Part2: Homoarginine Ameliorates Diabetic Nephropathy Independent Of Nitric Oxide Synthase-3
May 07, 2022
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3. RESULTS
3.1 Mouse characteristics
Diabetic wild type and NOS3- mice had significantly less body weight compared with non-diabetic mice after 24 weeks, which was significantly increased by homoarginine in diabetic NOS3-7 mice. Blood glucose was higher in all diabetic wild types and NOS3-7mice. Kidney/body weight ratio was significantly higher in diabetic NOS3-7 mice, while homoarginine treatment significantly reduced cardiac/body weight ratio in diabetic NOS3-mice. Systolic blood pressure was significantly increased in NOS3-7mice compared with wild type and showed a trend of reduction; albeit not significant by homoarginine treatment in diabetic NOS3-/ mice. (Table 1).
3.2 Homoarginine reduces albuminuria in diabetic NOS3-/- rmice
Diabetes significantly increased albuminuria in both wild type and NOS3-/ mice compared with normal, an effect significantly reduced by homoarginine treatment (Figure 1). Since the results observed in wild-type mice were consistent with our previous study(Wetzel et al.,2019)about the protective effect of homoarginine in DN, we only analyzed NOS3-/ mice for the remainder of this study.
3.3 Homoarginine reduces plasma BUN levels in diabetic NOS3-7-mice
We next evaluated the effects of homoarginine administration on plasma BUN levels in NOS3-mice. Plasma BUN levels were increased in diabetic mice and reduced by homoarginine treatment (Figure 2).
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3.4 NOS1 and NOS2 expressions are not altered in diabetic NOS3-mice
Next, we determined if diabetes and/or homoarginine altered the expression of other NOS isoforms in kidneys from NOS3 -mice. Our results show that neither diabetes nor homoarginine affected NOS1 or NOS2 gene expression in NOS3-7 mice (Figure 3).
3.5 Homoarginine reduces glomerular pathology and fibrosis in diabetic NOS3-mice
Analysis of PAS-stained glomerular sections showed that diabetic NOS3--mice were associated with increased glomerular volume, glomerular cellularity, glomerular basement membrane (GBM) thickness, and glomerular injury index compared with non-diabetic NOS3一mice; an effect significantly reduced using homoarginine treatment(Table 2 and Figure 4a). Furthermore, diabetic NOS3mice displayed an increased percent of fibrosis (using ImageJ)in Masson's trichrome (Figure 4b,c)-stained sections 24 weeks after diabetes, which was reduced by treatment with homoarginine.
3.6 Homoarginine reduces kidney fibrotic markers in diabetic NOS3-/- mice
Real-time PCR analysis show marked increases in kidney fibronectin (Figure 5a), TGFβ(Figure 5b), smooth muscle actin (Figure 5c), and E-cadherin (Figure 5d) following diabetes in NOS3-/mice. Homoarginine supplementation significantly blunted the increase in these fibrosis markers.
3.7 Homoarginine reduces glomerular macrophage infiltration in diabetic NOS3-/mice
Diabetic NOS3-/mice displayed a marked increase in in-filtrating kidney macrophages following diabetes (Figure 6)compared with non-diabetic mice. Homoarginine treatment significantly reduced macrophage infiltration 24 weeks after diabetes.
3.8 Homoarginine reduces cardiac fibrosis and improves cardiac function in mice diabetic NOS3-7-r
Diabetic NOS3-/ mice displayed an increased percent of fibrosis (using ImageJ)in Masson's trichrome (Figure 7a,b)-stained sections 24 weeks after diabetes, which was reduced by treatment with homoarginine. Furthermore, diabetic NOS3-7mice had a significant reduction in ejection fraction(EF)and fractional shrinking(FS)(Figure 7c-e)compared with non-diabetic controls at 22 weeks of diabetes, an effect significantly restored using homoarginine treatment.
3.9 Homoarginine restores MCU complex and its regulators in diabetic Ins2Akita mice
Kidney samples derived from our previous publication (Wetzel et al.,2019)were analyzed for the MCU complex and its regulators. Diabetic mice had a significant increase in kidney MCU and its positive regulator, MCUR1 expression, along with a reduction in kidney MCU negative regulator, MICU1 expression (Figure 8), compared with non-diabetic controls at 18 weeks of age in Ins2Ak mice, whereas diabetic mice treated with homoarginine supplementation restored MCU complex and its regulators to normal levels.
3.10 Homoarginine restores MCU complex mice and its regulators in diabetic NOS3-7-
Diabetic NOS3-/mice had a significant increase in kidney MCU(Figure 9a) along with a significant reduction in its negative regulator, MICUl expression(Figure 9b,c), compared with non-diabetic controls after 24 weeks of diabetes, whereas diabetic NOS3-mice treated with homoarginine supplementation restored MCU complex and its negative regulator to normal levels.
4 DISCUSSION
Diabetic nephropathy remains an important and unresolved complication of diabetes. Our previous publication demonstrated that homoarginine supplementation confers kidney protection in diabetic mouse models (Wetzel et al., 2019). However, the exact mechanism by which homoarginine mediates these effects was not clear. The current study shows that homoarginine supplementation mediates renal tissue protection in DN independent of NOS3 which will ultimately change our understanding of the mechanism(s)by which homoarginine induces renal and cardiac protection in DN. The protective effect of homoarginine in DN could be mediated via improving mitochondrial function.
Endothelial dysfunction, characterized by reduced bioavailability of NO, and increased oxidative stress is a hallmark characteristic of diabetes(Creager et al.,2003)and DN(Goligorsky et al.,2001). The role of NOS3 expression and action in DN is controversial; therefore although low or lack of NOS3 has been shown to exacerbate DN (Wang et al,2011; Zhao et al.,2006), a recent publication (Natarajan et al.,2019)showed that overexpression of NOS3 in Ins2Aki mice exacerbates DN probably via increased NOS3 uncoupling and oxidative stress rather than by increased renal NO production/action. Because homoarginine could be a substrate for NOS by increasing L-arginine(Hecker et al.,1991; Moali et al.,1998), it could also mediate its effect via NO action/production. However, our previous publication showed that oral arginine supplementation surprisingly did not prevent or reduce any markers of renal injury in diabetic mouse models, despite successfully increasing arginine levels in plasma and kidneys (You et al.,2014). Direct interaction between homoarginine levels and NO production is controversial. Homoarginine could reduce NO synthesis from arginine by(a)competition of arginine and homoarginine for cellular uptake by cationic amino acid transporters(CAT)(Chafai et al, 2017; Hagos et al.,2006);(b)by trans-stimulation of CAT, which in turn could increase cellular efflux of arginine; and (c) by com-petition of homoarginine and arginine for NOS(Pilz et al., 2015), although homoarginine is a less efficient substrate for this enzyme(Moali et al.,1998; Zwan et al,2013). Although homoarginine may increase arginine availability and thus NO synthesis by inhibition of arginase, this is unlikely because of the weak affinity of homoarginine for arginase (Marz et al,2010). Nonetheless, homoarginine may contribute to NO synthesis by serving as a substrate for NOSbecause homoarginine, even with its weaker affinity to NOS as compared with arginine, can be metabolized to NO and homocitrulline (Drechsler et al., 2011).
To examine the mechanism of homoarginine's protective effect in DN, we investigated the effects of homoarginine supplementation on kidney dysfunction, glomerular histopathological changes, and macrophage recruitment in NOS3-/-and their wild type littermate diabetic mouse model. Homoarginine supplementation significantly ameliorated diabetic albuminuria in diabetic wild type and NOS3-7mice, confirming our previous finding(Wetzel et al,2019), and was associated with reduced glomerular pathology and kid-ney fibrotic markers expression in diabetic NOS3-mice.
Taken together, our results indicate a direct role of homoarginine's protective effect in DN and support the concept that homoarginine mediates renal tissue protection independent of NOS3. Both cardiovascular morbidity and mortality are increased in patients with DN. In the heart, lower homoarginine levels are related to increased left ventricular thickening, lower EF, and higher NTproBNP(Bahls et al.,2018). Inversely, homoarginine supplementation preserves cardiac function in the murine model of post-myocardial infarction heart failure (Atzler et al.,2017). In the current study, we also demonstrate that the protective effect of homoarginine to improve cardiac function and reduce cardiac fibrosis is independent of NOS3 expression in a similar way to its effect on the kidney. We cannot however exclude the hemodynamic effect of homoarginine in DN given its trend to reduce blood pressure; albeit not significant.
The mechanism of the homoarginine protective effect appears to be linked to modulations of mitochondrial function regulated by the MCU complex and its regulators. Mitochondrial dysfunction plays a pivotal role in renal in-jury(Brooks et al.,2009;Coughlan &Sharma,2016;Hallan & Sharma,2016;Long et al..2016; Sharma,2015; Sun et al.2008;Wang et al.,2012;You et al.,2016;Yu et al 2006; Zhan et al.,2013).In the kidney, endothelial cells are especially vulnerable to mitochondrial dysfunction(Hong et al.,2012)and contribute to oxidative stress, persistent energy depletion, impairment of energy-dependent repair mechanisms, and cell death in kidney injury (Funk et al., 2010; Funk & Schnellmann, 2012).MCU is a multimeric complex involved in rapid Ca' uptake in mitochondria and has been shown to be dysregulated in cardiac pathology (Liao et al.,2015; Luongo et al.,2015; Nemaniet al,2018; Tomar et al,2019; Woods et al,2019). Mitochondrial Ca2+ uptake mediated by the MCU complex plays a critical role in signal transduction, bioenergetics, and cell death, and its dysregulation is linked to several human diseases. Although podocyte apoptosis has been linked to MCU dysregulation(Xu et al.,2018; Yuan et al.,2017), the role of the MCU complex and its interaction with homoarginine supplementation in DN are not known. Our current data in diabetic mice demonstrated that homoarginine administration significantly reduced the expression of MCU, increased the MCU-negative regulator (MICU1), and reduced the MCU-positive regulator (MCUR1)in DN, and this effect is independent of NOS3 expression. Therefore, we speculate that there is an association between mitochondrial Ca' signaling and adverse clinical outcomes in DN. Normalization of mitochondrial function is necessary to restore oxidative metabolism during DN. Additional studies are needed to confirm the direct effect of homoarginine on mitochondrial function.
In conclusion, our study demonstrates for the first time that the mechanism of renal and cardiac tissue protection using homoarginine supplementation in DN is independent of NOS3, and it is likely mediated by improving mitochondrial dysfunction. Results of our study may ultimately result in novel therapeutic interventions designed to augment homoarginine in the treatment of DN.
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