Background
Diabetic nephropathy is the leading cause of end-stage renal disease in the United States. In 2008, 44% of new cases of kidney failure were attributed to diabetes, and the numbers are expected to increase as the number of Americans with diagnosed diabetes has reached above 20 million – with another estimated 7 million people with undiagnosed diabetes [
1].
Hypertension is a major risk factor for renal disease progression in patients with diabetes. One of the most common causes of secondary hypertension is renal artery stenosis (RAS). Atherosclerosis, the main cause of RAS, shares many similar risk factors with diabetes type II, thereby making it likely for RAS to co-exist in diabetic type II patients. Indeed, in patients with type II diabetes and hypertension the incidence of RAS is between 17-44% [
2‐
4] and even subcritical RAS confers a significant risk for progression to renal failure [
3]. However, it is still unclear if this increased risk is due to hypertension alone or contributed by other factors that are induced during RAS. It is well recognized that RAS is associated with activation of the renin-angiotensin system which leads to systemic hypertension. We have previously demonstrated that in our unilateral RAS model, the decrease in blood flow to the stenotic kidney is associated with an increase in blood flow to the contralateral kidney [
5], raising the possibility that the contralateral kidney may be susceptible to hyperfiltration injury. However, few studies have directly addressed potential interactions between hyperfiltration and pathophysiologic activation of renin-angiotensin system in the development of diabetic renal disease.
We therefore sought to test the hypothesis that activation of the renin-angiotensin system and hyperfiltration interact to produce chronic injury in the contralateral, non-stenotic kidney of db/db mice. We demonstrate that db/db mice with RAS develop diffuse mesangial sclerosis in their contralateral kidney that is not observed in age matched db/db mice or in WT mice with RAS. Unilateral nephrectomy, infusion of Angiotensin II, or their combination in age-matched db/db mice failed to reproduce the glomerular and, in particular, the interstitial lesions observed in db/db mice subjected to RAS. Prophylactic administration of hydralazine and valsartan yield only modest attenuation of renal damage in the contralateral kidney of db/db mice with RAS, with no difference between the two interventions. We conclude that renovascular hypertension in diabetic db/db mice produced accelerated and progressive renal injury that cannot be explained by increase in blood pressure alone.
Methods
Animal models
C57BLKS (WT) (N = 50) and C57BLKS/JLepr (db/db) (N = 166) male mice, 5–6 weeks old, were obtained from Jackson Laboratory (Bar Harbor, ME). Induction of hypertension and RAS was performed using a modified cuff approach as previously described [
5,
6] at 6–7 weeks of age (N = 10 for each time points). Mice were studied at 2, 4 and 6 weeks post RAS induction. Sham surgeries (N = 5 for each time points) consisted of a flank incision and mobilization of the renal artery without placement of a cuff. To determine the effect of angiotensin II induced hypertension with or without hyperfiltration, unilateral nephrectomies or sham surgeries [
7,
8] were performed on db/db mice at 6–7 weeks of age as previously described. Osmotic mini-pump (Alzet model 2004, Durect, Cupertino, CA) loaded with Angiotensin II (1000 ng/kg/min) or PBS were inserted subcutaneously on the same day [
9‐
13]. To determine the effect of lowering blood pressure, Hydralazine (10 mg/kg/day) or angiotensin II receptor blocker Valsartan (8 mg/kg/day) was administered in drinking water of db/db mice with RAS on the day of the surgery (N = 10 per each treatment, N = 8 for vehicle treatment).
Blood pressures were measured on conscious acclimatized mice using tail cuff method (CODA System, Kent Scientific, Torrington, CT) three days prior to surgery and subsequently at two-week intervals. Mice were euthanized by exsanguination at 2, 4, and 6 weeks post-surgery. Kidneys and hearts were perfused with sterile PBS, excised, weighed, and either preserved immediately for histology, or shock frozen in liquid nitrogen for Western blotting and PCR analysis. All animal protocols were approved by the Mayo Clinic Institutional Animal Care and Use Committee.
Biochemical analysis
Blood was collected by tail bleed for serial measurements and finally by terminal bleed. The plasma fraction was separated by centrifugation upon collection and stored at -80°C until assay. Renin activity in plasma was assessed via production of angiotensin I from angiotensinogen using a commercially available GammaCoat Plasma Renin Activity 125I RIA kit (DiaSorin, Stillwater, MN), using porcine angiotensinogen (A2283, Sigma-Aldrich, St. Louis, MO) substrate. Urine albumin and creatinine were measured on spot urine sample using Albuwell and Creatinine kit (Exocell, Philadelphia, PA). Commercially available ELISA kits were used for the measurements of serum CCL2 and IL-6 (R&D Systems, Minneapolis, MN).
Histology and immunohistochemistry
Kidneys were fixed with 10% neutral buffered formalin and processed for histology or immunostaining using standard techniques. Histological section (5 μm thick) were prepared and stained with hematoxylin-eosin (H&E), Masson’s trichrome, periodic acid-Schiff, anti-fibronectin (Abcam, Cambridge, MA), and anti-F4/80 (AbD Serotec, Raleigh, NC). H&E slides were used to assess atrophy, glomeruli area and diameter. Atrophy was semi quantitatively assessed by a renal pathologist by assessing the relative surface area occupied by atrophic tubules compared to the total cortical surface area, as previously described [
14]. Mesangial matrix expansion was assessed in PAS sections with a 0–4 scale (0 = normal, 1 = mild mesangial matrix expansion, 2 = moderate matrix expansion with patent capillaries, 3 = severe matrix expansion with segmental capillary loop consolidation, 4 = severe matrix expansion with global capillary loop consolidation). Each glomerulus was scored positive or negative for fibronectin, and quantified as percent positive glomeruli over total glomeruli per tissue sections. Degree of fibrosis was quantified in trichrome sections by assessment of ratio of surface area of the cortical area (avoiding great vessels and glomeruli) at 200× magnification. Interstitial fibronectin deposition and renal microphage infiltration was similarly quantified in fibronectin and F4/80 sections respectively. All measurements and quantification were performed in a random blinded fashion using an Olympus BX50 microscope (Olympus America, Melville, NY), a Micropublisher 3.3 RTV camera (Q-Imaging, Surrey, BC), and the NIS Elements Imaging Software (Nikon Instruments, Inc., Melville, NY).
Transmission electron microscopy
For transmission electron microscopy, tissue was removed from the paraffin block and placed into warm xylene for 90 minutes, transferred to warm absolute ethanol for 30 minutes, then transferred to decreasing concentrations of ethanol to 60% then placed into Trump’s fixative (1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2) for overnight fixation. Tissue was then rinsed in 0.1 M phosphate buffer, pH 7.2, post-fixed in 1% osmium tetroxide for one hour, rinsed in distilled water, dehydrated, embedded in Spurs resin, and sectioned at 90 nm. Micrographs were taken on a Philips Technai 12 (FEI, Hilsboro, OR) operating at 80KV. Glomerular basement membrane measurement was performed by Mayo Clinic Electron Microscopy Core Facility in a random blinded fashion.
mRNA analysis
Total RNA was extracted with RNeasy Mini Plus kit (Qiagen, Valencia, CA) and reversed transcribed using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Gene expression analysis was determined by quantitative real-time PCR using CFX96 (Bio-Rad, Hercules, CA) and normalized to 18 s. The following primers were used: Ren1 forward 5’ –GAG GTA GCG ACC CGC AGC ATT AT- 3’; Ren1 reverse 5’ – GCG CTG CCT CCC AGG TCA AA- 3’; Ccl2 forward 5’ – AGC ACC AGC ACC AGC CAA CTC – 3’; Ccl2 reverse 5’ – TGG ATG CTC CAG CCG GCA ACT – 3’; Il-6 forward 5’ – TGG TGA CAA CCA CGG CCT TCC – 3’; 1 l-6 reverse 5’ – TAA GCC TCC GAC TTG TGA AGT GGT – 3’; 18 s forward 5’ – CTC AAC ACG GGA AAC CTC AC – 3’; 18 s reverse 5’ – CGC TCC ACC AAC TAA GAA CG – 3’.
Statistical analysis
Data are presented as mean ± SE. Comparisons between two groups were done using student t-test for parametric data and Mann–Whitney test for non-parametric data or data without normal distribution. To assess interactions between time points and multiple groups, two-way ANOVA followed by a Tukey adjustment for post-hoc comparison across different time points and treatment groups was used. For comparison across multiple groups, one-way ANOVA followed by a Tukey adjustment was used for post-hoc comparison of the measurements. P values <0.05 were considered significant. Statistical analyses were performed with Graphpad Prism 6 (GraphPad Software, La Jolla, CA).
Discussion
A role for hypertension in the development of renal lesions in db/db mice has not been clearly established [
20‐
25]. We found that db sham mice did not develop spontaneous hypertension, while db RAS mice develop hypertension to an extent that is similar to that observed in WT RAS mice, yet associated with transient but more prolonged increases in plasma renin activity and greater renal
Ren1 expression. This persistent increase in plasma renin activity in db RAS mice may reflect interactions between hemodynamic forces associated with renovascular hypertension and the diabetic mileau. Despite similar level of systolic blood pressure, the contralateral kidney of db RAS mice developed chronic renal injury characterized by development of mesangial matrix expansion, interstitial fibrosis, tubular atrophy, and interstitial inflammation, as opposed to the contralateral kidneys in a number of other strains of non-diabetic mice subjected to RAS [
5,
16,
26]. Glomerular histopathologic alterations in the contralateral kidney of db/db mice were striking, and reminiscent of those observed in progressive human diabetic nephropathy, with severe and diffuse mesangial matrix expansion, evident as early as 2 weeks following induction of hypertension. Mesangial matrix expansion consistently was far more extensive than in age-matched db sham mice, and was associated with
de novo glomerular fibronectin expression. Older db/db mice develop glomerular basement membrane thickening, but quantitative studies in this model have not yet been reported [
27‐
29]. We found an increase of glomerular basement membrane thickness in the contralateral db RAS kidney by six weeks post-surgery, as assessed by morphometric analysis of electron microscopic images, a well-recognized feature of evolving diabetic nephropathy. Glomeruli in these kidneys (but not in WT RAS) showed extensive effacement of visceral epithelial cell foot processes, a morphologic correlate of the progressive albuminuria observed in these mice. At all time-points, urine albumin excretion was significantly greater in db RAS than db sham mice. Based on these observations, we conclude that renovascular hypertension markedly accelerates renal disease progression in db/db mice as characterized by glomerular mesangial matrix expansion, progressive interstitial fibrosis and inflammation, and breakdown of the filtration barrier. This is in accordance with clinical observations indicating that progression of diabetic nephropathy is accelerated in patients with hypertension.
We infused db/db mice with angiotensin II for 4 weeks to address a potential role of angiotensin II induced hypertension on renal architecture in db/db mice. These mice developed hypertension to levels similar to those attained in db RAS mice, yet we observed a minimal increase in mesangial matrix deposition and no evidence of d
e novo glomerular fibronectin deposition. Nevertheless, db Ang-II developed albuminuria similar to that observed in db RAS mice and to that reported following angiotensin II infusion to non-diabetic mice [
30,
31]. Taken together, these observations suggest that the progressive and bilateral renal injury in db RAS mice is not mechanistically related to elevated angiotensin II levels alone, although angiotensin II plays a major role in development of albuminuria in this model [
7,
8]. This finding underscores a critical role for activation of the renin-angiotensin system in the development of albuminuria and provides a therapeutic rationale for the widespread use of renin-angiotensin inhibitors in treatment of chronic kidney disease.
We then sought to determine whether hyperfiltration associated with unilateral nephrectomy may underlie the progressive renal damage observed in the contralateral db RAS kidney. Unlike db RAS or db Ang-II mice, db UNX did not develop significant hypertension. Db UNX also did not develop increased urine albumin excretion that was observed in the db RAS or db Ang-II. However, as shown before, db/db mice with unilateral nephrectomy did develop greater glomerular mesangial matrix expansion than age-matched db/db mice with two kidneys [
8,
32], although its extent was less than that of db RAS mice. Although few investigators have directly reported the extent of interstitial fibrosis in this model, db/db mice evaluated at 14 – 18 weeks post UNX exhibited a modest increase in interstitial inflammation, interstitial volume, and number of tubules showing dilation or atrophy [
8,
32,
33]. In the current study, we find that db UNX mice, in striking contrast to db RAS mice, do not develop significant interstitial fibrosis or tubular atrophy at 4 weeks post UNX. Therefore, glomerular mesangial matrix expansion in db/db mice can be attributed at least in part to hemodynamic factors associated with hyperfiltration, whereas elevation of blood pressure appears to play a major role in development of albuminuria in db/db mice.
As Angiotensin-II induced hypertension and UNX alone only recapitulate some features of renal injury seen in the contralateral kidney of db RAS mice, we combined both in db/db mice. Remaining kidneys of db UNX + Ang-II mice developed all the features seen in the db RAS mice, namely mesangial expansion, interstitial fibrosis, tubular atrophy, and albuminuria, but the severity of injury observed in the contralateral kidney of db RAS mice was greater than that of db UNX + Ang-II mice.
To examine if hypertension was necessary for the development of progressive renal fibrosis in the contralateral kidneys of db/db mice, we treated them with ARB or the vasodilator hydralazine, which lowered blood pressure to levels similar to those observed in db sham mice without significant changes in plasma renin activity. Reduction of blood pressure was effective in reducing mesangial matrix expansion, fibronectin expression, interstitial fibrosis, and tubular atrophy in the contralateral kidney of db RAS mice. However, urine albumin excretion was significantly reduced by ARB only. Therefore, we conclude that hypertension plays an essential role for the development of chronic renal lesions in the contralateral kidney of db/db mice subjected to RAS, while increase level of angiotensin II plays a role in the development of albuminuria. Interestingly, while both drug treatments attenuate the development of renal injury, both do not abolish it. Given the less severe injury observed in the db UNX + Ang-II, these results point to some other factor independent of blood pressure elevation and hyperfiltration process that is mediated by the stenotic kidney, possibly by the activated RAAS.
We and other investigators have shown that the stenotic kidney experienced considerable oxidative stress [
14,
17] and produced substantial level of inflammatory cytokines [
18,
19]. Indeed, in comparison to the other models, contralateral kidney of db RAS exhibited significantly higher expression of the inflammatory chemokine CCL2 and the inflammatory cytokine IL-6, both of which represent prognostic of development of renal injury [
34‐
36]. Nonetheless, db RAS showed similar increased in serum CCL2 and IL-6 to db UNX + Ang-II. However, although serum levels of CCL2 might be elevated in diabetic patients, they are not associated to the development of albuminuria, renal macrophage influx, or interstitial fibrosis [
37‐
39]. Instead, both urine CCL2 and IL-6 excretion – reflecting production of these inflammatory molecules within the kidney itself – have been shown to correlate significantly with progression of renal injury [
37,
38,
40]. Furthermore, increased albuminuria may itself aggravate tubular injury and accelerate development of renal injury by increasing tubular CCL2 and IL-6 production [
41,
42].
Acknowledgments
We thank Kim Butters for assistance with primer design, Hui Tang, PhD for assistance with plasma renin content assay, Andrea McConico for assistance with ELISA assay, Mayo Clinic Electron Microscopy Core for assistance with EM imaging and morphometric analysis, Karen Lien for beautifully stained slides and Anuradha Krishnan, PhD for critical reading of this manuscript. These studies were supported by NIH/NIAID R01 AI100911-01, NIH/NHLBI P01 HL85307 and UL1 RR024150. S.P.H. is supported by NIH/NIGMS T32 GM 6584. This paper’s contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
Grant support
NIH R01-AI100911-01; P01-HL85307; T32 GM6584; UL1-RR024150.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SPH and JPG conceived the project. SPH, BEK and JPG designed the methods and the experiments. BEK performed all the animal surgeries. SPH carried out the laboratory experiments, analyzed the data, and interpreted the results. SPH and JPG wrote the manuscript. LOL and SCT gave technical support, conceptual advice, and critical review of the manuscript. All authors read and approved the final manuscript.