Potential effects and application prospect of angiotensin receptor-neprilysin inhibitor in diabetic kidney disease

Diabetic Kidney Disease (DKD), a microvascular complication of diabetes mellitus (DM) characterized by hyperfiltration, albuminuria and following progressive decline of renal function, is one of the main causes of end-stage renal disease (ESRD) and all-cause mortality in diabetic patients.1., 2. Despite the increasing progress in diagnosis and treatment, there was no significant decrease in the prevalence and mortality of DKD over the past two decades at the global level, and the proportion of DKD deaths relative to other types of chronic kidney disease (CKD) has been increasing.³ DKD is mediated by a series of pathophysiological mechanism associated with hyperglycemia, such as inflammation, oxidative stress and fibrosis. Renin-angiotensin-aldosterone system (RAAS) plays a vital role in pathogenesis and progression of DKD.⁴ RAAS inhibitors, mainly including angiotensin-converting enzyme inhibitor (ACEI) and angiotensin II receptor blocker (ARB), effectively ameliorate the pathologic conditions such as proteinuria and hypertension and consequently slow down the progression of DKD. Although ACEI and ARB have shown benefits in clinical trials and have been widely used in treatment of DKD, many patients treated with these drugs still had renal events.1., 5. Therefore, new strategy in DKD treatment is necessary.

Angiotensin receptor neprilysin inhibitor (ARNI) combines ARB and neutral endopeptidase inhibitor (NEPI). Sacubitril/valsartan (LCZ696, brand name Entresto) is the first agent in the ARNI class.⁶ In recent years, a number of studies have demonstrated its effect in cardiovascular protection, renal protection and blood pressure (BP) control, and probably in glycemic control. In this review, we will focus on the research advance in effect of ARNI on DKD and discuss its underlying mechanism and application value in DKD treatment

Neutral endopeptidase (NEP) or neprilysin is a zinc-dependent type II integral membrane metallopeptidase that degrades natriuretic peptides (NPs) and regulates a series of vasoactive peptides including angiotensin II (Ang II), bradykinin, endothelin-1(ET-1), substance P, adrenomedullin and amyloid.⁷ Neprilysin is widely distributed in the brain, vascular endothelial cells, smooth muscle cells, cardiomyocytes and neutrophils. Neprilysin is most abundant in the brush border of proximal renal tubular cells, so that neprilysin inhibition has a substantial effect on kidney. Based on physiological functions of NPs and other substrates, neprilysin inhibitor increases their bioavailability via attenuating their degradation.

Natriuretic peptides system, consisting of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), participates in fluid balance by natriuresis and vasodilation. In cardiovascular disease (CVD) and CKD, NPs play an important role against the overactivation of RAAS and sympathetic nervous system through cyclic guanosine monophosphate (cGMP)-dependent pathways.8., 9 In the kidney, ANP and BNP bind to natriuretic peptide receptor-A (NPR-A), activating its guanylyl cyclase (GC)-A.10., 11. After binding with ANP or BNP, GC-A elevates the intracellular level of cGMP, which then induces the activation of protein kinase (PK) G. The NPR-A/cGMP/PKG pathway promotes diuresis, not only by increasing ultrafiltration of glomerular membrane and glomerular hydrostatic pressure via hyperpolarization induced mesangial cells relaxation, but also by reducing sodium reabsorption at both the proximal tubule and inner medullary collecting duct.11., 12. In NPR-A/cGMP/PKG pathway, Na⁺ uptake is reduced through inhibition of Na⁺ channels in the kidney. These natriuretic and diuretic actions mainly occur in the medullary collecting duct.¹¹ The pGC-A/cGMP/PDE2 pathway is also reported to be involved in natriuresis caused by NPs.¹³ Besides cGMP and PDE-2, ANP elevates the level of serum nitrite/nitrate concentration and NO production, which correlates with the aldosterone synthesis.¹⁴ Additionally, NPs is reported to promote natriuresis and diuresis by modulating tubuloglomerular feedback and water-channel protein aquaporin-2 (AQP2) translocation.¹³

At first, studies on candoxatrilat (one of the NEPI drugs) were carried out to clarify clinical effects of NEP inhibitors, while no significant clinical benefit was observed.⁸ NEPI caused inhibition of Ang II degradation and compensatory activation of RAS and sympathetic nervous, indicating that NEPI should combine with RAS inhibitor for clinical treatment. Vasopeptidase inhibitor (VPI), which combined ACEI and NEPI, was the first combined RAS/NEP inhibitor. It has been shown that omapatrilat, the most widely studied VPI, reduced in the risk of death and hospitalization in patients with chronic heart failure (HF).¹⁵ In diabetic spontaneously hypertensive rats, omapatrilat attenuated albuminuria more effectively than perindopril, and high dose of omapatrilat completely ameliorated the rise in albuminuria.¹⁶ However, the development of omapatrilat as a therapeutic agent was discontinued because of the high rate of angioedema, which was likely due to the increased bradykinin activity.^6 17

ARNI combines positive effects of both NEPI and ARB. NEPI increases concentration of Ang II via the inhibition of Ang II degradation caused by neprilysin, which can be effectively solved by adding ARB. ARB inhibits RAAS system, leading to a reduction of glomerular capillary pressure and effective filtration pressure. ANP relaxes afferent arteriole, so that combining NEPI and ARB contributes to maintaining glomerular filtration pressure and glomerular filtration rate.18., 19. On the other hand, the afferent renal arterioles dilation caused by ANP may lead to glomerular hypertension that could promote glomerulosclerosis, while combination with ARB could inhibit the RAAS-driven vasoconstriction of the efferent arterioles and avoid the increase of intraglomerular pressure.²⁰ Compared with NEPI/ACEI, another kind of drug that inhibits RAAS and neprilysin, ARNI has an advantage in safety because it affects bradykinin activity less and mitigates the risk of angioedema.

Sacubitril/valsartan, the first angiotensin receptor neprilysin inhibitor, combines an ARB (valsartan) and an NEPI prodrug (sacubitril, or AHU377) in a 1:1 M complex, and LBQ657 (sacubitrilat) is the active metabolite of sacubitril that plays the role of neprilysin inhibition.⁶ Sacubitril/valsartan has been approved by U.S. Food and Drug Administration for the treatment of class II–IV HF with reduced ejection fraction (HFrEF) in 2015.²¹ The 2020 CCS/CHFS Heart Failure Guidelines Update introduced new evidence for ARNI in patients with HF with preserved ejection fraction (HFpEF).²² Besides HF, sacubitril/valsartan has been verified to be safe and effective for the treatment of hypertension.23., 24., 25., 26.

There have been studies indicating that ARNI has favorable effects on diabetic complications. In rats with diabetic retinopathy, ARNI could reduce capillary loss, cell apoptosis and levels of inflammatory markers more strongly than ARB.²⁷ Malek et al. found that in streptozotocin (STZ)-induced diabetic rats with diabetic cardiomyopathy, combination of NEPI and ARB not only inhibits inflammatory, profibrotic and apoptotic cascades, but also normalizes posttranslational histone modifications and histone acetyltransferase expression.²⁸ Moreover, the exposure of valsartan following administration of LCZ696 was much higher than valsartan alone.¹⁰ DKD and other diabetic complications, especially microvascular complications, share common mechanisms, raising the hypothesis that ARNI may have application potential in DKD.

Several studies have shown that NPs, especially ANP and BNP, is involved in the pathogenesis of renal dysfunction in DKD. ANP maintains glomerular filtration rate (GFR) via vascular smooth muscle cell relaxation, improvement of renal arterial perfusion and reduction of mesangial cells contraction.¹³ Besides, ANP can attenuate oxidative stress by elevating the antioxidant activities. The study by Staffel et al. showed that the NP/GC-A signaling could prevent podocyte injury and albuminuria, likely by suppressing of Transient receptor potential canonical (TRPC) channels.¹² ANP attenuates the diabetes-induced elevation of the renal function parameters, including serum creatinine, serum cholesterol, blood urea nitrogen (BUN), proteinuria, absolute kidney weight and renal collagen content.¹⁴ In diabetes and its complications, long-term excess of BNP protects renal glomerulus from injury.²⁹ ANP increases GFR, likely due to reduction of afferent arteriolar resistance. Ortola et al. found that in rats with early diabetes, elevated endogenous ANP levels contribute to glomerular hyperfiltration.³⁰

Preclinical studies have confirmed that ARNI normalized alteration of NPs and RAAS in kidney disease. In addition to RAAS inhibition effects of ARB, ARNI may play a role in renoprotection and blood glucose regulation by several potential mechanisms of NEP inhibition (Fig. 1). NEP inhibition affects the production and metabolism of ANP, BNP, cGMP and ET-1 in kidney, possibly in an organ-specific manner.³¹ It was reported that sacubitril/valsartan decreased serum ANP and urinary ET-1 excretion, while significantly increasing urinary ANP and nitric oxide in CKD models. This decrease in serum ANP may be at least partly due to the increase of renal NPR-C mRNA expression, which might potentiate the impaired renal response to ANP and the reduced degradation of ANP in the kidney.³² Moreover, sacubitril/valsartan blocks Ang II signaling through the Ang II type 1 (AT1) receptor, causing the compensatory upregulation of plasma renin and Ang I.⁹ Thiorphan/telmisartan, another combination of NEPI and ARB, was observed to reduce ACE and Ang II levels and elevate in Ang-(1–7) levels in rats with DKD, which suggested a normalization of RAAS component levels, showing reduced neprilysin levels, increased cGMP levels and activation of NPR-A/cGMP/PKG pathway.³³

Fig. 1. Potential mechanism of effect of ARNI on renoprotection and blood glucose control. ARNI, Angiotensin receptor-neprilysin inhibitor; NEPI, neutral endopeptidase inhibitor; ARB, angiotensin II receptor blocker; Ang II, angiotensin II; RAAS, renin-angiotensin-aldosterone system; NPs, natriuretic peptides; NPR-A, natriuretic peptide receptor-A; cGMP, cyclic guanosine monophosphate; PKG, protein kinase G; PDE, phosphodiesterases; GLP-1, glucagon-like peptide-1; GFR, glomerular filtration rate.

Besides RAAS and NPs, endothelin system is also involved in ARNI treatment. Increased endothelin contributes to disassembly of the podocyte actin cytoskeleton, apoptosis, and podocyte depletion in podocytes of diabetic kidney.³⁴ NEP plays an important role in the formation and breakdown of endothelin.⁸ Heerspink et al. demonstrated that the selective endothelin A receptor antagonist atrasentan reduced the risk of renal events in patients with type 2 diabetes mellitus (T2DM) at high risk of developing ESRD, suggesting a critical role of endothelin inhibition in protecting renal function.³⁵ Therefore, further exploration on the role of ARNI in endothelin system will be essential to clarify its effects on DKD. A study on HFrEF patients showed that treatment with LCZ696 was associated with decreased endothelin-1 plasma levels.³⁶ Effects of ARNI on the endothelin system is likely to depend on the degree of neprilysin inhibition: high-dose subsequently raised BP and increased the renal sodium-hydrogen exchanger 3 (NHE3) expression caused by ET-1 elevation, while a 10-fold lower dose did not affect circulating or urinary ET-1 levels.³⁷ It was demonstrated that too much NEP inhibition with RAAS inhibition may cause a rise in BP, which may be due to increased ET-1 and NHE3 protein abundance, and upregulated constrictor endothelin type B (ETB) receptors.³¹ Renal ET-1 elevation and the resulting NHE3 protein elevation may lead to a series of deleterious effects on kidney, including oxidative stress, inflammation, nephrin loss in podocytes and podocyte injury.31., 34. However, Roksnoer et al. reported that ARNI induce up-regulated ETB receptor-dependent vasodilation in renal medulla.³⁷ Thus, comprehensive assessment of the roles of ARNI in NPs, RAAS and endothelin system is needed, and a discussion of their interactions and impacts on processes of DKD is also necessary.

It has been reported that inflammation and oxidative stress could be attenuated by ARNI. In STZ induced diabetic rats, sacubitril/valsartan contributed to reduction of inflammatory markers TNF-α, IL-1β and IL-6, with an augmentation of anti-inflammatory IL-10 both in serum and in kidney tissue.³⁸ In rats with diabetes and hypertension, sacubitril/valsartan decreased gene expression of the macrophage marker Cd68 in renal cortex.³⁹ Oxidative stress is caused by an imbalance between the oxidation and antioxidant. Sacubitril/valsartan increased anti-oxidant enzymatic levels including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), endothelial nitric oxide synthase (eNOs) and glutathione-S-transferase (GST), while decreasing levels of oxidative stress markers including thiobarbituric acid reaction substances (TBARs), Gp91phox, nitrotyrosine and NADPH oxidase 4 (NOX4) in renal tissue, and these effects were more significant than valsartan alone.38., 40., 41. Transcription factors such as the nuclear factor kappa B (NF-κB) are activated in conditions of oxidative stress and inflammation. In different animal models of kidney disease, NF-κB pathway was attenuated by combination of NEPI and ARB, indicated by decreased NF-κB-p65 and downstream molecules MCP-1 and COX-2, with an increase of inhibitory subunit of NF kappa B alpha (IκBα) expressions.33., 38., 40. Additionally, sacubitril/valsartan could augment nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) and levels of its downstream gene products in renal tissues of CKD, while protein abundance of its repressor Keap1 was decreased, suggesting an activation of Nrf-2 pathway, which could inhibit oxidative stress and inflammation.⁴⁰

Proteinuria, notably albuminuria, is one of the main changes of DKD. To the best of our knowledge, it is controversial whether ARNI can reduce proteinuria and albuminuria more strongly than ARB. In TGR(mREN2)27 rats with diabetic nephropathy (DN), ARNI fully normalized both proteinuria and albuminuria while ARB only reduced albuminuria, and these changes are blood pressure-independent.³⁷ Compared with valsartan, sacubitril/valsartan in equivalent dose attenuated proteinuria more significantly in rat models with T2DM, CKD or early stage DN.32., 41., 42., 43. Similarly, it was found that sacubitril/valsartan reduces albuminuria more strongly than valsartan in hypertensive rats with diabetes.³⁹ Low dose sacubitril/valsartan was also reported to reduce proteinuria in Male Dahl SS rats with renal disease and salt-sensitive hypertension.⁴⁴ Habibi et al. emphasized its effect on reducing non-albuminuric proteinuria in rats with early DN, reflecting an amelioration of filtration barrier injury and glomerular dysfunction.⁴¹ Another study on CKD, by contrast, observed no significant differences between ARB and ARNI in reducing 24 h urine protein in the 5/6 nephrectomy rat models.⁴⁰ This discrepancy may be due to the difference in animal models, doses of drugs, methods of these experiments, and the limited sample size. Moreover, whether ARNI increase glomerular filtration and then aggravate proteinuria is also to be explored. Thus, further research with larger sample is required.

Podocyte injury and apoptosis lead to the destruction of the glomerular filtration membrane, correlating with proteinuria in DN.⁴⁵ Sacubitril/valsartan not only normalized the flatten of podocyte foot processes, but also prevented nephrin and podocin loss more effectively than valsartan.41., 43. The protection role of sacubitril/valsartan against podocyte injury may be attributed to reduction of glomerular TRPC6 channels and increase of renal ANP, inducing reduction of the nuclear factor of activated T cell (NFATc)-dependent regulator of calcineurin 1 (Rcan1), namely suppressing the TRPC6-NFATc-Rcan1 pathway, whereas valsartan alone doesn’t affect Rcan1.39., 46.

It was demonstrated that ARNI ameliorates histopathologic changes, including glomerulosclerosis, tubular injury and tubulointerstitial fibrosis, more significantly than ARB.32., 33., 37., 40., 41., 42. In renal tissues of diabetic rats, sacubitril/valsartan ameliorated diffused interstitial inflammation, vacuolarization of renal cells, necrosis, tubular degeneration and fragmented glomeruli, suggesting a reduction of renal injury characterized by necrosis and inflammatory cells infiltration.³⁸ Combination of NEPI and ARB also reduced c-PARP and c-Caspase, indicating reduction of renal cell apoptosis, and it was superior than ARB alone.³³ Additionally, significant inhibition of the increase in the mean glomerular volume was observed in histological sections of diabetic rats treated by sacubitril/valsartan.³⁸ ARNI alleviates increase of TGF-β, PAI-1 and α-SM actin, indicating the inhibition on renal fibrosis.33., 40. The various markers of renal fibrosis might be affected differentially by the severity of the damage and the underlying mechanism, so that a more comprehensive study on fibrosis is needed.⁴⁴

ARNI may have effects of ameliorating renal dysfunction. Sacubitril/valsartan attenuated increase of serum creatinine and BUN more significantly than valsartan in CKD.40., 47. Another study showed greater effect of sacubitril/valsartan in maintenance of renal function than valsartan alone.⁴² In diabetic TGR (mREN2)27 rats, sacubitril/valsartan preserved effective renal plasma flow and GFR, showing higher creatinine clearances compared with those treated with valsartan alone, whereas no change in filtration fraction was observed.³⁹ There are also researches showing that sacubitril/valsartan did not suppress the elevation in serum creatinine.³² Nevertheless, it was reported that the ratio of creatinine to cystatin c, which is associated with decreased kidney function indicating derangement in GFR, was significantly lower in rats with early DN when treated with sacubitril/valsartan, and the increase in cystatin c suggested the reversed hyperfiltration.⁴¹

There is evidence that ARNI is likely to affect insulin resistance and blood glucose regulation via modulation of NPs. In T2DM, elevated neprilysin activity was observed in tissues affected by vascular disease.²⁰ Insulin sensitivity and insulin secretion were hypothesized to relate to NP secretion, NP receptor expression and the post-receptor signaling.⁴⁸ Low ANP and BNP may aggravate insulin resistance by activation of renin-angiotensin system, which could downregulate the natriuretic peptide system, blunt the natriuretic action of ANP, and activate the sympathetic nervous system.29., 49. Moreover, ANP and BNP could promote adiponectin synthesis by adipocytes and subsequently improve glucose metabolism and insulin resistance by the AMPK signaling pathway.⁵⁰ Singh et al. reported that treatment with intermediate-dose ANP and high-dose ANP significantly attenuated the elevated serum glucose concentration and urine output when compared with the diabetic control group.¹⁴ Some other substrates of neprilysin, including incretin glucagon-like peptide-1 (GLP-1), ET-1, Angiotensin-(1–7) and bradykinin, are also proved to regulate glucose and metabolism.50., 51. Inhibition of neprilysin improves insulin sensitivity, β cell function and glucose tolerance in diabetic individuals by increasing levels of NPs, GLP-1 and bradykinin.50., 51., 52. For example, it was reported that the elevated active GLP-1 levels, reduced plasma DPP-4 activity and improved β cell function were correlated with improved blood glucose in high-fat-fed NEP-deficient mice.⁵⁰ However, neprilysin inhibition may attenuate the ability of angiotensin-(1–7) to promote insulin secretion via its cleavage to angiotensin-(1–2).⁵² Neprilysin inhibition may also increase levels of substrates that can affect β cell survival such as islet amyloid polypeptide and IL-1β, which promote amyloid deposition and β cell apoptosis.⁵² Therefore, NEPI should be combined with RAS blocker to avoid the potential deleterious effects of elevated Ang II levels. The safety and effectiveness of neprilysin inhibition should be further investigated in patients with diabetes.

Whether ARNI influence blood glucose and glycometabolism in DKD is still not clear. Sacubitril/valsartan tended to lower nonfasting blood glucose levels more strongly than valsartan, while in another study fasting glucose or HbA1c wasn’t reduced by sacubitril/valsartan. 39., 41. Some studies reported that hyperglycemia and metabolic disorder were substantially ameliorated by combination therapy of NEPI and ARB in diabetic rats, whereas others observed unaltered blood glucose after treatment. 28., 33., 37. Myakala et al. observed that sacubitril/valsartan but not valsartan decreased the blood glucose level in KKAy diabetic mice.⁴³ Besides, it was reported that sacubitril/valsartan was more effective in lowering plasma total cholesterol than valsartan.⁴¹ In STZ-induced hyperglycemic rats, there was a trend towards less cholesterol, triglycerides and LDL-C levels in the sacubitril/valsartan group compared to the valsartan group.⁵³ Progression of DKD is a long-term process and these experiments may be limited by brief duration, so that long-term studies are needed to find out the effect of ARNI on glycometabolism and lipometabolism.

To the best of our knowledge, there are no clinical trials that specifically focus on the action of ARNI on DKD. According to analysis of the Prospective Comparison of ARNI with ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure (PARADIGM-HF) trial, which enrolled 8399 patients with chronic HFrEF (including 3784 with diabetes), sacubitril/valsartan lowered the decline in estimated glomerular filtration rate (eGFR), and this favorable effect in patients with diabetes was twice as large as in those without diabetes, mainly due to the increase of cGMP.54., 55. The renal composite outcome and cardiovascular outcome occurred significantly less frequently in patients assigned to sacubitril/valsartan, despite a modest increase in the urine albumin creatine ratio (UACR) compared with enalapril.⁵⁵ A retrospective cohort study including 66 consecutive CKD patients with systolic dysfunction showed that sacubitril/valsartan significantly improved eGFR () of CKD stage 3b patients, and notably, had beneficial effect in older patients with a higher percentage of diabetes.⁵⁶ Since a greater reduction of systolic BP than valsartan and amelioration of HF were observed in the researches, it is important to identify whether the renoprotection effect of ARNI is independent of the drug’s positive effect on hemodynamics and HF.51., 57. Nevertheless, a series of studies on HF patients treated with sacubitril/valsartan showed a blood pressure-independent alleviation of eGFR decline, along with a reduction in composite renal outcome and the occurrence of ESRD when compared with valsartan.58., 59., 60. Some studies on HF patients showed an elevation of proteinuria.¹⁷ The increase in proteinuria and UACR mentioned above may be associated with a relaxation of contractile mesangial cells in the space between capillary endothelium and podocytes that contribute to the expansion of capillary surface area available for filtration, which may not result in a progressive loss of glomerular function.¹⁷ Increased intraglomerular pressure produced by natriuretic peptides and possibly attenuated tubular albumin reabsorption caused by cGMP were also involved.54., 60., 61. To evaluate renal effects caused by sacubitril/valsartan, there is a need to have more research on patients without HF.

The United Kingdom Heart and Renal Protection (UK HARP)-III trial, involving 414 patients with GFR 20–60 mL/min/1.73 m², showed that compared to irbesartan, sacubitril/valsartan had no additional effect on kidney function or albuminuria but more significantly reduced BP and biomarkers of cardiovascular risk (NT-proBNP and troponin I).⁶² The differences in results of these trials may be attributed to different determinants of kidney disease progression in a proteinuric CKD population compared to HF populations.⁶² The concomitant treatment with diuretics could also become an influencing factor.⁶³ A meta-analysis involving 3460 individuals with HF and CKD found that compared with ACEI or ARB, patients treated with ARNI showed significant increase in eGFR and decrease in systolic blood pressure (SBP), diastolic blood pressure (DBP) and NT-proBNP, but there were no difference in UACR.⁶⁴ However, Ito et al. observed a significant reduction in UACR, which was greater in patients with macroalbuminuria than in patients with normoalbuminuria or microalbuminuria.⁵⁷ These inconsistent results may be associated with characteristics of participants, dosage of drugs, durations, choice of comparison objects, and degree of renal dysfunction. To determine whether ARNI is effective for small, long-term changes in GFR, study of larger scale and longer follow-up in a patient population at high risk of CKD progression, with BP similarly managed and balanced across the study arms, is in need.⁶⁵

Some clinical studies considered sacubitril/valsartan’s effect on glucose metabolism. Compared with enalapril, HbA1c values were decreased with sacubitril/valsartan in HF patients with diabetes⁶⁶, but the difference in glycemic control appeared to be very small.⁵⁴ Vodovar et al. observed consistently increased plasma GLP-1 concentration after a treatment with sacubitril/valsartan for 3 months, regardless of clinical characteristics or antidiabetic treatment, which explained the antihyperglycemic effect of the drug.⁶⁷ M. Packer also found that the improvement in glycemic control may be caused by activation of endogenous GLP-1.⁶⁸ Furthermore, peripheral insulin sensitivity was improved by sacubitril/valsartan compared with those treated by amlodipine in obese patients with elevated BP.⁵¹

Safety and adverse effects of ARNI should be taken into consideration when evaluating its application potential in DKD. Reduction of renal function secondary to RAAS inhibition should be taken into consideration.⁶⁹ Treatment with ARB usually causes renal dysfunction accompanied effective reduction in BP, but in clinical studies LCZ696 only led to small and non-progressive increases in serum creatinine concentration, and this risk tended to be lower than valsartan alone.57., 60. ARNI causes NEP inhibition, which may impair amyloid β- peptide (Aβ) degradation and clearance, increasing the risk of Aβ-related disorders, so that treatment with ARNI may increase risk of cognitive dysfunction, age-related macular degeneration (AMD), Alzheimer’s disease (AD), and cerebral amyloid angiopathy (CAA).70., 71. Diabetic patients are already at higher risk of Alzheimer’s disease, so neuronal side effects should be attached great importance.²¹ Since AD progression is a long process, the potential impact of ARNI on the memory and cognitive function should be assessed in larger studies with longer periods.⁷¹ Side effects of acute ARNI treatment, such as hyperkalemia and hypotension, have also been reported.⁷⁰ Sacubitrilat is excreted through the kidney, so the accumulation of sacubitrilat due to kidney dysfunction may cause high concentration of sacubitrilat that leads to hypotension.⁶⁴ Some clinical studies showed a higher frequency of hypotensive events caused by ARNI compared with ARB.58., 64., 72. There are also case reports indicating that sacubitril/valsartan probably increase the risk of hypoglycemia in patients on concomitant insulin therapy, particularly in those with type 1 diabetes mellitus.73., 74. Since insulin is widely used in advanced DKD, it worth exploring the generality of hypoglycemia associated with ARNI.

Before applying ARNI to clinical treatment of DKD, an important problem to be solved is the absence of clinical studies that specifically focus on DKD patients. Present researches indicated that for DKD patients, ARNI is likely to be promising for reducing cardiovascular risk. However, most clinical studies on ARNI concentrate on patients with HF. The effects on DKD, particularly renal effects, are still to be worked out. Besides, all those clinical studies had a relatively short duration and excluded patients with severe kidney disease (eGFR <30 mL/min/1.73 m²). Therefore, there need large, comprehensive clinical trials focusing on DKD, which need to involve DKD patients of various stages, both with and without proteinuria. In addition, some researches on cardiovascular disease showed dose-dependence of ARNI.24., 26. Considering different dosage of drugs in different studies, whether dosage influence effect of the drug should be determined.

ARNI is hypothesized to improve insulin resistance and blood glucose control via augmentation of several substrates like NPs, GLP-1 and ET-1. It is worth exploring the role ARNI plays in metabolism, notably in glycometabolism. Furthermore, whether combining ARNI with new classes of antihyperglycemic drugs that slow progression of DKD (including sodium glucose cotransporter 2 (SGLT2) inhibitors, GLP1 receptor agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors) could provide additional renoprotection is to be determined.41., 67. For example, since GLP-1 is also a substrate of DPP-4, combining neprilysin inhibitor with DPP-4 inhibitor may help improve glucose metabolism.⁵²

ARNI exerts its influence through actions on NPs and RAAS. Compared with ARB, it additionally affects NPs via inhibiting degradation of NPs and other substrates such as ET-1. In accordance with previous studies, ARNI could manifest its renoprotective effects by improving natriuresis, ameliorating inflammation, oxidative stress and renal dysfunction, and slowing down glomerulosclerosis and tubulointerstitial injury of kidney. However, whether ARNI have a stronger beneficial effect than ARB on proteinuria (especially microalbuminuria) and blood glucose control, which play a vital role in progression DKD, is still unclear because of the lack of evidence. It is reported that in diabetic patients, ANP may induce microalbuminuria because of the augmented glomerular permeability, and endogenous BNP at a high physiological concentration might also induce hyperfiltration in the microalbuminuric stage, which may be potentiated by neprilysin inhibition.11., 75., 76. Thus, further research on the optimal stage for ARNI treatment in DKD is necessary. Moreover, further research on epigenetic mechanisms involved in the action of ARNI on DKD, such as histone acetylation and phosphorylation, is necessary. Histone acetylation plays an important role in diabetic complications: hyperglycaemia and its underlying genetic predisposition activate pathological cascades such as NF-κB and TGF-β signaling, which is correlated with increased histone acetylation, augmented inflammatory, fibrotic, and apoptotic gene transcription. Malek et al. reported that telmisartan and thiorphan combination therapy normalized histone acetylation (H2AK5Ac, H2BK5Ac, H3K9Ac, and H4K8Ac) and HATs (PACF and Ac-CBP) levels in rats with diabetic cardiomyopathy.²⁸ Therefore, although there are a number of researches on ARNI’s mechanism of action, more molecular mechanism is to be explored.