This study demonstrated that progressive renal injury in spontaneously hypertensive rats resulted in glomerulosclerosis and tubulointerstitial injury. This occurred despite the fact that hypertensive renal disease functions in concert to promote inflammatory responses[18–20]. Besides, a significant growth in pro-fibrotic proteins was shown on account of increased inflammation. Furthermore, there was a significant increase in autophagy, likely due to the importance of changes in indices of inflammation. It is well known that Golgi's apparatus breakdown is related to serious inflammation and fibrosis. Therefore, measures adopted to alleviate these pathologic processes may take effect in slowing down the progress of hypertensive renal disease.
One efficient and well-studied method in preventing progressive renal injury is the blockade of RAAS. The salutary roles of Renin-Angiotensin-Aldosterone System (RAAS) Blockade, in slowing the progress of hypertensive renal disease, are that they are brought by its antihypertensive and hemodynamic attributes and by its capacity of reducing inflammation and fibrosis. The binding of angiotensin II to its receptor grows generation of NLRP3 inflammasomes, leading to fibrosis, inflammation, and ultimately tissue damage. Hence, renoprotection is provided by RAAS blockade through mitigating this pathologic cascade of events. Therefore, unsurprisingly, in the present research treatment of spontaneously hypertensive animals with VAL led to serious amelioration of inflammation, autophagy, fibrosis and finally tubulointerstitial disease and glomerulosclerosis.
Another intriguing hidden mechanism by which preventing renal injury and progression of hypertensive renal disease may be realized is by augmentation of Natriuretic peptides (NPs). Natriuretic peptides hormone storage, in humans and mammals, as a family of natriureticpeptide forming, the members in NPs including brain natriuretic peptide(BNP), atrial natriuretic peptide(ANP), dendroaspis natriuretic peptide(DNP), ventricular natriuretic peptide (VNP), C-type natriuretic peptide(CNP), and micrurus natriuretic peptide (MNP), which are generated as pre-pro-peptides and experience cleavage to generate active peptides(BNP). ANP and BNP show an extensive scope of CV and renal roles, including natriuresis, diuresis and control of blood pressure. Renal sodium and water handling via inhibiting RAAS and antidiuretic hormone are regulated to mediate these activities. Enkephalinase (NEP) reduce the concentration of ANP and BNP, whilst enkephalinase inhibitors increase the content of ANP and BNP, thereby controlling heart failure. In addition, enkephalinase inhibitors can also increase ANP and urinary ANP content, whist also inhibiting the cGMP second messenger, in addition to exerting antioxidant, anti-inflammatory, anti-and fibrotic effects, whilst also inhibiting glomerulosclerosis and resisting renal tubular damage. While the inhibitory influence on the RAAS pathway is adopted to mediate several of the therapeutic roles of NPs, abundant evidence shows that NPs (especially ANP and BNP) have anti-autophagy, anti-inflammatory and antifibrotic attributes, which may greatly contribute to their renoprotective attributes. For instance, we have observed that ANP stops the activation of the nuclear element NF-κB, declines the generation of reactive nitrogen and halogen and reduces the generation and release of cytokines and chemokines.
In addition, it has been observed that ANP plays an inhibitory roles in producing inflammatory mediators in endothelial cells, macrophages, and cardiomyocytes. Besides, by comparing with control animals renal ablation in transgenic mice that overexpressed hepatic BNP reduces renal fibrosis and decreases protein abundance of main proteins in the fibrotic path and this role was independent of blood pressure.
The Ang (1–7) receptor inhibitor(A779) inhibits the effect of ARNI, which suppresses the NLRP3 Inflammasomes, up-regulated by Ang II. Pretreatment with ARNI significantly delays Ang II-induced inflammatory responses associated with up-regulated NLRP3 expression; this effect of ARNI is blocked by A779. Pretreatment human embryonic kidney cells (293T) with ARNI significantly inhibits NLRP3 expressions induced by Ang II; this effect is also blocked by A779[21]. Infusion of ARNI and A779 does not significantly change progressive renal injury in SHR groups. A779 in the SHR animals does not change the RAAS pathway and BNP expressions in relation to SHR group, while NLRP3 and casepase-1 expressions are significantly higher than SHR, VAL or ARNI groups. Infusion of A779 and ARNI combined for 12 weeks does not prevent progressive renal injury or inhibit inflammation and fibrosis of SHR rats.
In the aorta and in the primary aortic vascular smooth muscle cells of naturally hypertensive rats, we corroborated the results that the NLRP3 inflammasome was activated[22]. Moreover, there was enhanced expression of NLRP3 mRNA in renal biopsies of patients with various kidney diseases, including hypertensive nephrosclerosis[23]. NLRP3, caspase-1 and IL-1βexpression was elevated in T cells of hypertensive patients[24]. Our data and model simulations support this conclusion. Many studies agreed with our data, in other words reveal a great diversity between the hypertensive group (SHR) and the normal control group (SD). It shows that the inflammatory mechanism exerts a significant effect on the occurrence of hypertension and participates in the occurrence and development of hypertension, which verifies the possibility of this hypothesis. Finally, it was proven that hypertension is an immune mechanism theory: it also shows that the NLRP3 inflammasome, a component of the innate immune system, exerts a core effect on inflammatory response.
The inflammation, immune system and hypertension are associated with each other. The innate and adaptive immunity system triggers an inflammatory process is triggered, in which blood pressure may grow, promoting organ injury[25]. In an animal model of CKD, Cao et al illustrated that treatment with omapatrilat, an integrated neprilysin and ACE inhibitor, was related to great enrichments in blood pressure and proteinuria except as slowing the progress of tubulointerstitial fibrosis, glomerulosclerosis and renal injury[26]. Nevertheless, the improvement degree was not beyond that of ACE inhibition alone. In a similar research, Taal et al disclosed that omapatrilat treatment resulted in greater decreases in SBP and glomerular capillary pressure and this was related to a greater decrease in proteinuria, glomerulosclerosis and delayed progress of renal disease, by comparing with ACE inhibition alone[27]. In another research on nephron mass decrease and hypertension, Benigni et al compared the anti-hypertensive and reno-protective roles of combining omapatrilat plus enalapril compared to ACE inhibition alone. Their study also observed that treatment with omapatrilat led to a great decrease in proteinuria, glomerulosclerosis and tubulointerstitial fibrosis. Besides, declined renal synthesis of endothelin-1 and grown nitric oxide production leading to declined renal vasoconstriction were observed. Furthermore, greatly grown tubular ANP release in animals treated with omapatrilat was observed[28].
Meanwhile, we found that a kidney biopsy consistent with middle and advanced stage hypertensive nephropathy, showing a predilection for arteriola glomerularis afferens. The small arteries (arcuate arteries and interlobular arteries) and arterioles (afferent arterioles) took part in middle and advanced stage hypertensive nephropathy. Early pathological sections of hypertensive nephropathy revealed progressive fibrosis of arterial vessel walls. The pathological changes in the sections of middle and advanced hypertensive renal arterioles showed arcuate arteries (onion skin-like changes) in the afferent arterioles. The possible reason is that when the arterial vessel wall has obvious changes, the glomerulus will also have more obvious changes, and subsequently the following occur: renal tubules and renal interstitium change; the number of glomerular cells reduce; and the mesangial area is widened. So the pathological changes of hypertensive kidney damage are formed.
At present, a large number of basic studies have revealed that brain natriuretic peptide participates in the regulation of cardiac interstitial fibrosis by regulating the expression of fibrotic genes, acting on cytokines and inflammatory factors, and directly acting on myocardial fibroblasts. Our results show that global activation of the NLRP3 inflammasome leads to fibrosis, renal inflammation, and cell death. Activation of the NLRP3 inflammasome may be the mechanism leading to non-specific inflammatory damage and fibrosis in hypertension-damaged kidneys of rats. Of course, future experiments of this study can also use selective inhibition of NLRP3 to reduce renal inflammation in spontaneously hypertensive rats. Furthermore, fibrosis experiments to verify whether NLRP3-induced mechanisms can serve as therapeutic targets for nonspecific inflammation. Here, the authors only provide preliminary evidences that the activation of NLRP3 inflammasome may cause nonspecific immune inflammation and fibrosis in the kidneys.
Our findings highlight the importance of changes in the indices of inflammation when reducing the response of autophagy, fibrosis and influenced Golgi’s apparatus by the kidneys in hypertensive animals. Therefore, it is not surprising that in animals with hypertensive renal disease that were treated with ARNI we found a great improvement in the indices of inflammation, autophagy, and fibrosis, in addition to the fact that there was increased Golgi's apparatus breakdown beyond that which was discovered with ARB therapy alone. This was accompanied by a great enhancement in markers of renal function such as renal histology. These outcomes conform to previous research on the influence of ARNI on the progression of kidney pathology in animals suffering from hypertension.
Previous studies have demonstrated that Sacubitril/valsartan ameliorates oxidative stress, inflammation, and fibrosis, as well as enhances renal function beyond angiotensin receptor blockade in CKD[19]. In renal tissue samples from control rats (n = 6),CKD rats (n = 9), CKD rats treated with ARNI(N = 9), and CKD rats treated with VAL, representative Western blotting analyses and group data was used to depict the nuclear content of the p65 active subunit of NF-κB, and the protein abundance of MCP-1, iNOS and COX-2. Our study revealed a novel mechanism underlying sacubitril/valsartan improvement of renal function and histology and attenuated most of the molecular markers of inflammation, autophagy, fibrosis and Golgi’s apparatus beyond angiotensin receptor blockade in hypertensive renal disease. In the renal tissues samples of control rats (n = 5),SHR(n = 5), SHR rats treated with VAL (n = 5), SHR rats treated with ARNI(n = 5), and SHR rats treated with ARNI + A779(n = 5), representative Western blots analyses and group data depicts NLRP3 and Casepase-1 content of the NLRP3 inflammasome active subunit of inflammasome activation, as well as autophagy or autophagocytosis of LC3 and p62.
Recently, increasing evidence shows that the NLRP3/NF-κB pathway plays a major role in hypertensive renal disease, which works in concert to promote inflammatory response. Here, we found that the angiotensin receptor-neprilysin double blocker(which represses NLRP3 inflammasome activation) may provide a mechanistic and molecular basis for the new therapeutic use of inflammation in hypertension or progressive renal injury in hypertension. Moreover, it is generally recognized in published literature that the effect of the angiotensin receptor-neprilysin double blocker to block the inflammatory process is mediated by the NLRP3/NF-κB pathway in hypertensive nephropathy. This is, perhaps the most remarkable finding of the present study.
While similarities between the aforementioned mentioned researches and our current report are shown, there still remains great diversities. First and foremost, the latter research adopted a model of renal mass reduction, which utilises the removal of one kidney and the ligation of the blood supply to 2/3 of the contralateral kidney. This result represents the significant activation of the RAAS in severe hypertension, which can mainly contribute to progression of renal injury in this model. Hence, while these surveys aimed at accounting for this key features of the model, it is reasonable that treatment of hypertension and hemodynamic abnormalities exerted a main effect on the outcomes of these past researches. Nevertheless, the model in our research adopted surgical removal of renal tissue to cause renal mass decrease without renal artery ablation. While hypertension accompanies nephron mass decrease and hyperfiltration, related to this model, the degree of growth in blood pressure is not as serious as that discovered in models depending on the ligation of the renal artery. Nevertheless, a modest but statistically indifferent decline in the blood pressure of animals treated with ARNI was shown by comparing with the VAL group. Therefore, partial efforts of blood pressure control to the beneficial role of ARNI therapy cannot be excluded.
Another Another significant difference between previous studies and this study is their application of omapatrilat, whose clinical utility with ACE inhibition has been restricted because of the reduced breakdown of bradykinin production and increased risk of angioedema. Therefore, ARNI applies a varied generation of neprilysin inhibition, with an ARB. Clinical trials applying this agent in treating heart failure have displayed great improvements in mortality without an unacceptable risk of angioedema. It shall be noted that among patients with HF and preserved ejection fraction, therapy with ARNI is related to great preservation of eGFR by comparing with VAL therapy alone.
Nevertheless, the present research firstly evaluates the efficacy of ARNI in slowing the progress of renal disease in an animal model of hypertensive renal disease, and also offers several potential mechanisms in charge of the renoprotective attributes of this medication.
The association between autophagy and inflammation exerts significant effect on the pathogenesis of metabolic, non-communicable diseases[29]. The balance between too little and too much autophagy is key to the survival of cells because autophagy is associated with type 2-cell death programmed necrosis and autophagy. The mechanisms by which Inflammation mediates autophagy control and governs immune cell function remains unclear[29]. Our data shows the significant difference between the values from the control group and the SHR group. In all of P62 and LC3, this study has found great diversities between the SHR group and the VAL and ARNI group, but no difference between the VAL group and ARNI group. This observation is in good agreement with the report that autophagy was determined by P62 and LC3, and that ARNI administration showed a significant increase in autophagy compared with NC. Therefore, hypertension in SHR rats increased autophagy. The suppression of autophagy grows inflammation as cytokines are overexpressed, activating autophagy.
While the outcomes of this research are new, some limitations that should be mentioned are observed. Firstly, clinical studies are required to conform the in vivo outcomes in a well-accepted animal model of hypertensive renal disease. Second, a longer duration of subsequent and assessment of various doses of ARNI in treating hypertensive renal disease should be made in the future. Furthermore, although a great diversity between VAL and ARNI in regard to their influence on proteinuria was not observed, the effectiveness of this therapy in the setting of heavy proteinuria shall be assessed fully by further assessment of ARNI in an animal model of nephrotic syndrome. Besides, the blood pressure of hypertensive renal disease animals, treated with ARNI, was not greatly varied from those treated with VAL. In the end, the molecular paths by which neprilysin inhibition mediates anti-autophagy, anti-inflammation and antifibrotic roles shall be further elucidated by extra mechanistic research.
To sum up, ARNI treatment of animals with progressive renal injury in spontaneously hypertensive rats resulted in glomerulosclerosis and tubulointerstitial injury beyond that discovered with ARB therapy alone. This was reated to, and likely partly mediated by, a great reversal of the up-regulation of : inflammation; improvement of the anti-autophagy; profibrotic mediators; as well as the reduced breakdown of Golgi's apparatus in the kidney.