chronic bilateral renal denervation attenuates renal injury in a transgenic rat model of diabetic...

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Chronic bilateral renal denervation attenuates renal injury in a transgenic ratmodel of diabetic nephropathy

Yimin Yao,1,4 Ingrid C. Fomison-Nurse,1 Joanne C. Harrison,1 Robert J. Walker,2 Gerard Davis,3 andIvan A. Sammut1

1Department of Pharmacology, University of Otago, Dunedin, New Zealand; 2Department of Medicine, University of Otago,Dunedin, New Zealand; 3Department of Physiology, University of Otago, Dunedin, New Zealand; and 4Australian School ofAdvanced Medicine, Macquarie University, New South Wales, Australia

Submitted 1 November 2013; accepted in final form 2 June 2014

Yao Y, Fomison-Nurse IC, Harrison JC, Walker RJ, Davis G,Sammut IA. Chronic bilateral renal denervation attenuates renalinjury in a transgenic rat model of diabetic nephropathy. Am J PhysiolRenal Physiol 307: F251–F262, 2014. First published June 4,2014; doi:10.1152/ajprenal.00578.2013.—Bilateral renal denervation(BRD) has been shown to reduce hypertension and improve renalfunction in both human and experimental studies. We hypothesizedthat chronic intervention with BRD may also attenuate renal injuryand fibrosis in diabetic nephropathy. This hypothesis was examined ina female streptozotocin-induced diabetic (mRen-2)27 rat (TGR)shown to capture the cardinal features of human diabetic nephropathy.Following diabetic induction, BRD/sham surgeries were conductedrepeatedly (at the week 3, 6, and 9 following induction) in bothdiabetic and normoglycemic animals. Renal denervation resulted in aprogressive decrease in systolic blood pressure from first denervationto termination (at 12 wk post-diabetic induction) in both normogly-cemic and diabetic rats. Renal norepinephrine content was signifi-cantly raised following diabetic induction and ablated in denervatednormoglycemic and diabetic groups. A significant increase in glomer-ular basement membrane thickening and mesangial expansion wasseen in the diabetic kidneys; this morphological appearance wasmarkedly reduced by BRD. Immunohistochemistry and protein den-sitometric analysis of diabetic innervated kidneys confirmed thepresence of significantly increased levels of collagens I and IV,�-smooth muscle actin, the ANG II type 1 receptor, and transforminggrowth factor-�. Renal denervation significantly reduced protein ex-pression of these fibrotic markers. Furthermore, BRD attenuatedalbuminuria and prevented the loss of glomerular podocin expressionin these diabetic animals. In conclusion, BRD decreases systolic bloodpressure and reduces the development of renal fibrosis, glomerulo-sclerosis, and albuminuria in this model of diabetic nephropathy. Theevidence presented strongly suggests that renal denervation may serveas a therapeutic intervention to attenuate the progression of renalinjury in diabetic nephropathy.

renal denervation; diabetic nephropathy; (mRen-2)27 rat; fibrosis

DIABETIC NEPHROPATHY IS THE leading cause of end-stage renaldisease worldwide, and effective interventional strategies aredesperately required (49). The characteristics of diabetic ne-phropathy manifest as albuminuria, glomerular basementmembrane (GBM) thickening, mesangial hypertrophy, andtubulointerstitial fibrosis (8, 18, 44). Hypertension is often

present, forming a significant factor in the progression ofdiabetic nephropathy (56). Elevation of sympathetic nerveactivity (SNA) has been shown to occur in patients withdiabetic nephropathy (47) and is established as a major con-tributor in the onset of hypertension and renal failure (30, 58).Furthermore, the pharmacological inhibition of SNA at doseswhich do not affect hypertension has been shown to effectivelyreduce the progression of renal damage in both clinical diabeticnephropathy (52) and in experimental diabetic nephropathymodels (1). These findings appear to indicate that increasedSNA may instigate and even accelerate the development ofdiabetic renal injury independently of its hypertensive effect.

As renal nerves play a significant role in controlling SNA(48), diseased kidneys are therefore able to exert an excitatoryeffect on the SNA in patients with chronic renal failure (11,26). Earlier studies using rat models of experimental nephrop-athy have demonstrated that renal sensory nerve activity fromthe diseased kidneys to the brain is raised, resulting in anincreased posterior hypothalamic noradrenaline turnover andrenal SNA elevation (RSNA) (9, 10, 62–64). As the kidneysare richly supplied by renal sympathetic nerves, an increase inRSNA will further deteriorate the function of the diseasedkidneys by increasing renin synthesis and release from juxta-glomerular cells (28), thereby provoking an increase in renalNa� and water reabsorption (14) and renal vascular resistance(15), in what is described as a vicious cycle (60).

By effectively interrupting this excitatory cycle, bilateralrenal denervation (BRD) has reemerged as an effective therapyshown to delay the progression of hypertension. In severalclinical trials, BRD was demonstrated to provide long-termblood pressure reduction in patients with difficult-to-controland resistant hypertension (16, 31, 38, 43, 51, 53). The unex-pected failings of the latest clinical study (SYMPLICITYHTN-3) to show efficacy have, unfortunately, cast some doubton the value of renal denervation in resistant hypertension (55).However, BRD may have renoprotective effects in patientsbeyond simple blood pressure control. BRD has been found toimprove glucose tolerance and insulin sensitivity (43). In a ratmodel of early type 1 diabetes, BRD was also found to abolishglomerular hyperfiltration and prevent glomerular hypertrophyat 14 days after diabetic onset (42). Taken together, thesefindings suggested that renal nerves are involved in the earlyrenal pathological changes occurring during the developmentof diabetic nephropathy. However, the chronic effects of BRD

Address for reprint requests and other correspondence: I. A. Sammut, Dept.of Pharmacology and Toxicology, Univ. of Otago School of Medical Sciences,PO Box 56, Dunedin, Otago 9016, NZ (e-mail: [email protected]).

Am J Physiol Renal Physiol 307: F251–F262, 2014.First published June 4, 2014; doi:10.1152/ajprenal.00578.2013.

1931-857X/14 Copyright © 2014 the American Physiological Societyhttp://www.ajprenal.org F251

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on the progression of diabetic nephropathy have not been fullyexamined.

In this study, we examined the hypothesis that BRD con-ducted after the onset of hyperglycemia will preserve renalmorphology and function and reduce the severity of hyperten-sion and expression of renal fibrotic markers formed as aconsequence of diabetic induction. Furthermore, as previousreports have shown inconsistencies in the level of intrarenaldistribution of angiotensin II type 1 receptors (AT1R), partic-ularly as renin and angiotensin-converting enzyme content inthe rodent nephron may be variably altered in diabetes (35),this study also examined the effect of denervation on renalAT1R in diabetic nephropathy. The underlying features of thehypertensive transgenic (mREN-2)27 rat (TGR) model includeelevated prorenin levels and high tissue renin-angiotensin sys-tem activity that are critical to the development of diabeticnephropathy in humans (4). Previous reports have shown thatstreptozotocin (STZ)-induced diabetes in female heterozygousTGRs results in a rapid progression of the principal patholog-ical features of human diabetic nephropathy without the prom-inent effects of hypertension on renal pathology (3, 4, 32).Consequently, diabetic nephropathy was studied in this femaleTGR model over a 12-wk period following hyperglycemiconset at 6 wk of age. The present studies were performed toinvestigate the therapeutic benefit of BRD on the progressionof diabetic nephropathy in the female heterozygous, renin-overexpressing TGR model of STZ-induced hyperglycemia.

METHODS

Animal model and research design. All experimental proceduresinvolved in this project were conducted under the approval of theUniversity of Otago Animal Ethics Committee and in accordance withthe National Institutes of Health Guide for the Care and Use ofLaboratory Animals. Female heterozygous TGRs were obtained fromthe CARA Transgenic Facility at the University of Otago. Ratsreceived either an intravenous bolus of a citrate-buffered vehicle (0.05M) or STZ (55 mg/kg) to induce diabetes at 6 wk of age as described(32). Blood glucose levels were monitored and maintained at 20–25mM by daily administration of isophane (sc, 1–4 units). Animals inboth normoglycemic and diabetic groups were randomized to eitherBRD or sham denervation surgeries performed repetitively in eachanimal at postnatal weeks 9, 12, and 15 (Fig. 1). Systolic bloodpressure (SBP) and heart rate were measured weekly followinghabituation, using tail-cuff plethysmography and 24-h urine collec-tions obtained at specific intervals. Upon termination at postnatalweek 18, the animals were anesthetized (halothane-N2O2-O2 mix),and the kidneys were harvested and stored for further histological andbiochemical processing. In a separate set of treatment groups, theglomerular filtration rate (GFR) was measured using the inulin clear-

ance method and corrected to the left kidney weight as previouslydescribed (24).

BRD. As renal nerves in rats have been shown to fully reinnervatethe kidney within 3–6 wk after surgical denervation, it was necessaryto conduct repeat denervation surgeries (34, 45). At week 3 after STZadministration, a midline dorsal lumbar incision was made bilaterallyunder general anesthesia (halothane-N2O2-O2 mix), and each kidneyexposed by retracting the abdominal muscles. Positive identificationof the renal nerves was confirmed using a stimulator (10 V/10 Hz,1-ms square wave pulse) to produce temporary renal blanching.Subsequently, the renal arteries were stripped back of the surroundingnerve bundles surrounding the renal arteries and painted with 10%phenol in absolute ethanol to destroy any remnant nerves. Subsequentdenervation surgeries were similarly conducted. Sham renal denerva-tion in the intact animals was performed using identical procedures,with the renal nerves left intact.

Inulin clearance studies. Each animal was anesthetized with abolus injection of pentobarbitone sodium (70 mg/kg ip; NationalVeterinary Supplies). The left femoral vein was cannulated andinfused with 0.9% saline (18 ml·kg�1·h�1). The left carotid artery wasalso cannulated to allow access for blood sampling and mean arterialpressure monitoring during the experiment. A midline laparotomy wasconducted to allow cannulation of the left kidney ureter for urinecollection. On completion of the preparation, the 0.9% saline infusionwas replaced by a priming dose (2 ml) and then a sustained infusion(18 ml·kg�1·h�1) of inulin (15 mg/ml; Sigma, St. Louis, MO) andpentobarbitone sodium (0.44 mg/ml) in 0.9% saline. Following a 2-hequilibration period, urine was collected from the left ureter in two15-min periods. Blood plasma samples were collected from thecarotid artery at the beginning and end of each period. Inulin concen-tration in the plasma and urine samples was determined using amodification of the protocol described by Bojesen (7). Plasma andurine samples (50 �l) were deproteinized in 250 �l of NaOH (0.15 M)and neutralized in 1,700 �l of an H2SO4 (3.68 mM) and ZnSO4 (850mM) solution. The precipitated protein was removed by centrifugation(500 g, 15 min, 4°C). Subsequently, 500 �l of the supernatant wasincubated (100°C for 15 min) in a final ratio of 1:22.66 (vol/vol) to 2.5ml of the color reagent [20% diphenylamine in absolute ethanol(wt/vol) diluted in a 3:10 (vol/vol) H2SO4 (0.125 M) solution inabsolute ethanol]. The absorbance of each sample was read (� � 655nm) at room temperature, and inulin clearance is expressed as aplasma-to-urine ratio.

Histopathology. Freshly harvested kidneys were fixed in 10%neutral buffered formalin, routinely processed, embedded in paraffin,and sectioned using standard protocols. Periodic acid-Schiff’s stainingwas used to identify changes in basement membrane architecture andglycogen deposition in the kidneys. The glomerulosclerotic index(GSI) for each animal was determined by examining 150–200 glom-eruli in each section and scored in a double-blind manner usingtrained observers as previously described (32, 50): grade 0, normal;grade 1, sclerotic area up to one-quarter of total glomerular area; grade2, sclerotic area more than one-quarter and up to half of totalglomerular area; grade 3, sclerosis of more than half and up tothree-quarters of the glomerular area; and grade 4, sclerosis of morethan three-quarters of total glomerular area. Glomerulosclerosis wasdefined as glomerular basement membrane thickening, mesangialexpansion, and capillary occlusion. The GSI was calculated using thefollowing formula: GSI � (1 N1 � 2 N2 � 3 N3 � 4 N4)/(N0 � N1 � N2 � N3 � N4), where NX is the number of glomeruliin each grade of sclerosis.

Immunohistochemistry. Staining was performed on 5-�m micro-wave-fixed, paraffin-embedded sections using standard protocols. Af-ter a 2-h incubation with an animal-free blocker (Vector Laboratories,Burlingame, CA), the sections were incubated (15 h at 4°C) with thefollowing primary antibodies diluted in Tris-buffered saline: mouseanti-rat �-smooth muscle actin (SMA; 1:400; Sigma), rabbit anti-ratcollagen I (1:100; Rockland, Gilbertsville, PA), rabbit anti-rat colla-

6 9 12 15 18 weeks (age)

STZ/vehicle BRD/Sham Termination

Fig. 1. Experimental design and confirmation of successful diabetic inductionand chronic bilateral renal denervation (BRD). Streptozotocin (STZ; iv) wasadministered to 6-wk-old female transgenic rats (TGRs) allocated to thediabetic group. BRD or sham surgeries were performed, repetitively, at 9, 12,and 15 wk of age. Weekly systolic blood pressure recordings (Œ) and 24-hurine collections (o) were performed. At 18 wk of age, animals were eutha-nized, and kidneys were collected for immunohistochemical and Western blotanalysis.

F252 RENAL DENERVATION IN DIABETIC NEPHROPATHY

AJP-Renal Physiol • doi:10.1152/ajprenal.00578.2013 • www.ajprenal.org

gen IV (1:100; Abcam, Cambridge, UK), rabbit anti-rat NPHS2(podocin; 1:40; Abcam), rabbit anti-rat transforming growth factor(TGF)-�1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), mousemonoclonal AT1R (ab9391; 1:20, Abcam), and sheep anti-rat tyrosinehydroxylase (1:200; Novus Biologicals, Littleton, CO). The sectionswere then washed in Tris-buffered saline and incubated with eitherfluorescein (Molecular Probes, Leiden, The Netherlands)- or peroxi-dase (Pierce, Rockford, IL)-conjugated secondary antibodies. Immu-nohistochemical labeling for each protein of interest was photo-graphed in 10 separate fields/section using a Zeiss Axioplan 2 micro-scope with dedicated Axiovision v4.2 software (Carl Zeiss Vision).Semiquantitative analysis of DAB staining was conducted on themicrographs using Adobe Photoshop CS5 software (Adobe Systems,San Jose, CA) and calculated by the amount of pixels contained withina color spectrum using the “color range tool” preoptimized specifi-cally for DAB staining against a fixed intensity cut-off threshold inwhich the ratio of DAB stained pixels was calculated against back-ground in each field of interest (21, 39). Fluorescent labeling oftyrosine hydroxylase was photographed (40 objective) using a ZeissLSM 710 confocal microscope.

Western blot analysis. Frozen kidney cortical tissues were homog-enized, and protein concentrations were determined as previouslypublished (41) using a Lowry DC assay kit (Bio-Rad, Hercules CA).Sample protein concentrations were extrapolated using a proteinstandard curve with a Pearson correlation coefficient of �0.99.

Sample preparation, gel loading, electrophoresis, and transblottingprocedures were carried out using a Tris-glycine-buffered system(Invitrogen Life Technologies, Carlsbad, CA) according to themanufacturer’s instructions. Immunoblotting was performed usingstandard protocols with the following primary antibodies: rabbitanti-rat collagen I (1:400; Rockland), rabbit anti-rat collagen IV(1:1,000; Abcam), and rabbit anti-rat TGF-�1 (1:400; Santa CruzBiotechnology). To confirm equal protein loading, blots were re-probed with rabbit anti-rat �-tubulin (1:5,000; Sigma). A horseradishperoxidase-conjugated secondary antibody (1:1,000; Pierce) was usedto label the protein band, and blots were visualized using chemilumi-nescence (SuperSignal West Dura Extended Duration Substrate;Pierce). Densitometric scanning of protein bands of interest wasperformed using a GS-710 Calibrated Imaging Densitometer (Bio-Rad) and quantified by Quantity One software (Bio-Rad). Eachspecific protein band density was ratioed against the �-tubulin proteindensity quantified in the same sample.

Renal biochemical studies. The Na� concentration of the urinesamples was analyzed using a FP20 flame photometer (SEAC, Flor-ence, Italy) against a 100 mM NaCl standard solution, diluted (1:200)in a Li2CO3 (15 meqV) reference solution. Urinary albumin concen-tration was measured using an Albumin Blue Fluorescent Assay Kit(Active Motif, Carlsbad, CA) performed according to the manufac-turer’s instructions. Renal cortical norepinephrine levels were quan-tified at the terminal study point (12 wk post-diabetic induction) using

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Fig. 2. Physiological parameters in (mRen-2)27 TGRs from 5 to 18 wk of age. Values are means SE. Vertical dotted line represents the time point whenBRD/sham surgery was performed. Œ, Normoglycemic sham (NGInx); �, diabetic sham (DBInx); �, normoglycemic denervated (NGDnx); �, diabetic denervated(DBDnx). A: mean weekly blood glucose levels. B: absolute body weight change, C: systolic blood pressure. D: heart rate. E: absolute Na� excretion (UNaV).F: urine flow. In A, B, D–F: **P � 0.01 or ***P � 0.001 DBInx vs. NGInx, and *P � 0.05 or ***P � 0.001 DBDnx vs. NGDnx. In C: ***P � 0.001 NGInxvs. NGDnx, ***P � 0.001 DBInx vs. DBDnx and **P � 0.01 DBDnx vs. NGDnx. �P � 0.01, ��P � 0.01, or ��P � 0.001 vs. systolic blood pressure atbaseline (8.5 wk) within the group.

F253RENAL DENERVATION IN DIABETIC NEPHROPATHY


a commercial norepinephrine ELISA kit (BA E-5200, Labor Diag-nostika Nord) performed according to the manufacturer’s instructions.

Statistics. All values are expressed as means SE of n � 7–8individual experiments/group. Statistical analysis was conducted toexamine differences in responses between the four groups: normogly-cemic innervated (NGInx), normoglycemic denervated (NGDnx),diabetic innervated (DBInx), and diabetic denervated (DBDnx). Forsystolic blood pressure (SBP), heart rate, body weight, urine flow, andabsolute sodium secretion (UNaV), a two-way repeated measuresANOVA was used to provide a comparison between each of the twotreatment paradigms: NGInx vs. NGDnx, DBInx vs. DBDnx, NGInxvs. DBInx, and NGDnx vs. DBDnx. Significant interaction betweentwo groups in SPB and heart rate comparisons was further analyzedusing one-way repeated measures ANOVA followed by a Dunnettpost hoc comparison against an internal baseline measured at 8.5 wkpost-STZ administration (presurgical values). All other comparisonswere made with two-way ANOVA followed by a Bonferroni post hoccomparison. Statistics were performed using SigmaStat v3.5 software(Systat Software, San Jose, CA). Differences in group means wereconsidered statistically significant if P � 0.05.

RESULTS

Physiological parameters. Diabetic onset resulted in ele-vated blood glucose levels (Fig. 2A) and reduced body weightgain (DBInx vs. NGInx, P � 0.01; DBDnx vs. NGDnx, P �0.05, Fig. 2B) in both sham-operated and denervated TGRs.Repeated BRD had no impact on these parameters in eithernormoglycemic or diabetic groups.

As expected, SBP was elevated in the TGRs after week 6with no significant difference seen between the sham-operatednormoglycemic and diabetic groups over the duration of theexperiment. As shown in Fig. 2C, BRD intervention in bothnormoglycemic and diabetic animals produced a progressive,significant decrease in SBP compared with the sham-operated

groups, which was maintained over the 9-wk denervationperiod (NGInx vs. NGDnx, P � 0.001; DBInx vs. DBDnx,P � 0.001, Fig. 2C). Denervation, however, produced a sig-nificantly greater decrease in SBP in diabetic animals com-pared with the normoglycemic group (DBDnx vs. NGDnx,P � 0.01). Diabetic TGRs also had a lower heart rate comparedwith normoglycemic groups in both sham-operated (DBInx vs.NGInx, P � 0.001) and denervated groups (DBDnx vs.NGDnx, P � 0.001, Fig. 2D). Both diabetic groups exhibitedan increase in UNaV (Fig. 2E) and urine flow (Fig. 2F)following diabetic induction which persisted until termination(DBInx vs. NGInx, P � 0.001; DBDnx vs. NGDnx, P �0.001). Repeated BRD had no impact on these parameters ineither normoglycemic or diabetic animals.

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Fig. 3. Confirmation of successful chronic BRD. A: confirmation of surgicaldenervation in kidneys isolated from diabetic TGRs. Left: innervated kidneysection from diabetic TGR subjected to sham surgery showing tyrosinehydroxylase-positive fluorescent immunostaining (green) indicated by arrowswithin the adventitia of intrarenal arterioles, tubules, and glomeruli. Middle:denervated kidney section from a diabetic TGR subjected to repeated chronicBRD. Right: a negative control kidney section without tyrosine hydroxylaseprimary antibody. Nuclei were stained with 4,6-diamidino-2-phenylindole(blue). Scale bars � 50 �m. a, arteriole; g, glomerulus. B: renal cortical tissuenorepinephrine levels analyzed by ELISA. Values are means SE. ***P �0.001 vs. NGInx. ��P � 0.001 vs. DBInx.


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Fig. 4. Renal morphological changes in TGRs. A: morphological changes incortex and inner stripe of outer medulla in kidney sections stained withperiodic acid-Schiff. In NGInx and NGDnx kidneys, glomeruli appearance andtubular structure in the cortex and medulla appeared normal with no remark-able features. In the kidneys from the DBInx TGRs, most glomeruli showed aseverely thickened glomerular basement membrane (GBM) and mesangialhypertrophy; many cortical tubules were dilated with fibrotic casts deposited inthe lumen; inflammatory cell infiltration was seen in some areas of the kidney(arrows). In the medulla, severe fibrosis was observed extensively across theinterstitium and tubules (arrow). In DBDnx kidneys, the occurrence of glom-eruli with a thickened GBM and mesangial hypertrophy was reduced tonormoglycemic levels; a few cortical tubules were still dilated but withsignificantly less fibrotic casts appearing in the lumen; inflammatory infiltra-tion was not noticeable. In the medulla, some interstitial fibrosis was still seen(arrows); however, the frequency and the severity of fibrosis were reduced. B:glomerulosclerotic index in kidneys from TGRs subjected to diabetes and renaldenervation. Values are means SE. ***P � 0.001 vs. NGInx. �P � 0.05 vs.DBInx.



Confirmation of renal denervation. Positive tyrosine hydrox-ylase staining was found within cortical sections of sham-operated TGR kidneys indicating the presence of sympatheticinnervation (Fig. 3A). Successful denervation was confirmedby the absence of tyrosine hydroxylase staining and by thereduction of total norepinephrine levels in denervated renalcortical tissues (Fig. 3B). Denervation reduced cortical norepi-nephrine content significantly from 23 1.6 to 1.0 0.1ng/mg protein in the NGInx vs. NGDnx groups, respectively(P � 0.001), and from 37 2.6 to 1.7 0.6 ng/mg protein inthe DBInx vs. the DBDnx groups, respectively (P � 0.001).Renal norepinephrine content was also raised as a consequenceof diabetic induction in the sham-operated animals (P �0.001).

Renal morphological and functional parameters. Glomeru-lar structure appeared normal in the normoglycemic kidneys(Fig. 4A). In contrast, most glomeruli were sclerosed in kid-neys from diabetic sham-operated animals. This appearancewas attenuated in denervated diabetic kidneys. The glomerularpathology was reflected by the significantly higher GSI re-corded in kidneys isolated from sham-operated diabetic TGRscompared with the normoglycemic groups (1.94 0.47 DBInxvs. 0.56 0.10 NGInx, P � 0.001, Fig. 4B). This elevated GSIwas attenuated in the diabetic TGRs subjected to BRD (0.68 0.14 DBDnx vs. 1.94 0.47 DBInx, P � 0.001). Normal renalmedullary structure was observed in the normoglycemic TGRkidneys (Fig. 4A). In contrast, in diabetic sham-operated TGRkidneys, matrix expansion, GBM thickening, and lumen ex-pansion were observed among the renal tubules distributed atthe outer medullary region. Denervation of the diabetic TGRkidneys greatly reduced the extent of structural injury.

Podocin staining was diminished within the glomeruli ofsham-operated diabetic animals compared with the normogly-cemic groups (Fig. 5, A and B). Diabetic-induced podocin losswas prevented in TGRs subjected to BRD. Denervation did nothave any effect, however, on podocin staining in these hyper-tensive TGRs animals in the absence of diabetic insult. Thefunctional significance of this podocyte injury was demon-strated by the presence of albuminuria in the diabetic TGRs.

Urinary absolute albumin levels assessed after 12 wk of hy-perglycemia were raised in the diabetic sham-operated groupcompared with the normoglycemic groups (10.7 2.5 vs. 1.6 0.2 mg·24 h�1·kg�1, respectively, P � 0.001, Fig. 5A). Dia-betic animals subjected to BRD showed a significantly reducedurinary albumin level compared with the diabetic sham-oper-ated group (5.0 1.1 vs 10.7 2.5 mg·24 h�1·kg�1, respec-tively, P � 0.05).

Left kidney weight recorded at termination was raised in thediabetic TGRs compared with the normoglycemic TGRs (P �0.001, Fig. 6A). GFR (corrected for kidney weight) in thediabetic TGRs was significantly lower (P � 0.001, Fig. 6B)compared with the normoglycemic TGRs. BRD had no impacton kidney weight or GFR in either normoglycemic or diabeticTGRs.

Renal fibrotic and profibrotic markers. Weak collagen Iimmunolabeling was seen distributed in both renal cortex andmedulla in the normoglycemic TGRs (Fig. 7A). In the diabeticsham-operated group, elevated collagen I expression wasfound extracellularly at the GBM and in the interstitial space ofthe outer medulla. Intervention with BRD after the onset ofdiabetes reduced collagen I expression in both the cortex andmedulla. Differences in collagen I expression were confirmedby Western blot analysis showing a twofold higher expressionin the renal cortex of diabetic sham-operated TGRs (Fig. 7B),and this elevation was reduced in diabetic TGRs subjected toBRD. Collagen IV was weakly labeled in the normoglycemicTGR kidneys (Fig. 8A). Kidneys from the diabetic sham-operated group show elevated collagen IV expression withinthe glomeruli and in the interstitial space throughout the cortexand medulla. BRD intervention also successfully attenuated theextent of collagen IV expression found in diabetic TGRs.Protein analysis confirmed increased collagen IV expression inthe cortex of diabetic sham-operated TGRs compared with thenormoglycemic groups (Fig. 8B), and this elevated expressionwas also reduced in the denervated diabetic TGRs.

Very low levels of �-SMA were detected in the medulla ofnormoglycemic TGR kidneys (Fig. 9, A and B). In the diabeticsham-operated group, �-SMA was extensively expressed


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Fig. 5. Glomerular structural and functional integrity in TGRs. Podocin localization (A) and densitometric quantification of podocin positively stained regions(B) within glomeruli of renal cortical sections immunolabeled with anti-NPHS2 (podocin) antibody and counterstained with hematoxylin are shown. Strongpodocin immunohistochemical staining (indicated by arrows) is seen in the glomeruli of both normoglycemic sham-operated and denervated TGR kidneys.Glomerular staining intensity of podocin in sham-operated diabetic TGR was markedly reduced. Podocin labeling within glomeruli of diabetic TGRs subjectedto renal denervation was noticeably greater than in glomeruli isolated from the sham-operated diabetic TGRs. Podocyte injury was also confirmed by analyzing24-h urinary absolute albumin excretion (C) in TGRs before termination. Groups are defined as in Fig. 2. Values are means SE. **P � 0.01, ***P � 0.001vs. NGInx. �P � 0.05 vs. DBInx.



within the renal tubule and interstitial space at the outermedullary region (Fig. 9, C and E), and this diabetic-inducedelevation was attenuated in the medulla of diabetic TGRssubjected to BRD (Fig. 9, D and F).

TGF-�1 was elevated in both the cortex and medulla of thesham-operated diabetic TGR kidneys compared with nor-moglycemic TGRs (Fig. 10A). This expression was reduced inthe diabetic TGR kidneys subjected to BRD. Western blotanalysis showed a twofold higher expression in the renal cortexof the diabetic sham-operated group compared with normogly-cemic TGRs (Fig. 10B), and this elevation was reduced indiabetic TGRs subjected to BRD.

AT1R immunohistochemical studies in the normoglycemickidneys produced faint staining within the proximal tubulesand cortical and medullary collecting ducts (Fig. 11A). AT1Rimmunostaining became more pronounced within epithelialcells of the dilated cortical and outer medullary collectingtubules in the diabetic kidneys, producing a significant increasein AT1R immunostaining intensity relative to normoglycemicinnervated kidneys (Fig. 11B). Conversely, AT1R staining inthe rest of the nephron was reduced, particularly within regionsof extracellular matrix expansion. While chronic renal dener-vation failed to affect the low AT1R staining intensity withinthe normoglycemic group, the procedure resulted in an abruptloss of AT1R intensity in the diabetic animals.

DISCUSSION

The current study is the first to demonstrate that renal nervesplay an important role in the long-term development of struc-

tural damage seen in diabetic nephropathy. BRD interventionafter pancreatic �-cell destruction and diabetic onset attenuatedthe progression of diabetic nephropathy in this transgenicmodel of renin-overexpressing hypertensive rats. As expected,BRD reduced the severity of hypertension and renal catechol-amine levels in the normoglycemic animals. These parameterswere also successfully attenuated within the diabetic animals.However, while BRD had no effect on renal morphology orinjury markers in normoglycemic hypertensive groups, thisdenervation protocol clearly provided protection when hyper-tension was compounded by the presence of diabetes. Bothdiabetic-induced albuminuria and podocin loss but not hyper-glycemia were decreased by BRD intervention. BRD alsoreduced intrarenal AT1R expression within the collecting ductsand attenuated renal fibrosis in diabetic TGRs by reducing theexpression of the profibrotic inducer TGF-�1. This reductionwas supported by the attendant reduction in �-SMA andcollagens I and IV. This study assesses for the first time the

NGInx NGDnx DBInx DBDnx0.0

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Fig. 6. Left kidney weight (A) and glomerular filtration rate (B) in TGRs attermination. Groups are defined as in Fig. 2. Values are means SE. ***P �0.001 between indicated groups.

B NGInx NGDnx DBInx ~160 kDa

55 kDa

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-tubulin

DBInx


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Fig. 7. Collagen I localization and expression in the cortex and inner stripe ofouter medulla of kidney sections immunolabeled with collagen I antibody andcounterstained with hematoxylin. A: in normoglycemic innervated and dener-vated kidney sections, collagen I was weakly labeled and occasionally seen inthe glomerular basement membrane and medullary tubules. In the diabeticinnervated kidney sections, intense collagen I staining was found in the GBM(arrow) in most of the glomeruli; strong collagen I staining was also foundextensively in the interstitium (arrow) of the inner stripe of outer medullarytubules. BRD in the diabetic TGRs reduced collagen I labeling to normogly-cemic levels in the glomeruli and medullary tubules. Scale bars � 50 �m. B:Western blot analysis indicates an increased collagen I protein expression inthe diabetic innervated TGR kidneys. This elevation is attenuated in thediabetic TGR kidneys subjected to renal denervation. Values are means SE.*P � 0.05 vs. NGInx. �P � 0.05 vs. DBInx.



effect of renal denervation on the individual contribution ofdiabetes as well as hypertension in diabetic nephropathy.

The induction of diabetes in the hypertensive TGR animalsresulted in a reduced rate of growth compared with the nor-moglycemic animals. This pattern of body weight differencerecorded in the intact normoglycemic and diabetic TGRs isconsistent with earlier published data gained from the sameanimal (32). While this reduction in body weight gain has alsobeen observed in other diabetic rat models following STZadministration, our results indicate that the surgical interven-tion procedure itself had minimal impact on the animals’ healthand growth status (12). The 12-wk time course of this exper-imental model was carefully selected to ensure that the devel-opment of renal injury did not result solely from the effects ofchronic hypertension but as a consequence of the synergisticeffects of hyperglycemic induction. Applehoff and coworkers(3) clearly demonstrated that both diabetes and the ren-2 genemay contribute to the glomerular pathology described at 4 mo

in the male rat. Conversely, female TGRs develop lower levelsof hypertension (30–40 mmHg less) than their male littermatesand show no evidence of morphological changes in thenephron before 4–6 mo of age (4). Consequently, while SBPwas elevated in the intact female heterozygous TGR animalsover the 12-wk period, no evidence of renal morphologicalinjury or proteinuria was seen in the normoglycemic femaleTGRs. We found that BRD intervention reduced SBP and renalcortical catecholamine levels in the normoglycemic renin-overexpressing hypertensive rats. While heart rate was unaf-fected by denervation in either normo- or hyperglycemicgroups, BRD produced a more pronounced decrease in SBP inthe diabetic than in the normoglycemic animals, suggestingthat RSNA is highly important in maintaining elevated bloodpressure in the diabetic TGRs with renal injury. Indeed, renalcatecholamine levels were significantly raised in the diabeticfibrosed kidneys compared with the hypertensive only group,strongly supporting the hypothesis that diabetic renal injurycan elevate RSNA. In support of this premise, afferent renalnerve activity has also been shown to be elevated in the ratrenal injury model (10). Consequently, in innervated diabetichypertensive TGRs, the amplified afferent signals originatingfrom the kidneys may serve to sustain the elevation in systemicSNA and maintain an increased blood pressure, possibly tocompensate for a potential fall in cardiac output resulting fromdiabetic cardiomyopathy. In contrast, in denervated diabeticTGRs, permanent removal of renal afferent nerves suppressedSNA, resulting in a fall in SBP. Studies conducted by Campeseand colleagues (5, 9, 63) in the nephrectomized hypertensiverat renal injury model have shown that selective renal afferentdenervation can abolish the severity of hypertension and re-duce posterior hypothalamic noradrenaline content. Similarly,BRD was found to reduce blood pressure and attenuate thehypothalamic norepinephrine turnover in a neurogenic hyper-tensive rat model (62–64). These prior studies support thesuggestion that elevated afferent impulses from injured kidneyscan stimulate central sympathetic activity at the hypothalamiclevel, thereby evoking hypertension and may explain the anti-hypertensive effects of BRD observed in the present study.Further work on this model should help to clarify the specificrole of afferent renal nerve activity in the progression ofhypertension and renal failure.

Following diabetic onset, marked hyperglycemia and albu-minuria, combined with a reduction in the podocyte-specificprotein podocin, were observed in the innervated diabeticTGRs. The association between glomerular podocin loss andalbuminuria has also been found in both experimental modelsand in human diabetic nephropathy (37, 61). The reduction ofthe diabetes-induced albuminuria with BRD can be partiallyexplained by the reduction in blood pressure consequent withthis intervention (54). However, studies in cultured podocyteshave shown that the �-adrenoceptor agonist phenylephrine canincrease intracellular Ca2� concentration to alter podocyteshape and reduce foot processes, thus directly regulating podo-cyte functions (29). These in vitro studies strongly suggest thatelevated cortical norepinephrine levels recorded in the diabeticnephropathy animals may directly mediate podocyte injury,consequently permitting the development of albuminuria. Thissupports and may partially explain the mechanism throughwhich BRD attenuated podocyte injury in the diabetic kidneys.Indeed, BRD also reduced the severity of glomerulosclerosis

B

Collagen IV

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Fig. 8. Collagen IV localization and expression in the cortex and inner stripeof outer medulla of kidney sections immunolabeled with collagen IV antibodyand counterstained with hematoxylin. A: in sections from normoglycemicinnervated and denervated kidneys, collagen IV was weakly labeled in theglomeruli and medullary tubules. In the diabetic innervated kidney sections,intense collagen IV staining was found within the glomeruli; strong collagenIV staining was also found extensively in the interstitium of the medullarytubules. BRD in the diabetic TGRs reduced collagen IV labeling to normogly-cemic levels in the glomeruli and medullary tubules. Scale bars � 50 �m. B:Western blot analysis indicates increased collagen IV protein expression in thediabetic innervated TGR kidneys. This elevation was attenuated in the diabeticTGR kidneys subjected to renal denervation. Values are means SE. *P �0.05 vs. NGInx. �P � 0.05 vs. DBInx.



and preserved glomerular structure in diabetic TGRs. Selectiverenal afferent denervation was shown to reduce blood pressureand glomerulosclerosis severity in earlier studies of a hyper-tensive rat model of chronic renal failure conducted byCampese and colleagues (10). While glomerular protectionmay in part stem from the reduction in blood pressure conse-quent to BRD intervention, it is important to note, however,that in the absence of hyperglycemia, none of the hypertensiveanimals used in this study showed any evidence of proteinuriaor renal morphological damage. Evidence in support of thisantihypertensive-independent effect comes from other studiesby Nagasu and coworkers (46) showing that removal of sym-pathetic influence by acute renal denervation can directlyprotect glomerular structure in the absence of a blood pressure-lowering effect. Furthermore, the inhibition of RSNA usingpharmacological sympatholytic agents such as prazosin,timolol, guanethidine, and moxonidine at subantihypertensiveconcentrations has also been shown to preserve glomerularstructure in rat models of hypertensive renal failure (2, 22). Thepresence of �1-adrenoceptors within glomerular mesangialcells (6) suggests that the RSNA may play a direct role inmesangial dysfunction. Consequently, removal of this elevatedsympathetic influence on diabetic kidneys may protect thestructural and functional integrity of the glomerular mesan-gium.

The presence of renal cortical and medullary fibrosis asevidenced by the extensive deposition of glycogen and accom-panied by tubule dilation in the diabetic kidneys was confirmedby the elevated expression of renal fibrotic markers TGF-�1,�-SMA, and collagens I and IV. Repeated BRD at 3 wkfollowing diabetic onset suppressed the levels of these fibroticmarkers. Multiple mechanistic pathways may be involved inmediating the protective effect of BRD against diabetic renalfibrosis and warrants further investigation. Previous work con-ducted in other models of renal injury has shown that suppres-sion of renal sympathetic innervation by surgical denervationor moxonidine administration may be effective in suppressingrenal fibrotic markers such as TGF-�1 (2, 23, 57). Renalfibrogenesis induced by ureteric obstruction in a rat model hassimilarly been shown to be attenuated by denervation and by

selective �2-adrenoreceptor agonist administration; this protec-tion was reversed, however, by the infusion of a calcitoningene-related peptide and norepinephrine, elegantly demonstrat-ing that both renal afferent and efferent nerves are involved inthe formation of renal fibrosis (33). Inflammatory responses arealso involved in mediating the progression of diabetic renalinjury (19). As renal denervation in the anti-Thy-1.1-inducedglomerulonephritis model was shown to produce anti-inflam-matory and antifibrotic effects (57), it is conceivable that theprotection afforded by BRD against diabetic nephropathy inthe TGRs could partially be due to an anti-inflammatory effect.

Systemic renin angiotensin system activity can also beeffectively reduced by a BRD abrogation of renin release fromthe juxtaglomerular apparatus (20). Elevated norepinephrinerelease from renal sympathetic nerve terminals as seen in thediabetic TGRs group can induce TGF-� expression, therebyenhancing the extracellular matrix accumulation and profi-brotic actions of intrarenal angiotensin II at the renal tubules(13). BRD intervention in this study may consequently inhibitthe involvement of the intrarenal renin-angiotensin system inthe TGF-�-mediated profibrotic pathways observed in thediabetic kidneys. Unfortunately, this study was not set up totease out the effects of denervation on the catabolic action ofproinflammatory matrix metalloproteinases and other proteo-lytic enzymes on pathogenic extracellular matrix accumulation(40). The potential impact of BRD on metalloproteinasesspecific to each stage of albuminuria development remains tobe clarified.

While various studies have demonstrated the interstitial andintratubular localization of the intrarenal renin-angiotensinsystem, only a few studies have attempted to resolve theactivation status of this pathway in diabetes. Induction ofmoderate hyperglycemia using STZ in a rat model has beenshown to result in elevated systemic angiotensin II and in-creased AT1R immunostaining in the renal collecting ductswithin 20 days (25, 35). Examination of our animal kidneyssubjected to a longer time course of hyperglycemia (12 wk postSTZ) shows that diabetic induction also had the effect ofincreasing AT1R expression in the cortical and outer medullarycollecting duct of these renin-overexpressing TGRs. No

NGInx NGDnx DBDnxDBInx

Fig. 9. �-Smooth muscle actin (SMA) localization andexpression in the inner stripe of outer medulla of kidneysections immunolabeled with anti-�-SMA antibody andcounterstained with hematoxylin. Minimal �-SMA stainingwas seen in the medullary tubules of normoglycemic sham-operated (A) and denervated (B) kidneys. In the diabeticinnervated TGR kidneys, intense �-SMA staining wasextensively expressed in the interstitium of the medullarytubules (C and magnified in E). BRD in the diabetic TGRkidneys markedly decreased medullary �-SMA deposition(D and magnified in F). Scale bars � 50 �m.



change in AT1R immunostaining of the nephron was evidentfollowing BRD in the normoglycemic animals, suggesting thatwhile both denervation and reduction in hypertensive statusmay not have had any effect on the weak AT1R staining, AT1Rlocalization was not raised as a consequence of the hyperten-sion but as a result of diabetic induction. Significantly, BRDintervention in the diabetic TGR abrogated the AT1R stainingof collecting tubules. This finding is consistent with the otherdata presented here demonstrating that renal morphology andfibrotic makers are not markedly altered by denervation in thehypertensive only heterozygous female TGR. The significanceof the increased AT1R expression within the collecting tubulesmay be explained as an angiotensin II-mediated conservationof the Na�/water balance in the face of an increased urineoutput in diabetes (25). However, while BRD reduced AT1Rexpression, BRD did not restore UNaV and reduce output inthis TGR model.

Diabetic induction resulted in the development of renalhypertrophy and a decreased GFR after 12 wk in the presentmodel, indicating that renal function was at the declining phaseof the diabetic disease progression. However, both GFR reduc-tion (32) and elevation (59) have been reported at this sametime point following diabetic onset in the same diabetic TGRmodel. Type 1 diabetic induction in the TGR increased GFR at4 wk but reduced it by 50% after 12 wk of diabetes in TGRs.These studies suggest that the initial point of GFR decline isvariable, occurring at slightly different time points during thedisease progression in different batches of animals. In contrastto the renal morphological protection afforded by BRD indiabetic TGRs, no similar effect was observed with the GFRindices. It should be noted that GFR in the normoglycemichypertensive TGRs was also unaffected by BRD intervention.Studies conducted in a type 1 diabetic rat have indicated thatBRD can reverse GFR changes if conducted early (2 wk)within the disease progression (42). While this discrepancy ishard to explain, our observations are consistent with a recentclinical study, which found that GFR was unchanged by BRDtreatment in patients with moderate to severe chronic kidneydisease (27).

In conclusion, the present study conducted in renin-overex-pressing TGRs substantiates the significance of renal sympa-thetic nerves in the pathogenesis of diabetic nephropathy.While denervation clearly had no effect on renal morphologyor fibrotic marker status in hypertensive only animals, BRDsubstantially reduced albuminuria and attenuated the renalfibrosis and glomerulosclerosis consequences of hyperglyce-mia. BRD was conducted over a 12-wk period starting 3 wkafter the onset of early diabetic injury (36). Further work willbe needed to clarify whether this procedure can revert estab-lished diabetic nephropathy and show benefit over currentpharmacological interventions. Early experimental studies us-ing mesenchymal cell transplantation in type 1 diabetic micehave proved promising, indicating that both microalbuminuriaand renal structural injury may be reversed; however, thistherapy is still experimental (17). While reducing the progressof the disease, angiotensin-converting enzyme inhibitors fail toprevent angiotensin escape pathways from eventually provok-ing the renal manifestations of hyperglycemia. By attenuatingthe development of diabetic nephropathy, renal denervationmay consequently prove valuable as an additive to the currentpractice of renin-angiotensin-aldosterone blockade. The supe-riority of this combination therapy over monotherapy withquinapril has already been demonstrated in a renal ablationmodel (23). We conclude that the protection of renal indicesand structure reported in this study provides strong scientificsupport for the application of renal denervation procedures indiabetic nephropathy.

ACKNOWLEDGMENTS

The authors thank Prof. Zoltan Endre (Kidney Research Group, Universityof Otago, Christchurch, NZ) for valuable discussions on and use of thetransgenic (mRen-2)27 colony. The authors also thank Amanda Fisher (Uni-versity of Otago, Department of Pathology) and Andrew McNaughton (Uni-versity of Otago, University of Otago, Department of Anatomy) for technicalassistance with immunostaining and confocal microscopy.

GRANTS

These studies were supported by the Otago School of Medical Sciences,University of Otago.

B NGInx NGDnx DBInx DBInx ~25 kDa TGF- 1

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Fig. 10. Transforming growth factor (TGF)-�1 localization and expression inthe cortex and medulla of the kidney sections immunolabeled with anti-TGF-�1 antibody and counterstained with hematoxylin. A: in sections fromnormoglycemic innervated and denervated kidneys, TGF-�1 staining wasweakly labeled. In the diabetic innervated kidneys, intense staining was foundwithin the glomerular mesangium (thin arrows) and in the cytoplasm andluminal surface of the proximal and distal tubules (wide arrow); in the medulla,TGF-�1 was found in the interstitial space within the cytoplasm of themedullary tubules. BRD in the diabetic TGRs reduced TGF-�1 labeling tonormoglycemic levels in the glomeruli and medullary tubules. Scale bars � 50�m. B: Western blot analysis shows increased TGF-�1 protein expression inthe cortex of diabetic innervated TGR kidneys. This elevation is attenuated inthe diabetic TGRs subjected to renal denervation. Values are means SE.*P � 0.05 vs. NGInx. �P � 0.05 vs. DBInx.



DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: Y.Y., J.C.H., R.J.W., G.D., and I.A.S. providedconception and design of research; Y.Y., I.C.F.-N., and J.C.H. performedexperiments; Y.Y., I.C.F.-N., and G.D. analyzed data; Y.Y., J.C.H., G.D., andI.A.S. interpreted results of experiments; Y.Y. prepared figures; Y.Y. andI.A.S. drafted manuscript; Y.Y., I.C.F.-N., J.C.H., R.J.W., G.D., and I.A.S.approved final version of manuscript; J.C.H. and I.A.S. edited and revisedmanuscript.

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Fig. 11. A: ANG II type 1 receptor (AT1R) immunolabeling and localization in the cortex and outer medulla of normoglycemic and diabetic sectionscounterstained with hematoxylin. Faint AT1R labeling (brown DAB stain) was observed within the proximal and distal tubules of normoglycemic or diabeticTGRs. AT1R labeling was also weak within the collecting duct segments and connecting tubules of normoglycemic kidney sections but appeared prominent (seearrows) within the epithelial layer of the collecting tubules obtained from the intact DBInx group. Note the lack of DAB staining in the abnormally expandedmatrix around the dilated collecting tubules of the DBInx. AT1R labeling was strikingly reduced by denervation in the diabetic animals. Scale bars � 50 �m.B: percentage of tissue positively stained for DAB against background, recorded in 10 randomly selected cortical and medullary fields. Values are means SE(n � 7 - 8 individual animals/group). ***P � 0.0001 vs. NGInx.



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chronic bilateral renal denervation attenuates renal injury in a transgenic rat model of diabetic...

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