Abstract
Background. Aliskiren is a direct renin inhibitor (DRI) and provides an organ-protective effect in human and animal experiments. However, there is no current evidence of the effect of DRI on insulin resistance and metabolic abnormalities in type 2 diabetic animals.
Methods. We investigated the effects and molecular mechanism of aliskiren in db/db mice and cultured mesangial cells (MCs).
Results. Aliskiren treatment for 3 months at a dose of 25 mg/kg/day via an osmotic mini-pump did not induce significant changes in blood glucose levels, systolic blood pressure, serum creatinine and electrolyte levels. However, aliskiren treatment improved insulin resistance confirmed by insulin tolerance test and various biomarkers including homeostasis model assessment index levels and lipid abnormalities. The treated group also exhibited significant improvement in cardiac functional and morphological abnormalities including left ventricular hypertrophy, and induced phenotypic changes in adipose tissue. Aliskiren treatment also markedly decreased urinary albumin excretion, glomerulosclerosis and suppressed profibrotic and proinflammatory cytokine synthesis and improved renal lipid metabolism. In cultured MCs, high glucose stimulation increased MC renin concentration. Furthermore, renin treatment directly up-regulates synthesis of proinflammatory and profibrotic cytokines, which were abolished by prior treatment with aliskiren and angiotensin receptor (AT1) antagonist. These results suggest that the beneficial effect of aliskiren is mediated by an angiotensin-dependent mechanism.
Conclusions. Together, these results imply that aliskiren provides an organ-protective effect through improvement in insulin resistance and lipid abnormality, as well as direct anti-fibrotic effect in target organ in db/db mice. Aliskiren may be a useful new therapeutic agent in the treatment of type 2 diabetes mellitus and diabetic nephropathy.
Introduction
Dysregulation of the renin–angiotensin–aldosterone system (RAAS) has an important role in the development of target organ damage in diabetes mellitus [1–3]. Therefore, drugs targeting RAAS suppression have been most widely used for slowing or preventing target organ damage [1], although most drugs are not completely effective in preventing or reversing diabetic complications, suggesting an incomplete blockade of the RAAS. The beneficial effects of the ACE inhibitor and of ARB may be attenuated by the stimulation of renin that results from the negative feedback loop associated with decreased angiotensin II activation. Thus, direct renin inhibition provides a more logical approach to creating a complete blockade of RAAS activity without a rebound increase in plasma renin activity (PRA).
The blocking of the RAAS through the inhibition of renin was conceptualized years ago [4]. Aliskiren is the first drug of a new class of agents known as renin inhibitors, which directly inhibit renin in order to decrease PRA [5]. Unlike previous renin inhibitors, aliskiren has oral bioavailability and an extended half-life [6]. The most recent reports for aliskiren treatment revealed effective organ protection related to an anti-hypertensive effect [7–10]. Although aliskiren treatment through monotherapy or combination therapy with other RAAS blockades has been shown to have protective renal and cardiac benefits in human and animal studies, there is a lack of evidence on the effects of aliskiren on insulin resistance and renal lipid metabolism as a mechanism of target organ injuries in type 2 diabetic models.
Recent hypertension trials have reported a lower incidence of diabetes mellitus among patients treated with RAS inhibitor when compared with other classes of antihypertensive medications [11,12]. Although most of the patients from those studies were presented with hypertension or congestive heart failure, a 22% relative risk reduction for new-onset diabetes mellitus was demonstrated in a meta-analysis of randomized controlled trials [13]. In spite of these firm clinical data, the anti-diabetic mechanism of the RAS blockade has yet to be resolved. Multiple mechanisms have been suggested for the improvement of insulin sensitivity by RAS inhibition, such as improving insulin signalling pathways at the cellular level, improving insulin secretion by pancreatic beta cells, and modulation of adipocytokines in the adipose tissues [14–16].
Accumulation of excess lipids in non-adipose tissues induces cellular dysfunction, and this phenomenon, known as lipotoxicity, may play an important role in the pathogenesis of tissue injury [17,18]. Several mechanisms have been suggested in explaining lipotoxicity, such as a direct toxic effect of fatty acids or products of their metabolism, increased production of reactive oxygen species, ATP deficiency, and fatty acid-induced apoptosis [17]. Lipid deposition in the kidney is not a rare phenomenon, and previous reports suggest an important role for renal lipid metabolism in the pathogenesis of diabetic nephropathy [19,20]. Together, these findings suggest that RAS inhibition may improve insulin resistance and abnormal lipid metabolism in insulin resistance states.
In the present study, we investigated the mechanism behind the beneficial effect of aliskiren on diabetic vascular complications in type 2 diabetic mice. Because RAAS plays an important role in insulin resistance, we also examined the effect of aliskiren on insulin resistance and lipid metabolism. Considering that renal lipid metabolism is also important in the development of diabetic nephropathy, we also examined the change in renal lipid metabolism by direct renin inhibition. In addition, we conducted an in vitro study to define further the molecular mechanism of aliskiren.
Materials and methods
Animal studies
Six-week-old male diabetic db/db mice (C57BLKS/J-leprdb/leprdb) and male non-diabetic db/m mice (C57BLKS/J-leprdb/+) were purchased from the Jackson Laboratory. The mice were treated with aliskiren at the age of 8 weeks, and all mice were kept at controlled temperature (23 ± 2°C) and humidity (55 ± 5%) levels under an artificial light cycle. The mice were divided into three groups. The first group consisted of non-diabetic db/m mice as diabetic controls (n = 8), the second group was composed of untreated diabetic db/db mice (n = 7), and the third group was made up of diabetic db/db mice treated with aliskiren (SPP-100; Novartis Pharmaceuticals, Basel, Switzerland) for 3 months at a dose of 25 mg/kg/day through an osmotic mini-pump (n = 6). We used the ALZET model 1002 which was replaced every 2 weeks, and inserted into a different area on the back of the mice each time due to the high irritation caused by aliskiren on skin. Food and water intake, urine volume, body weight, fasting blood glucose concentration, and HbA1c level were measured every month. Plasma glucose levels were measured with the glucose oxidase method, while creatinine levels were determined by the modified Jaffe method. Plasma insulin and adiponectin levels were measured using an ELISA kit (Linco Research, St. Charles, MO, USA). The homeostasis model assessment index (HOMA-IR) was calculated using the formula of fasting glucose (mmol/L) × fasting insulin (mU/L)/22.5. Plasma triglyceride and cholesterol analyses were performed using a GPO-Trinder kit (Sigma, St. Louis, MO, USA). Plasma renin concentration (PRC) was measured using a radioimmunoassay kit (Gammacoat, Diasorin, Stillwater, MN, USA). Briefly, blood was collected into a 75-μL haematocrit tube containing EDTA. Plasma was separated by centrifugation and frozen until used for renin measurement. With the use of a 5-fold dilution of 10 μL of plasma, renin concentration was determined by radioimmunoassay. PRC was determined by measuring angiotensin I generated after addition of excess substrate, angiotensinogen (AGT). Samples were incubated with porcine AGT (1 mM) at 37°C for 1 h, followed by measurement of angiotensin I levels. Plasma aldosterone concentrations were measured with a radioimmunoassay kit (Coat-A-Count® kit, DPC, Los Angeles, CA, USA). Insulin tolerance testing (ITT) was conducted through an intraperitoneal injection of 0.75 units/kg of regular insulin to fasting mice and the subsequent measurement of blood glucose levels at 0, 30, 60, 90 and 120 min. Lipids from hepatic, adipose and renal cortical tissues were extracted by the Bligh and Dyer method [21]. Total cholesterol and triglyceride contents were measured using a commercial kit (Wako Chemicals, Richmond, VA, USA). To determine the amount of urinary albumin excretion, individual mice were separated in a metabolic cage, where urine was collected and measured for 24 h every month. The urinary albumin concentration was determined by a competitive ELISA (Shibayagi, Shibukawa, Japan), while urinary VEGF levels were measured by a commercial kit (Quantikine, R&D Systems, Minneapolis, MN, USA). Mice were killed under anaesthesia with intraperitoneal injections of sodium pentobarbital (50 mg/kg). All experiments were conducted in accordance with the NIH guideline and with the approval of the Korea University Institutional Animal Care and Use Committee.
To determine the cardiovascular effect of aliskiren, systolic blood pressures and echocardiograms were taken at the end of the study. Systolic blood pressure was measured in pre-warmed un-anaesthetized mice using tail-cuff plethysmography (LE 5001-Pressure Meter, Letica SA, Barcelona, Spain). Echocardiograms were performed in un-anaesthetized mice using a 13-MHz linear transducer (Vivid 7 ultrasound system, GE Healthcare Co., USA). Standard echocardiographic short- and long-axis views were obtained. Left ventricular function, ventricular size and wall thickness were measured from M-mode frames. Digital images were obtained and analysed offline. Fractional shortening was estimated as a systolic function from this formula: fractional shortening (%) = [(LVIDd − LVIDs)/LVIDd] × 100. LV mass index was calculated as a marker of LV hypertrophy corrected by a 10-g body weight from the following formula: 1.055 × [(IVSd + LVIDd + LVPWd)3 − (LVIDd)3]/10 g, where LVIDd and LVIDs are the LV internal dimensions in diastole and systole, respectively, IVSd represents interventricular septal thickness in diastole, and LVPWd stands for LV posterior wall thickness in diastole. To determine the total collagen content in cardiac tissue, ventricular tissue was homogenized in an ice bath and eluted by pepsin (1 mg/100 mg heart tissue) in 0.05 mol/L acetic acid containing 0.005 mol/L EDTA for 72 h at 25°C, spun down in a centrifuge at 4°C, and the supernatant was used for the collagen assay. Total soluble collagen was measured by the SircolTM soluble collagen assay kit (Biocolor, Belfast, Northern Ireland), following the manufacturer's instructions. Briefly, 1 mL of Sirius red reagent was added to 100 μL of test samples and mixed for 60 min at room temperature in a mechanical shaker. The collagen–dye complex was precipitated by centrifugation at 14 000 g for 10 min. To release the bound dye, 1 mL of alkali reagent (0.5 M NaOH) was added to the precipitate, and then, the absorbance was measured at 540 nm using an ELISA reader.
Light microscopy and immunohistochemistry
Cardiac, hepatic and adipose tissues were fixed for 48 h with 10% paraformaldehyde at 4°C. The tissues were then dehydrated, embedded in paraffin, cut into 4 μm thick slices, and stained with Masson's trichrome, and haematoxylin and eosin (H & E). The kidney tissues embedded in paraffin were cut into 4-μm-thick slices and were used for periodic acid–Schiff (PAS) and immunohistochemistry for type IV collagen, TGFβ1, PAI-1, and VEGF. A semi-quantitative score for the sclerosis index (SI) was used to evaluate the degree of glomerulosclerosis on PAS-stained sections as described previously [22]. Glomerular mesangial expansion was also semi-quantitatively scored. The percentage of mesangial matrix occupying each glomerulus was rated on a scale from 0 to 4 as follows: 0, 0%; 1, < 25%; 2, 25–50%; 3, 50–75%; and 4, > 75%. Histologic examination was carried out by a pathologist in an objective manner. More than 50 glomeruli were analysed in kidney sections of each mouse.
For immunohistochemical staining, renal tissue was sliced into 4-μm-thick sections and transferred to a 10 mmol/L citrate buffer solution at pH 6.0. The tissue was then heated at 80°C for 30 min to retrieve antigens for TGFβ1 staining. Alternatively, the sections were transferred to a 10-mM Tris–HCl buffer solution (pH 10.0) and microwaved for 10 min for VEGF or transferred to Biogenex Retrievit (pH 8.0) (InnoGenex, San Ramon, CA, USA) and microwaved for 10–20 min for antigen retrieval prior to PAI-1 staining. For type IV collagen staining, slides were treated with trypsin (one tablet/1 mL H2O) for 20 min for antigen retrieval. After washing in water, 3% H2O2 in methanol was applied to the slides for 20 min in order to block endogenous peroxidase activity. The slides were then incubated at room temperature with either 3% bovine serum albumin/3% normal goat serum (VEGF and type IV collagen) for 60 min, with 10% powerblock (PAI-1) for 15 min, or with 20% normal sheep serum (TGFβ1) for 30 min to block endogenous peroxidase activity. Then, the slides were incubated at 4°C overnight with rabbit polyclonal anti-TGFβ1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-VEGF antibody (1:100; Labvision Cor, Fremont, CA, USA), rabbit polyclonal anti-type IV collagen antibody (1:150; BioDesign International Inc., Sarco, ME, USA), and rabbit polyclonal anti-PAI-1 antibody (1:60; American Diagnostica, Stamford, CT, USA). After overnight incubation, the slides were incubated with a secondary antibody for 30 min. For colouration, slides were incubated at room temperature with a mixture of 0.05% 3,3'-diaminobenzidine containing 0.01% H2O2 and then counterstained with Mayer’s haematoxylin. Negative control sections were stained under identical conditions with a buffer solution that was substituted for the primary antibody. For evaluation of immunohistochemical staining results, glomerular fields were graded semi-quantitatively. Over 60 glomeruli were graded under high power (× 400), and an average score was calculated.
Mesangial cell cultures
Human mesangial cells (MCs) were isolated from the renal cortex of normal human kidneys undergoing nephrectomy due to renal cell carcinoma. The glomeruli were treated with collagenase and cultured in Dulbecco’s modified Eagle medium (DMEM) which contained 10% heat-inactivated fetal calf serum (FCS). The cells were grown in DMEM supplemented with 100 μg/mL of penicillin/streptomycin, 1% HEPES, 2 g sodium bicarbonate, 2 mM l-glutamine and 10% heat-inactivated FCS. To determine whether a high glucose environment increased the renin concentration, MCs were cultivated on 100-mm dishes and serum-restricted for 24 h. Afterwards, the cells were treated with 30 mM of d-glucose. Forty-eight hours later, the media were collected, and the cells were scraped from the dishes in the presence of extraction buffer (20 mM Tris–HCl, pH 7.4, 10 mM ethylenediaminetetraacetic acid, 5 mM ethylenegylcol tetraacetate, 5 mM β-mercaptoethanol, 50 μg/mL phenylmethyl sulphonyl fluoride, 10 mM benzamidine, and 0.1 μg/mL aprotinin) and homogenized. Cell lysates and conditioned media were centrifuged at 15 000 rpm for 10 min at 4°C, and the supernatants were collected. Renin concentrations in cell lysates and their conditioned culture media were measured as described previously. The renin amount in each condition was normalized to the total protein content determined by the BCA protein assay kit (Pierce, Rockford, IL, USA). Next, the direct effect of renin on TGFβ1, type IV collagen, VEGF and PAI-1 productions were evaluated. Mesangial cells were treated with or without recombinant human renin (Cayman Chemical, Ann Arbor, MI, USA) at a final concentration of 10 nM under normal glucose conditions. In some wells, aliskiren was added to the cells at a concentration of 100 nM 1 h before treatment with renin and high-concentration glucose. To evaluate whether the effect of renin is mediated by angiotensin II-dependent pathway or not, 10 μM of L158809 (AT1R blocker) was added to the cells 1 h before treatment with renin. Secreted VEGF and total soluble collagen were measured in cultured supernatants using a commercial ELISA kit (Quantikine, R&D Systems, Minneapolis, MN, USA) and the SircolTM soluble collagen assay kit (Biocolor, Belfast, N. Ireland), respectively. Supernatant levels of VEGF and collagen were expressed relative to the total protein concentration. All experimental groups were cultured in triplicate and harvested at 48 h for extraction of total RNA and protein.
Analysis of gene expression by real-time quantitative PCR
Total RNA was extracted from renal cortical tissues and experimental cells with Trizol reagent. Primers were designed for their respective gene sequences using Primer 3 software, while the secondary structures of the templates were examined and excluded using mfold software. The nucleotide sequences of all of the primers used in this study are shown in Supplementary Tables 1 and 2 (see online supplementary material for these tables). Quantitative gene expression was performed on a LightCycler® 1.5 system (Roche Diagnostics Corporation, Indianapolis, IN, USA) using SYBR Green technology. In 96-well, real-time PCR plates, 10 μL SYBR Green master mix was added to 1 μL of RNA (corresponding to 50 ng of total RNA) and 900 nM of forward and reverse primers for a total of 20 μL of reaction volume. Real-time RT–PCR was performed for 10 min at 50°C and 5 min at 95°C. Subsequently, 30 cycles were applied which consisted of denaturation for 10 s at 95°C and annealing with extension for 30 s at 60°C. The ratio of each gene and β-actin level (relative gene expression number) was calculated by subtracting the threshold cycle number (Ct) of the target gene from that of β-actin and raising two to the power of this difference. The specificity of each PCR product was evaluated by melting curve analysis, followed by agarose gel electrophoresis.
Statistical analysis
For parametrically distributed data, we used the analysis of variance (ANOVA) to compare the quantitative variables, and post-hoc analysis was performed using Bonferroni’s method. Data for mRNA of renal renin, renin receptor, CPT-1, urinary Na excretion, cardiac AT1 receptor, renin and TGFβ1 mRNA levels were not normally distributed. Because log transformation normalized this distribution, we used the log-transformed data for these variables. A significance level of 5% was chosen for all tests (P = 0.05). Statistical analyses were performed using SPSS for Windows, version 10.0 (SPSS, Inc., Chicago, IL, USA).
Results
Effect of aliskiren on insulin resistance in experimental animals
Table 1 shows the various biochemical results obtained for each experimental group. Fasting blood glucose level, body weight and HbA1c level were significantly higher in diabetic mice than in non-diabetic control mice during the study period (Supplementary Table 3; see online supplementary material for this table). However, treatment of diabetic mice with aliskiren had no effect on these parameters. Although organ weights including kidney, liver and fat showed higher levels in diabetic mice, they did not reach statistical significance (Supplementary Table 4; see online supplementary material for this table). As shown in Figure 1, diabetic mice exhibited marked insulin resistance, confirmed by an i.p. insulin tolerance test. Interestingly, aliskiren treatment significantly improved the insulin resistance state (Figure 1). Table 2 shows the changes in the metabolic parameters of the experimental animals. In accordance with insulin resistance, diabetic mice showed significantly higher levels of HOMA-IR, plasma cholesterol and triglyceride than the controls. Aliskiren treatment showed a significant improvement in these parameters. As shown in Figure 2, the diabetic group showed more severe hepatic steatosis, and cholesterol content. Aliskiren treatment did not show significant improvement in hepatic fat accumulation (Table 2). In addition, adipose tissue obtained from epididymal fat revealed that diabetic mice had larger immature adipocytes than those of non-diabetic controls. Treatment with aliskiren restored the phenotypes of these cells to small differentiated adipocytes (Figure 2, Supplementary Figure 4; see online supplementary material for a colour version of this figure).
Biochemical parameters in experimental animals
| Treatment groups | db/m + vehicle (n = 8) | db/db + vehicle (n = 7) | db/db + aliskiren (n = 6) |
|---|---|---|---|
| Body weight (g) | 33.88 ± 0.72 | 45.40 ± 4.38 | 43.00 ± 7.74 |
| SBP (mmHg) | 102.96 ± 1.10 | 101.34 ± 1.32 | 98.18 ± 2.60 |
| FBG (mmol/L) | 8.0 ± 0.4 | 33.8 ± 2.8*** | 29.7 ± 1.5*** |
| HbA1c (%) | 4.49 ± 0.24 | 8.39 ± 0.37*** | 8.88 ± 0.36*** |
| Creatinine (μmol/L) | 30.0 ± 1.0 | 42.0 ± 3.0*** | 45.0 ± 2.0*** |
| Plasma Na (mmol/L) | 142.1 ± 2.86 | 150.0 ± 3.53 | 147.5 ± 1.44 |
| Plasma K (mmol/L) | 3.44 ± 0.13 | 3.88 ± 0.26 | 3.92 ± 0.23 |
| Urine Na (μmol/day) | 73.40 ± 14.28 | 335.3 ± 45.97*** | 313.8 ± 72.44** |
| Urine K (μmol/day) | 128.4 ± 35.80 | 627.4 ± 56.88*** | 508.6 ± 96.18*** |
| Urine Na/K ratio | 1.83 ± 0.15 | 2.13 ± 0.37 | 1.76 ± 0.32 |
| PRC(ng AngI/mL/h) | 80.4 ± 11.25 | 45.1 ± 12.76* | 123.7 ± 25.3*,### |
| Ccr (μL/min/g BW) | 0.25 ± 0.05 | 0.21 ± 0.03 | 0.32 ± 0.07 |
| P-aldosterone (pg/mL) | 200.3 ± 58.6 | 231.3 ± 9.82 | 167.3 ± 10.3 |
| Treatment groups | db/m + vehicle (n = 8) | db/db + vehicle (n = 7) | db/db + aliskiren (n = 6) |
|---|---|---|---|
| Body weight (g) | 33.88 ± 0.72 | 45.40 ± 4.38 | 43.00 ± 7.74 |
| SBP (mmHg) | 102.96 ± 1.10 | 101.34 ± 1.32 | 98.18 ± 2.60 |
| FBG (mmol/L) | 8.0 ± 0.4 | 33.8 ± 2.8*** | 29.7 ± 1.5*** |
| HbA1c (%) | 4.49 ± 0.24 | 8.39 ± 0.37*** | 8.88 ± 0.36*** |
| Creatinine (μmol/L) | 30.0 ± 1.0 | 42.0 ± 3.0*** | 45.0 ± 2.0*** |
| Plasma Na (mmol/L) | 142.1 ± 2.86 | 150.0 ± 3.53 | 147.5 ± 1.44 |
| Plasma K (mmol/L) | 3.44 ± 0.13 | 3.88 ± 0.26 | 3.92 ± 0.23 |
| Urine Na (μmol/day) | 73.40 ± 14.28 | 335.3 ± 45.97*** | 313.8 ± 72.44** |
| Urine K (μmol/day) | 128.4 ± 35.80 | 627.4 ± 56.88*** | 508.6 ± 96.18*** |
| Urine Na/K ratio | 1.83 ± 0.15 | 2.13 ± 0.37 | 1.76 ± 0.32 |
| PRC(ng AngI/mL/h) | 80.4 ± 11.25 | 45.1 ± 12.76* | 123.7 ± 25.3*,### |
| Ccr (μL/min/g BW) | 0.25 ± 0.05 | 0.21 ± 0.03 | 0.32 ± 0.07 |
| P-aldosterone (pg/mL) | 200.3 ± 58.6 | 231.3 ± 9.82 | 167.3 ± 10.3 |
Values are expressed as means ± SEM.
FBG, fasting blood glucose; Na, sodium; K, potassium; PRC, plasma renin concentration; Ccr, creatinine clearance; P-aldosterone, plasma aldosterone concentration; n, number of mice in each group.
*P < 0.05 vs. db/m + vehicle; **P < 0.01 vs. db/m + vehicle; ***P < 0.001 vs. db/m + vehicle; ###P < 0.001 vs. db/db + vehicle.
Biochemical parameters in experimental animals
| Treatment groups | db/m + vehicle (n = 8) | db/db + vehicle (n = 7) | db/db + aliskiren (n = 6) |
|---|---|---|---|
| Body weight (g) | 33.88 ± 0.72 | 45.40 ± 4.38 | 43.00 ± 7.74 |
| SBP (mmHg) | 102.96 ± 1.10 | 101.34 ± 1.32 | 98.18 ± 2.60 |
| FBG (mmol/L) | 8.0 ± 0.4 | 33.8 ± 2.8*** | 29.7 ± 1.5*** |
| HbA1c (%) | 4.49 ± 0.24 | 8.39 ± 0.37*** | 8.88 ± 0.36*** |
| Creatinine (μmol/L) | 30.0 ± 1.0 | 42.0 ± 3.0*** | 45.0 ± 2.0*** |
| Plasma Na (mmol/L) | 142.1 ± 2.86 | 150.0 ± 3.53 | 147.5 ± 1.44 |
| Plasma K (mmol/L) | 3.44 ± 0.13 | 3.88 ± 0.26 | 3.92 ± 0.23 |
| Urine Na (μmol/day) | 73.40 ± 14.28 | 335.3 ± 45.97*** | 313.8 ± 72.44** |
| Urine K (μmol/day) | 128.4 ± 35.80 | 627.4 ± 56.88*** | 508.6 ± 96.18*** |
| Urine Na/K ratio | 1.83 ± 0.15 | 2.13 ± 0.37 | 1.76 ± 0.32 |
| PRC(ng AngI/mL/h) | 80.4 ± 11.25 | 45.1 ± 12.76* | 123.7 ± 25.3*,### |
| Ccr (μL/min/g BW) | 0.25 ± 0.05 | 0.21 ± 0.03 | 0.32 ± 0.07 |
| P-aldosterone (pg/mL) | 200.3 ± 58.6 | 231.3 ± 9.82 | 167.3 ± 10.3 |
| Treatment groups | db/m + vehicle (n = 8) | db/db + vehicle (n = 7) | db/db + aliskiren (n = 6) |
|---|---|---|---|
| Body weight (g) | 33.88 ± 0.72 | 45.40 ± 4.38 | 43.00 ± 7.74 |
| SBP (mmHg) | 102.96 ± 1.10 | 101.34 ± 1.32 | 98.18 ± 2.60 |
| FBG (mmol/L) | 8.0 ± 0.4 | 33.8 ± 2.8*** | 29.7 ± 1.5*** |
| HbA1c (%) | 4.49 ± 0.24 | 8.39 ± 0.37*** | 8.88 ± 0.36*** |
| Creatinine (μmol/L) | 30.0 ± 1.0 | 42.0 ± 3.0*** | 45.0 ± 2.0*** |
| Plasma Na (mmol/L) | 142.1 ± 2.86 | 150.0 ± 3.53 | 147.5 ± 1.44 |
| Plasma K (mmol/L) | 3.44 ± 0.13 | 3.88 ± 0.26 | 3.92 ± 0.23 |
| Urine Na (μmol/day) | 73.40 ± 14.28 | 335.3 ± 45.97*** | 313.8 ± 72.44** |
| Urine K (μmol/day) | 128.4 ± 35.80 | 627.4 ± 56.88*** | 508.6 ± 96.18*** |
| Urine Na/K ratio | 1.83 ± 0.15 | 2.13 ± 0.37 | 1.76 ± 0.32 |
| PRC(ng AngI/mL/h) | 80.4 ± 11.25 | 45.1 ± 12.76* | 123.7 ± 25.3*,### |
| Ccr (μL/min/g BW) | 0.25 ± 0.05 | 0.21 ± 0.03 | 0.32 ± 0.07 |
| P-aldosterone (pg/mL) | 200.3 ± 58.6 | 231.3 ± 9.82 | 167.3 ± 10.3 |
Values are expressed as means ± SEM.
FBG, fasting blood glucose; Na, sodium; K, potassium; PRC, plasma renin concentration; Ccr, creatinine clearance; P-aldosterone, plasma aldosterone concentration; n, number of mice in each group.
*P < 0.05 vs. db/m + vehicle; **P < 0.01 vs. db/m + vehicle; ***P < 0.001 vs. db/m + vehicle; ###P < 0.001 vs. db/db + vehicle.
Insulin tolerance test in experimental animals. Data are shown as mean ± SEM. *P < 0.05 vs. db/db + vehicle; **P < 0.01 vs. db/db + vehicle.
Metabolic parameters in experimental animals
| Treatment groups | db/m + vehicle (n = 8) | db/db + vehicle (n = 7) | db/db + aliskiren (n = 6) |
|---|---|---|---|
| Cholesterol (mmol/L) | 1.64 ± 0.24 | 3.90 ± 0.24*** | 2.78 ± 0.68*,# |
| Triglyceride (mmol/L) | 0.30 ± 0.06 | 1.48 ± 0.09*** | 1.20 ± 0.26*** |
| HDL-C (mmol/L) | 1.0 ± 0.01 | 2.0 ± 0.01** | 2.0 ± 0.01* |
| LDL-C (mmol/L) | 0.30 ± 0.10 | 0.42 ± 0.03 | 0.27 ± 0.01 |
| HOMA-IR | 0.40 ± 0.09 | 3.60 ± 1.35** | 0.90 ± 0.39# |
| Insulin (ng/mL) | 1.01 ± 0.24 | 2.17 ± 0.75 | 0.59 ± 0.24 |
| Adiponectin (μg/mL) | 8.79 ± 1.54 | 4.79 ± 1.14 | 4.57 ± 1.98 |
| Kid-Chol (μg/mg protein) | 6.42 ± 1.69 | 12.14 ± 2.04*** | 8.76 ± 1.12*,# |
| Kid-TG (μg/mg protein) | 12.32 ± 1.76 | 13.59 ± 2.49 | 9.17 ± 1.35 |
| Liv-Chol (μg/mg protein) | 5.54 ± 2.29 | 14.87 ± 3.39** | 10.29 ± 1.83 |
| Liv-TG (μg/mg protein) | 6.62 ± 4.21 | 14.56 ± 5.51 | 6.31 ± 2.07 |
| Fat-Chol (μg/mg protein) | 5.26 ± 1.06 | 5.33 ± 1.21 | 5.92 ± 1.79 |
| Fat-TG (μg/mg protein) | 21.11 ± 2.83 | 27.40 ± 5.17 | 36.76 ± 3.86 |
| Treatment groups | db/m + vehicle (n = 8) | db/db + vehicle (n = 7) | db/db + aliskiren (n = 6) |
|---|---|---|---|
| Cholesterol (mmol/L) | 1.64 ± 0.24 | 3.90 ± 0.24*** | 2.78 ± 0.68*,# |
| Triglyceride (mmol/L) | 0.30 ± 0.06 | 1.48 ± 0.09*** | 1.20 ± 0.26*** |
| HDL-C (mmol/L) | 1.0 ± 0.01 | 2.0 ± 0.01** | 2.0 ± 0.01* |
| LDL-C (mmol/L) | 0.30 ± 0.10 | 0.42 ± 0.03 | 0.27 ± 0.01 |
| HOMA-IR | 0.40 ± 0.09 | 3.60 ± 1.35** | 0.90 ± 0.39# |
| Insulin (ng/mL) | 1.01 ± 0.24 | 2.17 ± 0.75 | 0.59 ± 0.24 |
| Adiponectin (μg/mL) | 8.79 ± 1.54 | 4.79 ± 1.14 | 4.57 ± 1.98 |
| Kid-Chol (μg/mg protein) | 6.42 ± 1.69 | 12.14 ± 2.04*** | 8.76 ± 1.12*,# |
| Kid-TG (μg/mg protein) | 12.32 ± 1.76 | 13.59 ± 2.49 | 9.17 ± 1.35 |
| Liv-Chol (μg/mg protein) | 5.54 ± 2.29 | 14.87 ± 3.39** | 10.29 ± 1.83 |
| Liv-TG (μg/mg protein) | 6.62 ± 4.21 | 14.56 ± 5.51 | 6.31 ± 2.07 |
| Fat-Chol (μg/mg protein) | 5.26 ± 1.06 | 5.33 ± 1.21 | 5.92 ± 1.79 |
| Fat-TG (μg/mg protein) | 21.11 ± 2.83 | 27.40 ± 5.17 | 36.76 ± 3.86 |
Values are expressed as mean ± SEM.
HOMA-IR, homeostasis model assessment index; Kid-Chol, kidney cholesterol content; Kid-TG, kidney triglyceride content; Liv-Chol, liver cholesterol content; Liv-TG, liver triglyceride content; Fat-Chol, fat cholesterol content; Fat-TG, fat triglyceride content; n, number of mice in each group.
*P < 0.05 vs. db/m + vehicle; **P < 0.01 vs. db/m + vehicle; ***P < 0.001 vs. db/m + vehicle; #P < 0.05 vs. db/db + vehicle.
Metabolic parameters in experimental animals
| Treatment groups | db/m + vehicle (n = 8) | db/db + vehicle (n = 7) | db/db + aliskiren (n = 6) |
|---|---|---|---|
| Cholesterol (mmol/L) | 1.64 ± 0.24 | 3.90 ± 0.24*** | 2.78 ± 0.68*,# |
| Triglyceride (mmol/L) | 0.30 ± 0.06 | 1.48 ± 0.09*** | 1.20 ± 0.26*** |
| HDL-C (mmol/L) | 1.0 ± 0.01 | 2.0 ± 0.01** | 2.0 ± 0.01* |
| LDL-C (mmol/L) | 0.30 ± 0.10 | 0.42 ± 0.03 | 0.27 ± 0.01 |
| HOMA-IR | 0.40 ± 0.09 | 3.60 ± 1.35** | 0.90 ± 0.39# |
| Insulin (ng/mL) | 1.01 ± 0.24 | 2.17 ± 0.75 | 0.59 ± 0.24 |
| Adiponectin (μg/mL) | 8.79 ± 1.54 | 4.79 ± 1.14 | 4.57 ± 1.98 |
| Kid-Chol (μg/mg protein) | 6.42 ± 1.69 | 12.14 ± 2.04*** | 8.76 ± 1.12*,# |
| Kid-TG (μg/mg protein) | 12.32 ± 1.76 | 13.59 ± 2.49 | 9.17 ± 1.35 |
| Liv-Chol (μg/mg protein) | 5.54 ± 2.29 | 14.87 ± 3.39** | 10.29 ± 1.83 |
| Liv-TG (μg/mg protein) | 6.62 ± 4.21 | 14.56 ± 5.51 | 6.31 ± 2.07 |
| Fat-Chol (μg/mg protein) | 5.26 ± 1.06 | 5.33 ± 1.21 | 5.92 ± 1.79 |
| Fat-TG (μg/mg protein) | 21.11 ± 2.83 | 27.40 ± 5.17 | 36.76 ± 3.86 |
| Treatment groups | db/m + vehicle (n = 8) | db/db + vehicle (n = 7) | db/db + aliskiren (n = 6) |
|---|---|---|---|
| Cholesterol (mmol/L) | 1.64 ± 0.24 | 3.90 ± 0.24*** | 2.78 ± 0.68*,# |
| Triglyceride (mmol/L) | 0.30 ± 0.06 | 1.48 ± 0.09*** | 1.20 ± 0.26*** |
| HDL-C (mmol/L) | 1.0 ± 0.01 | 2.0 ± 0.01** | 2.0 ± 0.01* |
| LDL-C (mmol/L) | 0.30 ± 0.10 | 0.42 ± 0.03 | 0.27 ± 0.01 |
| HOMA-IR | 0.40 ± 0.09 | 3.60 ± 1.35** | 0.90 ± 0.39# |
| Insulin (ng/mL) | 1.01 ± 0.24 | 2.17 ± 0.75 | 0.59 ± 0.24 |
| Adiponectin (μg/mL) | 8.79 ± 1.54 | 4.79 ± 1.14 | 4.57 ± 1.98 |
| Kid-Chol (μg/mg protein) | 6.42 ± 1.69 | 12.14 ± 2.04*** | 8.76 ± 1.12*,# |
| Kid-TG (μg/mg protein) | 12.32 ± 1.76 | 13.59 ± 2.49 | 9.17 ± 1.35 |
| Liv-Chol (μg/mg protein) | 5.54 ± 2.29 | 14.87 ± 3.39** | 10.29 ± 1.83 |
| Liv-TG (μg/mg protein) | 6.62 ± 4.21 | 14.56 ± 5.51 | 6.31 ± 2.07 |
| Fat-Chol (μg/mg protein) | 5.26 ± 1.06 | 5.33 ± 1.21 | 5.92 ± 1.79 |
| Fat-TG (μg/mg protein) | 21.11 ± 2.83 | 27.40 ± 5.17 | 36.76 ± 3.86 |
Values are expressed as mean ± SEM.
HOMA-IR, homeostasis model assessment index; Kid-Chol, kidney cholesterol content; Kid-TG, kidney triglyceride content; Liv-Chol, liver cholesterol content; Liv-TG, liver triglyceride content; Fat-Chol, fat cholesterol content; Fat-TG, fat triglyceride content; n, number of mice in each group.
*P < 0.05 vs. db/m + vehicle; **P < 0.01 vs. db/m + vehicle; ***P < 0.001 vs. db/m + vehicle; #P < 0.05 vs. db/db + vehicle.
Representative histological findings of epididymal fat pads (A–C) and liver (D–F) in experimental animals: (A, D) db/m mice at 20 weeks old, (B, E) db/db mice at 20 weeks old, and (C, F) db/db mice treated with aliskiren for 3 months at 20 weeks old. H & E staining in adipose tissue and PAS staining in liver tissue (original magnification × 200).
Because aliskiren treatment improved insulin resistance and lipid abnormalities in diabetic mice, the effect of aliskiren on renal lipid metabolism was examined. As shown in Figure 3, diabetic mice showed significantly higher levels of expression of SREBP2 and HMG CoA reductase genes, which are involved in cholesterol synthesis. However, we could not find any significant changes in the expressions of other genes involved in lipid metabolism. Interestingly, aliskiren treatment restored the abnormal gene expression observed in diabetic mice. In accordance with these changes, renal cholesterol contents were significantly decreased in groups treated with aliskiren (Table 2 and Figure 3).
mRNA expression for renal lipid metabolism in renal cortical tissues in experimental animals. FAS, fatty acid synthase; ABCA1, ATP-binding cassette transporter-1; SREBP2, sterol regulatory element-binding protein-2; SREBP1c, sterol regulatory element-binding protein-1c; HMG-CoA reductase; FXR, farnesoid X receptor; CPT-1, carnitine palmitoyl transferase-1; ACO, acetyl-CoA oxidase. Data are shown as mean ± SEM. **P < 0.01 vs. db/m + vehicle; ***P < 0.001 vs. db/m + vehicle; ##P < 0.01 vs. db/db + vehicle; ###P < 0.001 vs. db/db + vehicle.
Effects of aliskiren on cardiovascular and renal protection in experimental animals
As shown in Table 1, systolic blood pressure, plasma aldosterone levels and electrolyte levels did not show significant differences among the three groups. Plasma creatinine levels were significantly higher in diabetic mice compared with non-diabetic controls, but creatinine clearance did not show significant difference. Aliskiren treatment did not show significant change in plasma creatinine and creatinine clearance. PRC showed significantly lower levels in diabetic mice, and aliskiren group showed significantly increased PRC levels (Table 1). Supplementary Table 5 (see online supplementary material for this table) shows the echocardiographic results from the experimental animals. Although systolic blood pressure was not different among the three groups, diabetic mice showed increased LVMI and decreased systolic function, reflected by fractional shortening, compared with the same parameters in non-diabetic mice (Figure 4A). Interestingly, aliskiren treatment significantly improved LVMI and fractional shortening (Figure 4A). These findings indicate that, in diabetic mice, aliskiren provides cardiac protective effects that are independent of blood pressure-lowering effects. These functional improvements were further confirmed by significantly decreased total collagen content in the cardiac tissue (Figure 4B). In addition, these functional improvements are also associated with improvements in the gene expressions of profibrotic and proinflammatory molecules in the cardiac tissue. These molecules were remarkably upregulated in type 2 diabetic mice, as shown in Figure 4C. Concerning RAS activation, there were no significant changes in the expression of RAS including AT1 receptor, AT2 receptor, renin and renin receptor (Figure 4D). As shown in Figure 4E, diabetic mice revealed more fibrotic changes, which were dramatically inhibited by aliskiren treatment.
Effects of aliskiren on cardiac functional and morphologic parameters. (A) Echocardiographic results for LVMI and fractional shortening. (B) Total collagen content in the heart. (C) mRNA expression in cardiac tissue for proinflammatory and profibrotic cytokines. (D) mRNA expression in cardiac tissue for RAS. (E) Representative histological findings of cardiac tissue. (Masson's trichrome stain; original magnification × 200). Data are shown as mean ± SEM. *P < 0.05 vs. db/m + vehicle; **P < 0.01 vs. db/m + vehicle; ***P < 0.001 vs. db/m + vehicle; #P < 0.05 vs. db/db + vehicle; ##P < 0.01 vs. db/db + vehicle.
Next, we investigated the effects of aliskiren on renal function. Urinary albumin excretion (UAE) and urinary excretion of VEGF levels in diabetic mice were significantly higher during the study period. Aliskiren treatment markedly attenuated UAE and urinary excretion of VEGF at the end of the study period (Figure 5A and B). Furthermore, aliskiren treatment considerably suppressed the profibrotic and proinflammatory gene expression, which were significantly up-regulated in diabetic kidneys (Figure 5C). Figure 6 shows the representative renal histologic changes in the experimental animals. Consistent with the significant attenuation of albuminuria, glomerulosclerosis and immunoreactivity for fibrotic molecules were significantly improved in the aliskiren treatment group (Figures 6 and 7).
Effects of aliskiren on urinary albumin and VEGF excretion, and mRNA expression in renal tissue. (A) 24-h urinary albumin excretion. (B) Urinary VEGF excretion. Urine VEGF levels were corrected by urine creatinine levels, and (C) mRNA expression for proinflammatory, profibrotic cytokines, renin and renin receptor. Data are shown as mean ± SEM. *P < 0.05 vs. db/m + vehicle; **P < 0.01 vs. db/m + vehicle; ***P < 0.001 vs. db/m + vehicle; #P < 0.05 vs. db/db + vehicle; ##P < 0.01 vs. db/db + vehicle; ###P < 0.001 vs. db/db + vehicle.
Representative renal histological findings in experimental animals: (A–E) db/m mice at 20 weeks old. (F–J) db/db mice at 20 weeks old, and (K–O) db/db mice treated with aliskiren for 3 months at 20 weeks old. Representative renal PAS staining (A, F, K), TGFβ1 immunostaining (B, G, L), type IV collagen immunostaining (C, H, M), PAI-1 immunostaining (D, I, N) and VEGF immunostaining (E, J, O). Original magnification × 100.
Effects of aliskiren on renal morphological changes. Glomerulosclerosis index, mesangial expansion score and glomerular immunostaining score for TGFβ1, type IV collagen, PAI-1 and VEGF. Data are shown as mean ± SEM. **P < 0.01 vs. db/m + vehicle; ***P < 0.001 vs. db/m + vehicle; ##P < 0.01 vs. db/db + vehicle; ###P < 0.001 vs. db/db + vehicle.
To further define the molecular mechanism of aliskiren, we next performed in vitro experiments. Firstly, we observed the expression pattern of renin receptor in glomerular cells, and found that abundant expression was observed in mesangial cells (data not shown). We performed an in vitro experiment to determine whether a high glucose environment increases renin production. As shown in Figure 8A, high glucose stimulation noticeably increased renin synthesis in MCs. Next, we examined the direct effect of renin and aliskiren to further elucidate the molecular mechanism of aliskiren’s beneficial effect. As expected, high glucose stimulation significantly increased the gene expressions of TGFβ1, type IV collagen, VEGF and PAI-1 (Figure 8B). Administration of renin in culture medium also up-regulated the expressions of TGFβ1, type IV collagen and VEGF without significant changes in PAI-1, which were abolished by aliskiren treatment (Figure 8B). It is of interest that prior treatment with aliskiren under high glucose conditions also decreased the expressions of TGFβ1, type IV collagen, VEGF and PAI-1. Since renin treatment induces pathologic changes through both angiotensin-dependent and angiotensin-independent mechanisms, we further evaluated whether prior treatment with AT1 receptor antagonist decreased renin-induced changes in the production of cytokines. Interestingly, prior treatment of AT1 receptor antagonist almost completely abolished renin-induced cytokine production (Figure 8B–D). In agreement with changes in gene expression, collagen and VEGF protein secretion showed similar patterns (Figure 8C and D).
Effects of high glucose on renin synthesis and profibrotic action of renin in cultured MCs. (A) Effect of high glucose on mesangial cell renin production. (B) Effect of high glucose and renin on TGFβ1, type IV collagen, PAI-1 and VEGF mRNA expressions. (C, D) Effect of high glucose and renin on collagen and VEGF protein secretions. Secretory collagen and VEGF protein were measured from culture supernatants by ELISA, and protein contents were corrected by the total cellular protein content. NG, normal glucose; HG, high glucose; alisk, aliskiren; AT1B, angiotensin receptor type 1 antagonist; Data are shown as mean ± SEM. *P < 0.05 vs. NG; **P < 0.01 vs. NG; ***P < 0.001 vs. NG; #P < 0.05 vs. HG; ##P < 0.01 vs. HG; ω P < 0.05 vs. renin; φ P < 0.01 vs. renin; γ P < 0.001 vs. renin.
Discussion
In the present study, we demonstrated that aliskiren treatment significantly improved insulin resistance, metabolic parameters and renal lipid metabolism in db/db mice. Aliskiren treatment also showed organ-protective effects such as improvement of functional and structural changes in the heart and kidney. We also provided evidence that aliskiren could inhibit renin-mediated activation of profibrotic cytokine synthesis through angiotensin-dependent mechanism in cultured mesangial cells.
Most clinical trials showed blood pressure-lowering efficacy of aliskiren in hypertensive patients [7,8,23]. Pilz et al. demonstrated a protective effect of aliskiren for both heart and kidney injuries in double transgenic rats for human renin and angiotensinogen genes [9]. In the recent clinical study on Aliskiren in the Evaluation of Proteinuria in Diabetes (AVOID), aliskiren, combined with losartan, has a significant anti-proteinuric effect that was independent of its blood pressure-lowering effect in patients with hypertension and diabetic nephropathy [24]. Kelly et al. illustrated that aliskiren had a similar efficacy to ACE inhibitor for reduction of urinary albumin and glomerulosclerosis in diabetic transgenic (mRen-2)27 rats [10]. Together, these results suggest a beneficial effect of renin inhibition in hypertensive and diabetic renal disease.
The most interesting finding in this study is an improvement in insulin resistance by aliskiren treatment. Aliskiren treatment significantly decreased plasma levels of HOMA-IR, lipid abnormalities, and insulin sensitivity confirmed by insulin tolerance test. Adipose tissue obtained from epididymal fat revealed that diabetic mice had larger immature adipocytes, which are more insulin resistant, than those of the non-diabetic controls. Furthermore, we observed that gene expression in adipose tissue was more inflammatory and showed more of an immature pattern in diabetic mice, and aliskiren treatment restored the phenotype to small differentiated adipocytes [25,26]. These results raise an interesting possibility that renin inhibition may be linked with insulin resistance and lipid abnormalities in type 2 diabetes.
Recent studies have theorized the potential mechanisms of renin in the pathogenesis of insulin resistance. Women with polycystic ovarian syndrome demonstrated insulin resistance and an increase in serum renin levels. These renin levels had a significant correlation with insulin resistance indices [27]. In addition, renin knockout mice revealed a lean, insulin-sensitive phenotype with resistance to diet-induced obesity [28]. Furthermore, renin inhibition attenuates insulin resistance, oxidative stress and pancreatic remodelling, and improves systemic insulin sensitivity in transgenic Ren2 rats that overexpress renin [29,30]. Together, these data suggest a possible link between renin activation and insulin resistance.
In this study, we found some discrepant results in the biomarkers of insulin resistance such as plasma levels of HOMA-IR, fasting blood glucose (FBG) and HbA1c. Although the reason for the difference between these variables is not clear, FBG is influenced by many factors including stressful condition and food intake, and HbA1c levels may be affected by several factors such as erythrocyte lifespan, iron deficiency, haemoglobin glycation rate and renal function. It may be possible that some of these factors induce the discrepant results. However, insulin tolerance test and HOMA-IR have been considered as one of the most important biomarkers for insulin resistance.
Another interesting finding in this study is the improvement in renal lipid metabolism by renin inhibition. In diabetic mice, mRNA expression levels of enzymes involved in cholesterol synthesis, such as HMG-CoA reductase and SREBP2, were significantly increased, whereas other enzymes involved in lipid metabolism did not show significant changes by renin inhibition. This finding agrees with previous reports that suggest the role of abnormal renal lipid metabolism in the pathogenesis of diabetic nephropathy [19,20]. Aliskiren treatment induced improvement in the alterations of renal lipid metabolism and subsequently reduced renal cholesterol contents. These results suggest that renin inhibition in part improves renal function via improvement of renal lipid metabolic abnormalities.
In the present study, we did not find significant changes in systolic blood pressure by aliskiren treatment. However, aliskiren treatment down-regulated inflammatory genes and total collagen content, which leads to improvement in LVMI and systolic function in the cardiac tissue. Surprisingly, we found that renin expression was also observed in cardiac tissues and mesangial cells because it has been considered that juxtaglomerular cell is the only site for renin synthesis. However, several pathological conditions activate the RAS including renin synthesis in the cardiac myocytes [31,32]. Furthermore, cardiac fibroblasts that are one of the major cell types in the heart express all components of RAS [33]. It has been shown that MCs synthesize, store and secrete both renin and prorenin, and recent reports suggest that high glucose stimuli activate all RAS components including angiotensinogen, ACE and renin, accompanied by angiotensin II generation [34,35]. In this study, we did not find any significant changes in the RAS in the heart irrespective of the presence or absence of diabetes mellitus. In addition, aliskiren treatment did not induce significant changes in the gene expression of RAS in the cardiac tissues.
In this study, we observed that aliskiren therapy significantly reduced urinary albumin excretion and ameliorated glomerulosclerosis without affecting systolic blood pressure. This suggests a beneficial effect of aliskiren that is independent of the haemodynamic mechanism. In addition, aliskiren treatment markedly suppressed the expressions of MCP-1, TGFβ1, type IV collagen and PAI-1 in the diabetic kidney. Our in vitro study showed that AT1 receptor antagonist almost abolished the renin-induced overproduction of proinflammatory cytokine, which implies that renin effect may be mediated through an angiotensin-dependent pathway. Furthermore, it is of interest that aliskiren treatment abolished urinary levels of VEGF excretion associated with reduction in renal VEGF synthesis. In line with the previous report [36], we observed that renal synthesis of VEGF was significantly increased in the diabetic kidney. Angiotensin II is a potent stimulus for VEGF synthesis in mesenchymal stem cells and mesangial cells [37,38]. Several current experimental studies suggest direct evidence of renin as a potential mediator of VEGF synthesis. Treatment of the (pro)renin receptor blocker ameliorates diabetes-induced retinal expression of VEGF in streptozotocin-induced AT1 receptor-deficient mice [39]. In addition, glomerular VEGF mRNA was increased 2-fold in transgenic (mRen-2)27 rats [40]. Collectively, these data suggest that VEGF is regulated by renin independent of angiotensin II. In this study, we observed that high glucose and renin stimulation significantly increased VEGF synthesis, which was inhibited by aliskiren treatment. This finding further supports the in vivo result that aliskiren decreases urinary excretion of VEGF and renal synthesis of VEGF in diabetic mice.
The limitation of this study is that we only measured blood pressure at the end of the study period using tail plethysmography without telemetric method, so we cannot exclude the possibility that the blood pressure was lower at any time during the study period in the aliskiren-treated group. Additional haemodynamic factors as well as non-haemodynamic ones may be operative in producing a beneficial effect of aliskiren treatment. In addition, we used db/db mice which have a defect in the leptin receptor in this experiment. Because humans with leptin receptor defect do not develop insulin resistance and diabetes, our results may not be relevant for humans.
In conclusion, aliskiren provides a protective effect against target organ damage in type 2 diabetic animal models through the improvement of metabolic alteration as well as through inhibition of the profibrotic processes in target organs. These findings suggest that aliskiren may be a useful new therapeutic agent in the treatment of type 2 diabetes mellitus and diabetic nephropathy.
We thank Novartis Pharmaceuticals, Basel, Switzerland for kindly providing aliskiren. This work was supported by a grant from the Brain Korea 21 project and a special grant from Korea University.
Conflict of intereststatement. None declared.
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