the effect of insulin treatment on rac1 expression in diabetic kidney
TRANSCRIPT
Renal Failure, 2013; 35(3): 396–402Copyright © Informa Healthcare USA, Inc.ISSN 0886-022X print/1525-6049 onlineDOI: 10.3109/0886022X.2013.764256
LABORATORY STUDY
The Effect of Insulin Treatment on Rac1 Expression in Diabetic Kidney
Mukaddes Gumustekin1, Serap Cilaker Micili2, Mualla Aylin Arici1, Meral Karaman3,Mehmet Ensari Guneli3 and Işil Tekmen2
1Department of Pharmacology, School of Medicine, Dokuz Eylul University, Izmir, Turkey; 2Department of Histology andEmbryology, School of Medicine, Dokuz Eylul University, Izmir, Turkey; 3Department of Laboratory Animal Science, Schoolof Medicine, Dokuz Eylul University, Izmir, Turkey
Abstract
This study was designed to evaluate the renoprotective effect of insulin on diabetic nephropathy through Rac1 inhibition.Twenty Wistar rats were divided into three groups: control (C), diabetic (D), and insulin-treated diabetic (D þ I). Diabeteswas induced by a single streptozotocin (STZ) injection (45 mg/kg i.p.) in adult male rats. Diabetic animals were treatedsubcutaneously with insulin (6 U/kg), or saline once a day for 8 weeks. Age-matched control rats received only saline. Thekidney tissue samples were analyzed by immunohistochemical staining for Rac1 and cleaved caspase-3 expressions andusing the TUNEL method for determining apoptotic cells. Diabetes increased the number of TUNEL (þ) cells and cleavedcaspase-3 and Rac1 expression levels in kidney. Administration of insulin for 8 weeks reduced Rac1 expression andameliorated histopathological changes in kidney of STZ-induced diabetes model. These results may suggest that therenoprotective effect of insulin at least partly results from inhibition of Rac1 overexpression.
Keywords: apoptosis, caspase-3, diabetic nephropathy, insulin, Rac1
INTRODUCTION
Nephropathy may occur about in one-third of diabeticpatients and it is the most common cause for end-stagerenal disease.1 Although there are various mechanismsthat may lead to pathological and functional changes indiabetic kidney, the exact mechanisms are not clearlyidentified. Recent studies indicate that apoptosis is oneof the hallmarks of diabetic nephropathy (DN).2
Hyperglycemia may lead to various pathologic pro-cesses including glycosylation of circulating and cellularproteins and overproduction of reactive oxygen species(ROS).1–3 Small GTPase Rac1 is an intracellular signal-ing molecule that influences multiple cellular functionssuch as controlling the organization of cytoskeleton, geneexpression, and cell proliferation. Rac1 is also a subunitof the plasma membrane NADPH-oxidase, leading tothe production of ROS. Rac1-induced ROS has beendemonstrated in a variety of cellular processes, includinggrowth, migration, transformation, and apoptosis.4,5
NADPH activation through Rac1 was shown on apopto-sis in cardiomyocytes of diabetic mice5 and intestinalepithelial cells.6 While previous studies demonstrated
that the NADPH-oxidase activation in the kidney inboth hyperglycemia and hypertension models,7,8 onlyone study presented in a congress by Yoshida et al.reported that hyperglycemia-induced Rac1 activationwas observed in cultured cells and kidney of diabeticmice.9
The treatment of DN includes the strict glycemic con-trol by insulin and other treatment modalities such asreducing blood pressure, weight and lipid control, andsmoking cessation. Intensive insulin treatment has beenshown in large prospective randomized studies to delaythe onset of DN and its progression in patients with type1 and type 2 diabetes.10 Insulin is known to preclude DNby lowering blood glucose level and thus preventingdamages caused by hyperglycemia.1,10 However,through mitogen-activated protein kinase (MAPK) andphosphoinositide-3 kinase (PI3K) signaling pathways,insulin has been reported to contribute to remodelingof the actin cytoskeleton of glomerular podocytes andprevent apoptosis in some tissues, including kidneys.1,11
The fact that small GTPase Rac1 plays a role in theorganization of the actin cytoskeleton and can induce
Address correspondence to Mukaddes Gumustekin, Department of Pharmacology, School of Medicine, Dokuz Eylul University, Izmir,Turkey. Tel.: þ90 232 4123906; Fax: þ90 232 2590541; E-mail: [email protected]
Received 16 October 2012; Revised 12 December 2012; Accepted 27 December 2012
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apoptosis in some tissues (including kidney) may raisethe question in minds whether renoprotective effect ofinsulin occurs at least partly via Rac1, besides loweringhyperglycemia.
Therefore, we aimed to investigate the renoprotectiveeffect of insulin onDN at least partly through Rac1 inhibi-tion. First, we evaluated the Rac1 expression levels andtheir association with apoptosis in kidneys of streptozoto-cin (STZ)-induced diabetic rats. Then we explored theeffect of insulin on Rac1 expression and apoptosis.
MATERIALS AND METHODS
AnimalsAdult male Wistar rats weighing 180–220 g were used inthe present study. The study was approved by the EthicsCommittee of the Research of Laboratory Animals,Dokuz Eylül University Medical School, and all proce-dures were performed according to the “Principles ofLaboratory animal care” by the “National Institute ofHealth” publication. All animals were kept in standar-dized conditions of temperature (21–22�C) and illumi-nation (12:12 L/D) and cages with mesh bottoms,providing free access to tap water and pelleted food.The animals were fasted for 12 h before the experiment,but had free access to water until the beginning of theexperiment.
Induction of Diabetes MellitusFreshly prepared STZ (Sigma Chemical Co, St. Louis,MO, USA) was dissolved in 0.02 M citrate buffer(pH ¼ 4.5) and administered intraperitoneally to therats at 45 mg/kg. The age-matched control groupreceived citrate buffer only. On the third or fourth dayafter STZ administration, serum glucose levels weremeasured using an Optium Xceed glucometer (AbbottDiabetes Care, Oxon, UK) with a drop of blood takenfrom the tail vein under diethyl ether anesthesia. Ratswith blood glucose levels higher than 300 mg/dL wereconsidered diabetic. Eight weeks after STZ administra-tion, chronic diabetes was considered to be developed.
Experimental Study DesignTwenty rats were divided into three groups as follows:STZ-induced diabetic rats (D, n¼ 7), age-matched con-trol rats (C, n ¼ 6), and insulin-treated diabetic rats(D þ I, n ¼ 7). While the insulin-treated diabetic rats(Dþ I) were treated with 6U/kg/day NPH insulin (whichis accepted to be effective for 18–24 h, Lilly, USA) sub-cutaneously (s.c.) for 8 weeks, diabetic group (D) wastreated with saline s.c. for the same period.12 The ratswere terminated by cervical dislocation and leftnephrectomy was performed immediately. The bloodsamples were obtained via cardiac puncture for measur-ing blood glucose level.
Histological examinationKidney tissues were fixed in 10% buffered formalin andembedded in paraffin wax. Five-micron-thick sectionswere stained with hematoxylin and eosin and periodicacid-Schiff (PAS). Histological findings were recorded indetail.
In situ terminal deoxynucleotidyl transferasebiotin-dUTP nick end labeling (TUNEL) assayApoptosis was evaluated by the in situ terminal-deoxynucleotidyl-transferase-mediated dUTP digoxi-genin nick end labeling (TUNEL) assay. A Dead EndColorimetric TUNEL system kit (In Situ Cell DeathDetection Kit1 Roche, Mannheim, Germany) was usedfor apoptotic cell detection. Sections were deparaffi-nized, rehydrated in graded alcohol and microwave inpretreated in trypsin solution at 37�C for 10 min (Roche;No. 10 109 819 001). After being washed in phosphate-buffered saline (PBS), the specimens were incubatedwith fluorescein-labeled deoxy-UTP and terminaldeoxynucleotidyl transferase at 37�C for 60 min.Subsequently, anti-fluorescein antibody-peroxidase(converter POD) solutions were applied to the slides.Sections were stained with diaminobenzidine, counter-stained with hematoxylin, and mounted with entellan.Detection of apoptotic cells was performed manuallyunder the light microscope at a magnification of �400.The apoptotic index was calculated as the percentage ofcells showing TUNEL positivity. The percentage ofTUNEL (þ) cells was determined by counting thepositive cells from five random fields in each rat. Twoindependent blinded observers performed all measure-ments for the source of kidney tissue.13
Immunohistochemical AssessmentImmunohistochemical staining was performed using thestreptavidin/biotin method. The immunohistochemistryprocedure was performed for Rac1 (sc-217, Santa CruzBiotechnology, 1/100 dilution) and cleaved caspase-3(9661, Cell Signaling, 1/300 dilution). Sections of 5 μmwere cut by microtome and were incubated at 60�Covernight and then dewaxed in xylene for 30 min. Afterrehydrating through a decreasing series of alcohol, sec-tions were washed in distilled water for 10 min. Theywere then treated with 10mMcitrate buffer (Cat No.AP-9003-125 Labvision) at 95�C for 5 min to unmask anti-gens by heat treatment. Then slides were rinsed threetimes for 2 min each with deionized water. Sections weredelineated using a Dako pen (Dako, Glostrup,Denmark) and incubated in a solution of 3% H2O2 for15 min to inhibit endogenous peroxidase activity. Theywere then incubated with normal serum blocking solu-tion for 30 min, after then incubated in a humid chamberovernight atþ4�Cwith antibodies. Afterwards, they werewashed three times for 5 min each with PBS, followed byincubation with biotinylated IgG and then with strepta-vidin–peroxidase conjugate (Invitrogen, Histostain-Plus
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Broad Spectrum, 85–9043). After washing three timesfor 5 min with PBS, sections were incubated for 5 minwith a 3, 3'-diaminobenzidine (DAB) substrate contain-ing diaminobenzidine (1718096, Roche) to detectimmunoreactivity and then with Mayer’s hematoxylin.Sections were covered with Entellan mounting (Merck,Germany) and were analyzed by light microscopy using aBH-2 microscope (Olympus, Tokyo, Japan). Controlsamples were processed in an identical manner, but inthe absence of primary antibody step. Immunolabelingintensity of Rac1 (tubulointerstitial and glomerularareas) was graded (with semiquantitative assay) indepen-dently by two observers blind to the experimental condi-tions on the following scale: mild (þ), moderate (þþ),and strong (þþþ) labeling. The percentage of cleavedcaspase-3 positive cells was determined by counting thepositive cells from five random fields in each specimen attubulointerstitial and glomerular areas.
Statistical AnalysisAll values were expressed as the mean � standard error(mean � SEM). The Kruskal–Wallis and Mann–Whitney U-test was used to compare staining intensityvalues between groups. Spearman correlation analysiswas performed between cleaved caspase-3 and Rac1expressions. All statistical procedures were performedby SPSS 15.0 software for Windows (Chicago, IL,USA). A value of p < 0.05 was considered significant.
RESULTS
Blood Glucose Levels and Body Weights of ExperimentalAnimalsEight weeks after STZ injection, rats exhibited increasedblood glucose levels and decreased body weights thancontrol rats, as expected. In the insulin-treated diabeticgroup, blood glucose levels were close to the controlgroup and their body weight did not decrease as in thediabetic group (Table 1).
HistologyNormal histological structure was observed in PAS-stained kidney tissue specimens of the control group(Figure 1A). Mesangial matrix expansion, loss of prox-imal tubulus brush border, inflammatory cell infiltrationin interstitial tissue, degeneration in the renal tubulus,and vacuolization were observed in diabetic group(Figure 1B). Moreover, these degenerative changes
were mainly prevented by the insulin treatment(Figure 1C).
Determination of Apoptotic CellsIn the diabetic group compared to the control group,TUNEL staining results obtained in the renal cortexwere 22.7 � 1.2% versus 7.4 � 0.4% in the tubulointer-stitial area and 13.5 � 0.7% versus 5.7 � 0.4% in theglomerular area, respectively (p < 0.001, Figures 1D–Eand 2). TUNEL (þ) cells in the insulin-treated diabeticgroup (13.2 � 0.6% in tubulointerstitial area and7.2 � 0.5% in glomerular area) were significantly lowerthan the diabetes group (p < 0.001, Figure 1F), but notidentical as the control group (Figure 2).
Immunohistochemical AnalysisImmunohistochemical staining of cleaved caspase-3showed weak expression (minimal positive staining) inkidney cortex tissues in the control group (tubulointer-stitial area 4.4 � 0.1 and glomerular area 2.3 � 0.2)(Figures 1G and 3). Cleaved caspase-3 immunoreactiv-ity were significantly increased in tubulointerstitial andglomerular mesangial areas in diabetic group (tubuloin-terstitial area 18.5 � 0.5 and glomerular area 5.3 � 0.2)(Figures 1H and 3). The kidney sections of insulin-treated diabetic rats (tubulointerstitial area 4.4 � 0.7and glomerular area 4.0 � 0.3) were significantly lowercompared to the diabetic group but similar immunoreac-tivity was seen when compared to the control group(Figures 1I and 3). Immunohistochemical staining ofRac1 showed weak expression (minimal positive stain-ing) in the kidney cortex tissues (tubulointerstitial area0.3 � 0.1 and glomerular area 0.2 � 0.1) of the controlgroup (Figures 1K and 4). In the diabetic group,increased Rac 1 expressions were observed in glomerularmesangial areas and tubulointerstitial areas (tubulointer-stitial area 1.9 � 0.1 and glomerular area 0.6 � 0.1)(Figure 1L, p < 0.001). Although some glomerularmesangial areas and tubulointerstitial areas also showedincreased immunoreactivity in insulin-treated diabeticgroup (tubulointerstitial area 0.5 � 0.1 and glomerulararea 0.4� 0.1), kidney specimens showed similar immu-noreactivity as the control group (Figures 1M and 4).
Correlation AnalysisIn the diabetic group, Rac1 expressions showed moder-ate correlation with tubular cleaved caspase-3 expression(r ¼ 0.383, p ¼ 0.021) and inverse correlation with
Table 1. Blood glucose levels and body weights of experimental animals.
C D D þ I p
Body weights (g) 230 � 6.6 188 � 11� 233 � 10.9 p < 0.01Blood glucose levels (mg/dL) 86.2 � 5.6 571.7 � 45.4�� 139.1 � 28.9 p < 0.001
Notes: The values expressed as mean � SEM. C, control; D, diabetic;Dþ I, insulin-treated diabetic group.�p < 0.01, compared with C and D þ I groups; ��p < 0.001, compared with C and D þ I groups
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glomerular cleaved caspase-3 expression (r ¼ �0.417,p ¼ 0.011). There was no correlation between cleavedcaspase-3 expression and Rac1 expression in tubuli andglomeruli of both control and insulin-treated diabetickidneys (Table 2).
DISCUSSION
In the present study, the inhibitory effect of insulin on thedevelopment of DN was indirectly shown in the STZ-induced diabetic rat model to be collaterally effectivealong with decreasing hyperglycemia, as well as
decreasing Rac1 expression and inhibiting apoptosis inthe kidneys, at least to a certain extent.
Hyperglycemia is the major factor responsible for thedevelopment and progression of diabetic complicationssuch as DN.1,2 The present study showed that 8 weekcourse of STZ-induced hyperglycemia caused histo-pathologic findings of DN including mesangial matrixexpansion, loss of proximal tubulus brush border,inflammatory cell infiltration in interstitial tissue, degen-eration in the renal tubulus, and vacuolization in kidneyof Wistar rats. Increased oxidative stress due to oxygen
Control
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TUNEL
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Rac1
(K)
(G)
(D)
(H) (I)
(M)(L)
(A) (B) (C)
(F)(E)
Diabetes Insulin-treated diabetes
Figure 1. Photomicrograph kidney sections from control (A), diabetic (B), and insulin-treated diabetic rats (C) PAS stain. It was observednormal morphologic structure in PAS-stained control group kidney specimens (A). Mesangial matrix expansion, loss of proximal tubulusbrush border, inflamatory cell infiltration in interstitial tissue, degeneration in renal tubulus, and vacuolization were observed in diabeticgroup (B). Insulin-treated diabetic group, these degenerative changes were mainly prevented (C). TUNEL (þ) cells and cleaved caspase-3expressions in diabetic (tubulointerstitial area and glomerular area) group (E and H) were increased according to control (D and G) andinsulin-treated diabetic groups (F and I). Rac1 immunoreactivity was showed moderate positivity in tubulointerstitial area and glomerulararea in diabetic group (L), weak positivity was found both in control (K) and insulin-treated diabetic groups (M).
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0Control Diabetes
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Diabetes + insulin
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Tubulointerstitial area
Glomerular area
Figure 2. Percentage (%) of TUNEL (þ) cells in kidney tissues.Notes: All data were presented mean � SEM. �p < 0.001, diabeticgroup versus control and insulin-treated diabetic group;��p < 0.001, insulin-treated diabetic group versus diabetic group.
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Control Diabetes Diabetes + insulin
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Figure 3. Percentage (%) of Cleaved caspase-3 cells in kidneytissues.Notes: All data were presented mean � SEM. �p < 0.001, diabeticgroup versus control and insulin-treated diabetic group;��p < 0.001, insulin-treated diabetic group versus diabetic group.
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free radical production induced by hyperglycemia playsan important role in the development and progression ofDN.1,7 Although multiple sources of ROS have beendemonstrated, NADPH oxidase is a pivotal determinantof the redox state in the kidneys and higher NADPHoxidase levels and activity in the kidney has been detectedin diabetes14,15 and in high glucose conditions.16,17
NADPH oxidase is a multicomponent enzyme complexand its subunit Rac1 is critical for gp91phox –NADPHoxidase activation, which is the major source of ROSproduction exerting cellular dysfunction and cell deathin the form of necrosis or apoptosis.6 It has been demon-strated that apoptosis may play an important role in thediabetic renal injury.2,18–20 However, previous studiesshowed that Rac1 is a critical molecule of ROS produc-tion and a likely cause of apoptosis.6,21
In this study, increased TUNEL (þ) cells in the glo-merular mesangium and tubular epithelial cells in thekidneys of diabetic rats were observed when comparedto control group, and these results were found compati-ble with the recent reports.17,20 In the diabetic group,cleaved caspase-3 expression was found higher in tubu-lointerstitial and glomerular areas compared to the con-trol group, and was consistent with the report by Satoet al. which stated that caspase-3 activities in the kidneysof diabetic rats at the 8th week were 1.5 times greaterthan those in the control group.20 It has been reportedthat glomerular apoptosis was minimal in rats having 4and 8 weeks of DM and was compatible with the
outcome of the present study and also showed signifi-cantly higher glomerular apoptosis values in rats with 6months of DMcompared to the controls.18 The presenceof apoptotic cells and increased cleaved caspase-3expression in the tubulointerstitial area that was evenhigher than glomerular area suggested that STZ-inducedDN model in the present study may affect primarilytubulointerstitial area at 8 weeks after diabetes induce-ment. The results obtained in the present study are com-patible with those of the study by Menini et al.11 whofound a significant increase in glomerular apoptosis at 4and 6 months in STZ-induced diabetic rats but not at 2months after diabetes inducement. Also, in our previousstudy, TUNEL-positive cells were found both in theglomerular mesangium and tubular epithelial cells inthe kidneys of diabetic rats. Our previous results seemto be in conflict with our present results but, our previousstudy had been performed in 14 weeks (4 months) afterSTZ-inducement of diabetes while the present study wasperformed with STZ-induced diabetic rats after 8 weeks(2 months) of inducement.19
Rac1 mediated renal injury was shown in salt,Angiotensin-II- and oxalate-induced renal injuriesin vivo and in vitro models.22,23 Shibata et al. revealedthat high salt diet caused renal Rac1 upregulation leadingto hypertension and renal injury in salt-sensitive Dahlrats.22 In addition, Thamilselvan et al. demonstratedthe role of Rac1 on oxalate-induced cell injury in epithe-lial cell line from pig kidney with properties of proximaltubular cells. Also, the beneficial effect of Rac1 inhibitorwas shown in the same study.23While Rac1 was shown tobe associated with nephropathy in nondiabetic experi-mental models in these studies, only one study byYoshida et al. reported hyperglycemia-induced Rac1activation in cultured cells and kidney of diabeticmice.9 However, we were not able to read the full textof their study, since it was not published yet, to ourknowledge.
In this study, Rac1 expression was higher in the kid-neys of diabetic rats compared to the control group. Also,Rac1 expression in the tubuli was significantly increasedand its distribution was similar to that of apoptoticregions. These present results were consistent withabove mentioned studies investigating renal injury innondiabetic experimental models as well as the studyconducted by Yoshida et al in diabetic kidneys.9
Although Rac1 expression was moderately correlatedwith tubular cleaved caspase-3 expression, there wasinverse correlation in glomeruli in the diabetic group.These results suggest that the increase in Rac1 expres-sions may be related to especially tubular apoptosis andalso may be associated with DN in 8 weeks STZ-induceddiabetic rat model.
The treatment of DN should include the normalizingglycemic control by insulin and the other treatments suchas reducing blood pressure, weight and lipid control, andsmoking cessation. Optimal glycemic control for type 1diabetes needs insulin treatment. Alsomost of the patients
Table 2. Results of correlation analysis between cleaved caspase-3expression and Rac1 expression among experimental groups.
C D D þ I
Tubulointerstitial area r ¼ 0.169 r ¼ 0.383� r ¼ 0.099p ¼ 0.331 p ¼ 0.021 p ¼ 0.646
Glomerular area r ¼ �0.229 r ¼ �0.417�� r ¼ 0.169p ¼ 0.185 p ¼ 0.011 p ¼ 0.430
Notes: C, control; D, diabetic; D þ I, insulin-treated diabeticgroup.
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Control Diabetes Diabetes + insulin
∗
Tubulointerstitial area
Glomerular area
Figure 4. Distribution of Rac1 expressions in kidney. The degreesof immunopositivity are indicated as: 0, negative staining; 1, weakstaining; 2, moderate staining; 3, strong staining.Notes: All data were presented mean � SEM. �p < 0.001, Diabeticgroup versus control and insulin-treated diabetic group.
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with type II diabetes ultimately will require insulin.1,10
Intensive insulin treatment that has been shown in variouslarge prospective randomized studies may delay the onsetof DN and its progression in patients with type 1 and type2 diabetes.10 Although the mechanisms of insulin in pre-venting diabetic complications are well known, themechanisms are unclear for developing DN.
It has been shown that insulin has an antiapoptoticeffect on some tissues including heart in ischemia-reoxygenation models and kidney in diabetic state.11,24
Therefore, in this present study, the effects of insulintreatment on apoptosis and on Rac1 expressions wereevaluated in the STZ-induced diabetic rats. In the dia-betic group, the histological changes induced by diabeteswere prevented by 8 week insulin treatment, as expected.TUNEL (þ) cells in the insulin-treated diabetic groupwere significantly lower than the diabetic group, but notat the same levels as the control group. Although insulintreatment decreased cleaved caspase-3 expressionsnearly 41% in tubulointerstitial area and 25% in glomer-ular area, it has not decreased the levels as in the controlgroup. This was also evidenced in the present study, sothat preventing an increase in TUNEL (þ) cells andcaspase-3 by insulin may be the result of its antiapoptoticaction. Unfortunately, there was not enough decrease inapoptosis in the insulin-treated group compared to thecontrol group, which may suggest that additional treat-ment to prevent DN is needed.
Rac1 immunoreactivity was significantly decreasedand there was no correlation between Rac1 and cleavedcaspase-3 expressions in the kidneys of insulin-treateddiabetic group. The decrease in Rac1 immunoreactivityin the insulin-treated group may suggest that preventingeffect of insulin in addition to hyperglycemia improvingeffect may, at least, partly result from the inhibition ofRac1 overexpression.
Moreover, the treatment modalities such as Rac1 inhi-bition may be promising in this area of research andfurther relevant studies are required to be conducted.The significant decreases in Rac1 expressions in tubu-lointerstitial area suggest that Rac1 inhibition may bemost effective in tubulointerstitial injury in DN.
Insulin treatment only delays DN development butnot in all patients. Therefore, other treatment modalitiessuch as Angiotensin converting enzyme inhibitors andAngiotensin receptor blockers related renin–angiotensinaldosterone system (RAAS) may be used to preventDN. The relation of Rac1 with RAAS system wasshown in several studies which suggest that mineralocor-ticoid receptor activation by Rac1 may be responsible forsalt sensitive and A-II induced renal injury.21–23
Insulin is known to prevent DN by lowering bloodglucose level and thus inhibiting hyperglycemia-related damages.1,10 However, through MAPK andPI3K signaling pathways, insulin has been reported tocontribute to remodeling of the actin cytoskeleton ofglomerular podocytes and prevent apoptosis in sometissues, including kidneys.1,11 Various roles of the
small GTPase Rac1, such as organization of the actincytoskeleton and induction of apoptosis in some tis-sues including kidneys, have been demonstrated todate.4,5,21–23 This is the first study that examined theeffects of 8 weeks insulin treatment on Rac1 expressionin the kidneys of a STZ-induced diabetic rat model. Inthis study, the relation between insulin and Rac1 in ratDN model is investigated and demonstrated.
Renoprotective effect of insulin is substantially due toimproving hyperglycemia, however, inhibition of Rac1overexpression by insulin is also recognized important inthis regard, at least to a certain extent. This present studydemonstrated that insulin inhibited Rac1 overexpressionin the kidneys and exhibited renoprotective effect, at leastpartially, in this way.
Taken together, these findings suggest that, targetingRac1 signaling pathwaymay be a new therapeutic approachin the management of DN. However, further studies arenecessary to understand the role of Rac1 in DN.
ACKNOWLEDGMENTS
This work was supported by Dokuz Eylul UniversityResearch Foundation (2010. KB. SAG. 019). Theauthors thank Prof. Dr. Semra Kocturk for kindly pro-viding us with the cleaved caspase-3 antibody. This studywas presented as a poster in the EPHAR 2012 which washeld in Granada, Spain, 17–21 July 2012.
Declaration of interest: The authors report no con-flicts of interest. The authors alone are responsible for thecontent and writing of this article.
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