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Renoprotective Effects of Direct Renin Inhibition in Glomerulonephritis

      Abstract

      The development of glomerulonephritis causes glomerular injury and renal dysfunction and is thought to increase renin release, thus activating the renin-angiotensin system (RAS). The aims of this study were to demonstrate activation of the intrarenal RAs and determine the effects of direct renin inhibition (DRi) on the progression of glomerulonephritis. Rats were treated with anti-Thy1.1 antibody with or without DRI, aliskiren (30 mg/kg/d). In the glomerulonephritic rats, protein, microalbumin excretion levels, urinary angiotensinogen excretion, glomerular expansion score and intrarenal transforming growth factor-β and plasminogen activator inhibitor-1 mRNA levels were augmented compared with control rats; however, hypertension was not observed in the glomerulonephritic rats, and aliskiren treatment did not modify their blood pressure. The increases in urinary protein (94.7 ± 13.0 mg/d) and microalbumin (7.52 ± 2.6 mg/d) excretion were reduced by aliskiren (43.6 ± 4.5 mg/d of protein and 2.57 ± 0.7 mg/d of microalbumin). Furthermore, the progression of glomerular expansion and elevation of intrarenal transforming growth factor-β and plasminogen activator inhibitor-1 levels were prevented by aliskiren. Importantly, aliskiren suppressed the augmentation of urinary angiotensinogen levels, the increased angiotensinogen expression in the kidneys and the increases in Ang II levels in renal medulla induced by the anti-Thy1.1 antibody. These results suggest that DRI with aliskiren prevents intrarenal RAS activation leading to mitigation of the development of glomerulonephritis. In addition, the renoprotective effects of DRI on glomerulonephritis occur in a blood pressure-independent manner. Accordingly, treatment with aliskiren may be an effective approach to treat glomerulonephritis and other intrarenal RAS-associated kidney diseases.

      Key Indexing Terms

      The renin-angiotensin system (RAS) plays an important role in the development of hypertension and renal diseases.
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      Anti-Thy1.1 glomerulonephritis is an established model of glomerulonephritis.
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      obvious mesangial cell proliferation, marked extracellular matrix accumulation, adhesion to Bowman's capsule, glomerulosclerosis and tubulointerstitial fibrosis are observed in the anti-Thy1.1 glomerulonephritis. These pathological changes were less severe in rats chronically treated with ACE inhibitors or AT1R blockers.
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      These findings indicate that activation of the intrarenal RAS plays a pivotal role in the progression of glomerulonephritis.
      The development of renin inhibitors has provided an opportunity to evaluate the effects of direct renin inhibition (DRI) as another means of RAS blockade. Aliskiren is the main inhibitor currently available.
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      ; however, the effects of aliskiren on intrarenal RAS activity and its renoprotective effects when aliskiren is systemically administrated have not been established. The central hypothesis of this study is that activation of the intrarenal RAS plays a crucial role in the development of glomerulonephritis. In accordance with the hypothesis, the following aims were targeted: (1) to demonstrate the activation of intrarenal RAS
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      using urinary angiotensinogen (uAGT) as an index of intrarenal Ang II levels, in rats with anti-Thy1.1 glomerulonephritis; and (2) to demonstrate that DRI with aliskiren suppresses the activation of the intrarenal RAS in the glomerulonephritis model and prevents the progression of renal injury, thus developing further a novel strategy for treatment of glomerulonephritis. The obtained results demonstrate that the intrarenal RAS is markedly activated during the early phases of glomerulonephritis and that chronic systemic administration of aliskiren attenuates the increase in the intrarenal RAS activity and prevents the glomerular injury in anti-Thy1.1 glomerulonephritis.

      MATERIALS AND METHODS

      Animals

      The experiments were performed on Sprague–Dawley and Fischer male rats (230–430 g). The animal experimental protocol was approved by the Animal Care and Use Committee of Tulane University. The rats were housed in a constant temperature room with 12-hour dark and 12-hour light cycle with free access to food and water.

      Anti-Thy1.1 Glomerulonephritis

      A monoclonal antibody against rat anti-Thy1.1, OX-7, was purchased from Cedarlane Laboratories (Burlington, NC).

      Experimental Design

      Rats were divided into 3 groups; control group (n = 11), anti-Thy1.1 glomerulonephritis group (n = 19) and anti-Thy1.1 glomerulonephritis treated with aliskiren (30 mg/kg/d, n = 16). One group of rats was kept in individual metabolic cages with free access to food and tap water when urine samples were collected. Administration of aliskiren was started 4 days before the injection of anti-Thy1.1 antibody using an osmotic mini pump (Alzet Osmotic Pump; Alza, Mountain View, CA). Anti-Thy1.1 (200 μg/100 g body weight) was injected via a tail vein at day 0. Rats in the control group received the same volume of vehicle (isotonic saline). Blood and tissue samples were collected at 14 days after starting treatments by conscious decapitation.

      Systolic Blood Pressure and Renal Function

      Systolic arterial BP was measured by a tail-cuff system as previously described. Preliminary studies demonstrated that aliskiren treatment alone does not alter systolic blood pressure (SBP) in normal rats. In this group, after 10 days of aliskiren treatment, SBP averaged 114 ± 2 mm Hg compared with control measurement of 116 ± 4 mm Hg (n = 6). Twenty-four-hour urine samples were collected at days −4, 3, 5, 7, 10 and 14. Urinary protein was measured by the Pyrogallol red method (Wako Chemical, Osaka, Japan). The level of microalbumin was determined by the rat albumin ELISA kit (ALPCO Diagnostics, Salem, NH). Plasma creatinine levels were measured by Jaffe method.

      Urinary and Plasma Angiotensinogen Measurements

      Urinary and plasma concentrations of AGT were measured by using a commercially available ELISA kit (IBL America, Minneapolis, MN), as routinely used in our laboratory.
      • Kobori H.
      • Katsurada A.
      • Miyata K.
      • et al.
      Determination of plasma and urinary angiotensinogen levels in rodents by newly developed ELISA.
      The results were normalized based on the 24-hour urine volumes and reported as uAGT excretion.

      Plasma Renin Activity Assay

      The blood samples were collected into tubes containing 5.0 mmol/L EDTA, and plasma renin activity (PRA) were assayed by using a commercially available kit (DiaSorin, Stillwater, MN). PRA was expressed as nanograms per milliliter per hour of generated Ang I.

      Ang II Measurements in Plasma, Kidney Cortex and Medulla

      For plasma Ang II determinations, blood (1 mL) was added to 100% methanol. The supernatant was transferred, dried and then assayed. For the kidney Ang II determinations, the right kidney was sectioned into cortex and medulla. Each section was weighed and immediately minced into ice-cold 100% methanol. The tissue was homogenized with a tissue tearor and then centrifuged as mentioned above. The soluble homogenates were transferred, vacuum dried, extracted and then assayed in the same manner as is routinely done in our laboratory.

      Histological Analysis

      Glomerular matrix expansion was evaluated by periodic acid-Schiff staining. Kidney tissues were fixed in 10% buffered formalin for 24 hours, embedded in paraffin and cut into 4 mm sections. Twenty glomeruli in a kidney section were randomly selected, and the periodic acid-Schiff staining positive score was analyzed by using Image Pro-plus software. Thereafter, an average of the positive score in 20 glomeruli was calculated as a glomerular matrix expansion score in each animal.

      Immunohistochemistry

      Formalin fixed kidney sections (4 mm) were deparaffinized with xylene and dehydrated with ethanol. The samples were heated at 100°C for 60 minutes in citrate buffer, and rat AGT was detected by using a rabbit primary antibody against rat AGT (IBL America), a Vectastain ABC kit (VECTOR laboratories, Burlingame, CA) and 3,3'-diaminobenzidine substrate kits. Samples were costained with hematoxylin before analysis. The staining was performed with an Autostainer Plus (Dako, Carpinteria, CA). Immunoreactivity was semiquantitatively evaluated in a blinded test as described in Histological Analysis section.

      Western Blotting

      Proteins were extracted from the renal cortex, and the protein concentrations were quantified by a bicinchoninic acid protein assays kit (Pierce, Rockford, IL). For AGT, 10 μg of total protein and, for transforming growth factor (TGF)-β1, plasminogen activator inhibitor (PAI)-1 and prorenin receptor (PRR), 60 μg of total protein were separated by using a 4% to 12% SDS-PAGE (Invitrogen Life Technologies, Grand Island, NY) and then transferred to a nitrocellulose membrane. The membranes were incubated with a rabbit polyclonal anti-AGT antibody (IBL America), anti-TGF-β1 antibody (Santa Cruz Biotechnology, Dallas, TX), anti-PAI-1 antibody (Abcam, Cambridge, MA) or anti-ATP6AP2 antibody (Sigma-Aldrich, Saint Louis, MO). A secondary fluorescent-goat anti-rabbit IgG antibody (IRDye 800 CW; Li-Cor Biosciences, Lincoln, NE) was used to detect the target proteins. GAPDH or β-actin levels were also determined by using a mouse monoclonal anti-GAPDH antibody (Santa Cruz Biotechnology) or a mouse monoclonal anti-b-actin antibody (Abcam). A secondary fluorescent-goat anti-mouse IgG antibody (IRDye 680 CW; Li-Cor Biosciences) was used for the detection of GAPDH and anti-β-actin. Immunoreactive proteins were detected by an Odyssey System (Li-Cor Biosciences).

      Quantitative Real-Time RT-PCR

      For total RNA isolation, approximately 10 mg of tissue stored in RNA later (Ambion, Grand Island, NY) was used. The tissues were homogenized, and total RNAs were isolated by using the RNeasy mini kit (Qiagen, Valencia, CA). Quantitative RT-PCR analysis was performed as described previously.
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      • Garner M.K.
      • et al.
      Divergent localization of angiotensinogen mRNA and protein in proximal tubule segments of normal rat kidney.
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      • et al.
      Glomerular angiotensinogen is induced in mesangial cells in diabetic rats via reactive oxygen species—ERK/JNK pathways.
      Twenty nanograms of total RNA was analyzed using the Mx3000P system (Stratagene, LaJolla, CA) with the Brilliant Single-Step QRT-PCR Master Mix II Kit (Stratagene). The results were normalized on the basis of the mRNA expression level of β-actin. The sequence of the primers used in the study were as follows: TGF-β1 forward primer, 5'-TACCATGCCAACTTCTGTC-3'; reverse primer, 5'-AAG-GACCTTGCTGTACTGTGT-3'; and probe, 6 FAM-CCCTAC ATTTGGAGCCTGGAC-BHQ1; PAI-1: forward primer, 5'-CTCCACAGCCATTCTAGTCT-3'; reverse primer, 5'-CCAT-GAAGAGGATTGTCTCT-3'; and probe, 6 FAM-ACCGA TCCTTTCTCTTTGTGGTTC-BHQ1; PRR: forward primer, 5'-ATCCTTGAGACGAAACAAGA-3'; reverse primer, 5'-AGCCAGTCATAATCCACAGT-3'; and probe, 6 FAM-ACACCCAAAGTCCCTACAACCTTG-BHQ1; and β-actin forward primer, 5'-ATC ATG AAG TGT GAC GTT GA-3' reverse primer, 5'-GAT CTT CAT GGT GCT AGG AGC-3' and probe, 5'/HEX/TCT ATG CCA ACA CAG TGC TGT CTG GT/BHQ2/3'.

      Statistical Analysis

      All data are expressed as mean ± SE. Statistical analyses were performed using a 1-way factorial analysis of variance with GraphPad PRISM software (GraphPad Software, Inc, La Jolla, CA). Statistically significant differences were defined as P < 0.05.

      RESULTS

      SBP and Biological Parameters

      All groups displayed similar food consumption, water intake, urine volumes, Na+ and K+ excretion rates (Table 1) and body weight (Table 1). SBP values (Table 1) were not altered by the injection of anti-Thy1.1 antibody and were not significantly different throughout the 14 days. Similarly, SBP was not significantly altered by aliskiren treatment. Plasma creatinine values were also not significantly different in the 3 groups (Table 1).
      Table 1Body weight, systolic blood pressure (SBP) and plasma creatinine concentration in control, anti-Thy1.1 and anti-Thy1.1 + aliskiren-treated rats
      0 Week1 Week2 Weeks
      Body weight
       Control251.0 ± 7.2305.5 ± 10.0338.5 ± 12.0
       Anti-Thy1.1 GN228.9 ± 3.5300.0 ± 3.6327.8 ± 5.3
       Anti-Thy1.1 GN + Aliskiren265.0 ± 5.0329.3 ± 10.5364.3 ± 11.5
      Time course of SBP (mm Hg)
       Control109 ± 1.0103 ± 1.5104 ± 0.6
       Anti-Thy1.1 GN112 ± 3.9106 ± 0.8103 ± 3.0
       Anti-Thy1.1 GN + Aliskiren109 ± 2.0104 ± 1.4105 ± 1.4
      Plasma creatinine (μg/100mL)
       Control394.8 ± 7.3
       Anti-Thy1.1 GN401.4 ± 73.8
       Anti-Thy1.1 GN + Aliskiren293.3 ± 18.6

      Effects of the Aliskiren Treatment on Proteinuria and Microalbuminuria

      In response to the anti-Thy1.1 antibody injection, proteinuria developed by day 3 after the anti-Thy1.1 antibody injection. The maximum augmentation of urinary protein excretion was observed at day 3 and decreased slightly at days 7 and 14 but remained elevated (Figure 1A). Aliskiren treatment significantly attenuated the increase in urinary protein excretion at days 3, 5 and 7 compared with the group treated with the anti-Thy1.1 antibody alone (P < 0.01 versus anti-Thy1.1 glomerulonephritis group) (Figure 1A). The microalbumin excretion levels in the rats injected with the anti-Thy1.1 antibody alone were also much higher than those observed in the rats injected with the anti-Thy1.1 antibody and treated with aliskiren (Figure 1B). The microalbumin levels returned to control levels in the aliskiren-treated group by day 14.
      Figure thumbnail gr1
      Figure 1Effects of aliskiren on urinary protein excretion (A) and urinary microalbumin excretion (B) in rats with glomerulonephritis and treated with aliskiren. Data are expressed as mean ± SE. Section (P < 0.05) and double section (P < 0.01) indicate significant difference compared with day −4. Asterisk (P < 0.05) and double asterisk (P < 0.01) indicate significant difference compared with the control group. Pound (P < 0.05) and double pound (P < 0.01) indicate significant differences compared with the anti-Thy1.1 glomerulonephritis group.

      Effects of Aliskiren Treatment on Glomerular Injury

      The most prominent change in the anti-Thy1.1 glomerulonephritis group was mesangial matrix accumulation evaluated at 14 days after antibody administration. The glomerular matrix expansion score was significantly higher in the anti-Thy1.1 glomerulonephritis group compared with the control group (Figure 2). Chronic treatment with aliskiren significantly suppressed the degree of glomerular matrix expansion.
      Figure thumbnail gr2
      Figure 2Effects of aliskiren on glomerular injury in anti-Thy1.1 glomerulonephritis rats. The sections were stained by periodic acid-Schiff (PAS)-stained sections. Original magnification, 200× (left). The positive staining (%) in glomeruli is shown in the right panel. Asterisk (P < 0.05) indicates significant difference compared with the control group. Pound (P < 0.05) indicates significant difference compared with the anti-Thy1.1 glomerulonephritis group.

      Effect of Aliskiren on RAS Activation in Anti-Thy1.1 Glomerulonephritis

      We examined the ability of aliskiren treatment to effectively suppress the activation of intrarenal RAS. uAGT excretion, AGT protein expression in the kidney and PRA levels were evaluated as markers for intrarenal RAS activity. The anti-Thy1.1 glomerulonephritis group exhibited markedly increased uAGT concentration (Figure 3A) and excretion (Figure 3B) on days 3 and 5. Treatment with aliskiren significantly suppressed the elevation of uAGT levels. In contrast, plasma AGT levels were not different among the 3 groups.
      Figure thumbnail gr3
      Figure 3Effects of aliskiren on urinary angiotensinogen (uAGT) elevation in anti-Thy1.1 glomerulonephritis rats. uAGT levels were measured by AGT ELISA: (A) uAGT concentration and (B) uAGT excretion levels. The results were normalized based on urine volume. Section (P < 0.05) and double section (P < 0.01) indicate significant differences compared with day −4. Asterisk (P < 0.01) indicates significant difference compared with the control group. Pound (P < 0.01) indicates significant difference compared with the anti-Thy1.1 glomerulonephritis group.
      Figure 4 shows Western blot results demonstrating augmentation of AGT protein in the kidneys of the anti-Thy1.1 glomerulonephritis groups even at 14 days after initiating treatment. Aliskiren treatment prevented the increase in intrarenal AGT expression levels (Figure 4A). Furthermore, immunohistological analyses demonstrated that the AGT protein expression in renal tubules was significantly higher in the anti-Thy1.1 group compared with the control group. Aliskiren treatment significantly attenuated the increase in AGT protein levels (Figure 4B). As shown in Figure 5, both PRA and plasma Ang II levels were significantly increased in the anti-Thy1.1 group. Aliskiren treatment prevented the elevation of PRA and plasma Ang II levels. As shown in Figures 5C and 5D, kidney cortex and medulla Ang II contents were significantly higher in the rats with glomerulonephritis compared with the control group. However, although chronic aliskiren treatment significantly attenuated the Ang II content in medulla, Ang II content in the kidney cortex was not significantly reduced. PRR mRNA (Figure 6A) and protein (Figure 6C) levels in cortex were decreased in anti-Thy1.1 group. Aliskiren treatment did not normalize the suppression of PRR expression. PRR mRNA levels in anti-Thy1.1 with or without aliskiren groups were not significantly different.
      Figure thumbnail gr4
      Figure 4Effects of aliskiren on angiotensinogen (AGT) expression level in the kidney: (A) AGT protein expression levels detected by Western blot and (B) AGT-immunoreactivity in kidney sections. Original magnification: 200X. Asterisk (P < 0.05) indicates significant difference compared with the control group. Pound (P < 0.05) indicates significant difference compared with the anti-Thy1.1 glomerulonephritis group.
      Figure thumbnail gr5
      Figure 5Effects of aliskiren on plasma renin activity (PRA), plasma Ang II levels and kidney Ang II levels in anti-Thy1.1 glomerulonephritis rats. PRA (A), plasma Ang II (B), renal cortical Ang II (C) and renal medullary Ang II (D) levels were determined by radioimmunoassay. Kidney Ang II levels were normalized based on kidney weight. Data are expressed as mean ± SE. Asterisk (P < 0.05) and double asterisk (P < 0.01) indicate significant difference compared with the control group. Pound (P < 0.05) and double pound (P < 0.01) indicate significant differences compared with the anti-Thy1.1 glomerulonephritis group.
      Figure thumbnail gr6
      Figure 6Effects of aliskiren on PRR mRNA and PRR protein augmentation in anti-Thy1.1 glomerulonephritis rats. PRR mRNA in cortex (A) and PRR mRNA in medulla (B) levels were measured by quantitative real time RT-PCR. PRR protein levels in kidney cortex (C) were measured by Western blot. Asterisk (P < 0.05) and double asterisk (P < 0.01) indicate significant difference compared with the control group.

      Effects of Aliskiren on TGF-β1 and PAI-1 Levels

      To determine the effects of aliskiren to mitigate the renal injury caused by the anti-Thy1.1 antibody, we quantified the expression of TGF-β mRNA and PAI-1 mRNA by qRT-PCR. The anti-Thy1.1 glomerulonephritis group showed elevation of TGF-β mRNA (Figure 7A) expression and PAI-1 mRNA (Figure 7B) expression compared with control rats. Chronic aliskiren treatment significantly attenuated the augmentation of TGF-β mRNA expression levels and PAI-1 mRNA expression compared with the anti-Thy1.1 alone (Figures 7A and 7B). As shown in Figure 7D, Western blot results demonstrate augmentation of PAI-1 protein in the kidney of the anti-Thy1.1 glomerulonephritis group and aliskiren treatment prevented this increase. However, TGF-β1 protein levels were not significantly increased by the anti-Thy1.1 antibody or altered further by aliskiren treatment.
      Figure thumbnail gr7
      Figure 7Effects of aliskiren on transforming growth factor (TGF)-β1 mRNA, plasminogen activator inhibitor (PAI)-1 mRNA, TGF-β1 protein and PAI-1 protein augmentation in anti-Thy1.1 glomerulonephritis rats. TGF-β1 mRNA (A) and PAI-1 mRNA (B) levels in the kidney cortex were measured by qRT-PCR. TGF-β1 protein (C) and PAI-1 protein (D) levels in the kidney were measured by Western blot. Asterisk (P < 0.01) indicates significant difference compared with the control group. Pound (P < 0.05) and double pound (P < 0.01) indicate significant difference compared with the anti-Thy1.1 glomerulonephritis group.

      DISCUSSION

      There is a growing recognition that an inappropriately activated intrarenal RAS exerts a cardinal role in mediating or facilitating the progressive renal injury that occurs in glomerulonephritis.
      • Kondo S.
      • Shimizu M.
      • Urushihara M.
      • et al.
      Addition of the antioxidant probucol to angiotensin II type I receptor antagonist arrests progressive mesangioproliferative glomerulonephritis in the rat.
      • Wagner J.
      • Volk S.
      • Haufe C.C.
      • et al.
      Renin gene expression in human kidney biopsies from patients with glomerulonephritis or graft rejection.
      • Nagamatsu T.
      • Oka T.
      • Nagao T.
      • et al.
      Effects of KD3-671, an angiotensin II type 1 receptor antagonist, on anti-thy-1 nephritis in rats.
      • Mii A.
      • Shimizu A.
      • Masuda Y.
      • et al.
      Angiotensin II receptor blockade inhibits acute glomerular injuries with the alteration of receptor expression.
      • Zhang J.
      • Gu C.
      • Noble N.A.
      • et al.
      Combining angiotensin II blockade and renin receptor inhibition results in enhanced antifibrotic effect in experimental nephritis.
      Indeed, various studies have reported that blockade of the RAS with either ACE inhibitors or Ang II AT1R blockers ameliorate or markedly reduce the proteinuria and progressive renal injury in glomerulonephritis.
      • Mahmood J.
      • Khan F.
      • Okada S.
      • et al.
      Local delivery of angiotensin receptor blocker into the kidney ameliorates progression of experimental glomerulonephritis.
      • Nagamatsu T.
      • Oka T.
      • Nagao T.
      • et al.
      Effects of KD3-671, an angiotensin II type 1 receptor antagonist, on anti-thy-1 nephritis in rats.
      • Peters H.
      • Ruckert M.
      • Gaedeke J.
      • et al.
      Angiotensin-converting enzyme inhibition but not beta-adrenergic blockade limits transforming growth factor-beta overexpression in acute normotensive anti-thy1 glomerulonephritis.
      • Zhang J.
      • Gu C.
      • Noble N.A.
      • et al.
      Combining angiotensin II blockade and renin receptor inhibition results in enhanced antifibrotic effect in experimental nephritis.
      • Urushihara M.
      • Kobori H.
      Angiotensinogen expression is enhanced in the progression of glomerular disease.
      • Nakamura T.
      • Obata J.-E.
      • Kimura H.
      • et al.
      Blocking angiotensin II ameliorates proteinuria and glomerular lesions in progressive mesangioproliferative glomerulonephritis.
      However, it has also been reported that ACE inhibition alone does not completely protect against the glomerular damage and renal fibrotic remodeling suggesting additional mechanisms.
      • Westerweel P.E.
      • Joles J.A.
      • den O.K.
      • et al.
      ACE inhibition in anti-thy1 glomerulonephritis limits proteinuria but does not improve renal function and structural remodeling.
      One specific model of glomerulonephritis that has been studied extensively is the anti-Thy1.1 antibody-induced glomerulonephritis. Several recent studies have addressed the possible effects of DRI on the development of glomerulonephritis. Zhang et al
      • Zhang J.
      • Gu C.
      • Noble N.A.
      • et al.
      Combining angiotensin II blockade and renin receptor inhibition results in enhanced antifibrotic effect in experimental nephritis.
      studied the effect of ACE inhibition alone or in combination with renin receptor inhibition. They demonstrated that ACE inhibition alone reduced the increased expression of TGF-β1, fibronectin and PAI-1 caused by injection of anti-Thy1.1 antibody. Suppressing the renin receptor with small interfering RNA alone also reduced the expression of fibrotic markers in a manner similar to ACE inhibition. Importantly, the combination of both treatments further reduced the expression of the fibrotic markers. The authors suggested that renin receptor activation could be responsible for the inability of ACE inhibition alone to completely suppress the expression of fibrotic markers and suggested that blocking renin or renin receptors might have additional therapeutic potential. However, our studies failed to show an increased PRR expression in response to anti-Thy1.1 treatment.
      Huang et al
      • Huang Y.
      • Yamamoto T.
      • Misaki T.
      • et al.
      Enhanced intrarenal receptormediated prorenin activation in chronic progressive anti-thymocyte serum nephritis rats on high salt intake.
      showed that giving a high salt diet to antithymocyte nephritic rats increased the severity of the kidney fibrosis. Although plasma renin and Ang II levels were suppressed, there were increases in tubular prorenin and renin, and there was redistribution of PRR from the cytoplasm to the apical membrane along with elevated prorenin and Ang II in collecting ducts and connecting tubules of the nephritic rats. The authors suggested the involvement of the PRR in the augmentation of intrarenal Ang II and progression of kidney fibrosis in nephritic rats fed a high salt diet. In this study, PRR levels were suppressed by injection of anti-Thy1.1 antibody in the kidneys, particularly in renal cortex. Because aliskiren treatment did not restore the PRR levels, the suppression of intrarenal PRR was not induced by renin inhibition. Thus, intrarenal PRR is unlikely to be involved in the development of glomerulonephritis, suggesting that the progression of glomerulonephritis occurs in an Ang II-dependent and PRR-independent manner.
      Sato et al
      • Sato A.
      • Piao H.
      • Nozawa Y.
      • et al.
      Local delivery of a direct renin inhibitor into the kidney ameliorates progression of experimental glomerulonephritis.
      evaluated the effects of direct local renin inhibition on the progression of renal injury in anti-Thy1.1 glomerulonephritis. They accomplished local blockade by subrenal capsular implantation of a collagen sponge containing aliskiren. This treatment significantly suppressed mesangial matrix expansion and ameliorated the glomerular sclerotic index. Although juxtaglomerular renin expression was enhanced, expression of a-smooth muscle actin and type 1 collagen were markedly decreased, thus suggesting an important renoprotective effect of aliskiren.
      Although these and other previous studies
      • Nakamura T.
      • Obata J.-E.
      • Kimura H.
      • et al.
      Blocking angiotensin II ameliorates proteinuria and glomerular lesions in progressive mesangioproliferative glomerulonephritis.
      have provided substantial support for the involvement of the intrarenal RAS in the renal injury that occurs in glomerulonephritis, there has not been definitive evidence for specific enhancement of intrarenal RAS during the induction and development of anti-Thy1.1 antibody glomerulonephritis. Specifically, the effects of systemically administered aliskiren on renoprotection and intrarenal RAS activation in glomerulonephritis had not been evaluated. In this study, we measured urinary protein excretion as a marker of renal injury and uAGT as an index of intrarenal and/or intratubular RAS activity. We observed that the greatest increases in urinary protein and microalbumin excretion occurred early at 3 to 5 days after injection of the antibody. Similarly, the greatest increase in uAGT excretion rates occurred in the 3-to 5-day period. Importantly, the rats treated with aliskiren had a marked attenuation of the increase in urinary protein and microalbuminuria excretion rates during the same period. Furthermore, the stimulation of uAGT caused by the antibody was suppressed to an even greater extent, thus demonstrating the efficacy of aliskiren to markedly reduce the extent of stimulation of the intrarenal RAS.
      To evaluate the longer-term effects of aliskiren to mitigate or prevent the renal injury caused by the antibody, the rats were maintained for 13 to 14 days before harvesting the blood and tissue samples. At this time point, both the protein and AGT excretion rates had decreased markedly and were either the same or only slightly higher than the control values. Nevertheless, histological examination of the kidneys revealed sustained renal injury with increased glomerular matrix expansion and increased expression of AGT protein and kidney cortex and medullary Ang II contents. In addition, TGF-β1 and PAI-1 mRNA levels were still elevated at this time point, but only PAI-1 protein levels were augmented. Importantly, the rats that were also treated with aliskiren had either a reduced degree of injury or complete suppression of the effects caused by the antibody. The mesangial expansion and intrarenal AGT protein expression were almost completely suppressed and increases in TGF-b1 and PAI-1 mRNA expression were significantly lower. Recent studies have suggested that intratubular Ang II is generated by medullary renin.
      • Prieto M.C.
      • Gonzalez A.A.
      • Navar L.G.
      Evolving concepts on regulation and function of renin in distal nephron.
      These findings support a suppressing effect of aliskiren on the increases in renal medullary Ang II observed in this study.
      As shown in Table 1, treatment with anti-Thy1.1 did not cause perceptible changes in SBP. Similarly, treatment with aliskiren did not modify the SBP values measured at 1 and 2 weeks after initiating treatment. Although Wagner et al
      • Wagner J.
      • Dechow C.
      • Morath C.
      • et al.
      Retinoic acid reduces glomerular injury in a rat model of glomerular damage.
      reported a substantial increase in SBP following treatment with anti-Thy1.1, this response did not occur in our study and has not generally been substantiated by other studies.
      • Peters H.
      • Wang Y.
      • Loof T.
      • et al.
      Expression and activity of soluble guanylate cyclase in injury and repair of anti-thy1 glomerulonephritis.
      ·
      • Peters H.
      • Ruckert M.
      • Gaedeke J.
      • et al.
      Angiotensin-converting enzyme inhibition but not beta-adrenergic blockade limits transforming growth factor-beta overexpression in acute normotensive anti-thy1 glomerulonephritis.
      ·
      • Mii A.
      • Shimizu A.
      • Masuda Y.
      • et al.
      Angiotensin II receptor blockade inhibits acute glomerular injuries with the alteration of receptor expression.
      ·
      • Huang Y.
      • Yamamoto T.
      • Misaki T.
      • et al.
      Enhanced intrarenal receptormediated prorenin activation in chronic progressive anti-thymocyte serum nephritis rats on high salt intake.
      ·
      • Wenzel U.O.
      • Wolf G.
      • Jacob I.
      • et al.
      Chronic anti-Thy-1 nephritis is aggravated in the nonclipped but not in the clipped kidney of Goldblatt hypertensive rats.
      Furthermore, although it may have been expected that aliskiren treatment would decrease SBP, this has not been observed in normal rats at doses that can normalize arterial pressure in Cyp1a1-Ren2 transgenic rats.
      • Huang L.
      • Howard C.G.
      • Mitchell K.D.
      Chronic direct renin inhibition with aliskiren prevents the development of hypertension in Cyp1a1-Ren2 transgenic rats with inducible ANG II-dependent hypertension.
      In summary, treatment of glomerulonephritis rats with aliskiren reduced or prevented the marked proteinuria and albuminuria, in addition to the increased uAGT levels that occurred in response to the anti-Thy1.1 antibody. Furthermore, the sustained renal injury that was demonstrated at 14 days was also substantially reduced by the aliskiren treatment. In addition, because the glomerulonephritis rats did not exhibit hypertension, one noteworthy finding in this study is that the renoprotective effect of aliskiren in the rats with glomerulonephritis occurs in a BP-independent manner. These results clearly indicate the effectiveness of aliskiren in reducing or preventing the stimulation of the RAS and the development of renal injury that develops in anti-Thy1.1 antibody induced glomerulonephritis. They provide additional evidence for the effectiveness of DRI beyond hypertension and diabetes.

      ACKNOWLEDGMENTS

      The authors thank Novartis Pharmaceutical, Co, for supplying the aliskiren. They also appreciate the technical assistance provided by Porcha Davis, MS, and Ayumi Kitano, MS.

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