1Department of Nephrology, University of Erlangen-Nuremberg, Erlangen; and 2Cardiovascular Research, Bayer HealthCare, Wuppertal, Germany
Submitted 6 August 2004 ; accepted in final form 19 November 2004
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ABSTRACT |
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matrix expansion; anti-Thy1 model; BAY 412272
Guanylyl cyclase is a ubiquitously distributed cytoplasmic enzyme that exists in two isoenzyme forms, a membrane-bound form stimulated by various peptides and the intracellular soluble guanylyl cyclase (sGC). The intracellular sGC is a heme-containing heterodimer, consisting of an (7388 kDa)- and a
(70 kDa)-subunit (19). The
1
1 isoenzyme is thought to be the major isoform. Previous studies have shown that sGC is expressed in various renal cell types in vivo, such as glomerular arterioles, granular cells, descending vasa recta, fibroblasts, podocytes, and MC (22, 35). Nitric oxide (NO) is a major sGC stimulator in vivo (1), even though the effects of NO are mediated by various physiological pathways (21). Previous studies demonstrated inhibition of MC proliferation (28) and matrix expansion (36) by NO treatment in vitro. Inhibition of NO production caused glomerulosclerosis, hypertension, and matrix expansion in healthy rats in vivo (4, 26) as well as aggravated anti-glomerular basement membrane nephritis in rats (9). In contrast, a NO-generating
-blocker prevented renal injury in the remnant kidney model (34).
Nevertheless, it is still unknown whether these beneficial NO-dependent effects in renal disease are due to stimulation of sGC or the result of other activated pathways. Recently, a novel group of pyrazolopyridine derivatives was developed (32), which demonstrated a highly specific, NO-independent stimulation of sGC in a cell-free environment as well as in endothelial cells and platelets (31). In vivo studies demonstrated potent vasodilation and a subsequent dose-dependent decrease in blood pressure in spontaneously hypertensive and normal rats, as well as inhibition of platelet aggregation (10, 33). In a canine model of congestive heart failure, treatment with the specific sGC stimulator BAY 412272 improved cardiac output (5). In contrast, nothing is known about the regulation and potential therapeutic role of sGC stimulation in inflammatory kidney disease.
In this study, we investigated the regulation and therapeutic potential of direct stimulation of sGC in experimental mesangial proliferative glomerulonephritis in the rat in vivo.
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METHODS |
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To ensure equivalent antibody binding in all rats and to avoid potential interference with the treatment, we started therapy no earlier than 1 h after disease induction. Animals received either 10 mg/kg body wt BAY 412272 dissolved in a solvent solution consisting of Transcutol P (Gattefosse, Saint-Priest, Cedex, France)/Cremophor EL (Sigma, Munich, Germany)/water (vol/vol/vol, 10:20:70) or an equal amount of the solvent solution via oral gavage. The first three applications were given within 18 h as loading doses, followed by daily doses from day 2 on. A 24-h urine collection was performed for assessment of proteinuria and creatinine clearance before a survival biopsy on day 2 and a euthanasia biopsy on day 6. On day 6, blood pressure was measured by tail plethysmography. In addition, the bleeding time in 0.9% saline solution at 37°C was assessed after a small incision on the lower side of the proximal rat tail. Blood was collected via puncture of the inferior caval vein in all animals before death under anesthesia by a combination of ketamine (100 mg/kg) and xylazine (5 mg/kg). Serum and the collected urine were stored at 70°C until analysis.
Four additional experiments were performed. One equivalent experiment in four rats per group was performed for the measurement of cGMP levels in isolated glomeruli indicating successful intracellular stimulation of sGC via BAY 412272 therapy within glomerular cells in anti-Thy1 nephritis in vivo. Therefore, on day 6 the left kidney was removed for isolation of glomeruli and consecutive analysis by Western blotting or radioimmunoassay, whereas the other kidney was fixed and processed for immunohistochemistry. A second separate experiment was performed in four rats per group to control for the effect of lowering of systemic blood pressure on development of mesangial proliferative glomerulonephritis. Starting 1 h after disease induction, one group received standard antihypertensive triple therapy via the drinking water consisting of reserpine (5 mg/l), hydralazine (80 mg/l), and hydrochlorothiazide (25 mg/l), whereas the other group received normal drinking water. On day 6, systolic blood pressure was measured, the animals were killed, and the kidneys were processed as described above. A third separate experiment was performed to investigate the effects and safety of direct sGC stimulation via BAY 412272 in healthy rats. Five rats per group either received BAY 412272 therapy or solvent solution analogous to the other experiments but without consecutive induction of anti-Thy1 nephritis. Glomeruli and tissues were harvested for further analysis as described above.
To verify changes in sGC protein expression in isolated glomeruli during anti-Thy1 nephritis, a fourth experiment was performed, where anti-Thy1 nephritis was induced in six rats as described above. Kidneys of three rats each on days 2 and 6 were removed and used for isolation of glomeruli and consecutive Western blot analysis. Isolated glomeruli of three normal, healthy rats served as controls.
Tissue processing and immunohistochemical staining. Renal biopsies were fixed in methyl Carnoy's solution or 3% paraformaldehyde, embedded in paraffin, and cut into 5-µm sections for indirect immunoperoxidase staining as described elsewhere (7, 12).
To perform immunoperoxidase staining, tissue sections were incubated with the following primary antibodies as indicated: a murine IgM monoclonal antibody (mAb) against the proliferating cell nuclear antigen (PCNA; 19A2; Coulter Immunology, Hialeah, FL) (12); ED-1, a murine IgG1 mAb to a cytoplasmatic antigen present in monocytes, macrophages, and dendritic cells (Serotec, Oxford, UK) (12); OX-7, a murine IgG1 mAb specific for MC (Serotec) (12); a polyclonal antibody to collagen IV (goat anti-human/bovine collagen IV; Southern Biotechnology Associates, Birmingham, AL) (12), or a monoclonal antibody against fibronectin (GIBCO, Invitrogen, Karlsruhe, Germany) (27); JG-12, a monoclonal antibody against the AGE-receptor for staining of glomerular capillaries (kindly provided by D. Kerjaschki, University of Vienna, Vienna, Austria) (18); PL-1, a murine monoclonal antibody against rat platelets (kindly provided by W. W. Baker, Groningen, The Netherlands) (2); a monoclonal antibody against the 1-subunit of sGC (Alexis Biochemicals, Gruenberg, Germany) (35).
Negative controls for immunostaining included either deleting the primary antibody or substitution of the primary antibody with equivalent concentrations of an irrelevant murine mAb or preimmune rabbit IgG. All tissue sections were incubated with primary antibodies overnight at 4°C. Afterward, specific biotinylated secondary antibodies (all by Zymed, San Francisco, CA) were applied, followed by peroxidase-conjugated avidin D (Vector Labs, Burlingame, CA) and color development with diaminobenzidine with nickel chloride for nuclear staining and otherwise without nickel. In the case of immunofluorescence staining, a secondary goat anti-rabbit antibody (Molecular Probes, Leiden, The Netherlands) was applied.
Expression of collagen IV, fibronectin, and the sGC 1-subunit was quantified using computer-assisted image-analysis software (MetaVue, Visitron Systems, Munich, Germany) in a blinded fashion. At least 50 glomerular cross sections were analyzed at 400-fold magnification. PCNA-positive nuclei, ED-1-positive cells, glomerular microaneurysms (13), as well as the number of all glomerular cells were counted separately in 50 consecutive glomeruli/section using 400-fold magnification in a blinded fashion. Mesangiolysis and platelet influx were assessed using 400-fold magnification in 50 consecutive glomeruli using a semiquantitative scoring system from 0 to 4, where 0 = 0%, 1 = 125%, 2 = 2650%, 3 = 5175%, and 4 = 76100% of the glomerular area mesangiolytic or positive for platelets, respectively. The same score was used for assessing collagen IV-positive areas only in the additional experiment comparing the effects of antihypertensive therapy with the placebo group. All results are given as means ± SD per glomerular cross section.
Immunohistochemical double staining. To determine the number of proliferating MC, double immunostaining for PCNA, a marker of cell proliferation, and for OX-7 (MC specific) was performed as described previously (12). The number of proliferating MCs is given as means ± SD per glomerular cross section.
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Apoptotic cells were detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay as described previously (11). Cells were regarded as TUNEL positive if their nuclei were stained black and displayed typical apoptotic morphology with chromatin condensation. The number of apoptotic cells was counted in 50 sequentially selected glomeruli and is given as the means number ± SD per glomerular cross section.
Glomerular preparations and measurement of cGMP. Glomerular preparations were made as described elsewhere (12). Glomerular extracts were homogenized on ice in homogenization buffer containing 5 x 104 mmol IBMX (Sigma, Seelze, Germany) to block the activity of phosphodiesterases and snap-frozen in liquid nitrogen. Measurement of cGMP was performed in triplicate using a commercial radioimmunoassay kit (IBL, Hamburg, Germany) as described before (33).
Western blot analysis.
Protein samples containing 20 µg of protein from isolated glomeruli were separated by 8% SDS-PAGE and blotted onto PVDF membranes. Blots were blocked for 12 h at 4°C in blocking solution containing 2% milk powder (Applichem, Darmstadt, Germany)/1% bovine serum albumin (Merck, Darmstadt, Germany). After being washed, blots were incubated with the primary antibody for 2 h, followed by horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences, Buckinghamshire, UK). Immunoreactive bands were detected on the basis of chemiluminescence using an enhanced chemiluminescence kit. -Actin was detected simultaneously as the loading control using an antibody from Abcam (Cambridge, UK). Quantitative analysis was performed using a computer-assisted system (AIDA Image Analyzer, Raytest, Straubenhardt, Germany).
Miscellaneous measurements. Urinary protein was measured colorimetrically using a commercial test kit (Bio-Rad, Hercules, CA) based on the Bradford dye-binding procedure (6, 30). Serum and urinary creatinine were measured using an autoanalyzer (Beckman Instruments, Brea, CA).
Statistical analysis. All values are expressed as means ± SD. Statistical significance (defined as a P < 0.05) was evaluated by Student's t-test.
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RESULTS |
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Antihypertensive effects of sGC stimulation do not affect glomerular cell proliferation.
Although the antihypertensive effect of sGC stimulation by BAY 412272 in our particular experiment did not reach a level of significance compared with placebo-treated anti-Thy1 animals (P = 0.07), the effect of a decrease in systolic blood pressure per se on development of anti-Thy1 nephritis was tested. Although the standard triple antihypertensive treatment used in this control experiment resulted in a significant 35% decrease of blood pressure on day 6, no differences in the glomerular proliferative response and matrix accumulation were detected compared with placebo-treated rats [PCNA-positive cells per glomerular cross section were 14.9 ± 1.6 vs. 14.2 ± 2.7, not significant (NS); glomerular score for collagen IV 2.6 ± 0.4 vs. 2.8 ± 0.2, NS].
sGC stimulation does not affect accumulation of inflammatory cells. Since other reports (10, 33) demonstrated that platelet aggregation can be inhibited by sGC stimulation via BAY 412272, we also investigated the glomerular accumulation of platelets (PL-1 positive) and monocytes/macrophages (ED-1 positive), which was equivalent in the BAY 412272 and placebo group at all time points during anti-Thy1 nephritis (Table 1).
BAY 412272 treatment in normal healthy rats does not affect either renal histology or function.
To investigate whether sGC stimulation alters renal histology and function in normal rats, five additional healthy rats each were treated with either BAY 412272 or placebo. Despite a 6-day treatment, no difference in the cGMP levels of glomeruli from BAY 412272- or placebo-treated rats (870 ± 343 vs. 892 ± 137fmol/l, NS) could be detected. It has to be considered that cGMP levels in glomeruli from healthy rats were up to 20-fold lower compared with glomeruli from rats with anti-Thy1 disease and in a femtomolar range. In addition, glomerular sGC expression by Western blot analysis was also equivalent in BAY 412272- and placebo-treated rats (1.9 ± 0.8 vs 1.3 ± 0.3 relative glomerular, sGC 1-subunit protein/
-actin, NS) (not shown). Serum creatinine (0.182 ± 0.02 vs. 0.182 ± 0.02 mg/dl, NS), creatinine clearance (2.9 ± 0.5 vs. 3.1 ± 0.2 ml/min, NS), as well as proteinuria (6.5 ± 0.7 vs. 7.8 ± 2.2 mg/24 h, NS) were equal in BAY 412272- and placebo-treated rats. Renal histology as assessed by periodic acid-Schiff staining was completely normal and unremarkable in both groups. These findings indicate that BAY 412272 treatment did not cause any obvious renal side effects in normal healthy rats.
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DISCUSSION |
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Consistent with previous studies (22, 35), we could demonstrate endogenous expression of sGC protein in the normal rat kidney and glomerulus. In addition, glomerular sGC protein was upregulated predominantly at the late proliferative phase in anti-Thy1 nephritis, and immunostaining revealed a typical mesangial pattern. Despite endogenously stimulated expression of sGC during the late proliferative phase of the anti-Thy1 model, selectively the functional activity of sGC could be markedly increased by daily oral treatment of rats with a selective sGC stimulator (BAY 412272) from day 1 on. Treatment with this highly potent compound increased glomerular cGMP levels as the downstream messenger molecule but not sGC expression levels, clearly demonstrating that BAY 412272 reached its intracellular pharmacological target in vivo. These findings are consistent with previously published data on BAY 412272 and its closely related compounds (3133) demonstrating NO-independent, but heme-dependent, enzyme activity mediated via a selective binding site located on the -subunit of sGC. Whereas these compounds can enhance stimulatory effects of NO on sGC, NO antagonism, as provided by in vivo application of L-NAME in a low-NO model of hypertension (33), did not interfere with its enzyme-stimulating potency. In contrast to the results in anti-Thy1 nephritic rats, we could not detect any increase in glomerular cGMP levels after BAY 412272 treatment in normal healthy rats. While, clinically, some bleeding tendency at death (during surgery) indicated BAY 412272 action in these animals, no renal phenotype was apparent in these rats as assessed by histological and functional parameters. The reason for these unaltered glomerular cGMP levels by BAY 412272 treatment in normal rats is most likely the detection limit of the assay. While glomerular cGMP levels in anti-Thy1 nephritic rats were increased
20-fold, glomerular cGMP levels in normal healthy rats were only in the femtomolar range, where potential differences are hard to detect and may be especially vulnerable to some degradation that cannot be prevented during the glomerular isolation process.
A major finding of the present study was that sGC stimulation reduces glomerular cell proliferation in anti-Thy1 nephritis. In this model, glomerular cell proliferation at the late proliferative phase (as represented by day 6 biopsies) is predominantly confined to the mesangium, whereas more than one-half of the proliferating glomerular cells at the early time point on day 2 are of endothelial and monocytes/macrophage origin (13). Our data suggest that the antiproliferative effect of sGC stimulation is exclusively confined to MCs, since double staining for PCNA- and OX-7-positive, proliferating MCs revealed a clear-cut decrease in the BAY 412272-treated rats associated with a reduced glomerular cell number on day 6 but no effect in the early phase of disease on day 2. In addition, microaneurysm formation being dependent on endothelial cell repair and the degree of mesangiolysis was not altered by this treatment. The reduction of glomerular hypercellularity is likely due to a sole inhibition of MC proliferation, because glomerular cell removal via apoptosis, the major pathway of resolution in this disease (3), was not altered by sGC stimulation. The fact that the decrease in systemic blood pressure per se does not interfere with the time course of mesangial proliferative glomerulonephritis is supported by the results of our control experiment using a standard triple regimen as an antihypertensive treatment as well as former studies in this particular nephritis model (37). Since platelet depletion in this model is associated with inhibition of glomerular cell proliferation (16) and sGC stimulation is involved in inhibition of platelet activation/aggregation, we cannot completely exclude indirect antiproliferative effects via sGC-stimulated platelets. Nevertheless, this interpretation is not supported by the results of our study, because BAY 412272 did not influence glomerular platelet aggregation during anti-Thy1 nephritis.
Besides its antiproliferative effect, sGC stimulation also markedly inhibited extracellular matrix accumulation, the second hallmark of renal disease progression, as indicated by collagen IV as well as fibronectin staining. Previous studies have frequently demonstrated a link between glomerular cell proliferation and matrix accumulation (8, 15), although glomerular matrix expansion and MC proliferation can also be dissociated (39). Further studies will have to differentiate between whether this antifibrotic effect of sGC stimulation is due to its antiproliferative action or possibly independent of it.
While the specific mechanisms mediating these antiproliferative and antifibrotic effects of sGC stimulation have to be investigated in future studies, cGMP produced in response to NO and natriuretic peptides has been demonstrated to regulate various genes involved in cellular proliferation and matrix production (24). In vascular smooth muscle cells, MC, and fibroblasts, mainly antiproliferative effects of cGMP involve inhibition of growth factor-induced Erk-1/2 activity, inhibition of the early growth response gene-1 (egr-1) (28), reduction of endothelin-1 synthesis, modulation of cell-cycle regulatory genes, and increased expression of MAP kinase phosphatase-1 (24). In addition, the NO-cGMP pathway has also been shown to inhibit expression of genes mediating matrix synthesis directly or indirectly via transforming growth factor-, the major profibrotic cytokine. Studies in cultured MC demonstrated that the NO-cGMP system is able to downregulate the profibrotic mediator connective tissue growth factor (17) as well as thrombospondin-1, the activator of the transforming growth factor-
procytokine complex after glucose stimulation (25, 38).
sGC stimulation also significantly reduced proteinuria, which is considered an important indicator and progression factor in renal disease, at both early and later time points of anti-Thy1 disease. This antiproteinuric effect does not seem to be dependent on its antiproliferative and antifibrotic action, because on day 2 no other parameter besides proteinuria was affected by sGC stimulation. Since the development of proteinuria is frequently caused by alterations of the basement membrane or podocytes, the protective effect on proteinuria in this model may rather reflect stimulation of the sGC system in podocytes than in the mesangium. Although information on the role of the cGMP signaling cascade is very limited, the sGC-cGMP system is expressed in podocytes and can be stimulated by NO as demonstrated by in vitro studies (20, 22).
These various beneficial effects of BAY 412272 therapy could have been caused by a general interference with the disease inducing antibody binding or with the injury induction that follows antibody binding, especially since NO has been shown to be important in mediating MC injury in vitro and in this model (23, 29). To avoid interference with antibody binding, BAY 412272 treatment was initiated after maximal antibody binding had already occurred (14, 16). The equal degree of mesangiolysis, microaneurysm formation, cellular proliferation/matrix accumulation, and influx of inflammatory cells early on day 2 in both groups further supports the conclusion that sGC stimulation by BAY 412272 neither affected disease induction/MC injury unspecifically, via interference with antibody binding, nor specifically, such as suggested for inhibition of NO synthase by NG-monomethyl-L-arginine (L-NMMA) treatment (23). An interesting question is whether sGC stimulation can successfully prevent inhibition of mesangiolysis in a situation where NO production is inhibited via L-NMMA; this may be investigated in the future. Nevertheless as discussed before, unspecific interference with the disease induction process must be excluded, when a treatment such as L-NMMA with alteration of MC injury is started before disease induction, as done in the study by Narita et al. (23). On the other hand, initiation of treatment after antibody binding may come too late if NO and sGC are already important for mesangiolysis within the first hours after disease induction. Although the beneficial effects of sGC stimulation seen in this model system are similar to earlier studies with NO donors (28, 36) and contrary to some studies using NO synthase inhibitors (4, 9, 26, 34), the design of our study is not suitable for differentiating the diverse NO-mediated pathobiological effects on kidney disease. In addition, the protective effects gained by pharmacological sGC stimulation cannot be just extrapolated for the situation of the endogenous sGC in anti-Thy1 nephritis, where a specific sGC blockade due to the lack of available inhibitors will not be easily achievable.
In conclusion, we established specific pharmacological stimulation of sGC within glomerular cells as an effective beneficial therapy in experimental mesangial proliferative glomerulonephritis, exerting antiproliferative, antifibrotic, and antiproteinuric properties, whereas effects regarding mesangiolysis and apoptosis were lacking. Treatment with an orally available sGC stimulator such as BAY 412272 thus may become a potential novel form of treatment for mesangial proliferative glomerulonephritis in humans, the most common form of glomerulonephritis in the Western world.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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