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J Am Soc Nephrol 13:108-116, 2002
© 2002 American Society of Nephrology

{alpha}-Lipoic Acid Attenuates Hyperglycemia and Prevents Glomerular Mesangial Matrix Expansion in Diabetes

Mona F. Melhem*, Patricia A. Craven{dagger}, Julia Liachenko{dagger} and Frederick R. DeRubertis*

Departments of *Pathology and {dagger}Medicine, VA Pittsburgh Healthcare System and University of Pittsburgh, Pittsburgh, Pennsylvania.

Correspondence to Dr. Frederick R. DeRubertis, VA Pittsburgh Healthcare System, 111-U University Drive C, Pittsburgh, PA 15240. Phone: 412-688-6000, ext. 4690; Fax: 412-688-6947; E-mail: frederick.derubertis{at}med.va.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Previous studies demonstrated that 2 mo of dietary supplementation with {alpha}-lipoic acid (LA) prevented early glomerular injury in non-insulin-treated streptozotocin diabetic rats (D). The present study examined the effects of chronic LA supplementation (30 mg/kg body wt per d) on nephropathy in D after 7 mo of diabetes. Compared with control rats, D developed increased urinary excretion of albumin and transforming growth factor ß, renal insufficiency, glomerular mesangial matrix expansion, and glomerulosclerosis in association with depletion of glutathione and accumulation of malondialdehyde in renal cortex. LA prevented or ameliorated all of these changes in D. Because chronic LA supplementation also attenuated hyperglycemia in D after 3 mo, its effects on renal injury were compared with treatment of rats with sufficient insulin to maintain a level of glycemic control for the entire 7-mo period (D-INS) equivalent to that observed with LA during the final 4 mo. Despite superior longitudinal glycemic control in D-INS, urinary excretion of albumin and transforming growth factor ß, glomerular mesangial matrix expansion, the extent of glomerulosclerosis, and renal cortical malondialdehyde content were all significantly greater, whereas cortical glutathione content was lower than corresponding values in D given LA. Thus, the renoprotective effects of LA in D were not attributable to improved glycemic control alone but also likely reflected its antioxidant activity. The combined antioxidant and hypoglycemic actions of LA both may contribute to its utility in preventing renal injury and other complications of diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Increased oxidative stress has been implicated in the pathogenesis of diabetic complications, including nephropathy (1,2). Markers of increased oxidative stress (1–6) and reduced levels of antioxidants (7–12) are found in blood and tissue in both human and experimental diabetes. Multiple factors (1,2), including ketosis (13), may mediate these changes. In vitro studies indicate that hyperglycemia per se directly enhances oxidative stress in cultured endothelial and mesangial cells, which are targets for injury in diabetes (1,2,14–17). On the basis of these and other observations, the efficacy of dietary antioxidant supplementation in the prevention or attenuation of diabetic complications has been examined. Several different antioxidants, including vitamin E (VE), vitamin C (VC), taurine, and {alpha}-lipoic acid (LA), have been reported to ameliorate renal injury in experimental diabetes (18–22). In human diabetes, there is evidence that short-term (3 to 4 mo), high-dose (1600 to 1800 IU/d) VE supplementation reduces proteinuria in type 1 and 2 patients with overt nephropathy (19) and decreases hyperfiltration in type 1 patients without overt nephropathy (23). However, in the streptozotocin (STZ) diabetic rat, VE supplementation has had variable effects on renal injury. Protection has been observed with high doses of VE (18,21), whereas exacerbation of renal injury may occur with low-dose VE supplementation in the STZ-diabetic rat (20), possibly as a result of its capacity to act as a pro-oxidant under some conditions of increased oxidant stress (24).

Short-term (3 mo) treatment with oral LA (300 to 600 mg/d) has also been reported to attenuate proteinuria in patients with either type 1 or 2 diabetes and overt nephropathy (19). LA significantly reduced malondialdehyde in the serum of patients with diabetes, consistent with an antioxidant action (25,26). We recently compared the effects of VE, VC, and LA on early glomerular injury in the 2-mo STZ-diabetic rat model (22). Doses of VE (100 IU/kg body wt) and VC (1 g/kg body wt) were used, which were sufficient to raise renal cortical levels of these vitamins substantially. Nevertheless, in diabetic rats, VE and VC supplementation did not uniformly suppress increases in urinary albumin excretion (UAE), fraction clearance of albumin (FCalb), glomerular volume, glomerular or tubular content of transforming growth ß (TGFß), or glomerular content of collagen ({alpha}1) IV (22). By contrast, dietary supplementation with LA (30 mg/kg body wt per d) for 2 mo prevented increases in each of these indices of early renal injury in the diabetic rats (22). These findings suggested that LA may be a more effective renoprotective agent than either VE or VC in diabetes. However, there has been no assessment of the ability of LA to prevent the development of mesangial matrix expansion and glomerulosclerosis, which characterize more advanced diabetic nephropathy and lead to loss of renal function.

Accordingly, in the present study, we examined the effects of dietary supplementation with LA on renal function and structure in 7-mo STZ diabetic rats. Because dietary supplementation with LA for 7 mo also resulted in a delayed (after 3 mo) but significant reduction in the magnitude of the hyperglycemia in non-insulin-treated diabetic rats, the effects of LA on parameters of advanced renal injury were compared with a group of diabetic rats that were treated with sufficient insulin to maintain a level of hyperglycemia for the entire 7-mo period equivalent to that present in the LA-treated diabetic rats during the final 4 mo of the study. The results indicate that the action of LA to prevent mesangial matrix expansion and glomerulosclerosis in diabetes was not attributable to its delayed attenuation of hyperglycemia alone but likely also reflected its antioxidant activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Induction of Diabetes and Treatment of Rats
Age- and weight-matched female Sprague-Dawley rats (180 to 200 g; Zivic Miller Laboratories, Pittsburgh, PA) received an intraperitoneal injection of either 60 mg/kg STZ in sterile 0.01 M citric acid/0.9% saline solution or the STZ vehicle. Glucose was determined by glucometer (Diascam-S; Home Diagnostic Inc, Eatontown, NJ) on blood samples obtained from tail veins 48 h after injection of STZ. Rats with blood glucose higher than 300 mg/dl were entered into the study as diabetics. Four groups of rats were studied for 7 mo after entry: (1) control (nondiabetic), (2) untreated diabetic, (3) diabetic given diet supplemented with LA, and (4) insulin-treated diabetic. Diet was supplemented with sufficient LA (initially 400 mg/kg) to maintain an average daily LA consumption of 30 mg/kg body wt. During the study, insulin-treated diabetic rats received daily ultralente insulin, 2 to 5 U/d subcutaneously, beginning 96 h after administration of STZ, as previously reported (26). Blood glucose was determined on tail vein samples by glucometer at 2-wk intervals in all groups. In the insulin-treated diabetic rats, the insulin dose was adjusted as necessary at 2-wk intervals to maintain blood glucose levels between 250 and 400 mg/dl. This approximated the range of blood glucose values found after the third month of the study in the non-insulin-treated diabetic rats that received LA supplementation. All rats were fed a standard rat diet ad libitum with or without LA supplementation and had free access to water. BP was measured at monthly intervals with an electrosphygmograph and microphone cuff (International Biomedical, Austin, TX). The week before the rats were killed, they were placed in metabolic cages and their urine was collected for 24 h for determination of inulin and albumin clearances. Inulin clearance was determined in conscious unrestrained rats using a subcutaneous osmotic minipump to deliver [14C] inulin, as described previously (22,26). Urine and plasma samples were frozen for determination of albumin and [14C] inulin at the conclusion of the urine collection. All rats were killed 7 mo after entry into the study protocol. Before resection, kidneys were perfused free of blood in situ with saline at 4°C. After weights were obtained, one kidney was fixed in buffered formalin for subsequent histologic examination. The second kidney was quick-frozen in liquid N2 and stored at -80°C for biochemical and enzymatic determinations.

Determination of Creatinine, Glycosylated Hemoglobin, Insulin, Albumin, TGFß, and Albumin and Inulin Clearances
Plasma creatinine was determined with a kit obtained from Sigma Chemical Co. (St. Louis, MO) (27). Glycosylated hemoglobin was determined on red blood cell hemolysates using a kit obtained from Sigma Chemical Co. The method uses an affinity resin column that separates glycosylated from nonglycosylated hemoglobin (27). Glycosylated and nonglycosylated hemoglobin are quantified by absorbance at 415 nm, and glycosylated hemoglobin is expressed as a percentage of total hemoglobin. Insulin levels were measured using a rat insulin RIA kit (Linco Research, St. Louis, MO). Total (active plus latent) plasma and urinary TGFß levels were determined after acid activation using a sandwich enzyme-linked immunosorbent assay kit obtained from Genzyme (Boston, MA). TGFß was not detectable in plasma or urine samples before acid activation. Albumin was determined by an enzyme-linked immunosorbent assay as described previously (22,26). Albumin and inulin clearances were calculated, and FCalb was expressed as the ratio of albumin to inulin clearance.

Determination of Renal Cortical Content of Reduced Glutathione, Malondialdehyde, and LA
Glutathione (GSH) was assayed in extracts of quick-frozen renal cortex by its ability to form a highly colored yellow anion when reacted with 5,5'-dithiobis(2-nitrobenzioic acid) as described previously (22,28). Renal cortical malondialdehyde (MDA) was determined in quick-frozen samples of renal cortex after extraction in 1.15% KCl as described previously (20,27). Samples were reacted with thiobarbituric acid and heated at 95°C for 60 min. After extraction with n-butanol and pyridine (15:1 vol/vol), absorbance of the organic layer was measured at 532 nm; 1,2,3,3-tetramethoxypropane (MDA) was used as a standard. LA was determined by HPLC in extracts of quick-frozen renal cortex, as previously reported (25,29). For LA, the lower limit of detection by this method in renal cortex was 0.2 ng/mg protein. Recovery of LA added to renal cortical homogenates exceeded 95%.

Glomerular Morphometrics and Histology
After formalin fixation, renal cortex was embedded in paraffin sectioned at 3 µm and stained with periodic acid Schiff reagent (PAS) or trichrome. Sections were coded and read by an observer who was unaware of the study group from which the section was derived. Twenty-five glomeruli were selected from each rat for analysis. A cross-sectional area of the glomerular tuft was determined, as previously reported in detail (21,22,27). The glomerular tuft area and the area of the tuft with positive PAS staining each was measured with a digital planimeter using a SAMBA 4000 image analyzer (Imaging Products International, Chantilly, VA) (21,22,27). Mesangial matrix fraction was calculated as the area of positive PAS staining, expressed as a function of total glomerular tuft area (31). The extent of glomerulosclerosis was also assessed by analysis of renal cortical sections stained with trichrome. Each glomerulus examined was scored for positive trichrome staining as follows: 0, mesangium trichrome negative, with presence or absence of faint linear staining in Bowman’s capsule and/or capillary basement membrane; +1, 1% to 10% of mesangial area trichrome positive; +2, 11% to 20% positive; +3, >20% of mesangial area positive.

Statistical Analysis
Significance of differences was determined by analysis of variance followed by Fisher’s multiple comparison test using Statview software (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1 shows sequential blood glucose values in the four study groups. During the first 3 mo after STZ, blood glucose levels of diabetic rats that received dietary LA supplementation (D-LA) did not differ from diabetic rats that received neither LA nor insulin (D) but were significantly higher than those of insulin-treated D (D-INS). By contrast, from 4 to 7 mo, blood glucose values of D-LA were significantly lower than those of D but did not differ from values of D-INS. Blood glucose values for all diabetic groups were significantly higher than those of nondiabetic groups throughout the study (Figure 1, Table 1). Consistent with the sequential blood glucose levels, at month 3, percentage of glycosylated hemoglobin (% GHb) of D-LA did not differ from that of D but was significantly higher than the % GHb of D-INS. % GHb of D-LA at month 7 was lower than D, but the difference did not achieve statistical significance; % GHb of D-LA and D-INS were comparable at 7 mo. GHb levels of all diabetic groups were significantly higher than the corresponding control values at 3 and 7 mo (Table 1).



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  		Figure 1. Sequential changes in blood glucose concentration in control ({square}), untreated diabetic rats ({diamond}), diabetic rats treated with insulin (Diabetic + Ins) to maintain moderate hyperglycemia ({circ}), and diabetic rats that received dietary {alpha}-lipoic acid (Diabetic + LA) supplementation ({triangleup}). Blood glucose was determined every 2 wk. The monthly values shown reflect the average of the two determinations performed during that month for each rat; the monthly average was entered as a single value for purposes of statistical analysis. Results represent mean ± SE; n = 10 rats in each group. *P < 0.05 comparing Diabetic + LA with Diabetic + Ins; #P < 0.05 comparing Diabetic + LA with untreated Diabetic. Values in all diabetic groups were significantly higher than control at all time points. Values in untreated diabetic rats were significantly higher than Diabetic + INS at all time points.

 

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  		Table 1. Effects of treatment of diabetic rats with LA or INS on BG, GHb, body weight, kidney weight, plasma insulin and creatinine, and BPa
 
Plasma insulin levels of D and D + LA at 7 mo both were significantly lower than control values but did not differ between the two diabetic groups. As a result of the lower blood glucose level in D-LA compared with D, the plasma insulin/glucose ratio in D-LA was significantly higher than in D. This ratio in either diabetic group was substantially lower than control (Table 1). Mean body weights of D and D-LA but not of D-INS were significantly lower than control. Mean body weight of D-LA, however, was higher than that of D, likely reflecting the attenuation of hyperglycemia in D-LA from month 4 through month 7. Absolute mean kidney weight of D-INS was significantly greater than that of any of the other study groups. Absolute mean kidney weight of D-LA was higher than that of D but did not differ from that of control (Table 1). When expressed as a function of body weight, mean kidney weight of all diabetic groups was significantly higher than that of the nondiabetic rats. Although mean kidney weight of D-INS was somewhat lower than the corresponding values for either D or D-LA when expressed as a function of body weight, these differences did not achieve statistical significance. Mean BP did not differ among the four study groups (Table 1). Plasma creatinine concentration in D was significantly higher than that of Control. By contrast, mean plasma creatinine levels of D-LA and D-INS did not differ from control and were significantly lower than the value for D after 7 mo of diabetes. Consistent with plasma creatinine levels (Table 1), inulin clearance was significantly lower in D compared with control (Figure 2). By contrast, in both D-LA and D-INS, mean inulin clearances were significantly higher than control after 7 mo of diabetes. FCalb was significantly higher than control in all of the diabetic groups (Figure 2). However, FCalb was markedly lower in D-LA compared with either D or D-INS after 7 mo of diabetes.



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  		Figure 2. Effects of treatment of diabetic rats with LA (Diabetic + LA) or insulin (Diabetic + Ins) on inulin (CIN) and fractional albumin (FCAlb) clearances. Results represent mean ± SE; n = 10 rats in each group. *P < 0.05 versus control; #P < 0.05 versus untreated diabetic rats; {Delta}P < 0.05 versus Diabetic + Ins.

 
As shown in Figure 3, mesangial matrix fraction was significantly increased in both D and D-INS but not in D-LA, compared with control. Mesangial matrix fraction in D-LA was lower than in either D or D-INS after 7 mo of diabetes. Matrix fraction in D-INS was lower than in D, but this difference did not achieve statistical significance. Representative PAS stains of renal cortical sections from rats in each study group are shown in Figure 4. Glomerulosclerosis, as reflected by the extent of positive trichrome staining in the mesangium (Table 2), was most advanced in D and of intermediate extent in D-INS, compared with control glomeruli (Table 2). The extent of glomerulosclerosis in diabetic rats that were treated with LA did not differ significantly from nondiabetic rats and was clearly less than that seen in diabetic rats that were treated with insulin.



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  		Figure 3. Effects of treatment of diabetic rats with LA (Diabetic + LA) or insulin (Diabetic + Ins) on mesangial matrix fraction. Mesangial matrix area was expressed as a fraction of the total glomerular tuft area in periodic acid Schiff (PAS)-stained renal cortical sections. Results represent mean ± SE; n = 10 rats in each group. Twenty-five glomeruli from each rat were examined to determine the matrix fraction, with the average of these 25 determinations entered as a single value for purposes of statistical analysis. *P < 0.05 versus control; #P < 0.05 versus diabetic rats; {Delta}P < 0.05 versus Diabetic + Ins.

 


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  		Figure 4. Representative photomicrographs of PAS-stained renal cortical sections from control (A and E), insulin-treated diabetic rats(B and F), LA-treated diabetic rats (C and G), and untreated diabetic rats (D and H) assessed at 7 mo. The individual glomeruli shown in A through D are magnifications (x60) of one of the glomeruli contained in the corresponding low-power (x30) illustrations of renal cortical sections from the same rat (E through H). Accumulation of PAS-positive matrix in the mesangium was assessed quantitatively as shown in Figure 3.

 

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  		Table 2. Effects of treatment of diabetic rats with LA or INS on glomerulosclerosisa
 
Tubulointerstitial histopathologic changes in renal cortex and medulla were not prominent in any of the diabetic rat groups. They consisted of focal areas of tubular cell dropout, atrophy, and vacuolization, as well as focal areas of interstitial inflammatory cell infiltration and fibrosis, with the latter assessed by trichrome staining. These focal changes were more widespread in D than in either D-INS or D-LA. Thus, both therapeutic interventions attenuated the modest degree of tubulointerstitial injury found. The current model did not permit an assessment of the relative efficacy of LA versus insulin in the suppression of structural changes induced by diabetes in these regions of the kidney as a result of the limited pathology found.

As shown in Table 3, urinary excretion of TGFß was increased in all of the diabetic groups compared with control. However, urinary TGFß in D-LA was significantly lower than values in either D or D-INS. Plasma TGFß concentrations were comparable in the study groups, consistent with a nephrogenous source of the higher urinary TGFß values in the diabetic groups.


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  		Table 3. Plasma TGFß concentration and urinary TGFß excretion in control and diabetic ratsa
 
As shown in Table 4 and consistent with earlier observations, LA was detectable only in renal cortical extracts from diabetic rats fed a diet supplemented with this agent. Renal cortical GSH content of both D and D-INS was significantly lower than the value in control. By contrast in D-LA, the GSH level in renal cortex was significantly higher than values in the other two diabetic groups and did not differ from the value of control (Table 2). MDA content of renal cortex of both D and D-INS but not that of D-LA was significantly higher than control. MDA content of the renal cortex of D-LA was significantly lower than the corresponding values in either D or D-INS.


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  		Table 4. Renal cortical content of LA, GSH, and MDA in control and diabetic ratsa
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Prolonged supplementation of the diet of STZ-diabetic rats with LA was associated with attenuation of both hyperglycemia and diabetic renal injury. Amelioration of hyperglycemia has been previously documented in both human and experimental diabetes treated with LA (32–35). Earlier studies demonstrating a hypoglycemic effect of LA in the STZ-diabetic rat have been acute (hours to days) and used high parenteral doses of this agent (34,35). No increases in plasma insulin levels were observed in the STZ-diabetic rats that were given LA compared with untreated diabetic rats (33–35), suggesting that LA does not act as an insulin secretagogue, reduce insulin clearance, or protect pancreatic ß-cells from injury in this model. Rather, these and other available data from experimental and human diabetes support an action of LA to increase insulin sensitivity as the primary mechanism for its hypoglycemic effect (32–40), including observations in the STZ-diabetic rat (34,35,40). LA has been shown to enhance glucose uptake in skeletal muscle and adipocytes, possibly by increasing GLUT4 protein content and/or its redistribution to the plasma membrane (37–40). Data in the current study are also consistent with an action of LA to increase insulin responsiveness in the STZ-diabetic rat. Although the STZ-diabetic rat is a model of insulinopenic diabetes, plasma insulin levels remained detectable (Table 1) and thus some level of ß-cell function persisted. There is evidence that insulin resistance also contributes to hyperglycemia in the STZ-diabetic rat (41). The findings that the absolute levels of plasma insulin were not different but the ratio of insulin to glucose were twofold higher in diabetic rats that were fed LA compared with untreated diabetic rats (Table 1) are consistent with but do not prove enhanced insulin responsiveness in the LA group. The basis for the delayed effect of dietary supplementation of LA on glucose levels observed in the present study is unclear. The same dose of LA that was used in the current study did not have a detectable hypoglycemic effect when fed to STZ-diabetic rats for only 2 mo in an earlier study (22), further confirmation of the delay in expression of the hypoglycemic effect of this dose of LA, at least when given orally. Of note, renal cortical levels of LA (8.9 ± 1.0 ng/mg protein) observed after supplementation of diabetic rats for 7 mo in the current study were approximately 60% higher (5.6 ± 0.6) than those observed earlier after only 2 mo of supplementation with the same dose of LA (22). Thus, it is possible that delayed expression of the hypoglycemic action of LA is linked to progressive cellular accumulation of LA, including possibly in insulin-responsive tissues such as muscle and fat (38,40). Whether the hypoglycemic effect of LA in diabetes involves its antioxidant activity or other mechanisms is uncertain (37,40). However, recent studies indicated that LA protects against oxidative stress-induced insulin resistance in muscle cells (42).

LA supplementation in the current study clearly prevented or delayed the development of advanced diabetic renal injury. Thus, compared with indices in untreated diabetic rats, supplementation of the diet of diabetic rats with LA attenuated albuminuria, loss of renal function, mesangial matrix expansion, and the development of glomerulosclerosis. Protection against renal injury in the LA-treated diabetic rats occurred despite persistent glomerular hyperfiltration. This dissociation has previously been noted with LA (22) and with numerous interventions that have ameliorated renal injury in diabetes, including dietary supplementation with other antioxidants (20,21) and angiotensin-converting enzyme inhibition (43). As previously proposed (43), increased intraglomerular pressure rather than hyperfiltration may be the key hemodynamic determinant of diabetic renal injury. The former may not have been increased in LA-treated rats despite persistent hyperfiltration, as is the case with angiotensin-converting enzyme inhibitors (43). Moreover, when expressed as a function of body weight, LA did not prevent the relative increase in renal mass induced by diabetes (Table 1). This suggests that the pathogenetic mechanisms that are responsible for renal hypertrophy at least in this model of diabetes may not be identical to those that mediate diabetic glomerulosclerosis.

The lower blood glucose levels induced by treatment of diabetic rats with LA represents an obvious confounding factor in the assessment of the mechanism(s) by which LA may have prevented renal functional and structural changes in the diabetic rats. However, comparison of the effects of LA on indices of renal injury to those of treatment of diabetic rats with a sufficient dose of insulin to maintain superior longitudinal glycemic control over a 7-mo period (Table 1, Figure 1) indicates that the renoprotective effects of LA are not adequately explained by its hypoglycemic action. Thus, with respect to corresponding parameters in D-INS, albuminuria, urinary TGFß excretion, mesangial matrix expansion, and the extent of glomerulosclerosis all were significantly attenuated in D-LA. The renoprotection conferred by LA supplementation occurred despite that glycemic control during the first 3 mo of diabetes was substantially worse in D-LA than in D-INS. In fact, glycemic control during this period did not differ from that of the untreated diabetic rats (Figure 1, Table 1).

Attenuation of renal injury by LA may be linked to its antioxidant activity. The antioxidant properties of both LA and its reduced form dihydrolipoic acid (DHLA), which is rapidly generated from LA in many tissues, are well established (25,44,45). The LA/DHLA redox couple can scavenge a number of free oxygen radicals, including hydroxyl radicals, single oxygen, and probably superoxide and peroxyl radicals (25,44,45). In addition, LA and/or DHLA chelate a number of transitional metals (25,44,45), recycle VC and VE, and increase cellular levels of GSH (25,44,45). LA has also conferred protection against ischemia-reperfusion injury in a number of in vitro and in vivo experimental models (25,45). As noted above, there is considerable evidence that increased oxidative stress may participate in the pathogenesis of diabetic complications, including nephropathy (1–12,18–22). In this regard, administration of LA has been reported to attenuate neuropathy in experimental and human diabetes in association with reduced markers of oxidative stress (29,46,47). Recent observations have also indicated that LA reduces markers of oxidative stress in retina (48) and renal cortex (22) from STZ-diabetic rats. In the present study, GSH levels were reduced and MDA levels were increased in renal cortex from both D and D-INS but not in D-LA, compared with controls (Table 3). Because measurement of MDA and GSH was made in renal cortical homogenates, the changes observed may reflect those that occur in renal tubules, glomeruli, or both. Although these parameters are not highly specific, the increases in MDA and reductions in GSH found in D and D-INS are consistent with enhanced renal oxidative stress in these diabetic groups that was prevented by treatment with LA.

Administration of several structurally different antioxidants has been found to alter early functional and biochemical abnormalities induced in kidney by diabetes (18–23). For the most part, however, these studies have been short-term and have measured surrogates of advanced diabetic nephropathy, such as albuminuria, glomerular hyperfiltration, and gene expression or content of TGFß or selected matrix proteins in glomeruli (18,19,21,23). This was the case in our earlier study with LA in 2 mo STZ-diabetic rats (22) in which, as noted above, we found that LA suppressed albuminuria, glomerular hypertrophy, and, as assessed by immunohistochemistry, glomerular and tubular accumulation of TGFß and glomerular content of collagen {alpha}1(IV). Indeed, the current study with LA represents only the second demonstration to our knowledge that dietary antioxidant supplementation can attenuate or delay mesangial matrix expansion, glomerulosclerosis, and loss of renal function in experimental diabetes. Amelioration of these indices of advanced diabetic nephropathy has previously been reported by Trachtman et al. (20) in STZ-diabetic rats that were fed a diet supplemented with taurine.

The precise mechanisms by which LA and other antioxidants alter diabetic renal injury are not known. In the present study, administration of LA markedly suppressed the increase in urinary TGFß in diabetic rats. Higher urinary TGFß in the diabetic groups did not correlate with higher plasma TGFß and thus presumably reflected increased nephrogenous TGFß production. There is evidence that this prosclerotic cytokine is a key mediator of glomerular mesangial expansion in diabetes (49,50). Previous studies with LA (25) and other exogenous antioxidants (30) in diabetic rats and with enhanced endogenous antioxidant activity induced by overexpression of Cu2+/Zn2+ superoxide dismutase (SOD) in transgenic mice with STZ diabetes (27) have correlated suppression of glomerular and tubular TGFß content with the renal protection observed. Studies in mesangial cells have demonstrated that several antioxidants prevent activation of protein kinase C and the increases in TGFß and matrix protein synthesis otherwise induced by culture in high-glucose media (15). In this regard, it is of interest that recent studies in cultured endothelial and mesangial cells have specifically linked enhanced cellular superoxide production to the increases in protein kinase C activity, TGFß, and collagen synthesis that occur in response to high glucose (16,17,27,51). In endothelial cells, overexpression of mitochondrial Mn2+ SOD prevented several metabolic responses implicated in hyperglycemic mediated cell injury, including activation of protein kinase C and increased expression of TGFß1 (16,17). Similarly, in glomerular mesangial cells, overexpression of either Mn2+ SOD (51) or cytoplasmic Cu2+/Zn2+ SOD (27) prevented the increases in cellular superoxide and collagen synthesis otherwise observed under hyperglycemic culture conditions. Moreover, as noted above, STZ-diabetic, transgenic mice that overexpress Cu2+/Zn2+ SOD are protected from early diabetic glomerular injury in association with suppression of glomerular accumulation and urinary excretion of TGFß (27). These observations implicate enhanced cellular superoxide production in diabetes as a potential mediator of renal and endothelial cell injury. Accordingly, the oxygen radical scavenging properties of LA/DHLA (23,44,45) may participate in its renoprotective actions.

Whatever the precise mechanism of its actions, the combined antioxidant and hypoglycemic effects of LA should be particularly advantageous and perhaps even synergistic in preventing renal injury and other diabetic complications. There are at least limited data to suggest that these actions of LA are expressed in humans with diabetes (20,23,29,32,36,47).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication April 10, 2001. Accepted for publication August 1, 2001.




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