Effects of Dietary Supplementation of Α-Lipoic Acid on Early Glomerular

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J Am Soc Nephrol 12: 124–133, 2001 Effects of Dietary Supplementation of ␣-Lipoic Acid on Early Glomerular Injury in Diabetes Mellitus

MONA F. MELHEM,* PATRICIA A. CRAVEN,† and FREDERICK R. DERUBERTIS† Departments of *Medicine and †Pathology, Veterans Affairs Medical Center and University of Pittsburgh, Pittsburgh, Pennsylvania.

Abstract. Antioxidants, in particular vitamin E (VE), have been mented D compared with age-matched nondiabetic controls. reported to protect against diabetic renal injury. ␣-Lipoic acid With the exception of inulin clearance, LA prevented or sig- (LA) has been found to attenuate diabetic peripheral neuropa- nificantly attenuated the increase in all of these glomerular thy, but its effects on nephropathy have not been examined. In parameters in D, as well as the increases in renal tubular cell the present study, parameters of glomerular injury were exam- TGF-␤ seen in D. At the dose used, VE reduced inulin clear- ined in streptozotocin diabetic rats after 2 mo on unsupple- ance in D to control levels but failed to alter any of the other mented diets and in diabetic rats that received the lowest daily indices of glomerular injury or to suppress renal tubular cell dose of dietary LA (30 mg/kg body wt), VE (100 IU/kg body TGF-␤ in D. VC suppressed urinary albumin excretion, frac- wt), or vitamin C (VC; 1 g/kg body wt), which detectably tional albumin clearance, and glomerular volume but not glo- increased the renal cortical content of each antioxidant. Blood merular or tubular TGF-␤ or glomerular collagen ␣1 (IV) glucose values did not differ among the diabetic groups. At 2 content. LA but not VE or VC significantly increased renal mo, inulin clearance, urinary albumin excretion, fractional cortical glutathione content in D. These data indicate that LA albumin clearance, glomerular volume, and glomerular content is effective in the prevention of early diabetic glomerular injury of immunoreactive transforming growth factor-␤ (TGF-␤) and and suggest that this agent may have advantages over high collagen ␣1 (IV) all were significantly increased in unsupple- doses of either VE or VC.

Oxidative and glycooxidative stress may participate in the VE reduced urinary protein excretion by 46% (22). In patients pathogenesis of diabetic complications, including nephropathy with type 1 diabetes mellitus without overt nephropathy, short- (1,2). In both diabetic patients and experimental models of term (4 mo), high-dose VE supplementation (1800 IU/d) de- diabetes mellitus, markers of increased oxidative stress have creased glomerular hyperfiltration (23). Koya et al. (19) ad- been identified in blood and tissues (1–6). In human and ministered d-␣-tocopherol intraperitoneally at a dose sufficient experimental models of type 1 diabetes mellitus, including the to increase renal cortical VE content and observed attenuation streptozocin (STZ) diabetic rat, ketosis may contribute to ox- of glomerular hyperfiltration and albuminuria in the STZ- idative stress (7). Reduced nonenzymatic antioxidant defenses diabetic rat. However, in this diabetic model, effects of VE on have also been reported in human and experimental diabetes nephropathy have been variable. Thus, dietary supplementation mellitus with lower plasma and/or cellular levels of vitamin C with 2000 IU VE/kg diet, a dose that also raised renal cortical (VC), vitamin E (VE), glutathione, and total plasma antioxi- VE levels, failed to prevent increases in albumin clearance in dant activity described (8–13). For these reasons, there has the STZ-diabetic rat (21), whereas low-dose dietary VE sup- been interest in the use of dietary antioxidant supplementation plementation (100 IU/kg diet) exacerbated renal injury in this as an intervention to attenuate diabetic complications (14–18). experimental model (20). The reasons for the different renal With respect to diabetic nephropathy, studies in human and responses to VE observed in STZ diabetes mellitus are uncer- experimental diabetes mellitus have reported beneficial effects tain. The exacerbation of renal injury observed with low-dose of administration of several different antioxidants (9,19–23). dietary supplementation of VE could be related to the recog- The renal responses to VE in diabetes mellitus have been the nized capacity of this agent to act as a prooxidant under some most extensively evaluated (19–23). In patients with types 1 conditions of increased oxidative stress (24). Beneficial effects and 2 diabetes mellitus with overt nephropathy, short-term (3 on diabetic nephropathy of VE and other antioxidants have mo) dietary supplementation with a high dose (1575 IU/d) of been observed with relatively high doses (9,19,21–23). In experimental diabetes mellitus in the case of VE and VC, these Received March 24, 2000. Accepted June 21, 2000. doses have been sufficient to raise renal cortical levels of the Correspondence to Dr. Frederick R. DeRubertis, Department of Veterans antioxidants (19,21), although it has not been established that Affairs Medical Center, University Drive C, Pittsburgh, PA 15240. Phone: this is essential to their renoprotective effects. Whether other 412-688-6000 x4690; Fax: 412-688-6947; E-mail: frederick.derubertis@ med.va.gov antioxidants can provide more consistent renal protection in diabetes mellitus and/or do so when used at relatively low 1046-6673/1201-0124 Journal of the American Society of Nephrology doses remains to be established. Copyright © 2001 by the American Society of Nephrology ␣-Lipoic acid (LA) is an endogenously produced coenzyme J Am Soc Nephrol 12: 124–133, 2001 ␣-Lipoic Acid in Diabetic Nephropathy 125 that plays an essential role in mitochondrial dehydrogenase effects of VC. Rats were started on the supplemented diets or drinking reactions (25). Its properties as an antioxidant have recently water 48 h after injection of STZ. Nondiabetic controls and unsupple- been reviewed (25). LA or its reduced form, dihydrolipoic acid mented diabetic rats were fed standard rat chow. Diabetic rats that (DHLA), quenches a number of oxygen-free radical species in received VC supplementation consumed an average of 1 g VC/kg both lipid and aqueous phase, chelates transition metals, and body wt per d. Diabetic rats that received VE supplementation consumed an average of approximately 100 IU (77 mg) VE/kg body wt prevents membrane lipid peroxidation and protein damage via per d. Diabetic rats that received LA supplementation consumed an interactions with VC and glutathione (25). LA participates in average of 30 mg/kg body wt per d. Diabetic rats that received the recycling of VC and VE, increases cellular levels of glu- standard rat chow consumed an average of 5 IU VE/kg body wt per d. tathione, and suppresses nonenzymatic glycation (25). Treat- Standard chow does not contain added VC or LA because rats syn- ment with LA reduces markers of oxidative stress in plasma of thesize both of these moieties. patients with diabetes mellitus and poor glycemic control (26). Blood glucose from tail vein samples and BP were determined at There is evidence in both human and experimental diabetes 2-wk intervals. BP was measured with an electrosphygmograph and mellitus that administration of LA ameliorates diabetic neu- microphone cuff (International Biomedical, Austin, TX). The week ropathy (27–30). In the STZ-diabetic rat, dose-response effects before the rats were killed, they were placed in metabolic cages and their urine was collected for 24 h for determination of inulin and of LA have been reported on parameters of neural injury 14 (27,28,30), with benefits described in response to daily doses albumin clearances. [ C] inulin clearance was determined in con- scious, unrestrained rats with the use of a subcutaneous osmotic of LA from 25 to 100 mg/kg body wt. Very limited data are minipump to deliver [14C] inulin as described previously (31). An available on the effects of LA on diabetic nephropathy. A aliquot of the urine was frozen for determination of albumin and [14C] nonblinded study of patients with types 1 and 2 diabetes inulin. Blood was also obtained from the tail vein for determination of mellitus with clinical nephropathy demonstrated a 50% reduc- [14C] inulin and albumin at the conclusion of the urine collection. All tion in proteinuria after 3 mo of oral LA administration (300 to rats were sacrificed 2 mo after entry into the study protocol. The 600 mg/d) that was associated with a reduction in serum kidneys were perfused free of blood in situ with ice-cold saline before malondialdehyde (22). However, there has been no systematic resection. After weights were obtained, one kidney was fixed in examination of LA actions on renal function or structure in buffered formalin for subsequent immunohistochemistry. The second Ϫ either human or experimental diabetes mellitus. Accordingly, kidney was quick-frozen in liquid N2 and stored at 80°C for immu- in the current study, we compared the effects of dietary sup- nohistochemistry and determination of VC, VE, LA, and glutathione plementation of LA with those of VE and VC on parameters of content. early glomerular injury in the STZ-diabetic rat. Each antioxidant was used at the lowest daily oral dose that detectably Determination of Albumin and Fractional Albumin increased renal cortical levels of that agent in the diabetic Clearance animals. Albumin was determined by an enzyme-linked immunosorbent assay as described previously (31). Rabbit anti-rat albumin (IgG Materials and Methods fraction) and peroxidase-conjugated rabbit anti-rat albumin were ob- Treatment of Rats tained from ICN Pharmaceuticals (Aurora, OH) and diluted 10,000 and 600 times, respectively. Heat-inactivated normal rabbit serum was Age- and weight-matched female Sprague-Dawley rats (180 to used as a blocker. Standard curves were linear between 0.5 and 40 ng 200 g; Zivic Miller Laboratories, Pittsburgh, PA) received an intra- of albumin per well. Addition of known standard amounts of albumin peritoneal injection of either 60 mg/kg STZ in sterile 0.010 M citric to urine from each of the rat groups resulted in complete recovery of acid/0.9% saline solution or the STZ vehicle; all rats were permitted added albumin. Albumin and inulin clearances were calculated, and ad libitum intake of food and water throughout the study. Glucose was fractional clearance of albumin was expressed as the ratio of albumin determined with a glucometer (Diascam-S; Home Diagnostics, Inc., to inulin clearance. Eatontown, NJ) on blood samples obtained from tail veins 48 h after injection of STZ. Rats with blood glucose higher than 300 mg/dl at 48 h were entered into the study as diabetics. Preliminary studies were Immunohistochemical Staining for TGF-␤ and conducted in diabetic rats to determine the lowest daily oral dose of Collagen ␣1 (IV) each antioxidant that resulted in a detectable increase in the renal Renal cortical sections (5 ␮m) for transforming growth factor-␤ cortical content of that agent after 2 wk of supplementation. In the (TGF-␤) staining were fixed in buffered formalin and blocked for 60 case of LA, the daily oral intakes tested were approximately 15, 30, min. They were then incubated overnight at 4°C with 50 ␮g/ml and 45 mg/kg body wt. Rats were then placed in one of four study affinity-purified polyclonal rabbit panspecific anti–TGF-␤ antibody groups as follows: group I, nondiabetic controls; group II, untreated (R and D Systems, Minneapolis, MN). This antibody reacts with diabetic rats; group III, diabetic rats whose drinking water was sup- TGF-␤1, TGF-␤2, TGF-␤1.2, TGF-␤3, and TGF-␤5. Of these, TGF- plemented with 1 g/L VC (L-ascorbic acid) plus 100 ␮M desferriox- ␤1, 2, and 3 have been reported to be produced by rat mesangial cells amine; group IV, diabetic rats whose diet was supplemented with 800 (32). Frozen renal cortical sections (5 ␮m) were used for assessment mg VE (d-␣-tocopherol acetate, 1.36 IU/mg)/kg diet; and group V, of collagen ␣1 (IV). Rabbit anti-mouse collagen ␣1 (IV) antibodies diabetic rats whose diet was supplemented with 400 mg/kg LA. Each were obtained from Chemicon International, Inc. (Temecula, CA). group contained eight rats. Desferrioxamine was added to drinking Nonspecific staining was assessed by replacing the primary antibody water that had been supplemented with VC to prevent oxidation of with affinity-purified, nonimmune, rabbit IgG (R and D Systems). ascorbate. Desferrioxamine is poorly absorbed when administered Sections were washed and further developed according to the direc- orally. Accordingly, it is doubtful that this agent altered cellular or tions of the manufacturer (Dako Corporation, Carpinteria, CA) using plasma iron compartmentalization or contributed to the systemic an LSAB2 kit that contained second antibody linked to avidin and 126 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 124–133, 2001 peroxidase conjugated to biotin. Immunohistochemical staining for Results TGF-␤ and collagen ␣1 (IV) were assessed quantitatively with a As shown in Table 1, mean blood glucose was significantly SAMBA 4000 image analyzer (Image Products International, Chan- elevated in all diabetic groups compared with controls, but tilly, VA) using specialized computer software (Immuno-Analysis, there was no significant effect of supplementation with VC, version 1.4, Microsoft, Richmond, WA), a color video camera, and a VE, or LA on blood glucose levels in the diabetic rats. Body Compaq computer. Four to five glomeruli per rat were assessed for area and intensity of staining as described previously (21). Results are weight was lower in all diabetic groups compared with nondi- presented as the labeling index, which represents the percentage of the abetic rats but did not differ significantly among the diabetic total examined glomerular area that stained positively. Staining inten- groups. Conversely, kidney weight was higher in the diabetic sity of positive areas was also assessed (mean optical density). A rats compared with control but did not differ significantly mean quick score was then calculated (mean optical density ϫ label- among the diabetic study groups. Inulin clearance was mod- ing index) for the glomeruli from each rat. estly but significantly elevated in unsupplemented diabetic rats and in diabetic rats supplemented with VC or LA compared Glomerular Volume with values in control rats. Inulin clearance was lower in diabetic rats supplemented with VE compared with unsupple- Glomerular volume (VG) was determined on 32 glomeruli per mented diabetic rats and was not different from values in group, as previously reported (21,31). Briefly, VG was calculated from glomerular cross-sectional area as determined on formalin-fixed nondiabetic rats. BP did not differ among any of the study tissue by light microscopy using a SAMBA 4000 image analyzer. The groups. ϭ 3/2 formula VG B/k(AG) , in which AG is the cross-sectional area of As shown in the upper panel of Figure 1, compared with the glomerulus, was used for the calculation. B ϭ 1.38 is the shape values in nondiabetic rats, 24-h urinary albumin excretion coefficient for spheres, and k ϭ 1.1 is a size distribution coefficient. (UAE) was significantly higher in diabetic rats that were re- Tissue sections for VG and immunohistochemistry were random- ceiving no supplementation and in diabetic rats that were ized and examined in a coded manner. Thus, the treatment groups receiving VE or VC. By contrast, supplementation with LA from which the renal sections came were not known to the pathologist. reduced UAE in diabetic rats to values that were not different from those in nondiabetic rats. As illustrated in the lower panel Assay of Renal Cortical Reduced Glutathione Content of Figure 1, fractional clearance of albumin was also signifi- Glutathione was assayed in extracts of quick-frozen renal cortex by cantly elevated in untreated diabetic rats and those that were its ability to form a highly colored yellow anion when reacted with receiving VE or VC compared with control values; supplemen- 5,5Ј-dithiobis(2-nitrobenzoic acid) (DTNB) as described previously tation of LA reduced fractional clearance of albumin to values (33). that were not different from those in nondiabetic rats. Although VC failed to suppress UAE or fractional albumin clearance of Determination of Renal Cortical Antioxidant Content diabetic rats to control levels, these indices were significantly Renal cortical ascorbic acid (34), ␣-tocopherol (34), and LA (35) lower in diabetic rats that were receiving VC than correspond- were determined by HPLC in extracts of renal cortex that had been ing values in unsupplemented diabetic rats. quick-frozen in liquid nitrogen. For LA, the lower limit of detection As illustrated in Figure 2, VG was significantly greater in by this method in renal cortex was 0.2 ng/mg protein (1 pmol/mg unsupplemented diabetic rats compared with values in control protein). Recovery of LA added to renal cortical extracts exceeded rats. Supplementation of diabetic rats with VC or LA reduced 95%. VG to values that were not different from those in control rats, whereas supplementation of diabetic rats with VE had no effect

Statistical Analyses on VG. Significance of differences was determined by ANOVA followed Figures 3 and 4 illustrate the influence of antioxidant sup- by the Fisher multiple comparison test using Statview software. plementation on glomerular immunoreactive TGF-␤ content.

Table 1. Effects of antioxidant supplementation on blood glucose, body weight, kidney weight, inulin clearance, and BP in diabetic ratsa

Blood Glucose Body Weight Kidney Weight Inulin Clearance BP (mg/dl) (g) (g) (ml/min) (mmHg)

Control 168 Ϯ 35 283 Ϯ 4 1.2 Ϯ 0.1 1.4 Ϯ 01 107 Ϯ 6 Diabetic 469 Ϯ 54b 247 Ϯ 9b 1.7 Ϯ 0.07b 1.8 Ϯ 0.2b 109 Ϯ 7 Diabetic ϩ VE 487 Ϯ 61b 243 Ϯ 14b 1.9 Ϯ 0.1b 1.2 Ϯ 0.2c 114 Ϯ 6 Diabetic ϩ VC 472 Ϯ 76b 206 Ϯ 8b,c 1.7 Ϯ 0.1b 1.7 Ϯ 0.2b 103 Ϯ 4 Diabetic ϩ LA 435 Ϯ 55b 234 Ϯ 18b 1.6 Ϯ 0.09b 1.9 Ϯ 0.3b 111 Ϯ 5

a Results shown are means Ϯ SEM of measurements on eight rats per group. VE, vitamin E; VC, vitamin C; LA, ␣-lipoic acid. b P Ͻ 0.05 versus control. c P Ͻ 0.05 versus unsupplemented diabetic rats. J Am Soc Nephrol 12: 124–133, 2001 ␣-Lipoic Acid in Diabetic Nephropathy 127

noreactive TGF-␤ to values that were not different from those in control rats. Calculated quick scores (labeling index ϫ intensity of positive staining) gave results in the study groups analogous to those shown in Figure 4 for labeling index, as did assessment by visual inspection of representative renal cortical sections (Figure 3). Figures 5 and 6 illustrate the influence of antioxidant supplementation on glomerular immunoreactive collagen ␣1 (IV) content. Representative examples of glomerular staining for collagen ␣1 (IV) in renal cortical sections are shown in Figure 5, and assessment of staining by quantitative image analysis is shown in Figure 6. Collagen ␣1 (IV) staining was markedly increased in basement membrane and mesangium of glomeruli from untreated diabetic rats compared with values in control rats as assessed by visual inspection of representative renal cortical sections (Figure 5) or quantitatively from the labeling index (Figure 6). Supplementation of diabetic rats with VC or Figure 1. Effects of antioxidant supplementation on 24-h urine albu- VE had no effect on glomerular collagen ␣1 (IV) immunore- min excretion (UAE) and fractional clearance of albumin. A 24-h activity, as shown in Figures 5 and 6. However, supplementa- urine sample was obtained 3 d before killing, and albumin was tion with LA markedly reduced glomerular immunoreactive determined by enzyme-linked immunosorbent assay. UAE is shown in collagen ␣1 (IV) of diabetic rats to levels that were not differ- the upper panel, and fractional albumin clearance is shown in the ent from those in control rats (Figures 5 and 6). Calculated lower panel. Fractional clearance of albumin is defined as the ratio of ␣ albumin to inulin clearance. Results are means Ϯ SEM of values from quick scores of glomerular collagen 1 (IV) staining gave eight rats in each group. *, P Ͻ 0.05 versus Control; $, P Ͻ 0.05 results analogous to those shown in Figure 6 for labeling index. versus None (untreated diabetic rats). Figure 7 shows representative tubulointerstitial histology and tubuloimmunoreactive TGF-␤ content in renal cortical sections of nondiabetic, untreated diabetic and LA-treated diabetic rats. The major histologic change observed in this region of the kidney in untreated diabetic rats compared with nondiabetic rats was tubular dilation. This was attenuated by LA (Figure 7) but not VC and VE (not shown). Focal areas of tubular cell atrophy and dropout were also observed in cortex from all of the diabetic groups. Interstitial inflammatory infil- trates and interstitial fibrosis (assessed by trichrome staining) were not observed in any of the study groups. Because of tubular dilation in the diabetic rats, quantitative image analysis of fixed areas of the tubulointerstitial region of the cortex, which included enlarged tubular lumens, did not accurately reflect obvious increases in immunochemical staining for TGF-␤ observed in cortical tubule cells in the untreated diabetic rats compared with controls (Figure 7). Accordingly, Figure 2. Effects of antioxidant supplementation on glomerular volume. Glomerular volume was determined by light microscopy on labeling indices and mean optical densities of tubulointerstitial formalin-fixed cortical sections from eight rats per group using a areas were not calculated. The tubular cell staining intensity for SAMBA 4000 image analyzer. Results are means Ϯ SEM of deter- immunoreactive TGF-␤ was graded semiquantitatively by vi- minations on 32 glomeruli per group. *, P Ͻ 0.05 versus Control. sual inspection of cortical sections from five rats from each study group using a scale of ϩ1toϩ4, with ϩ1 arbitrarily representing TGF-␤ staining intensity in tubule cells of non- Representative examples of glomerular staining for TGF-␤ in diabetic rats. There was a clear increase (ϩ3) over control in renal cortical sections from each study group are shown in the tubular cell content of immunoreactive TGF-␤ in untreated Figure 3, and assessment of glomerular staining by quantitative diabetes mellitus. Staining intensity for TGF-␤ was reduced to image analysis is shown in Figure 4. As assessed by quantita- control levels (ϩ1) in LA-treated diabetic rats (Figure 7) but tive analysis, the area positive for TGF-␤ staining (labeling not in those treated with VC or VE (not shown). index, Figure 4) was markedly expanded in glomeruli from Focal increases in tubular basement membrane staining in- untreated diabetic rats compared with nondiabetic rats. Sup- tensity for immunoreactive collagen IV were also observed in plementation of diabetic rats with VC or VE had no effect on the renal cortex of the diabetic groups, as assessed by visual this parameter. However, supplementation of diabetic rats with inspection. However, these apparent increases occurred pre- LA markedly reduced the labeling index of glomerular immu- dominantly in areas of tubular cell atrophy or dropout and thus 128 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 124–133, 2001

Figure 3. Effects of Antioxidant supplementation on immunoreactive transforming growth factor-␤ (TGF-␤). A representative example of glomerular immunohistochemical TGF-␤ staining in renal cortical sections from each study group is shown. (A) nondiabetic rat; (B) diabetic; (C) diabetic ϩ vitamin C (VC); (D) diabetic ϩ vitamin E (VE); (E) diabetic ϩ ␣-lipoic acid (LA); (F) diabetic with nonimmune rabbit IgG substituted for TGF-␤ antibody. J Am Soc Nephrol 12: 124–133, 2001 ␣-Lipoic Acid in Diabetic Nephropathy 129

Discussion Previous studies of the STZ-diabetic rat have shown that LA improves nerve blood flow and conduction velocity, reduces lipid peroxidation, increases nerve glutathione, and reduces protein glycation (27,28,30). LA administration also reduces symptoms of peripheral neuropathy and manifestations of au- tonomic neuropathy in patients with diabetes mellitus (29). However, only limited data are available on effects of LA on diabetic nephropathy (22). The results of the present study demonstrate that dietary supplementation with LA prevents early glomerular injury in the STZ-diabetic rat. Thus, the

increases in UAE, fractional albumin clearance, VG, glomerular immunoreactive TGF-␤, and collagen ␣1 (IV) observed in Figure 4. Assessment of glomerular immunohistochemical staining the untreated STZ-diabetic rat all were absent or markedly ␤ for TGF- of control, unsupplemented diabetic rats, and diabetic rats attenuated in diabetic rats that were treated with LA. At the supplemented with an antioxidant by quantitative image analysis. doses used in the current study, neither LA nor VC prevented Staining was assessed with a SAMBA 4000 image analyzer as described in the Materials and Methods section. Four to five glomeruli glomerular hyperfiltration in the diabetic rats, whereas both of per rat were studied by this method. Results represent means Ϯ SEM these agents suppressed albuminuria. By contrast, VE sup- of the labeling index; *, P Ͻ 0.05 compared with value in Controls. pressed inulin clearance in the diabetic rats to control values but did not reduce UAE or fractional albumin clearance in the diabetic rats. Consistent with earlier interventional studies in the STZ-diabetic rat (20,36), the current results with LA dem- may have been secondary to basement membrane collapse or onstrate that albuminuria in diabetes mellitus can be reduced contraction after cell loss. despite persistent glomerular hyperfiltration. LA but not VC or As illustrated in Figure 8, VC or VE content of renal cortex VE also reduced cortical tubular cell content of TGF-␤ in the of diabetic rats that were receiving no antioxidant supplemen- diabetic rats. To the extent that increases in tubular TGF-␤ tation were 20 and 35% lower, respectively, than correspond- contribute to the tubulointerstitial fibrosis observed later (4 to ing values in nondiabetic rats. However, these differences were 6 mo) in the course of STZ diabetes mellitus in the rat (37), LA not statistically significant. LA was not detectable in renal may also attenuate diabetic injury in this region of the kidney. cortex from control rats, untreated diabetic rats, or diabetic rats The mechanisms by which dietary supplementation of LA that received VC or VE (not shown). The lower limit of attenuates renal injury in diabetes mellitus is not established by detection of LA in renal cortical extracts by the methodology the current studies. LA and DHLA participate in the recycling used in the current study was 0.2 ng/mg protein. Thus, in all of of VC and VE (25). Renal cortical LA content was increased in the foregoing study groups, renal cortical content was below the rats that received LA supplementation. However, renal this level. Supplementation of diabetic rats with VC signifi- cortical VC and VE levels in diabetic rats did not differ from cantly increased renal cortical VC but not VE content. Con- corresponding values in untreated diabetic rats. Accordingly, versely, supplementation of diabetic rats with VE significantly the effects of LA were not attributable to an enhancement of increased VE but not VC content of renal cortex. In diabetic VC or VE availability in the renal cortex of the diabetic rats. rats that received LA supplementation (30 mg/kg body wt per As noted above, LA is converted to the dithiol DHLA intra- d), renal cortical LA content was 5.6 Ϯ 0.6 ng (27 Ϯ 3 cellularly (25). The latter is an especially powerful antioxidant pmol)/mg protein. Given the sensitivity of the LA assay used that, along with LA, increases cellular glutathione levels in (0.2 ng/mg protein), failure to detect increases in renal cortical vitro and in vivo (38,39). LA supplementation in the present LA in diabetic rats supplemented with 15 mg/kg body wt per study clearly increased glutathione levels in renal cortex of the d suggests that LA content of this tissue, at least in diabetes mellitus, does not increase linearly with dietary consumption. diabetic rats, which is consistent with previously reported Renal cortical VC or VE content of diabetic rats that received effects of LA in other tissues (38). By contrast to LA, at the LA supplementation did not differ from corresponding values doses tested in the present study, neither VC nor VE supple- in untreated diabetic rats. mentation altered renal cortical glutathione content. Thus, in- As illustrated in Figure 9, renal cortical content of reduced creases in renal cortical LA and glutathione content were glutathione was not different in unsupplemented diabetic rats correlated with reductions in UAE, VG, glomerular and tubular compared with the value in nondiabetic rats. Supplementation content of TGF-␤, and glomerular collagen IV in the diabetic of diabetic rats with VC or VE had no significant effect on rats but not with reductions in either renal mass or GFR. Of renal cortical glutathione content. However, renal cortical glu- note, improvement of parameters of peripheral nerve injury in tathione was clearly higher in the diabetic rats that received LA the STZ-diabetic rat model in response to LA is also associated supplementation compared with values in unsupplemented di- with an increase in the glutathione content of this tissue (28), abetic rats or those receiving VE or VC supplementation. suggesting that this intracellular antioxidant moiety may par- 130 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 124–133, 2001

Figure 5. Effects of antioxidant supplementation on immunoreactive collagen ␣1 (IV). A representative example of glomerular immunohistochemical staining in renal cortical sections from each group is shown. (A) nondiabetic rat; (B) diabetic; (C) diabetic ϩ VC; (D) diabetic ϩ VE; (E) diabetic ϩ LA; (F) diabetic with nonimmune rabbit IgG substituted for anti-collagen ␣1 (IV) antibody. ticipate in the protection of both the nerve and the kidney from expansion in diabetic nephropathy (40–43). Similar to in vivo injury in diabetes mellitus. results in experimental diabetes mellitus, in vitro studies in Glomerular TGF-␤ is increased in both human and experi- cultured glomerular mesangial cells (MC) have demonstrated mental diabetes mellitus, and this prosclerotic cytokine has that several structurally distinct antioxidants, including ␣-to- been implicated as a major mediator of glomerular mesangial copherol, n-acetyl cysteine, and taurine, can suppress increases J Am Soc Nephrol 12: 124–133, 2001 ␣-Lipoic Acid in Diabetic Nephropathy 131

Figure 6. Assessment of glomerular immunohistochemical staining for collagen ␣1 (IV) of control, unsupplemented diabetic rats, and diabetic rats supplemented with an antioxidant by quantitative image analysis. Analysis was conducted as described in the legend of Figure 4 for TGF-␤. Results represent means Ϯ SEM of the labeling index. *, P Ͻ 0.05 compared with value of Controls. in active and latent TGF-␤ and the subsequent increases in matrix protein synthesis induced by culture of MC with high concentrations of glucose, angiotensin II, or thromboxane (44,45). These observations have implicated oxidative mechanisms in the increases in TGF-␤ induced by high glucose and other agents in MC. Increased generation of reactive oxygen species occurs in response to high glucose and/or angiotensin II in MC and endothelial cells (46,47). There is evidence to indicate that high glucose and other stimuli signal increases in TGF-␤ and the subsequent increases in matrix protein production by MC at least in part through activation of the protein kinase C (PKC) system (45). In cultured MC, several antioxidants as well as PKC inhibitors blocked the increases in PKC, TGF-␤, and matrix protein synthesis induced by high glucose or thromboxane but did not prevent increases in matrix protein synthesis in response to exogenous TGF-␤ (45). Thus, suppression of the activation of glomerular PKC that is known to occur in vivo in diabetes mellitus (48) may represent one mechanism by which antioxidants prevent increases in glomerular TGF-␤ and matrix protein synthesis in this disorder. The present studies were not specifically designed to estab- lish the efficacy or potency of LA versus VC or VE in the prevention of diabetic renal injury, but they nevertheless suggest that LA may be relatively more effective than either VE or VC in this regard. Results of our current and earlier studies (21) with VE and VC indicate that the actions of the last two agents are dose related and that very high doses are required to modify indices of glomerular injury in the STZ-diabetic rat. In Figure 7. Histology and immunohistochemical TGF-␤ staining of the the present study, the lowest oral dose of VE that detectably tubulointerstitial region of renal cortex. Representative renal cortical increased (by 30%) renal cortical levels of the vitamin in the sections from nondiabetic rats (A), untreated diabetic rats (B), and LA-supplemented diabetic rats (C) are shown. diabetic rats was used. This represented a 20-fold increase in dietary VE consumption compared with diabetic rats that received a standard diet. At this relatively high dose, VE supplementation failed to attenuate albuminuria or the increases in 40-fold increase in dietary VE consumption and raised renal ␤ ␣ VG, glomerular TGF- , and collagen 1 (IV) accumulation. cortical VE levels approximately 60%, attenuated but did not ␤ Moreover, a twofold higher dose of VE, which represented a totally prevent increases in VG or glomerular TGF- content in 132 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 124–133, 2001

STZ-diabetic rats (28,30). These observations indicate that LA is highly effective in preventing renal injury in diabetes mellitus and in particular in the suppression of increases in glomerular and tubular TGF-␤ and glomerular matrix protein accumulation. LA may be more potent than either VE or VC in these actions.

Acknowledgments The authors gratefully acknowledge the technical support of Mark Barsic, Julia Liachenko, and Diane George. This work was supported by funds from the Department of Veterans Affairs.

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