. The sections were then washed in PBS again and mounted in buffered glycerol. To reveal the hidden antigenic determinants, ethanol-fixed specimens both from diabetic patients, normal controls and IgA nephropathy were treated with 6 M urea and 0.1 Mglycine HCI buffer, pH 3.5, at 4”C for 1 h according to the technique described by Yoshioka et al. [19]. The distribution of the various basement membrane components was determined using an Olympus immunofluorescence microscope. The intensity of the staining of both GBM and mesangium was scored between 0 and 10 by 3 persons not knowing the identity of the specimens examined. After scoring, the intensity of the staining in normal control specimens was adjusted and set to a value of 5. The relationship between glomerulosclerosis, proteinuria and fluorescence intensity was also studied. Regression-correlation analysis was done using Spearman's test. Results were considered significant when p < 0.05.
Results
The clinicopathological findings in patients with diabet ic nephropathy are summarized in table 1. Seven patients manifested nephrotic syndrome; these patients had de creased creatinine clearance. The glomerulosclerosis score was high and accompanied by diabetic nodules in patients with nephrotic syndrome.
Makino/1 keda/ Haramoto/Ota
Loss of HS-PG in Diabetic Nephropathy
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 1:04:55 PM
matrix HS-PG in EHS sarcoma implanted in diabetic mice. In rats with streptozotocin-induced diabetes, a 30-40% reduction has been reported in the synthesis of heparan sulfate and its incorporation into the GBM [9]. Including those mentioned above, most studies of this kind have been performed in experimentally diabetic rats or mice [7-12], We have recently raised specific antibodies against the HS-PG of rat GBM [16]. In this study we used renal biopsy tissues obtained from patients with diabetic nephropathy for delineation of the changes in extracellular matrices by employing antibodies directed against HS-PG as well as other basement membrane components.
Fig. 1. Immunofluorescence micro graphs of a normal glomerulus stained with antibodies to HS-PG(A). laminin (C). type IV collagen (E) and fibronectin (G) and a diabetic glomerulus stained with antibodies to HS-PG (B), laminin (D), type IV collagen (F) and fibronectin (H).
gial region. Fibronectin staining had a mesangial pattern with the interstitial region (fig. lG). No change in staining intensity or pattern was observed after urea treatment in the specimens both from patients with diabetes, IgA nephro pathy and normal controls.
4 17
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 1:04:55 PM
In normal control specimens, HS-PG appeared as an intense linear staining in the GBM; minimal staining for HS-PG was observed in the mesangial region and tubular basement membrane (fig. IA). Laminin (fig. 1C) and type IV collagen (fig. IE) appeared as intense linear staining in the GBM and tubular basement membrane and in the mesan
Diabetic nephropathy A ) H S-PG
B) Laminin
C) Type IV collagen
D) Fibronectin
Fig. 2. Relationship between glomerular sclerosis and extracellular matrix components in patients with diabetic nephropathy: HS-PG (A), laminin (B). type IVcollagen (C) and fibronec tin (D). The characteristics of the different regression lines were as follows: Ay = -0.78x + 5.59, r = -0.75, p < 0.02: B y = -0.73x + 5.34, r = -0.66, p < 0.05: C y = 0.11x + 6.82, r = 0.43, p < 0.5; D y=0.42x + 5.24, r=0.52, p< 0.5.
Capillary
HS-PG Laminin Type IV collagen Fibronectin
Mesangium
control
DM
control
DM
+ + + + + + + + +
+ + + + + + +
± + + + +
± ± + + + + + +
DM = Diabetes mellitus.
Three patients with minor glomerular abnormality (cases 1-3) had no abnormalities in the various components of the extracellular matrix. In patients with diabetic glomer ulosclerosis (cases4-10), a marked reduction in the intensity of staining of the glomerular capillary wall with anti-HSPG antibodies was observed (fig. IB). A minimal decrease in
418
the intensity of fluorescence on the glomerular capillary wall was observed in the tissues stained with antilaminin antibodies (fig. ID) which had been obtained from patients with advanced glomerulosclerosis. An increase in type IV collagen (fig. IF) and fibronectin (fig. IH) was observed in the mesangium of the sclerosing glomeruli. These indirectly immunofluorescent findings are summarized in table 2. In patients with IgA nephropathy, there were no changes in HS-PG, type IV collagen and fibronectin with a slight decrease in laminin. When the immunofluorescence and light-microscopic findings are compared in patients with diabetes, HS-PG is found to have decreased in an inverse proportion to the grade of sclerosis (fig. 2A). When the immunofluorescence findings and degree of proteinuria were compared in pa tients with diabetes, HS-PG decreased as proteinuria in creased (fig. 3A). On the other hand in patients with IgA nephropathy, no loss of HS-PG was observed even with progression of glomerulosclerosis (fig. 4A). There were no correlations between the glomerular HS-PG and the degree of proteinuria in patients with IgA nephropathy (fig. 5A).
M akino/lkeda/H aram oto/O ta
Loss of HS-PG in Diabetic Nephropathy
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 1:04:55 PM
Table 2. Summary of immunofluorescence findings in various glomerular extracellular matrix components in patients with diabetic nephropathy with heavy proteinuria
Diabetic nephropathy B) Laminin
C) Type IV collagen
D) Fibronectin
Score
A ) H S-PG
Fig. 3. Relationship between proteinuria and extracellular matrix components in patients with diabetic nephropathy: HS-PG (A), laminin (B), type IV collagen (C) and fibronectin (D). The characteristics of the regression lines were as follows: A y = -0.38x + 5.l8, r = -0.71, p
Nephron 1992:61:415-421
Third Department of Internal Medicine, Okayama University Medical School, Okayama, Japan
Heparan Sulfate Proteoglycans Are Lost in Patients with Diabetic Nephropathy
Key Words
Abstract
Diabetes mellitus Heparan sulfate proteoglycan Glomerular basement membrane Laminin Type IV collagen Fibronectin Proteinuria IgA nephropathy Glomerulosclerosis
The pathogenesis of diabetic nephropathy relative to the changes in the glomeru lar extracellular matrices was investigated. Renal tissues from 10 diabetic pa tients were immunostained with antibodies directed against heparan sulfate proteoglycans (HS-PGs), laminin, type IV collagen and fibronectin. Seven patients were nephrotic and had advanced glomerulosclerosis with nodular lesion, while the other 3 had no renal manifestations or minor glomerular tissue alterations. Controls included kidneys removed from patients with renal tumors and specimens obtained by renal biopsy from patients with IgA nephropathy. Relationships among proteinuria, intensity of fluorescence and glomerular changes were studied. In diabetes 3 patients with minor glomerular lesions were found to have no changes in various components of extracellular matrices. A marked reduction in the intensity of staining with anti-HS-PG antibodies was observed in renal specimens from patients with nodular glomerulosclerosis and proteinuria, while a mild decrease in the intensity of fluorescence was observed in tissues stained with antilaminin antibodies. An increase compared to normal control sample findings in type IV collagen and fibronectin was observed in the mesangium of sclerosing glomeruli. No loss of HS-PG was observed in patients with IgA nephropathy. These results indicate that glomerular extracellular matrix HS-PG is lost in association with diabetic nephropathy; this loss results in alteration of the charge-selective properties of glomerular capillaries. This altera tion may, in part, be the cause of the proteinuria associated with diabetic nephropathy.
Introduction
The glomerular basement membrane (GBM) and mes angial matrix are together referred to as the glomerular extracellular matrices. These extracellular matrices are formed by a group of proteins that include type IV collagen [1], laminin [2], proteoglycans [3], fibronectin [4], entactin [5] and nidogen [6].
Accepted : October 9.199!
The changes most characteristic of diabetes are observed in the glomerular extracellular matrices and include thick ening of the GBM and increase in the mesangial matrix. Changes in the glomerular extracellular matrix components also occur. Among these, the loss of heparan sulfate prote oglycan (HS-PG) is believed to play an important role in the etiology of diabetic nephropathy [7-15]. Rohrbach et al. [7, 8] reported a reduced level of synthesis of extracellular
Hirofumi Makino. MI) Third Department of Medicine Okayama University Medical School 2-5-1 Shikata-cho. Okayama 700 (Japan)
©1992S.Kargcr AG.Basel (K)28-2766/92/ 0614-0415S2.75/0
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 1:04:55 PM
Hirofumi Makino Shuji Ikecla Toshinori Haramoto Zensuke Ota
Table 1. Clinico pathological find ings in patients with diabetes mellitus
Patient No
Age years
Sex
Type of disease
Duration years
Proteinuria g/day
Q.r ml/min
Sclerosis grade
Nodule (glomeruli)
I 2 3 4 5 6 7 8 9 I0
59 54 5! 39 66 53 32 45 53 45
F F F M M M F M M M
NIDDM NIDDM NIDDM IDDM NIDDM NIDDM IDDM NIDDM NIDDM NIDDM
0.2 0.3 12.0 24.0 10.0 5.0 15.0 n.k. 16.0 n.k.
0.I 0.3 LI 4.0 3.5 6.0 4.0 4.9 7.5 5.0
68 102 II4 50 20 54 56 45 63 28
0 0 I 2 3 3 3 3 4 4
0 0 0 0 4/9 7 /2 1 7 /I7 4/12 6/18 8/15
IDDM = Insulin-dependent diabetes mellitus; NIDDM = non-insulin-dependent diabetes mellitus; n.k. = not known: Ccr= creatinine clearance.
Patients and Methods Ten patients with diabetic nephropathy participated in the study. Daily urinary protein was measured using Esbach methods. Creati nine clearances were also measured. Percutaneous renal biopsy was performed for each patient. For light-microscopic study, specimens were stained with hematoxylin and eosin, periodic acid-Schiff and periodic acid-silver methenamine. The degree of glomerulosclerosis observed was scored between 0 and 4 using the classification system of Gellman et al. [17], The presence or absence of diabetic nodules was determined for each glomerulus. As controls, normal-appearing parts of the kidney removed from patients with kidney tumor at surgery as well as specimens obtained by renal biopsy from 10 patients with IgA nephropathy were used. The degree of glomerulosclerosis observed under the light microscope was scored between 0 and 3 in patients with IgA nephropathy. The distribution of various glomerular components was studied using indirect immunofluorescence techniques as described previous ly [18]. In brief, the kidney biopsy specimen was immediately snapfrozen, and 4-um-thick unfixed cryostat sections were prepared. Antibasement-membrane-HS-PG antibody was prepared by immunizing
416
rabbits with rat renal basement membrane HS-PG. The authenticity of specificity has been described in our previous publication [16). Anti laminin antibody was purchased from EY Laboratories (San Mateo, Calif., USA), and anti-type-I V-collagen and antifibronectin antibodies were purchased from Advance Co. (Tokyo, Japan). The second anti body. antirabbit IgG conjugated with fluorescein isothiocyanate (FITC), was obtained from Cappel Co. (West Chester. Pa., USA). The cryostat sections were first stained for various GBM components, washed in phosphate-buffered saline (PBS) and then stained with antirabbit IgG conjugated with FITC. The sections were then washed in PBS again and mounted in buffered glycerol. To reveal the hidden antigenic determinants, ethanol-fixed specimens both from diabetic patients, normal controls and IgA nephropathy were treated with 6 M urea and 0.1 Mglycine HCI buffer, pH 3.5, at 4”C for 1 h according to the technique described by Yoshioka et al. [19]. The distribution of the various basement membrane components was determined using an Olympus immunofluorescence microscope. The intensity of the staining of both GBM and mesangium was scored between 0 and 10 by 3 persons not knowing the identity of the specimens examined. After scoring, the intensity of the staining in normal control specimens was adjusted and set to a value of 5. The relationship between glomerulosclerosis, proteinuria and fluorescence intensity was also studied. Regression-correlation analysis was done using Spearman's test. Results were considered significant when p < 0.05.
Results
The clinicopathological findings in patients with diabet ic nephropathy are summarized in table 1. Seven patients manifested nephrotic syndrome; these patients had de creased creatinine clearance. The glomerulosclerosis score was high and accompanied by diabetic nodules in patients with nephrotic syndrome.
Makino/1 keda/ Haramoto/Ota
Loss of HS-PG in Diabetic Nephropathy
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 1:04:55 PM
matrix HS-PG in EHS sarcoma implanted in diabetic mice. In rats with streptozotocin-induced diabetes, a 30-40% reduction has been reported in the synthesis of heparan sulfate and its incorporation into the GBM [9]. Including those mentioned above, most studies of this kind have been performed in experimentally diabetic rats or mice [7-12], We have recently raised specific antibodies against the HS-PG of rat GBM [16]. In this study we used renal biopsy tissues obtained from patients with diabetic nephropathy for delineation of the changes in extracellular matrices by employing antibodies directed against HS-PG as well as other basement membrane components.
Fig. 1. Immunofluorescence micro graphs of a normal glomerulus stained with antibodies to HS-PG(A). laminin (C). type IV collagen (E) and fibronectin (G) and a diabetic glomerulus stained with antibodies to HS-PG (B), laminin (D), type IV collagen (F) and fibronectin (H).
gial region. Fibronectin staining had a mesangial pattern with the interstitial region (fig. lG). No change in staining intensity or pattern was observed after urea treatment in the specimens both from patients with diabetes, IgA nephro pathy and normal controls.
4 17
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 1:04:55 PM
In normal control specimens, HS-PG appeared as an intense linear staining in the GBM; minimal staining for HS-PG was observed in the mesangial region and tubular basement membrane (fig. IA). Laminin (fig. 1C) and type IV collagen (fig. IE) appeared as intense linear staining in the GBM and tubular basement membrane and in the mesan
Diabetic nephropathy A ) H S-PG
B) Laminin
C) Type IV collagen
D) Fibronectin
Fig. 2. Relationship between glomerular sclerosis and extracellular matrix components in patients with diabetic nephropathy: HS-PG (A), laminin (B). type IVcollagen (C) and fibronec tin (D). The characteristics of the different regression lines were as follows: Ay = -0.78x + 5.59, r = -0.75, p < 0.02: B y = -0.73x + 5.34, r = -0.66, p < 0.05: C y = 0.11x + 6.82, r = 0.43, p < 0.5; D y=0.42x + 5.24, r=0.52, p< 0.5.
Capillary
HS-PG Laminin Type IV collagen Fibronectin
Mesangium
control
DM
control
DM
+ + + + + + + + +
+ + + + + + +
± + + + +
± ± + + + + + +
DM = Diabetes mellitus.
Three patients with minor glomerular abnormality (cases 1-3) had no abnormalities in the various components of the extracellular matrix. In patients with diabetic glomer ulosclerosis (cases4-10), a marked reduction in the intensity of staining of the glomerular capillary wall with anti-HSPG antibodies was observed (fig. IB). A minimal decrease in
418
the intensity of fluorescence on the glomerular capillary wall was observed in the tissues stained with antilaminin antibodies (fig. ID) which had been obtained from patients with advanced glomerulosclerosis. An increase in type IV collagen (fig. IF) and fibronectin (fig. IH) was observed in the mesangium of the sclerosing glomeruli. These indirectly immunofluorescent findings are summarized in table 2. In patients with IgA nephropathy, there were no changes in HS-PG, type IV collagen and fibronectin with a slight decrease in laminin. When the immunofluorescence and light-microscopic findings are compared in patients with diabetes, HS-PG is found to have decreased in an inverse proportion to the grade of sclerosis (fig. 2A). When the immunofluorescence findings and degree of proteinuria were compared in pa tients with diabetes, HS-PG decreased as proteinuria in creased (fig. 3A). On the other hand in patients with IgA nephropathy, no loss of HS-PG was observed even with progression of glomerulosclerosis (fig. 4A). There were no correlations between the glomerular HS-PG and the degree of proteinuria in patients with IgA nephropathy (fig. 5A).
M akino/lkeda/H aram oto/O ta
Loss of HS-PG in Diabetic Nephropathy
Downloaded by: King's College London 137.73.144.138 - 1/10/2019 1:04:55 PM
Table 2. Summary of immunofluorescence findings in various glomerular extracellular matrix components in patients with diabetic nephropathy with heavy proteinuria
Diabetic nephropathy B) Laminin
C) Type IV collagen
D) Fibronectin
Score
A ) H S-PG
Fig. 3. Relationship between proteinuria and extracellular matrix components in patients with diabetic nephropathy: HS-PG (A), laminin (B), type IV collagen (C) and fibronectin (D). The characteristics of the regression lines were as follows: A y = -0.38x + 5.l8, r = -0.71, p
