Animal model and therapeutic prospects for left ventricular hypertrophy and associated renal complication

Cardiac and renal dysfunction often co-occur and considerably worsen the prognosis leading to difficulty in therapy in left ventricular hypertrophy (LVH). The aim of this study was to elucidate changes in expression of human ortholog genes of hypertension, vascular and cardiac remodeling and hypertensive nephropathy phenotypes under normal, disease and gasotransmitter, H2S (hydrogen sulphide) and NO (nitric oxide) and combined (H2S+NO), treatment of rat myocardium and renal tissues. LVH rat models were generated and were treated with H2S and NO. Relative gene expression was quantified. Heart and renal physical indices were significantly modified under individual as well as combined H2S+NO treatment in control and LVH rats. Expression analysis revealed, hypertension, vascular remodeling genes ACE, TNFα and IGF1, mRNAs to be significantly increased (P<0.05) in myocardia and kidneys of LVH rats, while individual and combined H2S+NO treatment reduced gene expression to normal/near to normal values. The cardiac remodeling genes MYH7, TGFβ, SMAD4 and BRG1 expression were significantly up-regulated (P<0.05) in the myocardia of LVH and combined H2S+NO treatment, which recovered the normal/near to normal expression more effectively as compared to individual treatments. Interestingly, individual as well as combined H2S and NO treatment significantly decreased PKD1 expression in renal tissue, which was significantly up-regulated in LVH rats (P<0.05). The reduction in hemodynamic parameters and cardiac indices as well as alteration in gene expression on treatment in LVH rat model indicates important therapeutic potential of combined treatment with H2S+NO gasotransmitters in hypertension and cardiac hypertrophy associated with renal complications.


Introduction
Left ventricular hypertrophy (LVH) develops as an adaptive response to increased hypertension induced cardiac workload and volumetric overload. The hypertrophied myocardium adversely affects cardiac function and is a major factor contributing to heart failure [1]. Cardiac and renal dysfunction often co-occur and considerably worsen the prognosis; renal dysfunction can be causative and/or a complication of cardiovascular disease [1]. Physiological cardio-renal interactions can be explained in terms of systemic hemodynamics and renal-body fluid system for blood pressure control [2]. Activation of the sympathetic nervous system and reninangiotensin system (RAS) in response to decreased cardiac output causes volume expansion and restores renal blood flow [3]. Renal control of blood pressure has a near infinite feedback gain principle such that renal failure, as a consequence of heart failure, causes sodium and water retention, which further exacerbates the cardiac failure [2]. Hypertension induced LVH is associated with vascular remodeling, mediated by vasoactive molecules and endothelial dysfunction as prominent pathological features [4]. Moreover, gene expression changes in the myocardium following stimuli such as pressure overload results in expression of alternate isoform of sarcomeric proteins, which is a major factor contributing to cardiac remodeling and hypertrophied growth of the adult heart muscle [5].
The gasotransmitters hydrogen sulphide (H 2 S) and nitric oxide (NO) are increasingly being studied for their therapeutic potential in treating cardiovascular disease [6,7]. Endogenously H 2 S is produced by pyridoxal-5′-phosphate (PLP)-dependent enzymes [8], while NO is generated by nitric oxide synthases [9]. Several studies have demonstrated the pivotal role of H 2 S in protecting against cardiac hypertrophy by reducing oxidative stress [10], myocardial apoptosis [11], hypertrophy and collagen content [12] and down-regulation of angiotensin II levels [13]. The 4 development of cardiac hypertrophy in mice lacking nitric oxide synthases [14] and the attenuation of LVH in hypertensive rats following treatment with L-arginine to improve NO production [15] highlights the therapeutic role of NO in ameliorating LVH. Recently, a hypothetical model proposed by Yong et al. [16] and Nagpure et al. [17] have shown the existence of interaction between H 2 S and NO, due to the production of intermediate HSNO (thionitrous acid) and HNO (nitroxyl) gases [16], the synergistic effect elicited by H 2 S, NO and these intermediate product is proposed to lead to cardio protection, maintenance of vascular tone and reduced oxidative stress. Recent studies have also shown that the pyridoxal-5′-phosphate (PLP)-dependent enzymes that produce H 2 S are expressed in the kidney [18], where H 2 S shows a protective role against chronic renal failure [19], renal ischemia/reperfusion injury [20] and control renal tubular and vascular function [21]. NO plays a significant role in regulating renal hemodynamics and tubular function [22] and, therefore, maintenance of arterial pressure and vascular volume [23]. This notion is further supported by the implication of NO deficiency in the progression of hypertension and renal diseases [24]. However, the effects of modulatory control of combined therapy with gasotransmitters H 2 S and NO, for the treatment of cardiovascular and renal disease remains largely unexplored.
In the present study we investigated the modulation of the expression of specific genes in response to individual and combined treatment of H 2 S and NO gasotransmitters in the myocardium and renal tissue of control and LVH rat models. The studied genes are known to be involved in hypertension, vascular and cardiac remodeling and hypertensive nephropathy in human. 5 Animal experimentation protocols used in this study were approved by the Animal Research and Service Centre "ARASC" under the Animal Ethics Committee, Universiti Sains Malaysia "AECUSM" (approval number: 2012/76/364) and Ethic Review Board of the Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan.

Experimental Animals
Male Wistar-Kyoto (WKY) rats with body weight in the range of 180-200g were procured from ARASC and acclimatized for 5 days in the new environment with food and water available ad libitum. Isoprenaline (5mg/kg S/C every 72 hrs) was administered subcutaneously as 5 injections and caffeine (62mg/L) was given in drinking water to the WKY rats for 14 days to induce LVH [25]. The H 2 S donor NaHS (56μM/kg) was administered exogenously in daily intraperitoneal injections for 5 weeks [26], beginning 3 weeks prior to isoprenaline and caffeine administration, while the control groups received saline injections in a similar manner. L-arginine (1.25g/L) was given in drinking water as NO donor for 5 weeks [27].
Seventy two rats were divided into two groups, one for the cardiovascular investigations and renal functional study in acute experiment and the other group for molecular expression studies of the myocardium and renal tissues. Both groups were further divided into 8 sub-groups: Control, LVH, Control-H 2 S (NaHS treated control), Control-NO (L-arginine treated control), Control-H 2 S+NO (NaHS and L-arginine treated control), LVH-H 2 S (NaHS treated LVH), LVH-NO (L-arginine treated LVH) and LVH-H 2 S+NO (NaHS and L-arginine treated LVH); both H 2 S and NO were given separately to the same animal groups (n=48 i.e. 6 rats per group) for the acute studies and n=24 i.e. 3 rats per group for the molecular expression studies. 6 At the end of the treatment period, tail vein blood sample was taken from each rat and subjected to centrifugation at 2000 x g for 10 minutes [25]. Plasma H 2 S concentration was measured using a standard procedure [21,26]. NO level in the plasma was measured using a laboratory kit (NJJC Bio Inc., Nanjing, China) by following the manufacturer's instructions.

Acute experiment
The acute experiment (on anesthetized non-behaving animals) was performed according to previously reported protocol [25]. The rats were made to fast overnight, by removing food from their cages the night before the experiment, and anaesthetized by intraperitoneally administering 60mg/kg pentobarbitone sodium (Nembutal; CEVA Sante Animale, Libourne, France). A cannula was introduced into the trachea to facilitate ventilation. Following this the right carotid artery was cannulated (PP50, Portex, Kent, UK) and the cannula was connected to a pressure transducer (model P23 ID Gould, Statham Instruments, UK), which was in turn connected to a data acquisition system (PowerLab®, ADInstrument, Australia) to continuously monitor mean arterial pressure (MAP). The left jugular vein was cannulated (PP50, Portex, Kent, UK) to allow infusion of maintenance doses of anesthesia and saline as required. A mid-line abdominal incision exposed the left kidney, which was covered with saline soaked pads to prevent drying. A cannula (PP50, Portex, Kent, UK) was inserted into the iliac artery and attached to another fluid filled pressure transducer connected to the PowerLab system to measure iliac mean blood pressure. A laser Doppler flow probe (AD Instruments, Australia), positioned on the outermost layer of the left kidney cortex was used to record the renal cortical blood perfusion (RCBP). The MAP and RCBP were measured continuously for an hour before the rats were euthanized with an overdose of anesthetic and the hearts were harvested post-mortem, dried and weighed for heart and left ventricle (LV) indices to observe the induction of LVH and how treatment effects 7 the regression of LVH. Internal diameter of the LV chamber and myocardium thickness were measured using vernier caliper according to previously reported protocol [28]. Pulse wave velocity (PWV) was measured by taking the propagation time of the pulse wave from Power Lab data and propagation distance was measured manually by putting a thread from the insertion point of the carotid artery cannula to that of the iliac artery cannula [29].

RNA extraction and expression analysis in myocardia and renal tissues of control, LVH, H 2 S and NO treated rats
Target tissues were extracted, disrupted and homogenized as previously described [30]. Briefly, following cervical dislocation, heart muscle and renal cortex were harvested and preserved in  (Table 1). To determine the effect of H 2 S, NO and H 2 S+NO treatment on renal complication in LVH rats, expression of polycystic kidney disease gene; polycystic kidney disease 1 (PKD1; NM_001106352.1) was determined in renal tissue (Table 1) Relative quantification of target gene along with internal control was calculated using the comparative cycle threshold method (2 -ΔΔCT ).   10 The concentration of H 2 S and NO was significantly lower in the plasma of the LVH rats    (Table 3).  1A). PTGIS expression increased by 1.5 fold in heart tissue of LVH rats as compared to control and H 2 S and H 2 S+NO treatment showed significant downregulation, while NO treatment increased expression in heart tissue of LVH rats (P<0.05).

Treatment (H 2 S, NO and H 2 S+NO) ameliorates LVH progression
Contrarily, in renal tissue PTGIS expression was significantly down-regulated in LVH group as compared to control, however, individual as well as combined H 2 S and NO treatment upregulated PTGIS in LVH rats (P<0.05) (Fig 1B). RGS5 was 0.2-0.3 fold down-regulated in both heart and renal tissues of LVH as compared to control group (P<0.05) but there was no notable significant change in gene expression of RGS5 after H 2 S, NO and H 2 S+NO treatment in LVH rats ( Fig 1C). TNFα was significantly up-regulated in both renal and heart tissues of LVH WKY as compared to control (P<0.05), while H 2 S, NO and H 2 S+NO treatment significantly down-regulated TNFα expression in kidney (P<0.05), however, it's expression did not alter in response to H 2 S and H 2 S+NO treatment while NO treatment significantly increased expression in heart tissue of LVH rats ( Fig 1D). IGF1 expression was significantly up-regulated in myocardia and renal tissues of LVH group as compared to control while H 2 S, NO and combined H 2 S+NO treatment significantly down-regulated it normal or near to normal (P<0.05) (Fig 1E).

Change in cardiac remodeling gene expression in myocardia in response to H 2 S, NO, and H 2 S+NO treatment
Expression of cardiac remodeling genes was studied in the myocardia of LVH rats in response to treatment, MYH7, TGFβ1, BRG1 and SMAD4 expression was observed to be significantly upregulated in myocardia of LVH as compared to control group (all P<0.05) (Fig 2) 2).

Altered PKD1 expression in renal tissues of H 2 S, NO, and H 2 S+NO treated rats
Hypertension induced LVH is an important complication in polycystic kidney disease (PKD) [31] and to elucidate the effect of H 2 S and NO treatment on hypertensive nephropathy in LVH rats we studied PKD1 expression in LVH rats before and after treatment. About 3-fold increase in PKD1 expression was observed in renal tissue of LVH WKY as compared to control group (P<0.05). PKD1 expression was significantly down-regulated after treatment of LVH rats with H 2 S, NO and H 2 S and NO combined (P<0.05) (Fig 3).

Discussion
In this study we aimed to elucidate the modulation in expression of particular orthologous genes upon gasotransmitters, H 2 S and NO, treatment in LVH rats, these genes are known to have a role in hypertension, vascular and cardiac remodeling and hypertensive nephropathy in humans. The significant decrease in MAP, heart index, LV index and myocardium thickness, increase in RCBP, and modulated expression of ACE, PTGIS, IGF1, TNFα, TGFβ1 and PKD1 upon exogenous administration of H 2 S+NO, as compared to individual treatments, showed that combined treatment may be more effective in ameliorating LVH and its complications. 16 Comparison of the pattern of gene expression between all animal groups showed that gene expression was most dissimilar in control and LVH groups, while expression was most similar in control and H 2 S+NO treated LVH groups (Fig 4; S1 Table) thus indicating that H 2 S+NO treatment has promising therapeutic potential in LVH and associated complications. We previously reported an up-regulation in endothelial nitric oxide synthase (eNOS) and cystathione-gamma lyase (CSE) expression upon exogenous H 2 S administration only [30]. We also reported that individual NO treatment up-regulated eNOS expression in control and LVH rats and CSE expression in control rats, but down-regulated CSE expression in LVH rats [27]. In our previous report on H 2 S+NO treatment we showed that CSE mRNA levels significantly decreased in control but not in LVH rats, while eNOS mRNA levels significantly increased in both LVH and control groups). Extending our study to check H 2 S and NO plasma concentrations, we have found that these changes corresponded to the mRNA levels of their respective synthases. The synthesis of biochemical intermediates and modulation of the respective pathways for NO and H 2 S synthesis by the gasotransmitters may explain the synergistic efficiency of combined H 2 S and NO treatment in ameliorating LVH relative to individual treatments.
The effect of individual and combined treatment with H 2 S and NO was also seen on the expression of genes in LVH WKY rats. Induction of LVH significantly up-regulated ACE expression while H 2 S+NO treatment down-regulated ACE expression in kidney and heart tissues.
Previous studies have reported a two to three fold increase in Angiotensin II (Ang II) levels in 17 isoprenaline induced LVH WKY rats [30,32], which indicates an activation of RAS, resulting in hypertension and arterial stiffness in these LVH models. Being a human ortholog, DNA sequence variations in ACE affects circulating ACE concentration and is linked to the development of LVH in humans. The DD genotype of an insertion/deletion (I/D) polymorphism results in elevated ACE plasma levels and is associated with an increased LV mass index [33] and a 2350G>A polymorphism that affects plasma ACE levels is also associated with LVH [34].
Moreover, ACE inhibitors have been shown to be less effective in LVH regression in patients with the DD genotype [35]. The down-regulation of ACE expression upon H 2 S+NO treatment in the present study indicates that combined treatment may alleviate hypertension caused by either endogenous or exogenous RAS activation. It was also observed that H 2 S treatment in control WKY rats significantly up-regulates ACE expression, which indicates that there may be a difference in the effects elicited by exogenous treatment with gasotransmitters in diseased as opposed to normal settings.
PTGIS catalyzes prostacyclin synthesis from prostaglandin H2 and primarily found in smooth muscle and vascular endothelial cells [36]; prostacyclin suppresses smooth muscle cell proliferation and platelet aggregation and promotes vasodilation [37]. We found a significant upregulation of PTGIS expression in LVH rats as compared to control, while H 2 S+NO treatment down-regulated its expression in LVH rats. This is in line with already reported data in which Champetier et al. [38] observed a 1.9 fold increase in PTGIS expression in LV of rats with severe aortic regurgitation, which is associated with left ventricle eccentric hypertrophy, Harding et al. [39] however, reported an anti-hypertrophic nature of prostacyclin and its inhibitory effect on cardiac fibrosis. Genetic changes and epigenetic modifications are known to alter human PTGIS ortholog expression where promoter hypermethylation results in reduced expression of PTGIS 18 [40]. Based on our findings, there is a significant down-regulation of PTGIS in LVH kidney and combined H 2 S+NO treatment up-regulates expression, which is contrary to the gene expression changes in the myocardium. A critical role for prostacyclin in renal development has been shown previously and PTGIS-knockout mice showed severely abnormal glomerular, vascular and interstitial development [41]. There might be a role of epigenetics in differential gene expression between the two tissues, thus indicating the treatment effectiveness also against epigenetic changes in a tissue specific manner.
RGS5 is one of the regulators of G-protein signaling and it is expressed in vascular and cardiac muscle tissues [42] where it serves to inhibit several Gαi-and Gαq-mediated signaling pathways including those that are induced by Ang II [42,43]. RGS5 human ortholog has been shown to be associated with blood pressure control [44], while, our data has demonstrated a significant reduction in RGS5 mRNA, hence diminishing its protective role against Ang II mediated signaling, in the heart and kidney tissue of LVH WKY rats as compared to control group and there was no notable change in RGS5 expression following H 2 S+NO treatment. In addition, RGS5 expression in heart negatively correlated to the expression of ACE, TGFβ1 and IGF1 in the heart in the current study (Fig 4; S1 Table) IGF1 promotes cell division, it is an antiapoptotic factor in vascular and endothelial cells and promotes cardiac growth and function [45]. Donohue et al. [46] have reported that development of hypertension and hypertrophy was associated with increased IGF1 mRNA levels in rats.
Similarly, Li et al. [47,48] found mRNA and protein levels of the human ortholog to be elevated in idiopathic hypertrophic obstructive cardiomyopathy (HOCM) tissues. Increased IGF1 levels in hypertensive patients as opposed to normotensive individuals is associated with increase in pressure load [49] and is also shown to be positively associated with chronic renal disease [50].
Moreover, promoter polymorphisms in IGF1 have been shown to be significantly related to LV mass in male athletes [51]. We found IGF1 expression to be up-regulated in LVH and significant reduction in expression upon H 2 S+NO treatment. TNFα, an inflammatory cytokine has been shown to effect left ventricular remodeling and hypertrophy under hypertension [52] and is a crucial player in the hypertension and cardiac remodeling processes induced by Ang II [53].
TNF-knockout mice have displayed diminished hypertensive response on activation of RAS [54].  Table).
The heart muscle responds to cardiac workload and hemodynamic stress by activation of the fetal gene expression program, causing hypertrophic growth [57]. In rats, MYH7 (β-MHC) is the fetal isoform of myosin heavy chain that is shown to be up-regulated in cardiac hypertrophy [58], which is consistent with our findings, where we also observed its higher expression. This MYH7 overexpression was slightly down-regulated upon LVH regression after H 2 S+NO treatment, although this was not statistically significant. It has been shown by Lowes et al. [59] that β-MHC human ortholog expression increases and expression of α-MHC isoform is reduced in ventricular myocardium in failing hearts. TGFβ1 promotes cardiac remodeling and hypertrophy in response 20 to pressure overload [60]. It has been reported previously that an increase in cardiac TGFβ1 mRNA in response to AngII infusion in mice and TNFα deficient mice failed to elicit this increase [53]. On comparing hypertensive and normotensive individuals, Argano et al. [61] reported a polymorphism in human TGFβ1 ortholog affecting TGFβ1 sera levels which was found to be associated with hypertension, left ventricular hypertrophy and renal damage. SMAD proteins are transcription activators that are involved in the transduction of TGFβ1 signal [60].
SMAD4 is a crucial part of TGFβ1 signaling pathway [62] and it is reported to induce apoptosis and negative remodeling in hypertrophied myocardia [63]. expression did not change significantly following treatment. We also observed change in TGFβ1, MYH7, SMAD4 and BRG1 expression and moderate to strong correlation between these components (Fig 4; S1 Table).
Another interesting finding of the current study is the modulation in PKD1 expression in LVH rat, in response to treatment. PKD1 encodes polycystin-1 which is an integral membrane protein expressed in the renal tubular epithelium [66]. Studies have shown that altered PKD1 expression may lead to cyst formation in polycystic kidney disease (PKD) [67,68]. Cardiovascular abnormalities such as hypertension and LVH are frequently associated with autosomal-dominant PKD (ADPKD) [69]. We found PKD1 to be ~3-fold up-regulated on induction of LVH in WKY rats and individual treatments as well as combined treatment with H 2 S and NO significantly down-regulated PKD1 expression. Moreover, PKD1 expression showed a strong positive 21 correlation with ACE, TNFα and IGF1 expression in the kidney (Fig 4; S1 Table), the genes that are involved in hypertrophy and vascular remodeling in the heart as well as kidney.

Conclusion
In summary, in the present study, there was a modulation of expression of the hypertrophy, vascular and cardiac remodeling and hypertensive nephropathy genes in LVH rat model and combined treatment with H 2 S and NO, effectively normalized gene expression in most cases.
Human orthologs of these genes have crucial roles in the development of hypertension induced LVH and related complications, which thus highlights a promising therapeutic role for H 2 S and NO combined treatment. Taken together, the present data therefore suggest that combined treatment of H 2 S and NO may be used as a therapeutic intervention for renal and cardiac protection directly and/or indirectly (Fig 5).