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Aldose reductase inhibition by ponalrestat (statil) does not prevent proteinuria in long-term diabetic rats
] Diab Comp 1993; 7:233-240
Aldose Reductase Inhibition by Ponalrestat (Statil) Does Not Prevent Proteinuria in Long-Term Diabetic Rats A. S. Reddi G. N. Jyothirmayi
ABSTRACT The aldose reductase pathway has been implicated in the development of chronic complications of diabetes. In this study, we investigated the effect of an aldose reductase inhibitor, statil, on glomerular synthesis of heparan sulfate and albuminuria in male Wistar rats made diabetic with streptozotocin. Heparan sulfate is the predominant glycosaminoglycan (GAG) proteoglycan in the glomerular basement membrane (GBM). It confers a negative charge on the GBM, and its loss has been related to the presence of albumin in the urine. Diabetic rats
synthesized less glomerular heparan sulfate and excreted more albumin than normal rats. Glomerular sorbitol concentration was significantly higher in diabetic than in normal rats. Chronic treatment of diabetic rats with statil did n o t improve either heparan sulfate synthesis or albuminuria despite normalization of glomerular sorbitol content. The present study does n o t support the role of excess sorbitol in the development of glomerular abnormalities in this rat model of streptozotocin diabetes. (Journal of Diabetes and Its Complications 7;4:233-240, 1993).
INTRODUCTION
zotocin diabetic rats .6 Prevention of sorbitol accumulation by aldose reductase inhibitors (ARIs) yielded conflicting results on proteinuria and glomerular pathology. Some investigators showed either no improvement in proteinuria 7or glomerular abnormalities including thickening of the glomerular basement membrane (GBM) in diabetic 7-I° or galactose-fed normal rats.U Other investigators 12,13reported prevention of GBM thickening by ARIs in galactose-fed normal rats. Mauer et al. 14 demonstrated variable effects of sorbinil, an ARI, on glomerular structure in streptozotocin diabetic rats. Sorbinil decreased GBM width in diabetic rats fed 20% protein diet but had no effect on rats fed 50% protein diet. However, a significant reduction in mesangial volume by sorbinil was observed in the latter group of rats. In this study, we investigated the effect of an ARI, statil, on glomerular synthesis of heparan sulfate and albuminuria in long-term streptozotocin diabetic rats.
he polyol pathway has been suggested as a possible link between hyperglycemia and chronic complications of diabetes. 1 The enzyme, aldose reductase, which reduces glucose to sorbitol, has been demonstrated in a variety of tissues and cells, including glomerular epithelial cells 2 and cultured mesangial cells. 3 An increase in renal aldose reductase activity with enhanced gene expression has been reported in streptozotocin diabetic 4 and diabetic BB5(biobreeding) rats. Elevated levels of sorbitol have also been described in glomeruli from strepto-
T
Department of Medicine, UMDNJ-NewJersey Medical School, Newark, New Jersey, USA. Reprint requests to be sent to: Dr. A. S. Reddi, Department of Medicine, UMDNNew Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103. © 1993 Journal of Diabetes and Its Complications
0891-6632/93/$6.00
234
REDDIAND JYOTHIRMAYI
J Diab Comp 1993; 7:233-240
Heparan sulfate, as a predominant glycosaminoglycan (GAG) proteoglycan, confers a negative charge on the GBM, ls-18 and loss of this charge has been shown to cause proteinuria in diabetic as well as nondiabetic renal diseases: 6'~s MATERIALS AND METHODS
A total of 28 male Wistar rats (Charles River) weighing 70-90 g were used in the study. Following an overnight fast, diabetes was induced in 19 rats with a single intraperitoneal injection of streptozotocin in 0.1 M citrate buffer, pH 4.5, at a concentration of 60 mg/kg body weight. The remaining 9 rats received an equivalent amount of buffer and served as normal control rats. One week following induction of diabetes, 10 diabetic rats were allowed to drink tap water containing statil ad libitum at a concentration of 25 mg/L; the remaining 9 diabetic and 9 nondiabetic rats were given only tap water. Water was changed every day in all groups of rats. The drug treatment was continued for 20 weeks. The amount of statil that was consumed by each rat per day in mg was 5.38 + 0.34 (mean + SEM): All groups of rats were fed the Purina rodent chow 5001 ad libitum, with the following composition by weight:protein (23.4%), fat (4.5%), fiber (5.8%), sodium (0.4%), calcium (1%), phosphorus (0.61%), potassium (1.1%) with vitamin supplementation. The energy provided was 4.25 kcal/g. One week prior to killing, systolic blood pressures were measured in conscious rats by the tail-cuff method. At 4, 8, 12, 16, and 20 weeks, each rat was placed in a metabolic cage for 24-h urine collection, at which time the body weight and food and water intake were recorded. After measuring the total volume, the urine was centrifuged and used for determinations of albumin. Animals.
D e t e r m i n a t i o n of Glomerular G A G Synthesis. At the time of killing, each rat was anesthetized with a single intraperitoneal injection of Nembutal (5 mg/100 g) and bled from the abdominal aorta, and kidneys were removed. They were weighed and cortices sepa-
rated from medulla. Glomeruli were isolated from the cortex by differential sieving through 150-, 250-, and 63-~m meshes, 19 using ice-cooled 0.02 M phosphatebuffered saline, pH 7.2. Following centrifugation at 100 g, the glomeruli were washed twice with Krebs Ringer solution, and the washed pellet was uniformly suspended in the same solution. About 10,000 glomeruli were incubated in 2 mL of Krebs Ringer containing 0.15 mg/mL glutamine, 50 g/mL ascorbate, and 50 ,Ci/ mL 3SS-sulfate (specific activity: 788 mCi/mmole) in an atmosphere of 95% 02 and 5% CO2. Following incubation for 4 h at 37°C, protein synthesis was terminated by the addition of I rnM puromycin. The contents and further washings of the medium were transferred to a preweighed tube and centrifuged at 1000 g for 15 min. To the glomerular pellet was added 2 mL of acetone to remove lipids. This step was repeated twice, and the pellet was dried and weighed. The dried glomeruli were taken up in 2 mL of 0.1 M acetate buffer containing 5 mM EDTA, 5 mM cysteine, and 2.5 mg/ mL crystalline papain (Sigma) and incubated at 60°C for 24 h to release glycopeptides. 2°-22 Incubation was continued for another 24 h with addition of papain (1.5 mg/mL). At this time, I mg of carrier chondroitin sulfate (Sigma) was added. The GAGs were precipitated with 0.3 mL of 10% cetylpyridinium chloride. The precipitate was washed twice with sodium chloride-saturated 95% ethanol, then with 95% ethanol and dried. The GAGs were further purified by removing proteins and nucleic acids by precipitation twice with 10% trichloroacetic acid and supernatants combined. The pH of the supernatant was adjusted to 5 with 0.5M sodium acetate and GAGs were precipitated with 3 volumes of 95% ethanol. The precipitate was dried and then dissolved in a known volume of distilled water. An aliquot of this sample was used for quantitation of radioactivity, which represented counts in total GAGs. To identify individual GAGs, another aliquot was treated with 5 mg of testicular hyaluronidase (Sigma) in 0.15 M NaC1 and 0.1 M acetate buffer, pH 5.6, at
TABLE 1. PERTINENT INFORMATION ON NORMAL (N + H20), DIABETIC (D + H20), AND STATIL-TREATED DIABETIC (D + STATIL) RATS AT TIME OF SACRIFICE (20 weeks)
Rats
Body Blood Weight Pressure N (g) (ramHg)
Kidney Weight (g)
N+H20 D+H20 D+statil
9 >610 130" + 6 9 325 ± 31 149 ± 4 10 366 ± 26 144 + 6
3.90*+ 0.09 5.10 ± 0.33 4.84 ± 0.31
Values shown are means -1- SEM. * N + H 2 0 versus D+H20: p < 0.02-0.001. ** D + H 2 0 versus D+statih p < 0.01-0.005.
Protein Intake (g/day)
Plasma Glucose (raM)
K÷ (mM)
5.95* + 0.32 8.17"+ 0.72 3.38+ 0.24 10.66 ± 0.50 28.56± 3.61 4.02± 0.37 8.40 ± 0.49 22.56± 2.00 3.70± 0.28
Creatinine (~M)
Urine Volume (mL/day)
Glomerular Sorbitol (nM/g protein)
72.49± 3.54 40.22*± 3.38 67* :t: 5 85.75± 9.72 184.22"*± 11.55 103"*± 7 63.65± 6.19 147.10± 6.09 68 + 6
ALDOSE REDUCTASEINHIBITIONAND PROTEINURIA 235
J Diab Comp 1993; 7:233-240
14 13
- - ~ - - - sample + Nitrous acid ....~ .... tissue sample rd
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FIGURE 1 Elution patterns of 3SS-labeled heparan sulfate (standard) and testicular hyaluronidase-digested glomerular material from Sephadex G50 column before and after nitrous acid degradation.
6 4 3 2 1
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37°C for 24 h to degrade chondroitin sulfate. The digested material was placed on a Sephadex G50 column (5 x 550 mm) and eluted with 0.15M NaC1 in 10% ethanol. The included volume contained over 95% of the uronic acid due to the degraded carrier, and the radioactivity associated with this fraction represented the counts in chondroitin sulfate. The voided volume contained mostly heparan sulfate, and the radioactivity associated with this fraction represented counts in heparan sulfate. The presence of heparan sulfate in the void volume was verified by its resistance to testicular hyaluronidase and its degradation by nitrous acid (Fig=re 1).
2% SDS, and 5% 2-mercaptoethanol; 2-3 crystals of sucrose and 5 ~L of bromophenol blue (tracking dye) (0.005% bromophenol blue in 0.12M tris-HC1 p H 6.8) were added to each sample. The samples were heated at 70°C for 20 min, vortexed, and centrifuged at high speed for 2 min and were loaded on top of homogenous 10% slab gels (0.8% Bis and 30% Acrylamide). Following electrophoresis at 40 mA for 120 min per gel in an electrode buffer containing glycine, 10% SDS, and tris-base at p H 8.6, the gels were stained in Coomassie blue solution for 10-15 min (1.25 g of Coomassie blue in 454 mL of methanol-water 1:1 (v/v) and 46 mL of glacial acetic acid), destained in a solution made of 75 mL of glacial acetic acid, 250 mL of methanol, and 675 mL of water and analyzed for urinary protein patterns. Densitometric readings of the negative film were taken using 2202 Ultrascan Laser Densitometer and 2220 Recording Integrator (LKB Bromma).
Gel Electrophoresis. Urinary protein analysis of normal, untreated diabetic, and diabetic rats treated with statil was performed using thin layer sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis (SDS gradient PAGE) technique. Urine samples containing 10 pg of protein and standard samples containing 5 ~g of low molecular weight/high molecular weight (LMW/HMW) standard protein (Bio-Rad, RockviUe, NY) in a volume of 50 pL were prepared in 1 x sample buffer containing 0.625M tris-HC1 pH 7.1,
Determination of Plasma Glucose, Creatinine, and K+. Plasma glucose and creatinine were determined by the glucose oxidase and alkaline picrate methods, respectively, using the reagents supplied by Sigma
TABLE 2. GLOMERULAR GLYCOSAMINOGLYCANS (GAG) IN NORMAL (N + H20), DIABETIC (D + H20), AND STATIL-TREATED DIABETIC (D + STATIL) RATS
Glomerular Weight Rats
N
(rag)
N+H20 D+H20 D+statil
8 8 9
58 + 5 60 + 7 62 ± 6
~S-sulfate Incorporation into Total GAG DPM/mgGlomerular
Weight 265* + 39 140 + 18 123 ± 25
Values shown are means + SEM. * N+H20 versus D+H20: p K 0.02-0.001.
% 3ss Incorporation into Heparan DPM/glomerulus Sulfate 1.53" + 0.26 0.82 + 0.13 0.82 ± 0.17
85.10" + 2.37 65.62 + 3.29 71.10 ± 0.99
P/o35 S Incorporation into Chondroitin Sulfate
O
14.87" + 2.36 31.65 ± 3.75 28.90 ± 0.99
236
J Diab Comp 1993; 7:233-240
REDDI A N D JYOTHIRMAYI
70 r-
60
~t (N En
v
E
5O
D+H20
40
FIGURE 2 Twenty-four-hour urinary excretion of albumin in normal (N + H20), diabetic (D+ H20), and statil-treated diabetic (D + S) rats. Each point represents the mean + SEM. *N + H2O versus D + H20: p < O.02-0.001; **N + 1420 versus D + S: p < O.020.001.
D+S
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E
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Chemical Co. (St. Louis, MO). Plasma K ÷ was determined by flame photometry. Determination of Urinary Albumin. Albumin concentration in 24-h urinary samples was determined by the radioimmunoassay method of Brodows et al. 23 Determination of Glomerular Sorbitol. Sorbitol in isolated glomeruli was assayed by the colorimetric method using a commercially available kit (Boehringer Mannheim, Indianapolis, IN). This method utilizes sorbitol dehydrogenase, NAD, diaphorase, and iodonitrotetrazolium chloride. Glomerular sorbitol con-
D+S 109 8 7 6 5 4 3 2 1 D
N kOo .... - 9 2 -66
-45 -31 -2t
-t4
FIGURE 3 Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis patterns of urinary proteins from one normal (N), one diabetic (D), and ten statil-treated diabetic (D + S) rats. Urine samples were obtained after 20 weeks of treatment.
centration is expressed as nM/g protein. Statistical Analysis. Data were analyzed by one-way analysis of variance and expressed as mean + SEM. Wherever appropriate, the significance between two unpaired means was calculated by Student's t test, and a p value above 0.05 was considered nonsignificant. RESULTS Table I shows pertinent information on various groups of rats. Significantly lower body weight and higher blood pressure, kidney weight, protein intake, plasma glucose, 24-h urine volume, and glomerular sorbitol content were observed in untreated diabetic than in normal rats. However, no difference in plasma K ÷ and creatinine levels were found between the two groups of rats. Except for glomerular sorbitol content and urine volume, statil treatment did not alter any of the parameters in diabetic rats. No diabetic rat in either group died throughout the study. Table 2 shows the 35S-sulfate incorporation into total GAG, heparan sulfate and chondroitin sulfate by glomeruli isolated from various groups of rats. Previous study 24 showed that incubation for 4 h is enough for optimal incorporation of 35S-sulfate into glomeruli from both normal and diabetic rats. As evident, glomeruli from diabetic rats incorporated less radioactivity into both total GAG and heparan sulfate when compared to normal rats. This decrease was statistically significant w h e n expressed either per mg dry glomerular weight or per glomerulus. The incorporation into chondroitin sulfate was significantly increased in diabetic rats. Statil treatment did not normalize incorporation of the radioactivity either into total GAG, heparan sulfate, or chondroitin sulfate. The glomerular weight was similar in all groups of rats. Figure 2 shows 24-h albumin excretion in normal,
J Diab Comp 1993; 7:233-240
ALDOSE REDUCTASE INHIBITION AND PROTEINURIA
237
60
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FIGURE 4 Densitometric readings of protein patterns from FIGURE 3 from one normal (N + H20), one diabetic (D+ H20), and a mean of ten statil-treated diabetic (D + S) rats. Values are expressed as % area under the protein peak of different molecular weight proteins. diabetic, and statil-treated diabetic rats at various time intervals. As evident, albumin excretion was significantly higher in diabetic than in normal rats. Also, albumin excretion increased with duration of diabetes. Statil treatment did not have any effect on proteinuria at any time interval in diabetic rats. Urinary protein pattern on SDS gradient PAGE is shown in Figure 3. Protein corresponding to albumin (molecular weight 66 kDa) was faintly visible in normal rats. However, the presence of a band corresponding to 66 kDa is evident in the diabetic as well as in 7 of the 10 statil-treated diabetic rats. In addition, an unknown protein with a molecular weight between 45 and 55 kDa was predominantly present in the diabetic rat. This protein was persistent in all of the statiltreated diabetic rats (Figure 4). DISCUSSION A number of biochemical and histochemical methods have demonstrated the presence of GAG proteogly-
can in GBM. 15-18,2s Among GAG proteoglycans, heparan sulfate comprises about 85%, and the remaining 15% is chondroitin and dermatan sulfate, ls'26 Because heparan sulfate confers a negative charge on the GBM, clinically detectable proteinuria in diabetic and nondiabetic subjects and animals has been attributed to the loss of heparan sulfate from the GBM. 15-18"21,25,27Indeed, removal of heparan sulfate from the GBM of rats by heparinase caused increased permeability to ferritin as well as albumin. 2s,29Decreases in both synthesis and content of heparan sulfate have been reported in the kidneys of diabetic animals and human subjects by biochemical and histochemical methods.22,30-42 This study demonstrates a decrease in heparan sulfate synthesis by glomeruli from diabetic rats. Statil, at a dose that effectively prevented sorbitol accumulation in glomeruli of diabetic rats, did not normalize heparan sulfate synthesis. Furthermore, albuminuria, which increased with duration of diabetes in these
238 REDDIAND JYOTHIRMAYI
rats, was not prevented by statil. This result is consistent with the observations of Daniels and Hostetter, 7 Mauer et al. 14and those of K6rner et al. 43in long-term diabetic rats. An earlier study by Cohen et al. 44 reported undersulfation of GBM heparan sulfate in diabetic rats and lack of normalization by sorbinil. Variable effects of statil on urinary excretion of albumin have been reported in both diabetic h u m a n subjects and animals. Cohen et al. 45reported no improvement on albumin excretion in non-insulin-dependent diabetic subjects with microalbuminuria treated with statil for I month. Similarly, another study 46also failed to show any improvement in albuminuria in normoalbuminuric insulin-dependent diabetic subjects treated with statil for 6 months. In contrast, Blohm6 and Smith 47 reported a decrease in albumin excretion in insulin-dependent diabetic patients with incipient nep h r o p ath y who were treated with statil for 3 months. In streptozotocin diabetic48 and spontaneously diabetic BB 49 rats, improvement in proteinuria was observed following either short- or long-term treatment with statil. Our results show that statil did not improve albuminuria even after 4 or 20 weeks of treatment. A significant reduction either in albuminuria or proteinuria has been reported in diabetic rats by ARIs other than statil. Tolrestat, 5°,51 sorbinil, 52,53or A1576s4 have been shown to improve proteinuria in diabetic rats. It is possible that structural dissimilarities in ARIs may have accounted for this difference. In our study, adequate dosages of statil were administered because sorbitol accumulation was prevented in glomeruli from the drug-treated diabetic rats. It is not know n whether higher doses than these would lower proteinuria. In summary, the present study demonstrates a decrease in glomerular heparan sulfate synthesis and an increase in albuminuria in long-term diabetic rats. These abnormalities were not prevented by statil despite normalization of glomerular sorbitol concentration. This study fails to support the involvement of polyol pathway in the development of biochemical and functional glomerular abnormalities in the streptozotocin diabetic rat.
J Diab Comp 1993; 7:233-240
K, Shigeta Y: Evidence for existence of polyol pathway in cultured rat mesangial cells. Diabetes 36:240-243, 1987. 4. Ghahary A, Luo J, Gong Y, Chakrabarti S, Sima AAF, Murphy LJ: Increased renal aldose reductase activity, immunoreactivity, and mRNA in streptozotocin-induced diabetic rats. Diabetes 38:1067-1071, 1989. 5. Ghahary A, Chakrabarti S, Sima AAF, Murphy LJ: Effect of insulin and statil on aldose reductase expression in diabetic rats. Diabetes 40:1391-1396, 1991. 6. Beyer-Mears A, Ku L, Cohen MP: Glomerular polyol accumulation in diabetes and its prevention by oral sorbinil. Diabetes 33:604-607, 1984. 7. Daniels BS, Hostetter TH: Aldose reductase inhibition and glomerular abnormalities in diabetic rats. Diabetes 38:981-986, 1989. 8. Poulsom R, Boot-Handford RP, Heath H: The effects of long-term streptozotocin-diabeticrats with an aldose reductase inhibitor. Exp Eye Res 37:507-515, 1983. 9. Tilton RG, Pugliese G, Williamson JR: Diabetesinduced glomerular changes in rats are not prevented by sorbinil [Abstract]. Diabetes 38(suppl 2):94A, 1989. 10. Osterby R, Gundersen HJG: Glomerular basement membrane thickening in streptozotocin diabetic rats despite treatment with an aldose reductase inhibitor. J Diab Comp 3:149-153, 1989. 11. Das A, Frank RN, Zhang NL: Sorbinil does not prevent galactose-induced glomerular capillary basement membrane thickening in the rat. Diabetologia 33:515-521, 1990. 12. Chandler ML, Shannon WA, DeSantis L: Prevention of retinal capillary basement membrane-thickening in diabetic rats by aldose reductase inhibitors [Abstract]. Invest Ophthalmol Vis Sci 25:159, 1984. 13. Stribling D, Armstrong FM, Harrison HE: Aldose reductase in the etiology of diabetic complications. J Diab Comp 3:70-76, 1989. 14. Mauer SM, Steffes MW, Azar S, Brown DM: Effects of sorbinil on glomerular structure and function in longterm diabetic rats. Diabetes 38:838-846, 1989. 15. Farquhar MG: The glomerular basement membrane. A selective macromolecular filter, in Hay ED (ed). Cell Biology of Extracellular Matrix. New York, Plenum Press, 1982, pp. 335-378.
ACKNOWLEDGMENTS
16. Kanwar YS: Biology of disease. Biophysiology of glomerular filtration and proteinuria. Lab Invest 51:7-21, 1984.
This work was supported in part by a grant from Stuart Pharmaceuticals. We thank Marie Birthwright for typing this manuscript.
17. Timpl R, Dziadek M: Structure, development and molecular pathology of basement membranes. Int Rev Exp Pathol 29:1-112, 1986.
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J Diab Comp 1993; 7:233-240
M: Reversal of proteinuria by sorbinil, an aldose reductase inhibitor in spontaneously diabetic (BB) rats. Pharmacology 36:112-120, 1988. 54. Triton RG, Chang K, Pugliese G, Eades DM, Province MA, Sherman WR, Kilo C, Wflliamson JR: Prevention of hemodynamic and vascular albumin filtration changes in diabetic rats by aldose reductase inhibitors. Diabetes 37:1258-1270, 1989.
Aldose Reductase Inhibition by Ponalrestat (Statil) Does Not Prevent Proteinuria in Long-Term Diabetic Rats A. S. Reddi G. N. Jyothirmayi
ABSTRACT The aldose reductase pathway has been implicated in the development of chronic complications of diabetes. In this study, we investigated the effect of an aldose reductase inhibitor, statil, on glomerular synthesis of heparan sulfate and albuminuria in male Wistar rats made diabetic with streptozotocin. Heparan sulfate is the predominant glycosaminoglycan (GAG) proteoglycan in the glomerular basement membrane (GBM). It confers a negative charge on the GBM, and its loss has been related to the presence of albumin in the urine. Diabetic rats
synthesized less glomerular heparan sulfate and excreted more albumin than normal rats. Glomerular sorbitol concentration was significantly higher in diabetic than in normal rats. Chronic treatment of diabetic rats with statil did n o t improve either heparan sulfate synthesis or albuminuria despite normalization of glomerular sorbitol content. The present study does n o t support the role of excess sorbitol in the development of glomerular abnormalities in this rat model of streptozotocin diabetes. (Journal of Diabetes and Its Complications 7;4:233-240, 1993).
INTRODUCTION
zotocin diabetic rats .6 Prevention of sorbitol accumulation by aldose reductase inhibitors (ARIs) yielded conflicting results on proteinuria and glomerular pathology. Some investigators showed either no improvement in proteinuria 7or glomerular abnormalities including thickening of the glomerular basement membrane (GBM) in diabetic 7-I° or galactose-fed normal rats.U Other investigators 12,13reported prevention of GBM thickening by ARIs in galactose-fed normal rats. Mauer et al. 14 demonstrated variable effects of sorbinil, an ARI, on glomerular structure in streptozotocin diabetic rats. Sorbinil decreased GBM width in diabetic rats fed 20% protein diet but had no effect on rats fed 50% protein diet. However, a significant reduction in mesangial volume by sorbinil was observed in the latter group of rats. In this study, we investigated the effect of an ARI, statil, on glomerular synthesis of heparan sulfate and albuminuria in long-term streptozotocin diabetic rats.
he polyol pathway has been suggested as a possible link between hyperglycemia and chronic complications of diabetes. 1 The enzyme, aldose reductase, which reduces glucose to sorbitol, has been demonstrated in a variety of tissues and cells, including glomerular epithelial cells 2 and cultured mesangial cells. 3 An increase in renal aldose reductase activity with enhanced gene expression has been reported in streptozotocin diabetic 4 and diabetic BB5(biobreeding) rats. Elevated levels of sorbitol have also been described in glomeruli from strepto-
T
Department of Medicine, UMDNJ-NewJersey Medical School, Newark, New Jersey, USA. Reprint requests to be sent to: Dr. A. S. Reddi, Department of Medicine, UMDNNew Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103. © 1993 Journal of Diabetes and Its Complications
0891-6632/93/$6.00
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REDDIAND JYOTHIRMAYI
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Heparan sulfate, as a predominant glycosaminoglycan (GAG) proteoglycan, confers a negative charge on the GBM, ls-18 and loss of this charge has been shown to cause proteinuria in diabetic as well as nondiabetic renal diseases: 6'~s MATERIALS AND METHODS
A total of 28 male Wistar rats (Charles River) weighing 70-90 g were used in the study. Following an overnight fast, diabetes was induced in 19 rats with a single intraperitoneal injection of streptozotocin in 0.1 M citrate buffer, pH 4.5, at a concentration of 60 mg/kg body weight. The remaining 9 rats received an equivalent amount of buffer and served as normal control rats. One week following induction of diabetes, 10 diabetic rats were allowed to drink tap water containing statil ad libitum at a concentration of 25 mg/L; the remaining 9 diabetic and 9 nondiabetic rats were given only tap water. Water was changed every day in all groups of rats. The drug treatment was continued for 20 weeks. The amount of statil that was consumed by each rat per day in mg was 5.38 + 0.34 (mean + SEM): All groups of rats were fed the Purina rodent chow 5001 ad libitum, with the following composition by weight:protein (23.4%), fat (4.5%), fiber (5.8%), sodium (0.4%), calcium (1%), phosphorus (0.61%), potassium (1.1%) with vitamin supplementation. The energy provided was 4.25 kcal/g. One week prior to killing, systolic blood pressures were measured in conscious rats by the tail-cuff method. At 4, 8, 12, 16, and 20 weeks, each rat was placed in a metabolic cage for 24-h urine collection, at which time the body weight and food and water intake were recorded. After measuring the total volume, the urine was centrifuged and used for determinations of albumin. Animals.
D e t e r m i n a t i o n of Glomerular G A G Synthesis. At the time of killing, each rat was anesthetized with a single intraperitoneal injection of Nembutal (5 mg/100 g) and bled from the abdominal aorta, and kidneys were removed. They were weighed and cortices sepa-
rated from medulla. Glomeruli were isolated from the cortex by differential sieving through 150-, 250-, and 63-~m meshes, 19 using ice-cooled 0.02 M phosphatebuffered saline, pH 7.2. Following centrifugation at 100 g, the glomeruli were washed twice with Krebs Ringer solution, and the washed pellet was uniformly suspended in the same solution. About 10,000 glomeruli were incubated in 2 mL of Krebs Ringer containing 0.15 mg/mL glutamine, 50 g/mL ascorbate, and 50 ,Ci/ mL 3SS-sulfate (specific activity: 788 mCi/mmole) in an atmosphere of 95% 02 and 5% CO2. Following incubation for 4 h at 37°C, protein synthesis was terminated by the addition of I rnM puromycin. The contents and further washings of the medium were transferred to a preweighed tube and centrifuged at 1000 g for 15 min. To the glomerular pellet was added 2 mL of acetone to remove lipids. This step was repeated twice, and the pellet was dried and weighed. The dried glomeruli were taken up in 2 mL of 0.1 M acetate buffer containing 5 mM EDTA, 5 mM cysteine, and 2.5 mg/ mL crystalline papain (Sigma) and incubated at 60°C for 24 h to release glycopeptides. 2°-22 Incubation was continued for another 24 h with addition of papain (1.5 mg/mL). At this time, I mg of carrier chondroitin sulfate (Sigma) was added. The GAGs were precipitated with 0.3 mL of 10% cetylpyridinium chloride. The precipitate was washed twice with sodium chloride-saturated 95% ethanol, then with 95% ethanol and dried. The GAGs were further purified by removing proteins and nucleic acids by precipitation twice with 10% trichloroacetic acid and supernatants combined. The pH of the supernatant was adjusted to 5 with 0.5M sodium acetate and GAGs were precipitated with 3 volumes of 95% ethanol. The precipitate was dried and then dissolved in a known volume of distilled water. An aliquot of this sample was used for quantitation of radioactivity, which represented counts in total GAGs. To identify individual GAGs, another aliquot was treated with 5 mg of testicular hyaluronidase (Sigma) in 0.15 M NaC1 and 0.1 M acetate buffer, pH 5.6, at
TABLE 1. PERTINENT INFORMATION ON NORMAL (N + H20), DIABETIC (D + H20), AND STATIL-TREATED DIABETIC (D + STATIL) RATS AT TIME OF SACRIFICE (20 weeks)
Rats
Body Blood Weight Pressure N (g) (ramHg)
Kidney Weight (g)
N+H20 D+H20 D+statil
9 >610 130" + 6 9 325 ± 31 149 ± 4 10 366 ± 26 144 + 6
3.90*+ 0.09 5.10 ± 0.33 4.84 ± 0.31
Values shown are means -1- SEM. * N + H 2 0 versus D+H20: p < 0.02-0.001. ** D + H 2 0 versus D+statih p < 0.01-0.005.
Protein Intake (g/day)
Plasma Glucose (raM)
K÷ (mM)
5.95* + 0.32 8.17"+ 0.72 3.38+ 0.24 10.66 ± 0.50 28.56± 3.61 4.02± 0.37 8.40 ± 0.49 22.56± 2.00 3.70± 0.28
Creatinine (~M)
Urine Volume (mL/day)
Glomerular Sorbitol (nM/g protein)
72.49± 3.54 40.22*± 3.38 67* :t: 5 85.75± 9.72 184.22"*± 11.55 103"*± 7 63.65± 6.19 147.10± 6.09 68 + 6
ALDOSE REDUCTASEINHIBITIONAND PROTEINURIA 235
J Diab Comp 1993; 7:233-240
14 13
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37°C for 24 h to degrade chondroitin sulfate. The digested material was placed on a Sephadex G50 column (5 x 550 mm) and eluted with 0.15M NaC1 in 10% ethanol. The included volume contained over 95% of the uronic acid due to the degraded carrier, and the radioactivity associated with this fraction represented the counts in chondroitin sulfate. The voided volume contained mostly heparan sulfate, and the radioactivity associated with this fraction represented counts in heparan sulfate. The presence of heparan sulfate in the void volume was verified by its resistance to testicular hyaluronidase and its degradation by nitrous acid (Fig=re 1).
2% SDS, and 5% 2-mercaptoethanol; 2-3 crystals of sucrose and 5 ~L of bromophenol blue (tracking dye) (0.005% bromophenol blue in 0.12M tris-HC1 p H 6.8) were added to each sample. The samples were heated at 70°C for 20 min, vortexed, and centrifuged at high speed for 2 min and were loaded on top of homogenous 10% slab gels (0.8% Bis and 30% Acrylamide). Following electrophoresis at 40 mA for 120 min per gel in an electrode buffer containing glycine, 10% SDS, and tris-base at p H 8.6, the gels were stained in Coomassie blue solution for 10-15 min (1.25 g of Coomassie blue in 454 mL of methanol-water 1:1 (v/v) and 46 mL of glacial acetic acid), destained in a solution made of 75 mL of glacial acetic acid, 250 mL of methanol, and 675 mL of water and analyzed for urinary protein patterns. Densitometric readings of the negative film were taken using 2202 Ultrascan Laser Densitometer and 2220 Recording Integrator (LKB Bromma).
Gel Electrophoresis. Urinary protein analysis of normal, untreated diabetic, and diabetic rats treated with statil was performed using thin layer sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis (SDS gradient PAGE) technique. Urine samples containing 10 pg of protein and standard samples containing 5 ~g of low molecular weight/high molecular weight (LMW/HMW) standard protein (Bio-Rad, RockviUe, NY) in a volume of 50 pL were prepared in 1 x sample buffer containing 0.625M tris-HC1 pH 7.1,
Determination of Plasma Glucose, Creatinine, and K+. Plasma glucose and creatinine were determined by the glucose oxidase and alkaline picrate methods, respectively, using the reagents supplied by Sigma
TABLE 2. GLOMERULAR GLYCOSAMINOGLYCANS (GAG) IN NORMAL (N + H20), DIABETIC (D + H20), AND STATIL-TREATED DIABETIC (D + STATIL) RATS
Glomerular Weight Rats
N
(rag)
N+H20 D+H20 D+statil
8 8 9
58 + 5 60 + 7 62 ± 6
~S-sulfate Incorporation into Total GAG DPM/mgGlomerular
Weight 265* + 39 140 + 18 123 ± 25
Values shown are means + SEM. * N+H20 versus D+H20: p K 0.02-0.001.
% 3ss Incorporation into Heparan DPM/glomerulus Sulfate 1.53" + 0.26 0.82 + 0.13 0.82 ± 0.17
85.10" + 2.37 65.62 + 3.29 71.10 ± 0.99
P/o35 S Incorporation into Chondroitin Sulfate
O
14.87" + 2.36 31.65 ± 3.75 28.90 ± 0.99
236
J Diab Comp 1993; 7:233-240
REDDI A N D JYOTHIRMAYI
70 r-
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5O
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FIGURE 2 Twenty-four-hour urinary excretion of albumin in normal (N + H20), diabetic (D+ H20), and statil-treated diabetic (D + S) rats. Each point represents the mean + SEM. *N + H2O versus D + H20: p < O.02-0.001; **N + 1420 versus D + S: p < O.020.001.
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Chemical Co. (St. Louis, MO). Plasma K ÷ was determined by flame photometry. Determination of Urinary Albumin. Albumin concentration in 24-h urinary samples was determined by the radioimmunoassay method of Brodows et al. 23 Determination of Glomerular Sorbitol. Sorbitol in isolated glomeruli was assayed by the colorimetric method using a commercially available kit (Boehringer Mannheim, Indianapolis, IN). This method utilizes sorbitol dehydrogenase, NAD, diaphorase, and iodonitrotetrazolium chloride. Glomerular sorbitol con-
D+S 109 8 7 6 5 4 3 2 1 D
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FIGURE 3 Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis patterns of urinary proteins from one normal (N), one diabetic (D), and ten statil-treated diabetic (D + S) rats. Urine samples were obtained after 20 weeks of treatment.
centration is expressed as nM/g protein. Statistical Analysis. Data were analyzed by one-way analysis of variance and expressed as mean + SEM. Wherever appropriate, the significance between two unpaired means was calculated by Student's t test, and a p value above 0.05 was considered nonsignificant. RESULTS Table I shows pertinent information on various groups of rats. Significantly lower body weight and higher blood pressure, kidney weight, protein intake, plasma glucose, 24-h urine volume, and glomerular sorbitol content were observed in untreated diabetic than in normal rats. However, no difference in plasma K ÷ and creatinine levels were found between the two groups of rats. Except for glomerular sorbitol content and urine volume, statil treatment did not alter any of the parameters in diabetic rats. No diabetic rat in either group died throughout the study. Table 2 shows the 35S-sulfate incorporation into total GAG, heparan sulfate and chondroitin sulfate by glomeruli isolated from various groups of rats. Previous study 24 showed that incubation for 4 h is enough for optimal incorporation of 35S-sulfate into glomeruli from both normal and diabetic rats. As evident, glomeruli from diabetic rats incorporated less radioactivity into both total GAG and heparan sulfate when compared to normal rats. This decrease was statistically significant w h e n expressed either per mg dry glomerular weight or per glomerulus. The incorporation into chondroitin sulfate was significantly increased in diabetic rats. Statil treatment did not normalize incorporation of the radioactivity either into total GAG, heparan sulfate, or chondroitin sulfate. The glomerular weight was similar in all groups of rats. Figure 2 shows 24-h albumin excretion in normal,
J Diab Comp 1993; 7:233-240
ALDOSE REDUCTASE INHIBITION AND PROTEINURIA
237
60
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FIGURE 4 Densitometric readings of protein patterns from FIGURE 3 from one normal (N + H20), one diabetic (D+ H20), and a mean of ten statil-treated diabetic (D + S) rats. Values are expressed as % area under the protein peak of different molecular weight proteins. diabetic, and statil-treated diabetic rats at various time intervals. As evident, albumin excretion was significantly higher in diabetic than in normal rats. Also, albumin excretion increased with duration of diabetes. Statil treatment did not have any effect on proteinuria at any time interval in diabetic rats. Urinary protein pattern on SDS gradient PAGE is shown in Figure 3. Protein corresponding to albumin (molecular weight 66 kDa) was faintly visible in normal rats. However, the presence of a band corresponding to 66 kDa is evident in the diabetic as well as in 7 of the 10 statil-treated diabetic rats. In addition, an unknown protein with a molecular weight between 45 and 55 kDa was predominantly present in the diabetic rat. This protein was persistent in all of the statiltreated diabetic rats (Figure 4). DISCUSSION A number of biochemical and histochemical methods have demonstrated the presence of GAG proteogly-
can in GBM. 15-18,2s Among GAG proteoglycans, heparan sulfate comprises about 85%, and the remaining 15% is chondroitin and dermatan sulfate, ls'26 Because heparan sulfate confers a negative charge on the GBM, clinically detectable proteinuria in diabetic and nondiabetic subjects and animals has been attributed to the loss of heparan sulfate from the GBM. 15-18"21,25,27Indeed, removal of heparan sulfate from the GBM of rats by heparinase caused increased permeability to ferritin as well as albumin. 2s,29Decreases in both synthesis and content of heparan sulfate have been reported in the kidneys of diabetic animals and human subjects by biochemical and histochemical methods.22,30-42 This study demonstrates a decrease in heparan sulfate synthesis by glomeruli from diabetic rats. Statil, at a dose that effectively prevented sorbitol accumulation in glomeruli of diabetic rats, did not normalize heparan sulfate synthesis. Furthermore, albuminuria, which increased with duration of diabetes in these
238 REDDIAND JYOTHIRMAYI
rats, was not prevented by statil. This result is consistent with the observations of Daniels and Hostetter, 7 Mauer et al. 14and those of K6rner et al. 43in long-term diabetic rats. An earlier study by Cohen et al. 44 reported undersulfation of GBM heparan sulfate in diabetic rats and lack of normalization by sorbinil. Variable effects of statil on urinary excretion of albumin have been reported in both diabetic h u m a n subjects and animals. Cohen et al. 45reported no improvement on albumin excretion in non-insulin-dependent diabetic subjects with microalbuminuria treated with statil for I month. Similarly, another study 46also failed to show any improvement in albuminuria in normoalbuminuric insulin-dependent diabetic subjects treated with statil for 6 months. In contrast, Blohm6 and Smith 47 reported a decrease in albumin excretion in insulin-dependent diabetic patients with incipient nep h r o p ath y who were treated with statil for 3 months. In streptozotocin diabetic48 and spontaneously diabetic BB 49 rats, improvement in proteinuria was observed following either short- or long-term treatment with statil. Our results show that statil did not improve albuminuria even after 4 or 20 weeks of treatment. A significant reduction either in albuminuria or proteinuria has been reported in diabetic rats by ARIs other than statil. Tolrestat, 5°,51 sorbinil, 52,53or A1576s4 have been shown to improve proteinuria in diabetic rats. It is possible that structural dissimilarities in ARIs may have accounted for this difference. In our study, adequate dosages of statil were administered because sorbitol accumulation was prevented in glomeruli from the drug-treated diabetic rats. It is not know n whether higher doses than these would lower proteinuria. In summary, the present study demonstrates a decrease in glomerular heparan sulfate synthesis and an increase in albuminuria in long-term diabetic rats. These abnormalities were not prevented by statil despite normalization of glomerular sorbitol concentration. This study fails to support the involvement of polyol pathway in the development of biochemical and functional glomerular abnormalities in the streptozotocin diabetic rat.
J Diab Comp 1993; 7:233-240
K, Shigeta Y: Evidence for existence of polyol pathway in cultured rat mesangial cells. Diabetes 36:240-243, 1987. 4. Ghahary A, Luo J, Gong Y, Chakrabarti S, Sima AAF, Murphy LJ: Increased renal aldose reductase activity, immunoreactivity, and mRNA in streptozotocin-induced diabetic rats. Diabetes 38:1067-1071, 1989. 5. Ghahary A, Chakrabarti S, Sima AAF, Murphy LJ: Effect of insulin and statil on aldose reductase expression in diabetic rats. Diabetes 40:1391-1396, 1991. 6. Beyer-Mears A, Ku L, Cohen MP: Glomerular polyol accumulation in diabetes and its prevention by oral sorbinil. Diabetes 33:604-607, 1984. 7. Daniels BS, Hostetter TH: Aldose reductase inhibition and glomerular abnormalities in diabetic rats. Diabetes 38:981-986, 1989. 8. Poulsom R, Boot-Handford RP, Heath H: The effects of long-term streptozotocin-diabeticrats with an aldose reductase inhibitor. Exp Eye Res 37:507-515, 1983. 9. Tilton RG, Pugliese G, Williamson JR: Diabetesinduced glomerular changes in rats are not prevented by sorbinil [Abstract]. Diabetes 38(suppl 2):94A, 1989. 10. Osterby R, Gundersen HJG: Glomerular basement membrane thickening in streptozotocin diabetic rats despite treatment with an aldose reductase inhibitor. J Diab Comp 3:149-153, 1989. 11. Das A, Frank RN, Zhang NL: Sorbinil does not prevent galactose-induced glomerular capillary basement membrane thickening in the rat. Diabetologia 33:515-521, 1990. 12. Chandler ML, Shannon WA, DeSantis L: Prevention of retinal capillary basement membrane-thickening in diabetic rats by aldose reductase inhibitors [Abstract]. Invest Ophthalmol Vis Sci 25:159, 1984. 13. Stribling D, Armstrong FM, Harrison HE: Aldose reductase in the etiology of diabetic complications. J Diab Comp 3:70-76, 1989. 14. Mauer SM, Steffes MW, Azar S, Brown DM: Effects of sorbinil on glomerular structure and function in longterm diabetic rats. Diabetes 38:838-846, 1989. 15. Farquhar MG: The glomerular basement membrane. A selective macromolecular filter, in Hay ED (ed). Cell Biology of Extracellular Matrix. New York, Plenum Press, 1982, pp. 335-378.
ACKNOWLEDGMENTS
16. Kanwar YS: Biology of disease. Biophysiology of glomerular filtration and proteinuria. Lab Invest 51:7-21, 1984.
This work was supported in part by a grant from Stuart Pharmaceuticals. We thank Marie Birthwright for typing this manuscript.
17. Timpl R, Dziadek M: Structure, development and molecular pathology of basement membranes. Int Rev Exp Pathol 29:1-112, 1986.
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M: Reversal of proteinuria by sorbinil, an aldose reductase inhibitor in spontaneously diabetic (BB) rats. Pharmacology 36:112-120, 1988. 54. Triton RG, Chang K, Pugliese G, Eades DM, Province MA, Sherman WR, Kilo C, Wflliamson JR: Prevention of hemodynamic and vascular albumin filtration changes in diabetic rats by aldose reductase inhibitors. Diabetes 37:1258-1270, 1989.