Boldine Prevents Renal Alterations in Diabetic Rats

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2013 Article ID 593672 12 pageshttpdxdoiorg1011552013593672

Research ArticleBoldine Prevents Renal Alterations in Diabetic Rats

Romina Hernaacutendez-Salinas1 Alejandra Z Vielma1 Marlene N Arismendi1

Mauricio P Boric1 Juan C Saacuteez12 and Victoria Velarde1

1 Departamento de Fisiologıa Pontificia Universidad Catolica de Chile Alameda Bernardo OrsquoHiggins No 3406513677 Santiago Chile

2 Instituto Milenio Centro Interdisciplinario de Neurociencias de Valparaıso Valparaıso Chile

Correspondence should be addressed to Juan C Saez jsaezbiopuccl and Victoria Velarde vvelardebiopuccl

Received 22 September 2013 Accepted 6 November 2013

Academic Editor Raffaele Marfella

Copyright copy 2013 Romina Hernandez-Salinas et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Diabetic nephropathy alters both structure and function of the kidney These alterations are associated with increased levels ofreactive oxygen species matrix proteins and proinflammatory molecules Inflammation decreases gap junctional communicationand increases hemichannel activity leading to increased membrane permeability and altering tissue homeostasis Since currenttreatments for diabetic nephropathy do not prevent renal damage we postulated an alternative treatment with boldine an alkaloidobtained from boldo with antioxidant anti-inflammatory and hypoglycemic effects Streptozotocin-induced diabetic and controlrats were treated or not treated with boldine (50mgKgday) for ten weeks In addition mesangial cells were cultured undercontrol conditions or in high glucose concentration plus proinflammatory cytokines with or without boldine (100120583molL) Boldinetreatment in diabetic animals prevented the increase in glycemia blood pressure renal thiobarbituric acid reactive substances andthe urinary proteincreatinine ratio Boldine also reduced alterations in matrix proteins andmarkers of renal damage In mesangialcells boldine prevented the increase in oxidative stress the decrease in gap junctional communication and the increase in cellpermeability due to connexin hemichannel activity induced by high glucose and proinflammatory cytokines but did not block gapjunction channels Thus boldine prevented both renal and cellular alterations and could be useful for preventing tissue damage indiabetic subjects

1 Introduction

Diabetic nephropathy (DN) is considered a microvascularcomplication of diabetes It is characterized by a chronicinjury to renal tissue mainly glomerular structures DN is themajor complication associated with type I diabetes mellitusaffecting approximately 30ndash40 of diabetic patients and isthe leading cause of end-stage renal disease [1]

There are several factors involved in the developmentof diabetic glomerulopathy but the main initiator of thisdisease is chronic hyperglycemia which triggers nonen-zymatic protein glycosylation and increased production ofreactive oxygen species increasing oxidative stress and favor-ing coagulation and fibrotic events [2] Hyperglycemia alsosensitizes the vessel wall of efferent arterioles to the action ofvasoconstrictors which together with an increase in vascular

permeability lead to hyperfiltration Also in the progres-sion of DN persistent proteinuria glomerular hypertrophymesangial expansion and tubulointerstitial fibrosis occurwhich lead to partial or total loss of renal function [3 4]

In diabetes and in different cell types cultured in highglucose concentration (from now and on named as high glu-cose) an increase in the inflammatory response is observedThis response is usually initiated by cytokine release andsubsequent attraction of macrophages important actors inthe progression of DN [5 6] In this and other diseases whereinflammation is present the expression of connexins (Cxs)including Cx43 is altered and increased activity of Cx formedhemichannels is observed The latter alterations have beenlinked to a reduction in gap junction activity (essential forintercellular communication) and have been considered arisk factor for certain diseases [7 8]

2 Journal of Diabetes Research

In addition TNF-120572 and IL-1120573 two proinflammatorycytokines reduce the activity of gap junctions [9 10] andincrease the activity of hemichannels in cortical astrocytes[11ndash13] Even more FGF-1 a growth factor that is ele-vated during diabetes increases the activity of hemichannelsformed by Cx43 [14 15] In cocultures of astrocytes andmicroglia proinflammatory conditions decrease gap junc-tional communication and increase membrane permeabilityeffect attributed to an increased hemichannel activity [12]Finally it has been postulated thatCxs are increased in certaindiseases that involve renal damage [16] Consequently Cxhemichannels have been proposed to be possible targets fornew therapies

As mentioned above reactive oxygen species are increas-ed in diabetes and oxidative stress induces opening of Cx43hemichannels present in the cell membrane [17] Interest-ingly the effect of reducing agents such as dithiothreitoldepends on the redox state of hemichannels Dithiothreitolinduces closure of oxidized channels but opens hemichannelsunder resting conditions that are preferentially in a reducedstate [18] In this sense a blocking agent that closes hemichan-nels both in the oxidized and reduced state should provide acomparative advantage over those that only affect the activityof hemichannels by redox changes

Currently to prevent the progression of DN patients areadvised to strictly control their glycemia and blood pressure[19] but this treatment is not enough to improve renalfunction In this paper we propose the complementary use ofboldine an alkaloid obtained from Peumus boldus to preventthe development of DN This alkaloid has been proposedto be useful in the treatment of certain diseases involvingfree radicals such as atherosclerosis ischemia-reperfusionand inflammatory diseases among others [20] Furthermoreour preliminary studies have identified boldine as a Cxhemichannel blocker through amechanism not yet identifiedbut independent of its antioxidant property Therefore wepropose that treatment with boldine could preserve a normalglomerular filtration and a tubular function during diabetesby blocking Cx hemichannels

2 Methods

21 Boldine Purification The hydrochloride form of boldinewas prepared by Harting company (Santiago Chile) andwas obtained from boldorsquos bark (Peumus boldus MolinaMonimiaceae) following a procedure described by Urzuaand Acuna [21] with minor modifications The purity of thealkaloid was checked by HPLC (high-performance liquidchromatography) analysis (99) as described previously [22]

22 Animals and Experimental Procedures Male Sprague-Dawley (SD) rats (180ndash200 g) bred in the bioterium ofthe Facultad de Ciencias Biologicas Pontificia UniversidadCatolica de Chile were used Rats were separated in threegroups (1) diabetic rats that received a single injectionof streptozotocin (45mgKg body weight IP) (2) diabeticrats additionally treated with boldine (diabetic + boldine)(50mgkg body weight in 1mL via gavage daily) for the last

10 weeks of experimentation and (3) rats from the controlgroup that only received the vehicle for the drug (1mL 01Mcitrate buffer pH 45 by gavage daily) Animal protocolswere approved by the Bioethical Committee of the PontificiaUniversidad Catolica de Chile and were in accordance withthe Guide for the Care and Use of Laboratory Animalsendorsed by the American Physiological Society Animalswere kept for 15weeks in a roomwith an ambient temperature(22ndash25∘C) 12 12 hours light dark cycles and food and waterad libitum under the supervision of a veterinarian

23 Renal Function To determine physical parameters andrenal function animals were weighed and blood and urinesampleswere obtained every 5weeks To obtain urine samplesanimals were housed in individual metabolic cages for 24 hDuring this time period they were deprived of food but hadwater ad libitum

Blood samples were taken from the tail vein under isoflu-rane anesthesia To measure blood glucose Accutrend bloodstrips were used Proteinuria wasmeasured using themethodof Bradford [23] and creatininuria was measured using akinetic kit from Valtek Diagnostics Laboratory (SantiagoChile)

At the end of each treatment animals were sacrificedunder anesthesia (ketamine 90mgkgxylazine 10mgkg IP)Kidneys were removed and cut into 5mm slices Part ofthe tissue was immediately frozen in liquid nitrogen forprotein analysis and part was fixed in Bouinrsquos solution forimmunohistochemistry

24 Measurement of Osmolarity AnOsmomat 030 osmome-ter (Gonotec GmbH Berlin Germany) was used to measureosmolarity in 50 120583L of either plasma or urine according tothe manufacturerrsquos instructions

25 TBARSMeasurement Levels of thiobarbituric acid reac-tive substances (TBARS) were estimated using the methoddescribed by Ramanathan et al [24] with slight modifica-tions Kidney homogenate supernatantsweremixedwith SDS(8wv) TBA (08wv) and acetic acid (20 vv) andheated for 60min at 90∘C Precipitatedmaterial was removedby centrifugation and the absorbance of the supernatant wasdetermined at 532 nm Levels of TBARSwere calculated usinga calibration curve with malondialdehyde (MDA Sigma-Aldrich Saint Louis USA)

26 Blood Pressure Measurement Blood pressure was mea-sured in the tail of conscious rats using a computerized 20390CODA Kent Science instrument (Torrington CT) in a roomat 25∘Cwith noise and light control Rats were trained for onemonth before the experimental measurements

27 Tissue Processing and Immunohistochemical AnalysisKidney samples (5mm thick) were fixed by immersion inBouinrsquos solution for 24 h at room temperature Each tissuesample was dehydrated and embedded in Paraplast Plus(Monoject Scientific St Louis MO) and serial sections 5120583mthick were obtained with a rotary microtome and mounted

Journal of Diabetes Research 3

on glass slides Some of these sections were stained withhematoxylin-eosin (HampE) to evaluate tissuemorphology andwith periodic acid-Schiff (PAS) staining to identify glyco-gen and glycoproteins Immunostaining was also performedusing indirect immunoperoxidase technique to localize col-lagen III and 120572-smooth muscle actin (SMA) Briefly tissuesections were deparaffinized rehydrated washedwith 005Mphosphate buffered saline (PBS) pH 76 and incubated withprimary antibody against collagen III (1 500) or 120572-SMA(1 500) overnight at 22∘C The secondary antibody and thecorresponding peroxidase anti-peroxidase complex (PAP)were sequentially applied for 30min each at 22∘C Theimmunoperoxidase reaction was visualized by incubatingsections in 01 (wv) of 331015840-diaminobenzidine and 003hydrogen peroxide The antibodies and PAP complex werediluted in Tris-phosphate-saline (TPS) buffer containing025 Triton X-100 and 07 (wv) 120582-carrageenan The sec-tions were washed with PBS between incubations contrastedwith hematoxylin and dehydratedThe images were acquiredwith a Nikon Eclipse E600 microscope equipped with aNikon DXM1200 digital camera

28 Cell Culture The cell line MES-13 derived from mesan-gial cells (CRL-1927 from ATCC) was cultured for 48 hin a mixture of DMEM (containing either 5mmolL or25mmolL glucose) and F-12 (2 1) giving a final glucoseconcentration of 75mmolL for control and 20mmolL forhigh glucose respectively These media were supplementedwith 10 FBS 100 UmL penicillin 100120583gmL streptomycinand 0025 120583gmL Fungizone (amphotericin B) To the highglucose medium TNF-120572 (10 ngmL) and IL-1120573 (10 ngmL)were added before the last 6 h of culture to mimic a diabeticenvironment Boldine (100120583molL)was added during the last24 h of treatment

29 Dye Uptake and Fluorescence Time-Lapse Imaging MES-13 cells were seeded on glass coverslips and covered witha normal glucose medium When cells reached confluencethey were washed twice in PBS pH 74 and then bathedwith Lockersquos recording saline solution (in mmolL 154 NaCl54 KCl 23 CaCl

2 and 5 HEPES pH 74) with 5 120583molL

ethidium (Etd) bromide Cells cultured on glass coverslipswere placed on an upright Olympus microscope BX 51W1Iwith water immersion objectives Changes in fluorescencewere monitored using a digital camera (12 bits QimagingBurnaby BC Canada) and the program of acquisition andimage analysis was METAFLUOR software (Universal Imag-ingCo Downingtown PAUSA) Fluorescencewas recordedevery 30 seconds for 10min at room temperature (20ndash22∘C)In the analysis the regions of interest were placed overthe cell nuclei selected at random and these regions werebackground subtracted Average slope values were comparedusing GraphPad Prism software

210 Dye Coupling MES-13 cells were seeded on glasscoverslips covered with normal glucose medium and thefunctional state of gap junctions was tested by evaluating thetransfer of Etd from one microinjected cell into neighboring

cells Cells were placed in a recording saline solution (inmolL 154 NaCl 54 KCl 23 CaCl

2 5 HEPES and 5 glucose

pH 74) and cultures were observed using an inverted micro-scope equipped with xenon arc lamp and a Nikon B filter(excitation wavelength 450ndash490 nm emission wavelengthabove 520 nm) Etd (25mmolL) was microinjected througha glassmicroelectrode Twominutes after injection cells wereobserved to determine whether dye transfer occurred Theincidence of coupling corresponds to the percentage of casesin which dye spread occurred tomore than one neighbor cellThe coupling index was calculated as the average number ofcells to which the dye had spread divided by the number ofpositive cases In all experiments the intercellular couplingwas tested by injecting a minimum of 10 cells

211 Electrophoresis and Western Blot Analysis To determinerelative levels of Cx43 MES-13 cells were lysed in RIPA withprotease inhibitors (1mgmL aminocaproic acid 1mgmLbenzamidine 02mgmL SBTI and PMSF 3mmolL) andphosphatase inhibitors (0012mgmL sodium orthovanadate446mgmL sodium pyrophosphate and 42mgmL sodiumfluoride) Lysates were centrifuged and supernatants werecollected for Western blot analysis Protein concentrationswere determined by the method of Lowry et al [25]Protein samples (50 120583g) from cells cultured under differenttreatments were separated by electrophoresis in 10 SDS-polyacrylamide gel (SDS-PAGE) and in one lane a sampleof prestained molecular weight markers (MW) was resolvedProteins were transferred to a 045 120583m PVDF membranewhich was blocked at room temperature with Tris pH 745skim milk (wv)BSA 1 (wv) Then the PVDF membranewas incubated overnight at 4∘C with anti-Cx43 (monoclonalZymed 1 500) followed by incubationwith rabbit secondaryantibody conjugated to peroxidase (polyclonal Santa CruzBiotechnology 1 1000) for 1 hThenmembrane was strippedand reblotted with antibody anti T-ERK (polyclonal SantaCruz Biotechnology 1 4000) as loading control followingthe same procedure as before

Immunoreactive bands were visualized using a chemi-luminescent reagent (Western Lightning Perkin Elmer)according to the procedure described by the supplier andKodak films X-LS The bands detected were digitized andsubjected to densitometric analysis using the program ImageJ (NIH Washington DC USA)

212 Statistical Analysis Data sets obtained from differentgroups of rats or cells under the same condition werecompared with each other by one-way analysis of vari-ance (ANOVA) followed by a Bonferronirsquos post hoc testDifferences were considered significant if 119875 le 005 Theanalyses were performedwith GraphPad Prism 5 software forWindows (1992ndash2007 GraphPad Software)

3 Results

31 Boldine Prevents Hyperglycemia and Hypertension inDiabetic Rats In order to establish a diabetic model andto study how it is affected by treatment with boldine body


Table 1 Pathophysiological status of experimental animals

Control (C) Diabetic (D) Control boldine (CB) Diabetic boldine (DB)Glycemia (mgdL) 141 plusmn 8 371 plusmn 74

lowastlowastlowast

147 plusmn 8 253 plusmn 31dagger

Weight (g) 530 plusmn 28 223 plusmn 25lowastlowastlowast

458 plusmn 22 235 plusmn 19lowastlowastlowast

Urine volume (mL) 16 plusmn 2 28 plusmn 5lowast

15 plusmn 3 24 plusmn 3

Plasma osmolality (mOsmolKg) 290 plusmn 4 310 plusmn 8 300 plusmn 6 300 plusmn 8

Urine omoles (in 24 h) 17 plusmn 1 23 plusmn 4 15 plusmn 1 24 plusmn 3

MDA plasma (nM) 50 plusmn 6 117 plusmn 9lowastlowast

57 plusmn 7 121 plusmn 22lowastlowast

MDA urine (pmoles in 24 h) 167 plusmn 20 842 plusmn 218lowastlowast


Mean blood pressure (mmHg) 116 plusmn 5 147 plusmn 12lowast


Parameters observed in control (C) diabetic (D) and control or diabetic rats treated with 50mgKg boldine (CB and DB resp) measured at the end of theexperiment Values represent the average plusmn SEM of at least 5 animals per group lowast119875 le 005 lowastlowast119875 le 001 lowastlowastlowast119875 le 0001 compared to control dagger119875 le 005compared to diabetic using one-way ANOVA followed by Bonferronirsquos post hoc test

weight mean blood pressure blood glucose urine volumeosmolality and lipid peroxidation were measured in plasmaand urine of each rat from the three abovementioned groups

Since an increased level of plasma glucose is the mainfeature in diabetes this parameter was evaluated periodicallyPlasma glucose values from all groups of animals at the endof the experiment are summarized in Table 1 The controlgroup (C) had a normal glycemia whereas the glycemia ofthe diabetic group (D) was significantly higher Interestinglythe diabetic group treated with boldine (DB) had a glycemialower than that of the diabetic group not significantlydifferent from the control group

It is well documented that animalswith experimental typeI diabetes lose weight over time due to the loss of muscle andadipose tissue At the end of the experiment rats from bothdiabetic groups (D and DB) weighted significantly less thananimals of group C with no significant differences within thetwo diabetic groups (Table 1)

Total urinary volume in a 24 h collection was significantlyincreased in the diabetic rats as compared to the controlgroup Interestingly in the diabetic group treated with bol-dine the urinary volume was slightly reduced compared tothe values of the diabetic group (Table 1)

Since high concentrations of glucose were detected inblood and urine from diabetic rats we evaluated whetherthere were changes in osmolality All groups showed similarvalues in plasma osmolality and total osmoles excreted in theurine in 24 h (Table 1)

In diabetes oxidized lipids are increased due to anincrease in oxidative stress and dyslipidemia [5] TBARSvalues in both plasma and urine were significantly higherin D and DB groups when compared with the controlgroups (Table 1) Interestingly boldine which has a potentantioxidant capacity did not modify TBARS values in thesetwo fluids as evidenced when DB was compared to D

One of the alterations that frequently occurs as a result ofor associated with diabetes is hypertension For this reasonmean blood pressurewasmeasured in conscious rats Controlrats and rats treated with boldine showed normal bloodpressure (Table 1) whereas blood pressure was significantlyincreased in the diabetic group when compared to controlInterestingly group DB showed a blood pressure similar tocontrol animals (Table 1)

Prot

einu

riac

reat

inin

uria

ControlDiabeticDiabetic + boldine

20

15

5

0

10

lowastlowastlowastlowastlowastlowast

Figure 1 Boldine prevents kidney damage in diabetic rats Thegraph shows the ratio of proteinuriacreatininuria in control(white bar) diabetic (black bar) and diabetic rats treated with50mgKgday boldine (grey bar) Each bar represents the meanvalue plusmn SEM of 5 different animals lowastlowastlowast119875 le 0001 using the one-way ANOVA test followed by Bonferronirsquos post hoc test

32 Boldine Prevents Kidney Damage in Diabetic Rats Dia-betes and in particular hyperglycemia is a risk factor forrenal function characterized by dysfunction in glomerularfiltration rate (GFR) To evaluate renal function the ratiobetween urinary proteins and urinary creatinine in thedifferent groups was evaluated Diabetic rats had significantlyhigher ratio (148 plusmn 12) when compared to control animals(17 plusmn 04) whereas diabetic rats treated with boldine showedvalues that were not significantly different from control (34plusmn10) but were significantly lower than group D (Figure 1)

33 Boldine Prevents RenalMorphological Changes in DiabeticRats In diabetes excess blood glucose triggers the increasein oxidative stress producing a homeostatic imbalance at thecellular level that alters the renal histology The structure ofkidneyswas altered in groupD as evaluated byHampE stainingwhich demonstrated dilated tubules (arrow head) increasedinterstitial matrix and thickening of basement membrane


C

D

lowast

lowast

lowast

lowast

DBlowast

(a)

C

D

lowast

lowastlowast

DB

lowast

100120583m

(b)

Figure 2 Boldine prevents the structural alterations in kidneys from diabetic rats Serial kidney sections (5120583m) were either stained with (a)hematoxylin and eosin or (b) with PAS staining The top panels correspond to kidney sections from control rats (C) the middle panels arefromdiabetic rats (D) and the bottompanels are fromdiabetic rats treatedwith 50mgKgday boldine (DB) lowast indicate clear cells arrowheadsindicate dilated tubules Note close spatial correspondence between clear cells and PAS staining Each picture is representative of at least 5different animals

in the glomerulus as compared to control (Figures 2(a) and2(b)) In addition clear cells (asterisks) were present in distaltubulesThese alterations were largely absent in kidneys fromgroup DB in which changes were restricted mainly to thepresence of few clear cells (Figure 2(a)) To determine thecontent of these clear cells we performed PAS staining thatlabels carbohydrates with an intense pink color Both D andDB groups were positive for this staining (Figure 2(b)) andlabeling colocalized with clear cells detected inHampE stainingPAS staining was abundant in group D very infrequent ingroup DB and absent in group C (Figure 2 middle bottomand top panels resp)

34 Boldine Reduces Protein Markers of Renal Damage inDiabetic Rats In diabetes an increase in kidney interstitialmatrix proteins is observed [26] Therefore we evaluatedif boldine prevents this modification in diabetic rats Sec-tions from group C stained for collagen III showed adiscreet brown staining which was limited mainly to thebasal lamina and blood vessels (Figure 3(a) top panel) Incontrast group D showed a large increase in brown staininglocated mainly in peritubular areas (Figure 3(a) middlepanel) Interestingly groupDB showed a staining very similarto that detected in control animals (Figure 3(a) bottompanel)


C

D

DB

(a)

DB

C

D

100120583m

(b)

Figure 3 Boldine decreasesmarkers of kidney damage in diabetic rats Panels show immunohistochemistry for collagen III (a) and 120572-smoothmuscle actin (SMA) (b) Top panels show fields of kidney sections from control (C) rats Central panels show fields of kidney sections fromdiabetic rats (D) and the bottom panels are from diabetic rats treated with 50mgKgday boldine (DB) Each picture is representative of atleast 5 different animals Arrowheads show staining in vascular tissue

Presence of myofibroblasts characterized by the reactiv-ity to120572-SMA has been detected in the interstitiumof diabetickidneys [27 28] As expected kidneys from group C onlypresented 120572-actin from smooth muscle (120572-SMA) stainingin blood vessels more specifically in smooth muscle cells(Figure 3(b) top panel arrowhead) However kidneys fromgroupD in addition to the vascular labeling showed a strong

labeling in peritubular areas (Figure 3(b) middle panel)Thisreactivity was clearly reduced in the DB group (Figure 3(b)bottom panel)

35 Boldine Prevents the Increase in Lipid Peroxidation inKidneys from Diabetic Rats and in Cells in a Diabetic Environ-ment We also quantified TBARS in renal homogenates as


6

4

2

0


lowastlowast

MD

A (120583

mol

g)

Figure 4 Boldine prevents lipid peroxidation in kidneys fromdiabetic rats Graph showing levels of TBARS (MDA) in kidneysfrom control rats (white bar) diabetic rats (black bar) and diabeticrats treated with 50mgKgday boldine (grey bar) Values areexpressed as 120583mol of MDA per g of renal tissue Each bar representsthe mean value plusmn SEM of tissue from 7 independent animals lowast119875 le005 using the one-way ANOVA test followed by Bonferronirsquos posthoc test

an indication of the tissue redox status TBARS from groupD (41 plusmn 04 120583molg of kidney) were significantly higher thanthose in control samples (27plusmn01 120583molg) whereas in groupDB TBARS were similar to control values (29 plusmn 02 120583molg)(Figure 4) Similarly TBARS were measured in the super-natant of mesangial cells grown for 48 h in control mediumor in high glucose medium plus proinflammatory cytokinesmimicking the diabetic environment The content of TBARSin supernatant of cells grown in medium containing highglucose and proinflammatory cytokines was twice that foundin cells cultured under control conditions (60 plusmn 05 versus28 plusmn 06 120583molg protein) (Figure 5) However the increasein TBARS was fully prevented in cells maintained in highglucose and proinflammatory cytokines treated with boldinefor the last 24 h (Figure 5)

36 Boldine Prevents the Increase in Membrane Permeabilityof Cells Grown in aDiabetic Environment Under resting con-ditions Cx hemichannels of different cells in culture show alow open probability but in the presence of proinflammatoryconditions they open even at negative membrane potentialand in the presence of extracellular divalent cations [29ndash31] contributing to a deregulation in the electrochemicalgradient across the cell membrane In diabetes proinflam-matory cytokines are increased [10] For this reason it wasexpected that activity of renal Cx hemichannels could beincreased Since it is difficult to evaluate the activity ofhemichannels in a tissue we estimated the Cx hemichannelactivity by determining the dye uptake rate from time-lapsemeasurements of Etd uptake in cultured mesangial cells(Figure 6(a)) The rate of Etd uptake (slope) of control cells

6

8

4

2

0

Normal glucoseHigh glucose + cytokines High glucose + cytokines + boldine

lowastlowast

MD

A (120583

mol

g)

Figure 5 Boldine prevents lipid peroxidation in mesangial cellsgrown in high glucose and proinflammatory cytokines Graph show-ing levels of TBARS (MDA) in the culturemediumofmesangial cellscultured for 48 h under normal glucose (white bar) high glucose+ cytokines (TNF-120572 (10 ngmL) and IL-1120573 (10 ngmL)) (black bar)or high glucose + cytokines treated with 100120583M boldine during thelast 24 h (grey bar) Values are expressed as 120583mol of MDA per g ofprotein in the culture medium Each bar represents the mean valueplusmn SEM of 4 independent experiments lowastlowast119875 le 001 using one-wayANOVA test followed by Bonferronirsquos post hoc test

incubated in normal glucosewas 017plusmn001AUmin whereascells grown in high glucose plus cytokines showed a slopeof 025 plusmn 005AUmin which was significantly higher thanthat of control cells (Figure 6(b)) In cells incubated in highglucose + cytokines and treated with 100 120583molL boldine therate of Etd uptake was not different from that of control cells(013 plusmn 001AUmin)

37 Boldine Prevents the Reduction in Cell Coupling via GapJunctions in Mesangial Cells Cultured in a Diabetic Envi-ronment Gap junctions mediate direct intercellular com-munication of ions and small molecules including secondmessengers [32]This type of intercellular communications isfrequently reduced by proinflammatory conditions [12 29ndash31] Mesangial cells cultured for 48 h in high glucose pluscytokines showed a decreased coupling incidence of 57 plusmn 8from a control value of 80 plusmn 8 However in cells incubatedwith 100 120583molL boldine for 24 h the coupling incidence was95 plusmn 3 indicating that boldine reverted the reduction incoupling induced by these diabetic conditions (Figure 7(a))In addition in cells cultured in high glucose plus cytokinesand boldine the number of coupled cells (coupling index)increased to 44 plusmn 07 compared to 13 plusmn 02 in cells grownin normal glucose and 09plusmn02 in cells grown in high glucosefor 48 h (Figures 7(b) and 7(c))

38 Boldine Increases the Relative Levels of Cx43 in MesangialCells Grown in High Glucose Plus Proinflammatory CytokinesIn inflammatory conditions changes in the expression of Cxs



Ethi

dium

upt

ake (

AU)

3

2

0

1

Time (min)0 2 4 6 8 10

lowast

(a)

Ethi

dium

upt

ake r

ate (

AUm

in)

04

03

02

01

00


lowastlowast

(b)

Figure 6 Boldine reduces the increase in Etd uptake induced by high glucose + cytokines in mesangial cells (a) The graph shows time-lapse measurements of fluorescence intensity of Etd bound to nucleic acids of mesangial cells cultured for 48 h under normal glucose (whitetriangle) high glucose + cytokines (TNF-120572 (10 ngmL) and IL-1120573 (10 ngmL)) (black circles) and high glucose + cytokines treatedwith 100 120583MBoldine for the last 24 h (grey square) (b) Each bar represents themean value of the rate of Etd uptake obtained from time-lapse curves Meanplusmn SEM for 4 independent experiments Normal glucose (White bar) high glucose + cytokine (black bar) or high glucose + cytokine with100120583M boldine (grey bar) for 24 h lowast119875 le 005 using one-way ANOVA test followed by Bonferronirsquos post hoc test

have been found [33] Cx43 is one of the most importantgap junction subunits found in the glomerulus [34] For thisreason the level of this protein was evaluated in mesangialcells cultured under diabetic conditions In MES-13 cellsmaintained in a diabetic environment a slight but not signifi-cant increase in relative protein levels of Cx43 (143plusmn20)wasobserved (Figure 8) Interestingly in cells incubated in highglucose + cytokines in the presence of 100 120583molL boldinethe increase in Cx43 levels was even higher (199 plusmn 26) andstatistically different from control (Figure 8)

4 Discussion

Previous studies have reported that boldine prevents theinflammatory response in rats [35] and treatment of diabeticrats with boldine reduces blood glucose levels [36] anddecreases oxidative stress [37] This study confirmed thehypoglycemic and antioxidant effects of boldine in diabeticrats however the most innovative findings of this researchare the protective effect of boldine on renal parenchyma indiabetic rats and its inhibitory effect on Cx Hemichannels

The reduction in glycemia observed in diabetic ratstreated with boldine could be due to an inhibitory effecton 120572-glucosidase [38] This enzyme acts upon 14-alphabonds breaking down starch and disaccharides to glucoseIn addition boldine might reduce blood glucose by its actionas an insulin sensitizer helping to increase glucose utilizationin skeletal muscle [36] or by reducing glucose reabsorption inrenal tubules

The elevated blood pressure observed in diabetic ratscould be due to vascular damage and renal damage caused

by the increase in oxidative stress The preclusion of anincrease in blood pressure in the diabetic group treated withboldine is in agreement with this notion since boldine hasbeen postulated as a potent antioxidant [39] The findingthat boldine prevented the increased tissue TBARS levels butnot urinary TBARS in diabetic rats was not expected Thisobservation might have at least two explanations One ofthem is that in diabetes in the absence of boldine TBARScould accumulate in the cell membrane of renal cells whencompared to urine or plasma Alternatively it is possible thatkidneys are capturing by reabsorption and accumulating intorenal cells filtered TBARS generated in other organs and forthis reason it is easier to observe significant differences inkidney than in body fluids In contrast in the presence ofboldine the accumulation of TBARS in renal tissue is lowerbut they are still eliminated to the urine

In addition to the effect in oxidative stress there are sev-eral other ways by which boldine could prevent the increasein blood pressure First boldine might act as inhibitor ofacetylcholinesterase [38] If this is so boldine could increaseacetylcholine concentration at the vascular wall that in turnmay contribute to the relaxation of blood vessels and conse-quently to the reduction in blood pressure Second boldinemight prevent renal damage which could help maintainingthe normal balance of the vasoactive systems present inkidneys Two major hormonal systems involved in bloodpressure regulation are present in kidneysThey are the reninangiotensin system that induces vasoconstriction and waterand salt retention and the kallikrein kinin system whichinduces vasorelaxation and water and sodium excretion [1940] The main enzymes of these systems are localized intubular segments of the kidney By preventing impairment


100

80

40

20

0

60

Cou

plin

g in

cide

nce (

)


lowast

(a)

Cou

plin

g in

dex

8

6

4

2

0

lowastlowast

lowastlowastlowast


(b)

20120583m

(c)

Figure 7 Boldine increases gap junctional coupling betweenmesangial cells Coupling incidence (a) and coupling index (b) were evaluated inconfluent mesangial cell cultures for 48 h under different conditions using the dye (Ethidium bromide) coupling technique normal glucose(white bar) high glucose + cytokine (TNF-120572 (10 ngmL) and IL-1120573 (10 ngmL)) (black bar) or high glucose + cytokine treated with 100120583Mboldine for the last 24 h (grey bar) Each bar represents the mean value plusmn SEM of 5 independent experiments In each experiment the dyewas microinjected into at least 10 cells lowastlowast119875 le 001 lowastlowastlowast119875 le 0001 using the one-way ANOVA test followed by Bonferronirsquos post hoc test (c)Etd was microinjected into the brightest cell (arrow) and diffused to neighboring cells Normal glucose (left bottom) high glucose + cytokine(middle bottom) and high glucose + cytokine and treated with 100 120583Mboldine (right bottom) for 24 hThe top panels are the correspondingphase views Scale bar = 20 120583m

in renal tubular epithelia boldine might contribute favorablyto maintain a normal balance of these two systems andthus a normal pressure is maintained in these animals Insupport of this notion in diabetic rats treated with boldineproteinuria and creatininuria remainedwithin normal rangesand morphological alterations in kidney such as thickeningof the glomerular basement membrane tubular dilation andaccumulation of glycogen in the distal tubules were largelyprevented Also a decrease in fibrosis and inflammatory cellinfiltration were observed in the group of diabetic rats treatedwith boldine

Although the kidney receives an important proportion ofthe cardiac output this tissue is very sensitive to hypoxiawhich produces a fast death of tubular cells activatingan inflammation response characterized by macrophageinfiltration and matrix accumulation Therefore boldine

could also be protecting the renal tissue by preventing thehypoxia-induced damage Several authors have postulated animportant action of Cx43 in preconditioning [41] In thissense the increase in Cx43 in response to boldine couldexplain in part the protective effect of this alkaloid

We propose that prevention of the damage is in part dueto the blockade of hemichannels and increased gap junctionactivity As it has been reported inflammatory conditionslead to increased opening of hemichannels promoting theloss in homeostasis and impaired cell function [12 29ndash3142] Our results show that mesangial cells kept in a diabeticenvironment presented an increase in hemichannel activityand a loss of cell communication via gap junctions whichwasprevented by treatment with boldine resulting in an increasein the number of coupled cells an effect that is essential forthe proper function of the kidney The further increase in


T-ERK

250

200

150

100

50

0


lowast

Cx43

T-E

RK (

of c

ontro

l)

Cx43

Figure 8 Boldine increases the relative Cx43 levels in mesangialcells grown in high glucose plus proinflammatory cytokines Graphshowing the relative levels of Cx43 under different conditions nor-mal glucose (white bar) high glucose + proinflammatory cytokines(TNF-120572 (10 ngmL) and IL-1120573 (10 ngmL)) (black bar) or highglucose + cytokines treated with 100120583M boldine (grey bar) for24 h Each bar represents the mean value plusmn SEM of 4 independentexperiments lowast119875 le 005 using the one-way ANOVA test followed byBonferronirsquos post hoc test

Cx43 protein levels in the presence of boldine could accountfor newly synthesized gap junctions and explain the increasein the number of coupled cells

In conclusion the alterations found in diabetic rats ashyperglycemia hypertension and renal damage were pre-vented in diabetic rats treated with boldine suggesting thatthe harmful effects generated by increased oxidative stressand proinflammatory state in diabetes were diminished withboldine not only by its antioxidant action but also due tothe maintenance of cellular homeostasis by preventing theopening of hemichannels and keeping cells coupled to eachother

Ethical Standards

Animal protocols were approved by the ethical committeeof the Pontificia Universidad Catolica de Chile and were inaccordancewith theGuide for theCare andUse of LaboratoryAnimals endorsed by the American Physiological Society

Conflict of Interests

Romina Hernandez-Salinas Alejandra Z Vielma MarleneN Arismendi Mauricio P Boric Juan C Saez and Victoria

Velarde declare that there is no conflict of interests regardingthe publication of this paper

Authorsrsquo Contribution

The experiments were conceived and designed by RominaHernandez-Salinas Juan C Saez Victoria Velarde andMauricio P Boric They were performed by RominaHernandez-Salinas Alejandra Z Vielma and Marlene NArismendi The data was analyzed by Romina Hernandez-Salinas Juan C Saez Victoria Velarde and Mauricio PBoric The reagents materials and analysis tools werecontributed by Romina Hernandez-Salinas Juan C Saezand Victoria Velarde The paper was written by RominaHernandez-Salinas Juan C Saez and Victoria Velarde

Acknowledgments

The expert technical assistance of Kenji Shoji Paola SotoLeonardo Cigarra Carlos Cespedes and Marıa Alcoholadois gratefully acknowledged This study was supported byGrants from Fondo de Fomento al Desarrollo Cientıfico yTecnologico (FONDEF)D07I1086 andAnillo ACT71 (to Vic-toria Velarde and Juan C Saez) and a PhD Fellowship fromthe Chilean Comision Nacional de Investigacion Cientıficay Tecnologica (CONICYT) (to Romina Hernandez-Salinas)The data of this paper are from a thesis submitted in partialfulfillment of the requirements for the degree of Doctor inPhysiological Sciences (Romina Hernandez-Salinas) at thePontificia Universidad Catolica de Chile

References

[1] T Nakagawa K Tanabe B P Croker et al ldquoEndothelialdysfunction as a potential contributor in diabetic nephropathyrdquoNature Reviews Nephrology vol 7 no 1 pp 36ndash44 2011

[2] M C Thomas ldquoAdvanced glycation end productsrdquo Contribu-tions to Nephrology vol 170 pp 66ndash74 2011

[3] R Choi B H Kim J Naowaboot et al ldquoEffects of ferulic acidon diabetic nephropathy in a rat model of type 2 diabetesrdquoExperimental and Molecular Medicine vol 43 no 12 pp 676ndash683 2011

[4] J Kang X-S Dai T-B Yu B Wen and Z-W Yang ldquoGlycogenaccumulation in renal tubules a key morphological change inthe diabetic rat kidneyrdquo Acta Diabetologica vol 42 no 2 pp110ndash119 2005

[5] K Alexandraki C Piperi C Kalofoutis J Singh A AlaverasandA Kalofoutis ldquoInflammatory process in type 2 diabetes therole of cytokinesrdquo Annals of the New York Academy of Sciencesvol 1084 pp 89ndash117 2006

[6] J-W Yoon and H-S Jun ldquoCellular and molecular pathogenicmechanisms of insulin-dependent diabetes mellitusrdquo Annals ofthe New York Academy of Sciences vol 928 pp 200ndash211 2001

[7] M Fernandez-Cobo C Gingalewski and A de Maio ldquoExpres-sion of the connexin 43 gene is increased in the kidneys and thelungs of rats injected with bacterial lipopolysacchariderdquo Shockvol 10 no 2 pp 97ndash102 1998

[8] J H-C Lin N Lou N Kang et al ldquoA central role of connexin43 in hypoxic preconditioningrdquoThe Journal of Neuroscience vol28 no 3 pp 681ndash695 2008


[9] H E Gonzalez E A Eugenın G Garces et al ldquoRegulationof hepatic connexins in cholestasis possible involvement ofKupffer cells and inflammatory mediatorsrdquoAmerican Journal ofPhysiology Gastrointestinal and Liver Physiology vol 282 no 6pp G991ndashG1001 2002

[10] W Meme C-F Calvo N Froger et al ldquoProinflammatorycytokines released from microglia inhibit gap junctions inastrocytes potentiation by 120573-amyloidrdquoThe FASEB Journal vol20 no 3 pp 494ndash496 2006

[11] J E Contreras H A Sanchez E A Eugenin et al ldquoMetabolicinhibition induces opening of unapposed connexin 43 gap junc-tion hemichannels and reduces gap junctional communicationin cortical astrocytes in culturerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no1 pp 495ndash500 2002

[12] M A Retamal N Froger N Palacios-Prado et al ldquoCx43hemichannels and gap junction channels in astrocytes areregulated oppositely by proinflammatory cytokines releasedfrom activated microgliardquo The Journal of Neuroscience vol 27no 50 pp 13781ndash13792 2007

[13] L Vergara X Bao E Bello-Reuss and L Reuss ldquoDo connexin43 gap-junctional hemichannels activate and cause cell damageduring ATP depletion of renal-tubule cellsrdquo Acta PhysiologicaScandinavica vol 179 no 1 pp 33ndash38 2003

[14] J M Garre M A Retamal P Cassina et al ldquoFGF-1 inducesATP release from spinal astrocytes in culture and opens pan-nexin and connexin hemichannelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no52 pp 22659ndash22664 2010

[15] K A Schalper N Palacios-Prado M A Retamal K F ShojiA D Martınez and J C Saez ldquoConnexin hemichannel compo-sition determines the FGF-1-induced membrane permeabilityand free [Ca2+]i responsesrdquo Molecular Biology of the Cell vol19 no 8 pp 3501ndash3513 2008

[16] J Toubas S Beck A-L Pageaud et al ldquoAlteration of connexinexpression is an early signal for chronic kidney diseaserdquoAmerican Journal of Physiology Renal Physiology vol 301 no1 pp F24ndashF32 2011

[17] M A Retamal C J Cortes L Reuss M V L Bennett andJ C Saez ldquoS-nitrosylation and permeation through connexin43 hemichannels in astrocytes induction by oxidant stressand reversal by reducing agentsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no12 pp 4475ndash4480 2006

[18] M A Retamal K A Schalper K F Shoji M V L Bennett andJ C Saez ldquoOpening of connexin 43 hemichannels is increasedby lowering intracellular redox potentialrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 104 no 20 pp 8322ndash8327 2007

[19] A A Taylor H Siragy and S Nesbitt ldquoAngiotensin receptorblockers pharmacology efficacy and safetyrdquo Journal of ClinicalHypertension vol 13 no 9 pp 677ndash686 2011

[20] H Speisky and B K Cassels ldquoBoldo and boldine an emergingcase of natural drug developmentrdquo Pharmacological Researchvol 29 no 1 pp 1ndash12 1994

[21] A Urzua and P Acuna ldquoAlkaloids from the bark of Peumusboldusrdquo Fitoterapia vol 54 no 4 pp 175ndash177 1983

[22] H Speisky B K Cassels S Nieto A Valenzuela and L JNunez-Vergara ldquoDetermination of boldine in plasma by high-performance liquid chromatographyrdquo Journal of Chromatogra-phy vol 612 no 2 pp 315ndash319 1993

[23] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[24] L Ramanathan N P Das and Q-T Li ldquoStudies on lipidoxidation in fish phospholipid liposomesrdquo Biological TraceElement Research vol 40 no 1 pp 59ndash70 1994

[25] O H Lowry N J Roserbrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[26] A Mathew R Cunard and K Sharma ldquoAntifibrotic treatmentand other new strategies for improving renal outcomesrdquo Con-tributions to Nephrology vol 170 pp 217ndash227 2011

[27] M Essawy O Soylemezoglu E CMuchaneta-Kubara J Short-land C B Brown and A M El Nahas ldquoMyofibroblasts andthe progression of diabetic nephropathyrdquo Nephrology DialysisTransplantation vol 12 no 1 pp 43ndash50 1997

[28] J Li X Qu and J F Bertram ldquoEndothelial-myofibroblasttransition contributes to the early development of diabetic renalinterstitial fibrosis in streptozotocin-induced diabetic micerdquoAmerican Journal of Pathology vol 175 no 4 pp 1380ndash13882009

[29] J A Orellana D E Hernandez P Ezan et al ldquoHypoxia inhigh glucose followed by reoxygenation in normal glucosereduces the viability of cortical astrocytes through increasedpermeability of connexin 43 hemichannelsrdquo Glia vol 58 no 3pp 329ndash343 2010

[30] J A Orellana K F Shoji V Abudara et al ldquoAmyloid 120573-induceddeath in neurons involves glial and neuronal hemichannelsrdquoThe Journal of Neuroscience vol 31 no 13 pp 4962ndash4977 2011

[31] J A Orellana X F Figueroa H A Sanchez S Contreras-Duarte V Velarde and J C Saez ldquoHemichannels in the neu-rovascular unit and white matter under normal and inflamedconditionsrdquo CNS and Neurological Disorders vol 10 no 3 pp404ndash414 2011

[32] G Kanaporis P R Brink and V Valiunas ldquoGap junction per-meability selectivity for anionic and cationic probesrdquoAmericanJournal of Physiology Cell Physiology vol 300 no 3 pp C600ndashC609 2011

[33] P Klee F Allagnat H Pontes et al ldquoConnexins protectmouse pancreatic 120573 cells against apoptosisrdquo Journal of ClinicalInvestigation vol 121 no 12 pp 4870ndash4879 2011

[34] F Hanner C M Sorensen N-H Holstein-Rathlou and JPeti-Peterdi ldquoConnexins and the kidneyrdquo American Journal ofPhysiology Regulatory Integrative and Comparative Physiologyvol 298 no 5 pp R1143ndashR1155 2010

[35] N Backhouse C Delporte M Givernau B K Cassels AValenzuela andH Speisky ldquoAnti-inflammatory and antipyreticeffects of boldinerdquo Agents and Actions vol 42 no 3-4 pp 114ndash117 1994

[36] T-C Chi S-S Lee and M-J Su ldquoAntihyperglycemic effect ofaporphines and their derivatives in normal and diabetic ratsrdquoPlanta Medica vol 72 no 13 pp 1175ndash1180 2006

[37] Y Y Jang J H Song Y K Shin E S Han and C S LeeldquoProtective effect of boldine on oxidative mitochondrial dam-age in streptozotocin-induced diabetic ratsrdquo PharmacologicalResearch vol 42 no 4 pp 361ndash371 2000

[38] A Mollataghi E Coudiere A H A Hadi et al ldquoAnti-acetylcholinesterase anti-120572-glucosidase anti-leishmanial andanti-fungal activities of chemical constituents of Beilschmiediaspeciesrdquo Fitoterapia vol 83 no 2 pp 298ndash302 2012


[39] J Fernandez P Lagos P Rivera and E Zamorano-PonceldquoEffect of boldo (Peumus boldus Molina) infusion on lipoper-oxidation induced by cisplatin in mice liverrdquo PhytotherapyResearch vol 23 no 7 pp 1024ndash1027 2009

[40] C P Vio S Loyola andVVelarde ldquoLocalization of componentsof the kallikrein-kinin system in the kidney relation to renalfunction state of the art lecturerdquo Hypertension vol 19 no 2pp II10ndashII16 1992

[41] G Lu H K Haider A Porollo and M Ashraf ldquoMitochondria-specific transgenic overexpression of connexin-43 simulatespreconditioning-induced cytoprotection of stem cellsrdquo Cardio-vascular Research vol 88 no 2 pp 277ndash286 2010

[42] D Johansen V Cruciani R Sundset K Ytrehus and S-OMikalsen ldquoIschemia induces closure of gap junctional channelsand opening of hemichannels in heart-derived cells and tissuerdquoCellular Physiology and Biochemistry vol 28 no 1 pp 103ndash1142011


In addition TNF-120572 and IL-1120573 two proinflammatorycytokines reduce the activity of gap junctions [9 10] andincrease the activity of hemichannels in cortical astrocytes[11ndash13] Even more FGF-1 a growth factor that is ele-vated during diabetes increases the activity of hemichannelsformed by Cx43 [14 15] In cocultures of astrocytes andmicroglia proinflammatory conditions decrease gap junc-tional communication and increase membrane permeabilityeffect attributed to an increased hemichannel activity [12]Finally it has been postulated thatCxs are increased in certaindiseases that involve renal damage [16] Consequently Cxhemichannels have been proposed to be possible targets fornew therapies

As mentioned above reactive oxygen species are increas-ed in diabetes and oxidative stress induces opening of Cx43hemichannels present in the cell membrane [17] Interest-ingly the effect of reducing agents such as dithiothreitoldepends on the redox state of hemichannels Dithiothreitolinduces closure of oxidized channels but opens hemichannelsunder resting conditions that are preferentially in a reducedstate [18] In this sense a blocking agent that closes hemichan-nels both in the oxidized and reduced state should provide acomparative advantage over those that only affect the activityof hemichannels by redox changes

Currently to prevent the progression of DN patients areadvised to strictly control their glycemia and blood pressure[19] but this treatment is not enough to improve renalfunction In this paper we propose the complementary use ofboldine an alkaloid obtained from Peumus boldus to preventthe development of DN This alkaloid has been proposedto be useful in the treatment of certain diseases involvingfree radicals such as atherosclerosis ischemia-reperfusionand inflammatory diseases among others [20] Furthermoreour preliminary studies have identified boldine as a Cxhemichannel blocker through amechanism not yet identifiedbut independent of its antioxidant property Therefore wepropose that treatment with boldine could preserve a normalglomerular filtration and a tubular function during diabetesby blocking Cx hemichannels

2 Methods

21 Boldine Purification The hydrochloride form of boldinewas prepared by Harting company (Santiago Chile) andwas obtained from boldorsquos bark (Peumus boldus MolinaMonimiaceae) following a procedure described by Urzuaand Acuna [21] with minor modifications The purity of thealkaloid was checked by HPLC (high-performance liquidchromatography) analysis (99) as described previously [22]

22 Animals and Experimental Procedures Male Sprague-Dawley (SD) rats (180ndash200 g) bred in the bioterium ofthe Facultad de Ciencias Biologicas Pontificia UniversidadCatolica de Chile were used Rats were separated in threegroups (1) diabetic rats that received a single injectionof streptozotocin (45mgKg body weight IP) (2) diabeticrats additionally treated with boldine (diabetic + boldine)(50mgkg body weight in 1mL via gavage daily) for the last

10 weeks of experimentation and (3) rats from the controlgroup that only received the vehicle for the drug (1mL 01Mcitrate buffer pH 45 by gavage daily) Animal protocolswere approved by the Bioethical Committee of the PontificiaUniversidad Catolica de Chile and were in accordance withthe Guide for the Care and Use of Laboratory Animalsendorsed by the American Physiological Society Animalswere kept for 15weeks in a roomwith an ambient temperature(22ndash25∘C) 12 12 hours light dark cycles and food and waterad libitum under the supervision of a veterinarian

23 Renal Function To determine physical parameters andrenal function animals were weighed and blood and urinesampleswere obtained every 5weeks To obtain urine samplesanimals were housed in individual metabolic cages for 24 hDuring this time period they were deprived of food but hadwater ad libitum

Blood samples were taken from the tail vein under isoflu-rane anesthesia To measure blood glucose Accutrend bloodstrips were used Proteinuria wasmeasured using themethodof Bradford [23] and creatininuria was measured using akinetic kit from Valtek Diagnostics Laboratory (SantiagoChile)

At the end of each treatment animals were sacrificedunder anesthesia (ketamine 90mgkgxylazine 10mgkg IP)Kidneys were removed and cut into 5mm slices Part ofthe tissue was immediately frozen in liquid nitrogen forprotein analysis and part was fixed in Bouinrsquos solution forimmunohistochemistry

24 Measurement of Osmolarity AnOsmomat 030 osmome-ter (Gonotec GmbH Berlin Germany) was used to measureosmolarity in 50 120583L of either plasma or urine according tothe manufacturerrsquos instructions

25 TBARSMeasurement Levels of thiobarbituric acid reac-tive substances (TBARS) were estimated using the methoddescribed by Ramanathan et al [24] with slight modifica-tions Kidney homogenate supernatantsweremixedwith SDS(8wv) TBA (08wv) and acetic acid (20 vv) andheated for 60min at 90∘C Precipitatedmaterial was removedby centrifugation and the absorbance of the supernatant wasdetermined at 532 nm Levels of TBARSwere calculated usinga calibration curve with malondialdehyde (MDA Sigma-Aldrich Saint Louis USA)

26 Blood Pressure Measurement Blood pressure was mea-sured in the tail of conscious rats using a computerized 20390CODA Kent Science instrument (Torrington CT) in a roomat 25∘Cwith noise and light control Rats were trained for onemonth before the experimental measurements

27 Tissue Processing and Immunohistochemical AnalysisKidney samples (5mm thick) were fixed by immersion inBouinrsquos solution for 24 h at room temperature Each tissuesample was dehydrated and embedded in Paraplast Plus(Monoject Scientific St Louis MO) and serial sections 5120583mthick were obtained with a rotary microtome and mounted














3 Results





lowastlowastlowast






















Prot

einu

riac

reat

inin

uria


20

15

5

0

10






C

D

lowast

lowast

lowast

lowast

DBlowast

(a)

C

D

lowast

lowastlowast

DB

lowast

100120583m

(b)





C

D

DB

(a)

DB

C

D

100120583m

(b)






6

4

2

0


lowastlowast

MD

A (120583

mol

g)




6

8

4

2

0


lowastlowast

MD

A (120583

mol

g)







Ethi

dium

upt

ake (

AU)

3

2

0

1

Time (min)0 2 4 6 8 10

lowast

(a)

Ethi

dium

upt

ake r

ate (

AUm

in)

04

03

02

01

00


lowastlowast

(b)



4 Discussion







100

80

40

20

0

60

Cou

plin

g in

cide

nce (

)


lowast

(a)

Cou

plin

g in

dex

8

6

4

2

0

lowastlowast

lowastlowastlowast


(b)

20120583m

(c)







T-ERK

250

200

150

100

50

0


lowast

Cx43

T-E

RK (

of c

ontro

l)

Cx43




Ethical Standards







Acknowledgments


References


























































3 Results





lowastlowastlowast






















Prot

einu

riac

reat

inin

uria


20

15

5

0

10






C

D

lowast

lowast

lowast

lowast

DBlowast

(a)

C

D

lowast

lowastlowast

DB

lowast

100120583m

(b)





C

D

DB

(a)

DB

C

D

100120583m

(b)






6

4

2

0


lowastlowast

MD

A (120583

mol

g)




6

8

4

2

0


lowastlowast

MD

A (120583

mol

g)







Ethi

dium

upt

ake (

AU)

3

2

0

1

Time (min)0 2 4 6 8 10

lowast

(a)

Ethi

dium

upt

ake r

ate (

AUm

in)

04

03

02

01

00


lowastlowast

(b)



4 Discussion







100

80

40

20

0

60

Cou

plin

g in

cide

nce (

)


lowast

(a)

Cou

plin

g in

dex

8

6

4

2

0

lowastlowast

lowastlowastlowast


(b)

20120583m

(c)







T-ERK

250

200

150

100

50

0


lowast

Cx43

T-E

RK (

of c

ontro

l)

Cx43




Ethical Standards







Acknowledgments


References
















































lowastlowastlowast






















Prot

einu

riac

reat

inin

uria


20

15

5

0

10






C

D

lowast

lowast

lowast

lowast

DBlowast

(a)

C

D

lowast

lowastlowast

DB

lowast

100120583m

(b)





C

D

DB

(a)

DB

C

D

100120583m

(b)






6

4

2

0


lowastlowast

MD

A (120583

mol

g)




6

8

4

2

0


lowastlowast

MD

A (120583

mol

g)







Ethi

dium

upt

ake (

AU)

3

2

0

1

Time (min)0 2 4 6 8 10

lowast

(a)

Ethi

dium

upt

ake r

ate (

AUm

in)

04

03

02

01

00


lowastlowast

(b)



4 Discussion







100

80

40

20

0

60

Cou

plin

g in

cide

nce (

)


lowast

(a)

Cou

plin

g in

dex

8

6

4

2

0

lowastlowast

lowastlowastlowast


(b)

20120583m

(c)







T-ERK

250

200

150

100

50

0


lowast

Cx43

T-E

RK (

of c

ontro

l)

Cx43




Ethical Standards







Acknowledgments


References














































C

D

lowast

lowast

lowast

lowast

DBlowast

(a)

C

D

lowast

lowastlowast

DB

lowast

100120583m

(b)





C

D

DB

(a)

DB

C

D

100120583m

(b)






6

4

2

0


lowastlowast

MD

A (120583

mol

g)




6

8

4

2

0


lowastlowast

MD

A (120583

mol

g)







Ethi

dium

upt

ake (

AU)

3

2

0

1

Time (min)0 2 4 6 8 10

lowast

(a)

Ethi

dium

upt

ake r

ate (

AUm

in)

04

03

02

01

00


lowastlowast

(b)



4 Discussion







100

80

40

20

0

60

Cou

plin

g in

cide

nce (

)


lowast

(a)

Cou

plin

g in

dex

8

6

4

2

0

lowastlowast

lowastlowastlowast


(b)

20120583m

(c)







T-ERK

250

200

150

100

50

0


lowast

Cx43

T-E

RK (

of c

ontro

l)

Cx43




Ethical Standards







Acknowledgments


References














































C

D

DB

(a)

DB

C

D

100120583m

(b)






6

4

2

0


lowastlowast

MD

A (120583

mol

g)




6

8

4

2

0


lowastlowast

MD

A (120583

mol

g)







Ethi

dium

upt

ake (

AU)

3

2

0

1

Time (min)0 2 4 6 8 10

lowast

(a)

Ethi

dium

upt

ake r

ate (

AUm

in)

04

03

02

01

00


lowastlowast

(b)



4 Discussion







100

80

40

20

0

60

Cou

plin

g in

cide

nce (

)


lowast

(a)

Cou

plin

g in

dex

8

6

4

2

0

lowastlowast

lowastlowastlowast


(b)

20120583m

(c)







T-ERK

250

200

150

100

50

0


lowast

Cx43

T-E

RK (

of c

ontro

l)

Cx43




Ethical Standards







Acknowledgments


References














































6

4

2

0


lowastlowast

MD

A (120583

mol

g)




6

8

4

2

0


lowastlowast

MD

A (120583

mol

g)







Ethi

dium

upt

ake (

AU)

3

2

0

1

Time (min)0 2 4 6 8 10

lowast

(a)

Ethi

dium

upt

ake r

ate (

AUm

in)

04

03

02

01

00


lowastlowast

(b)



4 Discussion







100

80

40

20

0

60

Cou

plin

g in

cide

nce (

)


lowast

(a)

Cou

plin

g in

dex

8

6

4

2

0

lowastlowast

lowastlowastlowast


(b)

20120583m

(c)







T-ERK

250

200

150

100

50

0


lowast

Cx43

T-E

RK (

of c

ontro

l)

Cx43




Ethical Standards







Acknowledgments


References















































Ethi

dium

upt

ake (

AU)

3

2

0

1

Time (min)0 2 4 6 8 10

lowast

(a)

Ethi

dium

upt

ake r

ate (

AUm

in)

04

03

02

01

00


lowastlowast

(b)



4 Discussion







100

80

40

20

0

60

Cou

plin

g in

cide

nce (

)


lowast

(a)

Cou

plin

g in

dex

8

6

4

2

0

lowastlowast

lowastlowastlowast


(b)

20120583m

(c)







T-ERK

250

200

150

100

50

0


lowast

Cx43

T-E

RK (

of c

ontro

l)

Cx43




Ethical Standards







Acknowledgments


References














































100

80

40

20

0

60

Cou

plin

g in

cide

nce (

)


lowast

(a)

Cou

plin

g in

dex

8

6

4

2

0

lowastlowast

lowastlowastlowast


(b)

20120583m

(c)







T-ERK

250

200

150

100

50

0


lowast

Cx43

T-E

RK (

of c

ontro

l)

Cx43




Ethical Standards







Acknowledgments


References














































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Boldine Prevents Renal Alterations in Diabetic Rats

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Transcript of Boldine Prevents Renal Alterations in Diabetic Rats