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Tetrahydrobiopterin (BH4) Supplementation Prevents the Cardiorenal Effects of Diabetes in Mice by Reducing Oxidative Stress, Inflammation and Fibrosis

Background: The effects of diabetes on the cardiovascular system as well as in the kidney are profound, which include hypertrophy and fibrosis. Diabetes also induces oxidative stress, at least in part due to the uncoupling of nitric oxide synthase (NOS); this is a shift in NO production toward superoxide production due to reduced levels of the NOS cofactor tetrahydrobiopterin (BH4). With this in mind, we tested the hypothesis that BH4 supplementation may prevent the development of diabetic cardiomyopathy and nephropathy. Methods: Diabetes was induced in Balb/c mice with streptozotocin. Then, diabetic mice were divided into two groups: one group provided with BH4 (sapropterin) in drinking water (daily doses of 15 mg/kg/day, during eight weeks) and the other that received only water. A third group of normoglycemic mice that received only water were used as the control. Results: Cardiac levels of BH4 were increased in mice treated with BH4 (p = 0.0019). Diabetes induced cardiac hypertrophy, which was prevented in the group that received BH4 (p < 0.05). In addition, hypertrophy was evaluated as cardiomyocyte cross-sectional area. This was reduced in diabetic mice that received BH4 (p = 0.0012). Diabetes induced cardiac interstitial fibrosis that was reduced in mice that received BH4 treatment (p < 0.05). We also evaluated in the kidney the impact of BH4 treatment on glomerular morphology. Diabetes induced glomerular hypertrophy compared with normoglycemic mice and was prevented by BH4 treatment. In addition, diabetic mice presented glomerular fibrosis, which was prevented in mice that received BH4. Conclusions: These results suggest that chronic treatment with BH4 in mice ameliorates the cardiorenal effects of diabetes,, probably by restoring the nitroso–redox balance. This offers a possible new alternative to explore a BH4-based treatment for the organ damage caused by diabetes.


Introduction
Diabetes mellitus is one of the most common chronic diseases worldwide, and continues to increase in numbers and significance, with characteristics of an epidemic, as modern lifestyles lead to reduced physical activity and increased obesity [1]. Diabetic cardiomyopathy is the manifestation in the myocardium of the alterations produced by the altered homeostasis of glucose metabolism, independent of coronary artery disease [2]. This cardiomyopathy is initially characterized by diastolic dysfunction and cardiac hypertrophy, with preserved ejection fraction. As diabetes progresses, systolic dysfunction and reduced ejection fraction develop. This process of cardiac deterioration in diabetes

Experimental Model and Protocol
Diabetes was induced in Balb/c mice (n = 30, male, 30-40 g) with the intraperitoneal injection of three doses (100, 100, and 200 mg/kg) of streptozotocin (Sigma, St. Louis, MO, USA) in 10 mM citrate buffer, pH 4.5. The control group received an injection of citrate. Then, diabetic mice were divided into two groups: one group provided with BH 4 (sapropterin, Inpheno, InnoPharmax, Inc., Taipei City, Taiwan, Lot # 6P001) in drinking water (daily doses of 15 mg/kg/day, during eight weeks), and the other received only water. A third group of normoglycemic mice that received only water were used as the control. The protocol was approved by the Bioethics Committee of Universidad de Talca (# 2015-087-DG). Mice were kept in the animal facility of the institution, at room temperature (22 • C), under a 12 h light/dark cycle.

Sample Collection and Storage
At the end of the eight weeks, animals were induced anesthesia with ketamine 90 mg/kg and 10 mg/kg xylazine. Then, a midline incision was made and blood was obtained from the cava vein. After blood withdrawing, the heart and kidney were extracted.

Plasma Biochemical Measurements
For plasma biochemical measurements, we used a brain natriuretic peptide (BNP) Kit ELISA mouse (Elabscience Biotech Co., Ltd., Wuhan, China). Plasma glucose was determined using a kit from Valtek (Santiago, Chile). Insulin was determined using an ELISA from EMD Millipore, Billerica, MA, U.S.A.

Histological Analyses
Cardiac and renal sections were obtained for pathology analysis. For this, hearts and kidneys were fixed in Bouin solution. Then, pieces of the organs were dissected, dehydrated in alcohol-xylol solutions and included in Paraplast. In a microtome, 5 µm sections were obtained and mounted in 0.1% polylysine-treated slides. After this, sections were rehydrated and prepared for hematoxylin-eosin, Masson's trichrome, and periodic acid Schiff's staining.

TUNEL Assay
Cardiac sections were probed with a Click-iT™ TUNEL Colorimetric IHC Detection Kit (Catalogue N • C10625, Thermo Fisher Scientific Inc., Carlsbad, CA, USA), for detection of apoptotic cells, as previously described [25,26].

Tetrahydrobiopterin (BH 4 ) Quantification
Plasma and cardiac BH 4 were determined as previously described [25,28] using a differential oxidation of biopterins protocol. Briefly, samples were submitted either to acidic or basic conditions (pH 3 or 9). Then, samples were oxidized with iodine. BH 2 and BH 4 contents were quantified by HPLC using HPLC (Perkin Elmer series 200, Waltham, MA) with fluorescence detection with excitation at 350 nm and emission at 450 nm.

Western Blot
Cardiac proteins were prepared as previously described for Western blot analysis [29]. Cardiac homogenates (30 µg) were mixed with loading buffer and summited to SDS-PAGE in 7.5% gels. Then, proteins were electro-transferred to nitrocellulose membranes. After blocking with Tween-buffered saline solution supplemented with 5% nonfat milk, membranes were incubated overnight at 4 • C with specific antibodies antinitrotyrosine (Badrilla, Leeds, U.K.). For NOS, SDS-PAGE was performed in nonreducing conditions of the loading buffer, and electrophoresis was run with the chamber immersed in ice. After electro-blotting, nitrocellulose membranes were incubated with anti-NOS1 antibody (Cell Signaling, Danvers, MA, USA) or NOS3 (BD Biosciences, Franklin Lakes, NJ, USA).

Statistical Analysis
Data are presented as means ± SEM, compared using ANOVA (normally distributed data) or Kruskal-Wallis test (nonparametric data) with Tukey's or Dunn's post hoc tests for comparisons between groups. A value of p < 0.05 was considered statistically significant.

Results
Three groups of mice were used: a group of normoglycemic mice, a group of streptozot ocin-induced diabetic mice, and a third group of diabetic mice that received sapropterin (BH 4 ) in drinking water, for eight weeks. At the end of the experimental period, mice were euthanized, and blood and organs were collected. Morphometric and blood parameters are presented in Table 1. These data confirmed the presence of hyperglycemia and reduced insulin levels in streptozotocin-treated mice. Additionally, cardiac hypertrophy was appreciated. Of these parameters, only cardiac hypertrophy was prevented by sapropterin treatment. Next, we evaluated plasma levels of BH 4 ( Figure 1) . These were reduced in diabetic mice compared with those in normoglycemic controls, and increased toward normal in mice treated with sapropterin (39.1 ± 5.7 control, 12.5 ± 5 diabetic mice, and 22.9 ± 8.4 pmol/L in diabetic mice treated with BH 4 , p < 0.05), as well as the ratio between BH 4 and its oxidized form BH 2 : 3.6 ± 1 control, 1.1 ± 0.6 diabetic mice, and 12.7 ± 4.1 in diabetic mice treated with BH 4 , p < 0.05 diabetic mice vs. diabetic mice + BH 4 . Intracardiac (intra-atrial) levels of BH 4 were significantly increased in sapropterin-treated mice (8.8 ± 2.2 control, 9.6 ± 4.7 diabetic mice, and 209.7 ± 99.9 pmol BH 4 mg/protein in diabetic mice + BH 4 , p = 0.0019).
Next, we evaluated the impact of sapropterin treatment on cardiac oxidative stress ( Figure 2). For this, we evaluated the levels of nitrotyrosine on intracardiac proteins by Western blot. This assay showed a significant increase in the content of nitrated proteins in diabetic mice compared with that of normoglycemic controls (p < 0.001), and this content was reduced in sapropterin-treated mice. In addition, we evaluated the levels of NOS1 presented as the dimer or monomer. Under reduced levels of BH 4 , NOS was unable to stabilize as a dimer and, as a consequence, was found in its monomeric form. Using SDS-PAGE under nonreducing conditions, the forms could be appreciated by Western blotting. This analysis showed that in our conditions, both the monomer and dimer of NOS1 were present. The NOS1 dimer and monomer levels were similar in the control and diabetic hearts. Nevertheless, the treatment with sapropterin increased the levels of dimer to monomer in diabetic hearts (p < 0.05). In the case of NOS3, the presence of the monomer was almost indistinguishable from that of the dimer. Neither of these constitutively expressed NOS showed changes in their expression levels (p > 0.05). These data suggested that sapropterin treatment was able to reduce intracardiac oxidative stress, probably independent of changes in NOS1 activity.

Figure 1.
Oral administration of sapropterin restores levels of tetrahydrobiopterin (BH4). Left, plasma levels of BH4 in control, diabetics, and diabetic mice that received sapropterin (BH4) in drinking water. Right, intracardiac BH4 levels in control (black), diabetic mice (red), and diabetic mice that received BH4 (green). n = 6 in each group. * p < 0.05 vs. control and diabetics.
Next, we evaluated the impact of sapropterin treatment on cardiac oxidative stress ( Figure 2). For this, we evaluated the levels of nitrotyrosine on intracardiac proteins by Western blot. This assay showed a significant increase in the content of nitrated proteins in diabetic mice compared with that of normoglycemic controls (p < 0.001), and this content was reduced in sapropterin-treated mice. In addition, we evaluated the levels of NOS1 presented as the dimer or monomer. Under reduced levels of BH4, NOS was unable to stabilize as a dimer and, as a consequence, was found in its monomeric form. Using SDS-PAGE under nonreducing conditions, the forms could be appreciated by Western blotting. This analysis showed that in our conditions, both the monomer and dimer of NOS1 were present. The NOS1 dimer and monomer levels were similar in the control and diabetic hearts. Nevertheless, the treatment with sapropterin increased the levels of dimer to monomer in diabetic hearts (p < 0.05). In the case of NOS3, the presence of the monomer was almost indistinguishable from that of the dimer. Neither of these constitutively expressed NOS showed changes in their expression levels (p > 0.05). These data suggested that sapropterin treatment was able to reduce intracardiac oxidative stress, probably independent of changes in NOS1 activity.

Cardiac Remodeling
Diabetes induced cardiac hypertrophy, evaluated as the ratio of heart weight/tibia length, which was prevented in the group that received BH4: (7.6 ± 1.03 g/mm control,

Apoptosis and Inflammatory Cells
Because there is significant cardiac damage in chronic diabetes, both apoptosis and the presence of inflammatory cells has been described in the diabetic myocardium. Apoptosis was evaluated as the presence of TUNEL-positive cells in cardiac sections ( Figure 4A). Diabetes induced an increase in the percentage of TUNEL + cardiomyocytes compared with normoglycemic hearts, but this increase was not modified in the hearts from sapropterin-treated mice. We also evaluated the presence of infiltrative inflammatory cells by immunofluorescence of F4/80, a cell surface marker that is present in macrophages ( Figure 4B). Diabetic hearts showed an increase in the number of macrophages present in the myocardium compared with normoglycemic controls. This number was significantly decreased in the hearts of sapropterin-treated mice.

Apoptosis and Inflammatory Cells
Because there is significant cardiac damage in chronic diabetes, both apoptosis and the presence of inflammatory cells has been described in the diabetic myocardium. Apoptosis was evaluated as the presence of TUNEL-positive cells in cardiac sections ( Figure 4A). Diabetes induced an increase in the percentage of TUNEL + cardiomyocytes compared with normoglycemic hearts, but this increase was not modified in the hearts from sapropterintreated mice. We also evaluated the presence of infiltrative inflammatory cells by immunofluorescence of F4/80, a cell surface marker that is present in macrophages ( Figure 4B). Diabetic hearts showed an increase in the number of macrophages present in the myocardium compared with normoglycemic controls. This number was significantly decreased in the hearts of sapropterin-treated mice.

Renal Changes
Next, we evaluated the impact of sapropterin treatment on diabetic nephropathy, evaluating glomerular morphology ( Figure 5).

Renal Changes
Next, we evaluated the impact of sapropterin treatment on diabetic nephropathy, evaluating glomerular morphology ( Figure 5).
Next, we evaluated the degree of macrophage infiltration and the expression of αsmooth muscle actin. Macrophage infiltration was evaluated by immunofluorescence staining of the cell surface marker F4/80 ( Figure 6). Renal sections of control normoglycemic mice did not show the presence of infiltrating inflammatory cells, neither in the glomeruli nor in the peritubular interstitium. On the contrary, F4/80-positive cells were extensively found in the peritubular space of diabetic mice. This infiltration was dramatically reduced in the diabetic kidneys from mice treated with sapropterin. Next, we evaluated the degree of macrophage infiltration and the expression of α-smooth muscle actin. Macrophage infiltration was evaluated by immunofluorescence staining of the cell surface marker F4/80 ( Figure 6). Renal sections of control normoglycemic mice did not show the presence of infiltrating inflammatory cells, neither in the glomeruli nor in the peritubular interstitium. On the contrary, F4/80-positive cells were extensively found in the peritubular space of diabetic mice. This infiltration was dramatically reduced in the diabetic kidneys from mice treated with sapropterin.
In addition, we evaluated the presence of myofibroblasts as a marker of initial fibrosis (Figure 7). For this, renal sections were analyzed for α-smooth muscle actin (α-SMA). As expected, in the kidneys from normoglycemic control mice, there was no evidence of the presence of myofibroblasts in the peritubular space. On the contrary, the renal sections of diabetic mice showed the presence of these cells in the peritubular space mainly. Kidneys from sapropterin-treated mice showed almost no signal for α-SMA. In addition, we evaluated the presence of myofibroblasts as a marker of initial fibrosis (Figure 7). For this, renal sections were analyzed for α-smooth muscle actin (α-SMA). As expected, in the kidneys from normoglycemic control mice, there was no evidence of the presence of myofibroblasts in the peritubular space. On the contrary, the renal sections of diabetic mice showed the presence of these cells in the peritubular space mainly. Kidneys from sapropterin-treated mice showed almost no signal for α-SMA.
These results suggested that sapropterin treatment reduced macrophage infiltration and peritubular fibrosis in the diabetic kidneys. Overall, these results suggested that chronic treatment with BH 4 in mice ameliorates the cardiorenal effects of diabetes , probably by restoring the nitric oxide production. This offers a possible new alternative to explore a BH 4 -based treatment for the organ damage of diabetes. Figure 6. Renal tubular macrophage infiltration is prevented by oral administration of BH4 to diabetic mice. Representative confocal images of renal sections probed for F4/80 (green), a marker of macrophages, in normoglycemic control, diabetic, and diabetic mice treated with sapropterin (BH4). Middle panel, corresponding section counterstained with propidium iodide (P.I., red) for nuclei. Right panel, merge of both F4/80 and P.I: signals. Images were obtained at 40× magnification. Scale bar indicates 10 µm.  These results suggested that sapropterin treatment reduced macrophage infiltration and peritubular fibrosis in the diabetic kidneys. Overall, these results suggested that chronic treatment with BH4 in mice ameliorates the cardiorenal effects of diabetes, probably by restoring the nitric oxide production. This offers a possible new alternative to explore a BH4-based treatment for the organ damage of diabetes.

Discussion
Our present results showed that oral administration of sapropterin (BH 4 ) for one month to diabetic mice was able to ameliorate some pathological changes in both the heart and kidneys, two of the main organ targets of diabetes. Notably, BH 4 administration reduced cardiac hypertrophy and fibrosis, while preventing glomerular hypertrophy in the kidney. These effects were associated with the reduction in oxidative stress, but apparently independent of NOS1 recoupling. Previous reports have shown that eNOS uncoupling is an important source of ROS in the diabetic kidney [30][31][32][33][34].
Reduced levels of tetrahydrobiopterin were attributed to the reduced expression of guanosine5 -triphosphate cyclohydrolase I (GTPCH), a rate-limiting enzyme in the synthesis of BH 4 [35]. Experiments where GTPCH was overexpressed then reverted the phenotypes associated with diabetic nephropathy [32] and cardiomyopathy [36,37].
In the diabetic heart, NOS uncoupling has emerged as an important source of ROS [36][37][38][39], in a way that appears as an important therapeutic target to prevent the development of diabetic cardiomyopathy. Our results agree with those obtained by recent studies regarding the role of NOS1 uncoupling in the heart, which indicate that BH 4 supplementation or genetic modifications that lead to the increased intracellular production of BH 4 has a beneficial impact on the left ventricular function of diabetic mice. This effect is achieved by improving intracellular calcium handling, hence left ventricular systolic and diastolic mechanics. Interestingly, some of the positive effects of BH 4 supplementation in the diabetic heart appear to be independent of NOS recoupling [39]. Our results are consistent with those findings because we did not observe significant recoupling of NOS1 in the diabetic heart after BH 4 treatment, although we observed reduction in cardiac oxidative stress. It was suggested that this protective effect may be exerted by a metabolic action of NOS1, increasing the expression of insulin-independent glucose transporters (GLUT-1), which improved myocardial energetics [39]. In addition, it was also shown that BH 4 exerts its beneficial effects in diabetic cardiomyopathy by activating peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) signaling by interacting with calcium/calmodulindependent protein kinase kinase 2 (CaMKK2). These effects are also independent of NOS1 activity [40].
It was described in a model of cardiac hypertrophy by transverse aortic constriction that BH 4 supplementation inhibits macrophage infiltration in the myocardium, probably by reducing the inflammatory signaling [41]. Interestingly, this protection is also independent of NOS uncoupling, which is consistent with a role of BH 4 in macrophages biology. The exact mechanism through which BH 4 mediates these anti-inflammatory effects remains to be determined, but it has been reported that BH 4 is important for the macrophage functions, dependent and independent of iNOS [42,43].
We also observed a reduction in the number of macrophages present in the diabetic myocardium after treatment with BH 4 . Nevertheless, we did not evaluate the origin of these macrophages. The recent literature indicates the presence of at least four types of macrophages in the heart, with one being the infiltrating-monocytes-derived macrophages [44][45][46]. Particularly, a study using streptozotocin-induced diabetes in mice showed that cardiac macrophages producing interleukin 1β play an important role in the generation of arrhythmias in the diabetic heart [47]. A recent study documented that BH 4 deficiency in macrophages increased the production of interleukin 1β and the inflammatory profile of these cells [48].
In the context of the diabetic patient, it is relevant to consider both the development of cardiomyopathy and nephropathy. Here, we showed that BH 4 treatment was able to prevent the cardiac damage associated with the initial stages of diabetic cardiomyopathy, reducing macrophage infiltration, fibrosis, and hypertrophy, and similar effects were observed in the kidney at the glomerular and tubular levels. Interestingly, even though we observed a general beneficial effect of BH 4 supplementation on renal morphology in diabetic mice, BH 4 mesangial expansion was not modified. This is consistent with a previous report that suggested that BH 4 may induce mesangial proliferation [49]. Plasma BH 4 concentration was postulated as a predictor of renal function in diabetic patients [50].
Fibrosis has been identified as a common factor in cardiorenal syndrome [51,52], which is a reciprocal interaction between cardiac and renal dysfunction in several pathological states, including diabetes [53,54]. Here we verified that fibrosis affected both organs and was prevented by BH 4 administration.
Importantly, sapropterin, a form of BH 4 , is available and approved for use in humans in phenylketonuric patients [55]. This opens the possibility that this drug may be considered for use in diabetes clinical trials.

Conclusions
The results of the present study indicated that chronic oral administration of sapropterin (BH 4 ) in mice ameliorates the morphological changes produced by diabetes in the heart and the kidney, probably by reducing oxidative stress, reducing inflammation, and the fibrotic processes that occur in the myocardium and at the glomerular and peritubular space in the kidney. This offers a possible new alternative to explore a BH 4 -based treatment for the organ damage caused by diabetes.