AJP - Renal Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 293: F1691-F1698, 2007. First published August 8, 2007; doi:10.1152/ajprenal.00235.2007
0363-6127/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/F1691    most recent
00235.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cruz, C.
Right arrow Articles by Pedraza-Chaverri, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cruz, C.
Right arrow Articles by Pedraza-Chaverri, J.

Renoprotective and antihypertensive effects of S-allylcysteine in 5/6 nephrectomized rats

Cristino Cruz,1 Ricardo Correa-Rotter,1 Dolores Javier Sánchez-González,2 Rogelio Hernández-Pando,3 Perla D. Maldonado,4 Claudia María Martínez-Martínez,2 Omar Noel Medina-Campos,5 Edilia Tapia,6 Diana Aguilar,3 Yolanda I. Chirino,5 and José Pedraza-Chaverri5

1Departamento de Nefrología y Metabolismo Mineral and 3Departamento de Patología Experimental, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; 2Departamento de Biología Celular, Escuela Médico Militar, Universidad del Ejército y Fuerza Aérea; 4Laboratorio de Patología Vascular Cerebral, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez; 5Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México; and 6Departamento de Nefrología, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico

Submitted 18 May 2007 ; accepted in final form 6 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Progressive renal damage and hypertension are associated with oxidative and nitrosative stress. On the other hand, S-allylcysteine (SAC), the most abundant organosulfur compound in aged garlic extract (AG), has antioxidant properties. The effects of SAC and AG on blood pressure, renal damage, and oxidative and nitrosative stress were studied in five-sixths nephrectomized rats treated with SAC (200 mg/kg ip) and AG (1.2 ml/kg ip) every other day for 30 days. Proteinuria and serum creatinine and blood urea nitrogen concentrations were measured on days 0, 5, 10, 15, and 30, and systolic blood pressure was recorded on days 0, 15, and 30. The degree of glomerulosclerosis and tubulointerstitial damage, the immunostaining for inducible nitric oxide synthase, 3-nitrotyrosine, poly(ADP-ribose), and the subunits of NADPH oxidase p22phox and gp91phox, and the activity of SOD were determined on day 30. SAC and AG reduced hypertension, renal damage, and the abundance of inducible nitric oxide synthase, 3-nitrotyrosine, poly(ADP-ribose), p22phox, and gp91phox and increased SOD activity. Our data suggest that the antihypertensive and renoprotective effects of SAC and AG are associated with their antioxidant properties and that they may be used to ameliorate hypertension and delay the progression of renal damage.

aged garlic; renal injury; hypertension; superoxide anion

CHRONIC RENAL FAILURE is associated with oxidative stress (52), the mechanism of which remains to be elucidated. Rats with five-sixths nephrectomy (5/6NX) are a widely studied model of chronic renal failure characterized by progressive renal injury, proteinuria, oxidative and nitrosative stress, and hypertension (3, 4, 28, 34, 46, 50–52). Tempol, an SOD mimetic, ameliorates hypertension and reduces 3-nitrotyrosine (3-NT) abundance and enhanced urinary excretion of nitric oxide (NO·) metabolites in rats with 5/6NX, suggesting a role of superoxide anion (O2bullet–) and an inactivation of NO· with O2bullet– in this experimental model (51). Consistent with these findings, downregulation of CuZn SOD and Mn SOD, enzymes that metabolize O2bullet– to H2O2, and upregulation of gp91phox, a subunit of the O2bullet–-producing enzyme NADPH oxidase, in kidneys from these rats favor O2bullet– production (51). In these animals, O2bullet– production enhances formation of 3-NT, a marker of nitrosative stress (7). Although it is controversial, inducible nitric oxide synthase (iNOS) expression is enhanced in rats with 5/6NX (3), which may lead to enhanced NO· production and enhanced 3-NT abundance. Additionally, the inhibition of iNOS with L-N6-(1-iminoethyl)-lysine or aminoguanidine ameliorated renal damage, suggesting a critical role of iNOS in the progressive renal dysfunction and structural injury in this experimental model (3).

In this context, the use of antioxidants may prevent progression of renal injury in rats with 5/6NX. It has been recently shown that vitamin E and N-acetylcysteine, two well-known antioxidants, have beneficial effects in rats with remnant kidney (48, 49).

On the other hand, S-allylcysteine (SAC), a water-soluble, nontoxic garlic compound and the most abundant organosulfur compound in aged garlic extract (AG) (26), has antioxidant properties in vivo (16, 18, 32, 38, 43, 44) and in vitro (12, 19, 22, 23). AG is commercially available and has been widely studied for its high antioxidant properties and health-protective potential (2, 5, 9, 17, 23, 36).

In addition, in vitro studies have demonstrated that SAC is able to scavenge O2bullet– (23, 32, 33), H2O2 (32, 33), hydroxyl radicals (·OH) (23, 33), and peroxynitrite anion (ONOO–) (22, 33, 35). Furthermore, Geng et al. (12) showed that SAC may block H2O2-induced nuclear factor-{kappa}B (NF-{kappa}B) activation. In this context, the findings of Fujihara et al. (11), who showed that chronic inhibition of NF-{kappa}B ameliorates renal damage in rats with remnant kidney, are relevant.

Some effects of SAC and AG on NO· production and blood pressure have been previously described. Kim et al. reported that SAC inhibits NO· production, iNOS expression, and NF-{kappa}B activation in the murine macrophage cell line RAW264.7 (23) and that SAC in the diet reduces mortality, with decreased incidence of stroke in stroke-prone spontaneously hypertensive rats (21). In addition to its antioxidant properties, AG is able to reduce blood pressure in spontaneously hypertensive rats (15). Taking into account the above-described information, we hypothesized that SAC and AG may reduce oxidative and nitrosative stress, progression of structural kidney injury, and systemic hypertension in 5/6NX rats. To establish a potential mechanism of action of SAC and AG in this model, we evaluated the following markers: 1) iNOS, which synthesizes copious amounts of NO· (14), 2) 3-NT formation as a marker of ONOO– generation and nitrosative stress (14, 34, 51), 3) poly(ADP-ribose) (pADPR), a marker of poly(ADP-ribose) polymerase (PARP) activation, a nuclear enzyme activated by oxidative and nitrosative stress (24), and 4) gp91phox and p22phox, subunits of NADPH oxidase, an enzyme that synthesizes O2bullet– (13).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. SAC was synthesized by the reaction of L-cysteine with allyl bromide and purified by recrystallization from ethanol-water, as previously described (27, 33). AG (Kyolic) was obtained from Wakunaga (Mission Viejo, CA). Xanthine, xanthine oxidase, and nitro blue tetrazolium (NBT) were purchased from Sigma Chemical (St. Louis, MO). Blood urea nitrogen (BUN) and creatinine concentrations were measured using commercial kits (Sera-Pak Plus Creatinine and Urea, Bayer, Tarrytown, NY). H2O2 was purchased from Mallinckrodt Baker (Xalostoc, México). Pentobarbital sodium was obtained from Pfizer. Mouse monoclonal antibodies against 3-NT (catalog no. 189542) were purchased from Cayman Chemical (Ann Arbor, MI), mouse monoclonal antibodies against pADPR (catalog no. SA-216) from Biomol (Plymouth Meeting, PA), and goat polyclonal antibodies against p22phox (catalog no. sc-11712) and gp91phox (catalog no. sc-5827), mouse monoclonal antibodies against iNOS (catalog no. sc-7271), and rabbit polyclonal antibodies against endothelial NOS (eNOS; catalog no. sc-654) from Santa Cruz Biotechnology (Santa Cruz, CA). The following secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA): biotin-streptavidin peroxidase (SP)-conjugated AffiniPure donkey anti-mouse IgG (catalog no. 715-065-151), biotin-SP-conjugated AffiniPure goat anti-rabbit IgG (catalog no. 111-065-003), and biotin-SP-conjugated AffiniPure donkey anti-goat IgG (catalog no. 705-065-003). Declere (catalog no. CMX633C) was obtained from Cell Marque (Hot Springs, AR), ABC Elite Vectastain kit (catalog no. PK-6101) from Vector Laboratories (Orton Southgate, Peterborough, UK), and diaminobenzidine substrate (catalog no. K3466) and Mayer's hematoxylin (Lillie's modification; catalog no. S3309) from DakoCytomation (Carpentaria, CA). All other chemicals were reagent grade.

Animals and procedures. The experimental protocol was approved by the local animal care committee (CINVA, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubrián). Male Wistar rats (280–300 g body wt) were anesthetized with pentobarbital sodium (50 mg/kg ip), and a ventral laparotomy was performed under aseptic conditions. The right kidney was removed, and a portion (two-thirds) of the left kidney was acutely infarcted by ligation of two first-order branches of the main renal artery (50). Recovery from anesthesia and from the surgical procedure was complete within 24 h. Rats were divided into three groups as follows: 5/6NX, 5/6NX + AG (1.2 ml/kg ip, every other day) (31), and 5/6NX + SAC (200 mg/kg ip, ever other day) (25). Rats were maintained in stainless steel metabolic cages for collection of urine samples. Samples of venous blood were taken from the tail of the rats 5, 10, 15, and 30 days after 5/6NX for serum creatinine and BUN determinations. Systolic blood pressure (SBP) was measured by a noninvasive tail-cuff method (model 179 Blood Pressure Analyzer, IITC, Woodland Hills, CA) on days 0, 15, and 30. Sham-operated (sham) rats were also studied on days 0, 15, and 30. At the end of the study, rats were anesthetized by an injection of pentobarbital sodium (30 mg/kg ip), and blood samples were taken from the aorta. The kidneys were excised and frozen in liquid nitrogen and stored at –80°C. The experimental protocol was approved by the Local Animal Care Committee (CINVA) of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán.

Biochemical markers of renal injury. Serum creatinine and BUN concentrations were measured with an autoanalyzer (Technicon RA-1000, Bayer). Urine protein concentration was measured by a previously described method (30).

Activity of antioxidant enzymes. SOD activity in kidney homogenates was assayed by a previously reported method (42) based on NBT reduction to formazan. The amount of protein that inhibits NBT reduction to 50% of maximum was defined as 1 unit of SOD activity. Results are expressed as units per milligram of protein. Catalase activity was measured by a previously described method, with H2O2 used as substrate (41).

Histological and immunohistochemical analyses. For light microscopy, kidney tissue was fixed by immersion in buffered formalin (pH 7.4) and embedded in paraffin. For histological analysis, kidney sections (3 µm) were stained with hematoxylin and eosin and with Masson's trichrome to stain collagen as an indicator of fibrosis. Interstitial fibrosis was determined in five random fields of the cortical area at x200 magnification (total area 4.1 x 105 µm2) with use of a computer-assisted color image analyzer (Qwin-500, Leica, Milton Keynes, Cambridge, UK). The same instrument was used to determine glomerulosclerosis by measurement of the extent of fibrosis identified by Masson's trichrome stain in 20 randomly selected glomeruli from each animal. The fraction of fibrosis was obtained by normalization of the area of the Masson's trichrome-positive material in each glomerulus to the whole glomerular area.

For immunohistochemistry, paraffin was removed from the kidney sections (3 µm), which were then boiled in Declere to unmask antigen sites; the endogenous activity of peroxidase was quenched with 0.03% H2O2 in absolute methanol. Kidney sections were incubated overnight at 4°C with a 1:100 dilution of anti-iNOS, a 1:250 dilution of anti-eNOS, a 1:70 dilution of anti-3-NT, a 1:50 dilution of anti-gp91phox, a 1:50 dilution of anti-p22phox, and a 1:200 dilution of anti-pADPR antibodies in PBS. Bound antibodies were detected with an avidin-biotinylated peroxidase complex ABC kit (Vectastain) and diaminobenzidine substrate. After they were appropriately washed in PBS, the slides were counterstained with hematoxylin. All specimens were examined by light microscopy (Axiovert 200M, Carl Zeiss). For automated morphometry analysis, the percentage of positive cells (stained brown) was determined with a computerized image analyzer (model KS-300 3.0, Carl Zeiss), which automatically detects positive cells and determines the number of positive cells per field. Five random fields per kidney were studied at x100 magnification (total area 1 x 106 µm2) for comparison of the different groups. All sections were incubated under the same conditions and in the same run, so immunostaining was comparable among the different experimental groups. For the negative control, preimmune goat serum, instead of the primary antibodies, was used (40, 47).

Statistical analysis. Values are means ± SE. Data were analyzed with Prism (version 3.02, GraphPad, San Diego, CA) by one- or two-way analysis of variance followed by Dunnett's post test or Bonferroni's multiple comparisons method, as appropriate. Pearson's correlation coefficient (r) was used for testing variables of interest. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Serum creatinine and BUN concentrations were increased in 5/6NX compared with sham rats. Both markers reached a peak value on day 5 and then decreased but remained high until the end of the study (day 30) in 5/6NX, 5/6NX + SAC, and 5/6NX + AG rats. SAC and AG ameliorated the increase in serum creatinine and BUN concentrations on days 5, 10, 15, and 30 (Fig. 1).


Figure 1
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 1. Time-course study of serum creatinine (A) and blood urea nitrogen (B) in sham ({blacksquare}), 5/6 nephrectomized (5/6NX, {square}), S-allylcysteine (SAC)-treated 5/6NX (5/6NX + SAC, {circ}), and aged garlic extract (AG)-treated 5/6 NX (5/6NX + AG, bullet) rats. Values are means ± SE (n = 6). !=P < 0.001 vs. sham. *,+P < 0.05 vs. 5/6NX.

 
Proteinuria increased in 5/6NX compared with sham rats. Proteinuria increased by day 10, reached a peak value on day 15, and decreased but remained high on day 30 in 5/6NX rats. This increase was significantly less in SAC- and AG-treated rats on days 10, 15, and 30 (Fig. 2A). The increase in SBP in 5/6NX rats on days 15 and 30 was significantly prevented by AG and SAC (Fig. 2B).


Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 2. Time-course study of proteinuria (A) and systolic blood pressure (SBP, B) in sham ({blacksquare}), 5/6NX ({square}), 5/6NX + SAC ({circ}), and 5/6NX + AG (bullet) rats. Values are means ± SE (n = 6). !=P < 0.001 vs. sham. *,+P < 0.001 vs. 5/6NX.

 
Kidneys from the sham-operated rats showed normal structure (Fig. 3). The kidney histology of remnant rats of all groups on day 30 showed extensive fibrosis, mild chronic tubulointerstitial inflammatory infiltrate, and thickening of the muscular layer and decrease in luminal diameter of many intermediate-sized arteries and arterioles. Numerous convoluted proximal tubules showed extensive tubular damage characterized by atrophy and detachment of epithelial cells, with active epithelial regeneration and luminal proteinaceous casts. Multiple glomeruli showed different grades of fibrosis and mesangial cell hyperplasia (Fig. 3). The immunohistochemical detection of 3-NT showed strong immunostaining in the tubular epithelium, mesangial and visceral epithelial glomerular cells, endothelium, and smooth muscle cells from the vascular walls (Fig. 3). 3-NT immunostaining was weak in kidneys from sham rats.


Figure 3
View larger version (120K):
[in this window]
[in a new window]

  		Fig. 3. Representative histological (Masson's trichrome stain) and immunohistochemical detection of 3-nitrotyrosine (3-NT) in kidney sections after 30 days of 5/6NX and effect of SAC and AG. Interstitial fibrosis, glomerular sclerosis (*), and vascular wall thickening (arrow) are extensive in 5/6NX rats; many cortical tubules show epithelial atrophy and tubular casts. Tissue damage and interstitial fibrosis are less extensive and there are fewer glomerular lesions in 5/6NX + SAC and 5/6NX + AG rats. Sham rats show normal structure. 3-NT immunostaining is strong in epithelial tubular cells (arrows), mesangial and visceral epithelial cells from damaged glomeruli (*), vascular walls (arrowheads), and interstitial cells in 5/6NX rats. 3-NT immunoreactivity is weaker in 5/6NX + SAC and 5/6NX + AG rats and scarce in sham rats. Magnification x100.

 
In contrast to the untreated 5/6NX rats, the morphometry study showed that AG or SAC significantly improved renal damage, reducing the area affected by fibrosis sixfold in glomeruli and tubular interstitium. 3-NT immunostaining was less intense in these AG- or SAC-treated animals than in untreated 5/6 NX rats (Figs. 3 and 4, Table 1).


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. Effect of AG and SAC on percentage of glomerulosclerosis (A) and tubulointerstitial damage (B) in rats killed on day 30. Values are means ± SE (n = 4). !=P < 0.001 vs. sham. *,+P < 0.001 vs. 5/6NX.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Quantitative data from immunohistochemistry studies

 
Similar to their effects on 3-NT, AG and SAC decreased the abundance of iNOS and p22phox (Fig. 5, Table 1) and gp91phox and pADPR (Table 1) on day 30 in all groups. SOD and catalase activities decreased significantly in 5/6NX rats (Fig. 6). AG and SAC significantly increased SOD activity but had no effect on catalase activity (Fig. 6). eNOS immunostaining remained unchanged in all groups (data not shown).


Figure 5
View larger version (102K):
[in this window]
[in a new window]

  		Fig. 5. Representative immunohistochemical detection of inducible nitric oxide synthase (iNOS) and p22phox in kidney sections after 30 days of 5/6NX and effect of SAC and AG. iNOS immunostaining is strong in epithelial tubular cells (arrows), mesangial and visceral epithelial cells from damaged glomeruli (*), and interstitial cells in 5/6NX rats, weaker in 5/6NX + SAC and 5/6NX + AG rats, and scarce in sham rats. p22phox immunostaining in interstitial cells (arrows) and mesangial cells from glomerulus (*) is strong in 5/6NX rats, less intense in 5/6NX + SAC and 5/6NX + AG rats, and scarce in sham rats. Magnification x400.

 

Figure 6
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 6. SOD (A) and catalase (B) activity in renal cortex on day 30 in sham, 5/6NX, 5/6NX + SAC, and 5/6NX + AG rats. Values are means ± SE (n = 6). !=P < 0.001 vs. sham. *,+P < 0.001 vs. 5/6NX.

 
Correlation studies. As expected, SBP showed a significant correlation with glomerulosclerosis (r = 0.86, P = 0.0004) and tubulointerstitial damage (r = 0.91, P = 0.0001). SBP also displayed a positive correlation with 3-NT (r = 0.806, P = 0.0002), iNOS (r = 0.68, P = 0.0036), gp91phox (r = 0.775, P = 0.0004), p22phox (r = 0.889, P < 0.0001), and pADPR (r = 0.57, P = 0.02) and a negative correlation with SOD (r = –0.786, P = 0.0002; Fig. 7).


Figure 7
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 7. Correlation analysis between SBP and 3-NT (A), gp91phox (B), p22phox (C), and SOD (D) in 5/6NX ({square}), 5/6NX + AG (bullet), and 5/6NX + SAC ({circ}) rats.

 
Glomerulosclerosis correlated positively with tubulointerstitial injury (r = 0.91, P < 0.0001), 3-NT (r = 0.92, P < 0.0001), gp91phox (r = 0.78, P = 0.0029), p22phox (r = 0.83, P = 0.0008), and pADPR (r = 0.81, P = 0.0014) and negatively with SOD (r = –0.82, P = 0.001; Fig. 8). Tubulointerstitial injury displayed a positive correlation with 3-NT (r = 0.91, P < 0.0001), iNOS (r = 0.75, P = 0.0048), gp91phox (r = 0.75, P = 0.0046), p22phox (r = 0.81, P = 0.0013), and pADPR (r = 0.68, P = 0.015) and a negative correlation with SOD (r = –0.68, P < 0.015).


Figure 8
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 8. Correlation analysis between glomerulosclerosis and 3-NT (A), gp91phox (B), p22phox (C), and SOD (D) in 5/6NX ({square}), 5/6NX + AG (bullet), and 5/6NX + SAC ({circ}) rats.

 
3-NT immunostaining correlated positively with p22phox (r = 0.89, P < 0.0001), gp91phox (r = 0.86, P < 0.0001), iNOS (r = 0.75, P = 0.0003), and pADPR (r = 0.75, P = 0.0003) and negatively with SOD (r = –0.8072, P < 0.0001; Fig. 9). iNOS immunostaining positively correlated with gp91phox (r = 0.55, P = 0.021) and pADPR (r = 0.66, P = 0.0039) and negatively with SOD (r = –0.54, P = 0.02). Finally, gp91phox, p22phox, and pADPR correlated negatively with SOD (r = –0.83, P < 0.0001; r = –0.73, P = 0.0005; and r = –0.61, P = 0.0074, respectively).


Figure 9
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 9. Correlation analysis between 3-NT and p22phox (A), gp91phox (B), iNOS (C), and SOD (D) in 5/6NX ({square}), 5/6NX + AG (bullet), and 5/6NX + SAC ({circ}) rats.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Renal damage in 5/6NX rats has been clearly associated with oxidative and nitrosative stress, characterized by 1) enhanced 3-NT formation, 2) downregulation of CuZn SOD and Mn SOD, 3) upregulation of gp91phox, 4) enhanced urinary excretion of NO· metabolites (51), and 5) enhanced iNOS expression (3). Our results suggest an imbalance in O2bullet– and NO· metabolism favoring their production. The above-mentioned alterations may lead to an enhanced ONOO– production and 3-NT formation. Interestingly, the scavenging of O2bullet– with tempol (51) reduced hypertension in these rats, suggesting a role of O2bullet– not only in renal injury, but also in the development of hypertension. In fact, oxidative and nitrosative stress has been involved in the pathogenesis of hypertension (10, 53). Considering the previously discussed background, we explored the effect of SAC and AG on rats with remnant kidneys. SAC and AG have demonstrated an antioxidant capacity, as well as tissue protective effects, in several experimental conditions (9, 16, 21, 22, 31, 32, 38, 43). In the present study, the following properties of AG and SAC are remarkable and support their use in rats with 5/6NX: 1) SAC and AG scavenge O2bullet– (23, 31–33), 2) SAC scavenges ONOO– (22, 33) and prevents hemolysis induced by this anion (35), 3) SAC inhibits iNOS expression and enhances NO· production from endothelial cells (23), 4) SAC prevents NF-{kappa}B activation (12), and 5) AG ameliorates high blood pressure in hypertensive rats (15) and increases NO·· production (37). AG, one of the best-known garlic preparations, is formed during garlic aging (up to 20 mo). During this time, unstable and highly odorous compounds in fresh garlic are converted to more stable and much less odorous compounds (1). The chemical composition of AG is different from that of whole garlic (26): the most abundant compounds in AG are alliin, cycloalliin, SAC, and S-allylmercaptocysteine, and the most abundant compounds in whole garlic are alliin and {gamma}-glutamylcysteines (26).

In the present work, the development of oxidative and nitrosative stress in rats with remnant kidney was confirmed by the formation of 3-NT and pADPR, the upregulation of iNOS, p22phox, and gp91phox, and the decrease in SOD and catalase activities.

Our data clearly show that SAC and AG dramatically reduced renal injury and hypertension in 5/6NX rats. The renoprotective effect was clearly and significantly associated with a decrease in abundance of 3-NT, gp91phox, p22phox, and pADPR and an increase in SOD activity. 3-NT is a marker of ONOO–, a powerful oxidant and nitrating agent, and nitrosative stress (7, 34, 51); pADPR is a marker of PARP activation secondary to oxidative and nitrosative stress (24). These data strongly suggest that the protective effect of SAC and AG in 5/6NX rats is associated with their antioxidant properties.

The decrease in 3-NT abundance may be secondary to the SAC- and AG-induced decrease in iNOS, gp91phox, and p22phox abundance and the increase in SOD activity. These combined effects should reduce the availability of NO· and O2bullet–, the substrates for ONOO– formation. 3-NT abundance showed a positive correlation with p22phox, gp91phox, and iNOS and a negative correlation with SOD activity.

The mechanism(s) by which SAC and AG decrease the abundance of iNOS, gp91phox, and p22phox and the increase in SOD activity is not clear. Our data suggest that iNOS activation is involved in renal injury in this experimental model, as reported by Bautista-Garcia et al. (3). In addition, our results are also in good agreement with those of Vaziri et al. (52), who suggested that NADPH oxidase is a source of O2bullet– in 5/6NX rats. The activation of NADPH oxidase also has been observed and implicated in the pathogenesis of several renal diseases (13, 20, 29). It has also been suggested that PARP activation is involved in the pathogenesis of 5/6NX. In fact, PARP activation has been implicated in several nephropathies (6, 8, 24, 39, 45). The use of PARP inhibitors may help clarify the role of PARP in 5/6NX rats.

The antihypertensive effect of AG in 5/6NX rats is consistent with the hypotensive effect of AG in spontaneously hypertensive rats (15). The SAC- and AG-induced reduction in oxidative and nitrosative stress may be associated with this effect. Our results clearly show that hypertension correlates positively not only with renal structural damage, but also with the immunostaining of iNOS, 3-NT, gp91phox, and p22phox, and negatively with SOD activity. Our data are consistent with the fact that SAC and AG modulate NO· production (21, 37). To our knowledge, this is the first report of the antihypertensive effect of SAC. Kim et al. (21) found a protective effect of SAC in stroke-prone spontaneously hypertensive rats, despite persistent high blood pressure.

In summary, SAC and AG showed marked antihypertensive and renoprotective effects in 5/6NX rats. These protective effects were clearly associated with the correction of the imbalance between O2bullet– production and the decrease in oxidative and nitrosative stress markers. These data suggest that SAC and AG may be used to ameliorate hypertension and delay the progression of renal injury.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Consejo Nacional de Ciencia y Tecnologia Grant 40009M.


   ACKNOWLEDGMENTS
 
The Axiovert 200 M confocal microscope was donated by Fundacion Gonzalo Rio Arronte IAP México.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Pedraza-Chaverri, Facultad de Química, Departamento de Biología, Edificio F, Segundo Piso, Laboratorio 209, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, Mexico DF, México (e-mail: pedraza{at}servidor.unam.mx)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Amagase H, Petesch BL, Matsuura H, Kasuga S, Itakura Y. Intake of garlic and its bioactive components. J Nutr 131: 955S–962S, 2001.[Abstract/Free Full Text]
  2. Banerjee SK, Mukherjee PK, Maulik SK. Garlic as an antioxidant: the good, the bad and the ugly. Phytother Res 17: 97–106, 2003.[CrossRef][Web of Science][Medline]
  3. Bautista-Garcia P, Sanchez-Lozada LG, Cristobal-Garcia M, Tapia E, Soto V, Avila-Casado MC, Marquez-Velasco R, Bojalil R, Franco M, Herrera-Acosta J. Chronic inhibition of NOS-2 ameliorates renal injury, as well as COX-2 and TGF-beta1 overexpression, in 5/6 nephrectomized rats. Nephrol Dial Transplant 21: 3074–3081, 2006.[Abstract/Free Full Text]
  4. Bobadilla NA, Tack I, Tapia E, Sanchez-Lozada LG, Santamaria J, Jimenez F, Striker LJ, Striker GE, Herrera-Acosta J. Pentosan polysulfate prevents glomerular hypertension and structural injury despite persisting hypertension in 5/6 nephrectomy rats. J Am Soc Nephrol 12: 2080–2087, 2001.[Abstract/Free Full Text]
  5. Borek C. Garlic reduces dementia and heart-disease risk. J Nutr 136: 810S–812S, 2006.[Abstract/Free Full Text]
  6. Chatterjee PK, Zacharowski K, Cuzzocrea S, Otto M, Thiemermann C. Inhibitors of poly(ADP-ribose) synthetase reduce renal ischemia-reperfusion injury in the anesthetized rat in vivo. FASEB J 14: 641–651, 2000.[Abstract/Free Full Text]
  7. Chirino YI, Orozco-Ibarra M, Pedraza-Chaverri J. Papel del peroxinitrito en diferentes enfermedades. Rev Invest Clin 58: 350–358, 2006.[Web of Science][Medline]
  8. Cuzzocrea S, Mazzon E, Dugo L, Serraino I, Di Paola R, Britti D, De Sarro A, Pierpaoli S, Caputi A, Masini E, Salvemini D. A role for superoxide in gentamicin-mediated nephropathy in rats. Eur J Pharmacol 450: 67–76, 2002.[CrossRef][Web of Science][Medline]
  9. Dillon SA, Lowe GM, Billington D, Rahman K. Dietary supplementation with aged garlic extract reduces plasma and urine concentrations of 8-iso-prostaglandin F2{alpha} in smoking and nonsmoking men and women. J Nutr 132: 168–171, 2002.[Abstract/Free Full Text]
  10. Escobales N, Crespo MJ. Oxidative-nitrosative stress in hypertension. Curr Vasc Pharmacol 3: 231–246, 2005.[CrossRef][Medline]
  11. Fujihara CK, Antunes GR, Mattar AL, Malheiros DM, Vieira JM Jr, Zatz R. Chronic inhibition of nuclear factor-{kappa}B attenuates renal injury in the 5/6 renal ablation model. Am J Physiol Renal Physiol 292: F92–F99, 2007.[Abstract/Free Full Text]
  12. Geng Z, Rong Y, Lau BH. S-allyl cysteine inhibits activation of nuclear factor {kappa}B in human T cells. Free Radic Biol Med 23: 345–350, 1997.[CrossRef][Web of Science][Medline]
  13. Gill PS, Wilcox CS. NADPH oxidases in the kidney. Antioxid Redox Signal 8: 1597–1607, 2006.[CrossRef][Web of Science][Medline]
  14. Goligorsky MS, Brodsky SV, Noiri E. NO bioavailability, endothelial dysfunction, and acute renal failure: new insights into pathophysiology. Semin Nephrol 24: 316–323, 2004.[CrossRef][Web of Science][Medline]
  15. Harauma A, Moriguchi T. Aged garlic extract improves blood pressure in spontaneously hypertensive rats more safely than raw garlic. J Nutr 136: 769S–773S, 2006.[Abstract/Free Full Text]
  16. Herrera-Mundo MN, Silva-Adaya D, Maldonado PD, Galvan-Arzate S, Andres-Martinez L, Perez-de la Cruz V, Pedraza-Chaverri J, Santamaria A. S-allylcysteine prevents the rat from 3-nitropropionic acid-induced hyperactivity, early markers of oxidative stress and mitochondrial dysfunction. Neurosci Res 56: 39–44, 2006.[CrossRef][Web of Science][Medline]
  17. Ide N, Lau BH. Aged garlic extract attenuates intracellular oxidative stress. Phytomedicine 6: 125–131, 1999.[Web of Science][Medline]
  18. Ide N, Lau BH. S-allylcysteine attenuates oxidative stress in endothelial cells. Drug Dev Ind Pharm 25: 619–624, 1999.[CrossRef][Web of Science][Medline]
  19. Imai J, Ide N, Nagae S, Moriguchi T, Matsuura H, Itakura Y. Antioxidant and radical scavenging effects of aged garlic extract and its constituents. Planta Med 60: 417–420, 1994.[CrossRef][Web of Science][Medline]
  20. Khanna AK, Pieper GM. NADPH oxidase subunits (NOX-1, p22phox, Rac-1) and tacrolimus-induced nephrotoxicity in a rat renal transplant model. Nephrol Dial Transplant 22: 376–385, 2007.[Abstract/Free Full Text]
  21. Kim JM, Chang N, Kim WK, Chun HS. Dietary S-allyl-L-cysteine reduces mortality with decreased incidence of stroke and behavioral changes in stroke-prone spontaneously hypertensive rats. Biosci Biotechnol Biochem 70: 1969–1971, 2006.[CrossRef][Medline]
  22. Kim JM, Lee JC, Chang N, Chun HS, Kim WK. S-allyl-L-cysteine attenuates cerebral ischemic injury by scavenging peroxynitrite and inhibiting the activity of extracellular signal-regulated kinase. Free Radic Res 40: 827–835, 2006.[CrossRef][Web of Science][Medline]
  23. Kim KM, Chun SB, Koo MS, Choi WJ, Kim TW, Kwon YG, Chung HT, Billiar TR, Kim YM. Differential regulation of NO availability from macrophages and endothelial cells by the garlic component S-allyl cysteine. Free Radic Biol Med 30: 747–756, 2001.[CrossRef][Web of Science][Medline]
  24. Kiss L, Szabo C. The pathogenesis of diabetic complications: the role of DNA injury and poly(ADP-ribose) polymerase activation in peroxynitrite-mediated cytotoxicity. Mem Inst Oswaldo Cruz 1: 29–37, 2005.
  25. Krause RJ, Glocke SC, Elfarra AA. Sulfoxides as urinary metabolites of S-allyl-L-cysteine in rats: evidence for the involvement of flavin-containing monooxygenases. Drug Metab Dispos 30: 1137–1142, 2002.[Abstract/Free Full Text]
  26. Lawson LD. The composition and chemistry of garlic cloves and processed garlic. In: The Science and Therapeutic Application of Allium sativum L. and Related Species, edited by Koch HP, Lawson LD. Baltimore, MD: Williams & Wilkins, 1996.
  27. Lawson LD, Wang ZJ, Hughes BG. {gamma}-glutamyl-S-alkylcysteines in garlic and other Allium spp.: precursors of AGE-dependent trans-1-propenyl. J Nat Prod 54: 436–444, 1991.[CrossRef]
  28. Lim CS, Vaziri ND. Iron and oxidative stress in renal insufficiency. Am J Nephrol 24: 569–575, 2004.[CrossRef][Web of Science][Medline]
  29. Louhelainen M, Merasto S, Finckenberg P, Lapatto R, Cheng ZJ, Mervaala EM. Lipoic acid supplementation prevents cyclosporine-induced hypertension and nephrotoxicity in spontaneously hypertensive rats. J Hypertens 24: 947–956, 2006.[Web of Science][Medline]
  30. Lowry OH, Rosebrough NJ, Farr A, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  31. Maldonado PD, Barrera D, Medina-Campos ON, Hernandez-Pando R, Ibarra-Rubio ME, Pedraza-Chaverri J. Aged garlic extract attenuates gentamicin induced renal damage and oxidative stress in rats. Life Sci 73: 2543–2556, 2003.[CrossRef][Web of Science][Medline]
  32. Maldonado PD, Barrera D, Rivero I, Mata R, Medina-Campos ON, Hernandez-Pando R, Pedraza-Chaverri J. Antioxidant S-allylcysteine prevents gentamicin-induced oxidative stress and renal damage. Free Radic Biol Med 35: 317–324, 2003.[CrossRef][Web of Science][Medline]
  33. Medina-Campos ON, Barrera D, Segoviano-Murillo S, Rocha D, Maldonado PD, Mendoza-Patiño N, Pedraza-Chaverri J. S-allylcysteine scavenges singlet oxygen and hypochlorous acid and protects LLC-PK1 cells of potassium dichromate-induced toxicity. Food Chem Toxicol 45: 2030–2039, 2007.[CrossRef][Web of Science][Medline]
  34. Modlinger PS, Wilcox CS, Aslam S. Nitric oxide, oxidative stress, and progression of chronic renal failure. Semin Nephrol 24: 354–365, 2004.[CrossRef][Web of Science][Medline]
  35. Morihara N, Ide N, Sumioka I, Kyo E. Aged garlic extract inhibits peroxynitrite-induced hemolysis. Redox Rep 10: 159–165, 2005.[CrossRef][Web of Science][Medline]
  36. Morihara N, Sumioka I, Ide N, Moriguchi T, Uda N, Kyo E. Aged garlic extract maintains cardiovascular homeostasis in mice and rats. J Nutr 136: 777S–781S, 2006.[Abstract/Free Full Text]
  37. Morihara N, Sumioka I, Moriguchi T, Uda N, Kyo E. Aged garlic extract enhances production of nitric oxide. Life Sci 71: 509–517, 2002.[CrossRef][Web of Science][Medline]
  38. Numagami Y, Ohnishi ST. S-allylcysteine inhibits free radical production, lipid peroxidation and neuronal damage in rat brain ischemia. J Nutr 131: 1100S–1105S, 2001.[Abstract/Free Full Text]
  39. Obrosova IG, Julius UA. Role for poly(ADP-ribose) polymerase activation in diabetic nephropathy, neuropathy and retinopathy. Curr Vasc Pharmacol 3: 267–283, 2005.[CrossRef][Medline]
  40. Orozco-Ibarra M, Medina-Campos ON, Sanchez-Gonzalez DJ, Martinez-Martinez CM, Floriano-Sanchez E, Santamaria A, Ramirez V, Bobadilla NA, Pedraza-Chaverri J. Evaluation of oxidative stress in D-serine induced nephrotoxicity. Toxicology 229: 123–135, 2007.[CrossRef][Web of Science][Medline]
  41. Pedraza-Chaverri J, Granados-Silvestre MD, Medina-Campos ON, Hernandez-Pando R. Effect of the in vivo catalase inhibition on aminonucleoside nephrosis. Free Radic Biol Med 27: 245–253, 1999.[CrossRef][Web of Science][Medline]
  42. Pedraza-Chaverri J, Maldonado PD, Medina-Campos ON, Olivares-Corichi IM, Granados-Silvestre MA, Hernandez-Pando R, Ibarra-Rubio ME. Garlic ameliorates gentamicin nephrotoxicity: relation to antioxidant enzymes. Free Radic Biol Med 29: 602–611, 2000.[CrossRef][Web of Science][Medline]
  43. Perez-Severiano F, Rodriguez-Perez M, Pedraza-Chaverri J, Maldonado PD, Medina-Campos ON, Ortiz-Plata A, Sanchez-Garcia A, Villeda-Hernandez J, Galvan-Arzate S, Aguilera P, Santamaria A. S-allylcysteine, a garlic-derived antioxidant, ameliorates quinolinic acid-induced neurotoxicity and oxidative damage in rats. Neurochem Int 45: 1175–1183, 2004.[CrossRef][Web of Science][Medline]
  44. Perez-Severiano F, Salvatierra-Sanchez R, Rodriguez-Perez M, Cuevas- Martinez EY, Guevara J, Limon D, Maldonado PD, Medina-Campos ON, Pedraza-Chaverri J, Santamaria A. S-allylcysteine prevents amyloid-beta peptide-induced oxidative stress in rat hippocampus and ameliorates learning deficits. Eur J Pharmacol 489: 197–202, 2004.[CrossRef][Web of Science][Medline]
  45. Racz I, Tory K, Gallyas F Jr, Berente Z, Osz E, Jaszlits L, Bernath S, Sumegi B, Rabloczky G, Literati-Nagy P. BGP-15—a novel poly(ADP-ribose) polymerase inhibitor—protects against nephrotoxicity of cisplatin without compromising its antitumor activity. Biochem Pharmacol 63: 1099–1111, 2002.[CrossRef][Web of Science][Medline]
  46. Romero F, Rodriguez-Iturbe B, Parra G, Gonzalez L, Herrera-Acosta J, Tapia E. Mycophenolate mofetil prevents the progressive renal failure induced by 5/6 renal ablation in rats. Kidney Int 55: 945–955, 1999.[CrossRef][Web of Science][Medline]
  47. Sanchez-Gonzalez DJ, Moro MA, Castillo-Henkel C, Herrera-Gonzalez N, Hernandez-Pando R, Larios-Medina FJ, Cobilt R, Blanco JA, Pedraza-Chaverri J, Villanueva C. Ozone exposure induces iNOS expression and tyrosine nitration in rat aorta. Environ Toxicol Pharmacol 17: 1–7, 2004.[CrossRef]
  48. Shimizu MH, Coimbra TM, de Araujo M, Menezes LF, Seguro AC. N-acetylcysteine attenuates the progression of chronic renal failure. Kidney Int 68: 2208–2217, 2005.[CrossRef][Web of Science][Medline]
  49. Tain YL, Freshour G, Dikalova A, Griendling KK, Baylis C. Vitamin E reduces glomerulosclerosis, restores renal neuronal NOS, and suppresses oxidative stress in the 5/6 nephrectomized rat. Am J Physiol Renal Physiol 292: F1404–F1410, 2007.[Abstract/Free Full Text]
  50. Tapia E, Franco M, Sanchez-Lozada LG, Soto V, Avila-Casado C, Santamaria J, Quiroz Y, Rodriguez-Iturbe B, Herrera-Acosta J. Mycophenolate mofetil prevents arteriolopathy and renal injury in subtotal ablation despite persistent hypertension. Kidney Int 63: 994–1002, 2003.[CrossRef][Web of Science][Medline]
  51. Vaziri ND, Dicus M, Ho ND, Boroujerdi-Rad L, Sindhu RK. Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency. Kidney Int 63: 179–185, 2003.[CrossRef][Web of Science][Medline]
  52. Vaziri ND, Oveisi F, Ding Y. Role of increased oxygen free radical activity in the pathogenesis of uremic hypertension. Kidney Int 53: 1748–1754, 1998.[CrossRef][Web of Science][Medline]
  53. Vaziri ND, Rodriguez-Iturbe B. Mechanisms of disease: oxidative stress and inflammation in the pathogenesis of hypertension. Nat Clin Pract Nephrol 2: 582–593, 2006.[CrossRef][Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/F1691    most recent
00235.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cruz, C.
Right arrow Articles by Pedraza-Chaverri, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cruz, C.
Right arrow Articles by Pedraza-Chaverri, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.