The combination of a neprilysin inhibitor (sacubitril) and angiotensin-II receptor blocker (valsartan) attenuates glomerular and tubular injury in the Zucker Obese rat

The impact of obesity and diabetes on kidney function and structure is well recognized due to increased incidences of diabetes-associated chronic kidney disease (CKD) and end stage renal disease (ESRD) [1‐3]. The progression of CKD is associated with persistent activation of the renin-angiotensin system (RAS), with consequent development of hyperfiltration, proteinuria, glomerular mesangial expansion, and tubulointerstitial fibrosis, resulting ultimately in decreased glomerular filtration rate (GFR) [3, 4]. Moreover, pre-diabetic individuals with impaired glucose tolerance and insulin resistance are at higher risk for developing CKD [5]. The standard of care for the management of kidney injury involves administration of angiotensin-converting enzyme inhibitors (ACEi) or angiotensin II (Ang-II) type 1 receptor (AT₁R) blockers (ARB) [6‐9]. However, in many subjects with obesity and diabetes, there is a relentless progression of CKD despite the use of these inhibitors, suggesting a critical need for developing additional therapeutic strategies beyond ARB and ACEi. In this regard, one of the attractive new targets for treating kidney injury involves neprilysin, an endopeptidase that is positively associated with cardiovascular (CV) morbidity and mortality in HF patients [10]. Neprilysin metabolizes several small vasoactive peptides, including atrial (ANP) and brain (BNP) natriuretic peptides, Ang-II, bradykinin and endothelin-1, among others [11]. Natriuretic peptides (NP) are well known to induce cGMP synthesis that promotes natriuresis, diuresis and vasodilation and have been shown to inhibit mesangial cell proliferation and kidney fibrosis [12‐15]. Earlier attempts to reduce the degradation of NPs utilizing an inhibitor of neprilysin alone (NEPi) fell short due to an increase in Ang-II levels that promote sodium retention, vasoconstriction and cardiac fibrosis [11]. Subsequently, NEPi were combined with ACEi; however their combination increased the risk of angioedema due to inhibition of bradykinin degradation, precluding its clinical utility [11].

The next iteration involved combination of NEPi with an ARB so that the latter suppresses the effects of increased Ang-II and the accompanying vasoconstrictor and aldosterone promoting effects. Subsequently, in July 2015, the FDA approved Entresto (LCZ696), a combination of the NEPi, sacubitril (sac), and the ARB, valsartan (val), for treatment of heart failure (New York Heart Association, class II–IV) with reduced (≤ 40%) ejection fraction (HFrEF). Analysis of the PARADIGM-HF (Prospective Comparison of ARNI with ACE inhibition to Determine Impact on Global Mortality and Morbidity in Heart Failure) trial, demonstrated that Entresto reduced hospitalization for HF and death from CV causes, compared to enalapril [16‐21].

Much recent interest has begun to focus on potential benefits of Entresto on kidney function. In this regard, a recent post hoc analysis of PARADIGM-HF reported that Entresto slowed the rate of decrease in estimated glomerular filtration rate (eGFR) and exhibited favorable effects on CV and kidney outcomes in HFrEF patients with or without CKD, compared to enalapril [22]. More recently, results of the UK HARP-III (United Kingdom Heart and Renal Protection-III) trial, designed to examine the effects of Entresto on kidney function and cardiac biomarkers in patients with moderate to severe CKD, reported comparable renoprotection with Entresto and irbesartan [23]. Another recent secondary analysis of the PARADIGM-HF trial investigating the course of renal disease in patients with type 2 diabetes mellitus (T2DM) indicated that Entresto significantly slowed the rate of decline in eGFR compared to enalapril and this positive result could not be explained by improvement in glycemia or the effect of the drug on the clinical course of HFrEF [24]. However, little is known regarding the underlying mechanisms of sac/val on glomerular and tubular injury, as well as renal microvascular dysfunction, in the setting of obesity and diabetes with early DN, conditions often associated with diastolic dysfunction or heart failure with preserved ejection fraction (HFpEF).

In this study, we examined the effects of sac/val in a model of early stage DN characterized by moderate glomerular and tubular injury [25]. We utilized the Zucker Obese (ZO) rat that displays hyperphagia-induced obesity, brought on by the absence of the leptin receptor, as well as, renal hyperfiltration, hypertension, dyslipidemia, oxidative stress and proteinuria [26‐29]. Additionally, the ZO rat is a well characterized model of cardiac diastolic dysfunction and aortic stiffness [30‐33]. We hypothesized that sac/val imparts greater kidney functional and structural protections compared to either val monotherapy or hydralazine, the latter being an antihypertensive medication (blood pressure control group) that does not specifically target the RAS. Indeed, supporting our hypothesis, sac/val more effectively suppressed both glomerular and tubular injury compared to val or hydralazine.

Sacubitril/valsartan (sac/val) was originally approved to treat HFrEF; however, a recent post hoc analysis from the PARADIGM-HF trial suggested significant improvements in GFR and CV outcomes with this drug combination, despite causing an increase in urine albumin-to-creatinine ratio [22]. Therefore, some recent pre-clinical studies have begun to explore the mechanisms underlying the renoprotective effects of this drug combination. In this regard, two recent studies demonstrated superior renoprotection with sac/val compared to val monotherapy in rat models of severe CKD due to subtotal nephrectomy [56, 57]. In the present study, we examined the effects of sac/val on glomerular, i.e., filtration barrier injury, as well as, tubular remodeling and kidney fibrosis, along with associated changes in biochemical parameters in ZO rats, a translationally relevant model of early stage DN. Our findings suggest that sac/val exerts similar effects on kidney fibrosis compared to val alone. However, the suppression of hyperfiltration and proteinuria due to glomerular and tubular injury appears to be well controlled in sac/val compared to val monotherapy.

We evaluated control of glycemia, BP, dyslipidemia and GFR by both sac/val and val. Not surprisingly, sac/val or val alone did not lead to significant reductions in fasting glucose or HbA1c; however, HbA1c was highest in ZOV (5.5 ± 0.5%) and lowest in ZOSV (4.4 ± 0.3; Table 1), suggesting that the addition of sacubitril to val may confer a more favorable metabolic response compared to val monotherapy in the diabetic ZO rat. Interestingly, recent clinical data demonstrated modest improvement in HbA1c levels with sac/val, but only after one (~ 0.26%) and three (~ 0.40%) years of follow up [58]. The brief duration of treatment in the present study and the fact that the principle mode of action of each drug in this combination is not known to directly regulate blood glucose, may explain why no significant improvement was observed in glycemia. Therefore, long-term studies of the efficacy of sac/val for treatment of DN are warranted. Moreover, each treatment exhibited differential effects on molecular and biochemical parameters, filtration barrier injury, GFR and glomerular and tubular remodeling, despite comparable lowering of MAP by all three treatments. This suggests that the renoprotection afforded by sac/val in this preclinical model may be independent of BP reduction.

Dyslipidemia is also known to contribute to nephropathy [59]. A previous study in male ZO rats treated with the ARB telmisartan, reported improvement in progression of DN and a tendency towards normalized plasma cholesterol and triglycerides [60]. Herein, we observed that sac/val was more effective in lowering plasma total cholesterol compared to val or hydralazine. Since hypercholesterolemia in diabetes is usually associated with increased oxidized LDL and renal injury [57, 61], it is possible that the improvement in plasma total cholesterol in ZOSV may have contributed, in part, to amelioration of kidney injury.

The observation that untreated ZO rats (ZOC) had lower plasma cystatin c indicates hyperfiltration, which is consistent with early stage DN in obese humans [3]. Our study shows a significant increase in cystatin c in sac/val treated rats indicating that the combination therapy reversed hyperfiltration. This initial reversal of hyperfiltration and reduction in GFR is consistent with inhibition of RAS activation [62], supporting the use of ARBs or ACEi in suppressing the eventual decline in GFR in diabetic patients. Recently, sodium glucose co-transporter 2 (SGLT2) inhibitors have also been shown to decrease GFR and exert renoprotection in patients with DN with baseline hyperfiltration [63, 64]. There is an apparent inconsistency between our preclinical study and results of the PARADIGM HF trial showing improvement in GFR that is likely a consequence of long treatment duration (44 months) [24]. Thus, a limitation of our study is that we only measured kidney function, i.e., GFR, during the early hyper filtrating stage of DN. We posit that a longer term treatment and follow up would likely result in protection from decline in GFR in the ZO rats treated with sac/val as observed in PARADIGM HF.

With respect to proteinuria, the magnitude of suppression of proteinuria by the combination therapy was slightly greater than val alone, suggesting that the addition of NEPi on top of an ARB elicits additional renoprotective properties. Similar to albuminuria, non-albuminuric proteinuria also reflects filtration barrier injury and glomerular dysfunction. It is also recognized as a sensitive marker of tubular injury in the setting of DN with minimal kidney injury [65]. In this study, the albuminuria was not markedly increased in ZOC, nor was it decreased in any of the treatment groups. Supporting these observations on proteinuria, the ultrastructural findings indicated healthier podocytes and their foot processes with sac/val than val alone, and this was associated with rescue of nephrin and podocin expression, suggesting that the combination therapy is more effective at improving glomerulopathy in relation to (non-albuminuric) proteinuria.

In addition to glomerulopathy, DN is characterized by tubulopathy [66]. A number of biomarkers have been validated to detect existing tubular injury or its reversal following treatment of acute kidney injury in both humans and preclinical models [67]. We observed increases in β-NAG and GTT in untreated ZO rats (ZOC) and none of the treatments affected their levels. Therefore, we examined additional sensitive biomarkers of early tubular injury. Out of eight injury biomarkers that we evaluated (Table 2), five were elevated in the untreated ZO rats with early stage DN. In this injury profile, clusterin and KIM-1 stand out as sensitive biomarkers, and their increased levels in urine indicate tubular necrosis. Of note, urinary levels of clusterin and KIM-1 have been shown to increase during early stages of DN [68‐71]. Importantly, we and others have shown their suppression following treatment with ARBs [69, 72]. Moreover, sac/val has recently been shown to suppress tubular injury markers, including KIM-1, in dogs with cardiorenal syndrome [73]. Supporting these observations, our data show that sac/val suppressed both clusterin and KIM-1, while val alone suppressed only clusterin (Table 2). On the other hand, hydralazine had no significant effect on any of the markers. Thus, our data demonstrating an increase in clinically relevant tubule injury markers in diabetic rats (ZOC) and their suppression with sac/val (ZOSV), further support the renoprotective effects of sac/val. Though we have not formally identified a specific injury profile or network linked to early stage DN, nor alteration of such a construct with therapeutic intervention, other studies have highlighted the role of metabolic dysregulation as an underlying cause for progression of kidney disease, including fibrosis. This topic has recently been reviewed emphasizing the significance of a metabolomics approach in identifying molecular mechanisms contributing to CKD [74]. Future studies on metabolomics are warranted when exploring renal outcomes with sac/val treatment.

Activation of the RAS and the accompanying increase in NADPH oxidase-mediated oxidative stress is implicated in progression of DN [25, 75]. We and others have reported that ARBs are renoprotective in rat genetic [72, 76] and nutritional models of nephropathy [77]. Kidney fibrosis, comprising both periarterial and interstitial fibrosis, is also a determinant of progression of DN. In this study sac/val and val, as well as hydralazine, reduced kidney fibrosis, and this inhibition occurred in parallel with suppression of oxidative stress, AT₁R expression and NOX2. These findings suggest that AT₁R antagonism and reduced oxidative stress, either by use of ARBs or hemodynamic effects, might have contributed to suppression of kidney fibrosis. In this regard, we have previously reported therapeutic suppression of kidney fibrosis and improvement in filtration barrier injury in association with a decrease in oxidative stress in ZO and Ren2 rats, as evidenced by an decrease in NADPH oxidase activity and 3-NTY accumulation [27, 72, 76].

In this study, compared to ZLC, 3-NTY expression was significantly elevated in ZOC, but was markedly suppressed in the three treated groups (ZOSV, ZOV and ZOH), with associated decreases in mesangial expansion and intertubular and periarteriolar fibrosis, suggesting the role for oxidative stress in kidney fibrosis. We previously proposed that, in the setting of metabolic syndrome/diabetes, elevated tissue Ang-II induces NOX-mediated oxidative stress and loss of nephrin and podocin, leading to podocyte effacement and loss of slit pores diaphragms, and that these protein deficiencies and accompanying ultrastructural abnormalities are prerequisite to proteinuria/albuminuria [26]. In this regard, RAS inhibitors, such as ARBs and ACE inhibitors, have been reported to ameliorate loss of nephrin and podocin in the diabetic kidney of humans and rodents [48, 78].

In this study, AT₁R, NOX2 and NOX4 gene expression and oxidative stress increased in untreated ZO rats (ZOC) and these effects occurred in concert with decreases in podocin and nephrin expression. Although AT₁R expression and oxidative stress decreased similarly in all treatment groups, the rescue of nephrin and podocin expression occurred only in rats treated with sac/val (ZOSV), compared to val (ZOV). It is also noteworthy that sac/val was more effective at suppressing NOX4 expression compared to val and hydralazine. However, RRI, some urinary tubular injury markers and ultrastructural features were not consistently associated with treatment-related suppression in oxidative stress and fibrosis. Therefore, the renoprotective effects of sac/val may only be due, in part, to reductions in glomerular and tubular nitroso-oxidative stress. These findings suggest that other factors/pathways contribute to the efficacy of combination therapy. In this regard, recent studies have shown a role for a cGMP dependent pathway in improving podocin expression and filtration barrier [79, 80]. Natriuretic peptides act mainly through increase in cGMP thereby possibly contributing to improved renoprotection by sacubitril on filtration barrier proteins compared to val monotherapy.

High BP has been shown to contribute to proteinuria. Given that hydralazine reduced MAP, but not proteinuria, compared to sac/val, suggests that BP reduction, although important, may not be the primary determinant contributing to improvements in proteinuria by this combination. Further, mesangial expansion occurred in ZOC and was significantly reduced in ZOSV, ZOV and ZOH. However, val monotherapy (ZOV) was more effective in preventing the expansion of mesangium compared to sac/val or hydralazine. As such, treatment-related protection from mesangial expansion appears to play a lesser role in preventing glomerular injury in this model of early stage DN.

The RRI may be useful in predicting subclinical CV and kidney vascular disease in diabetes [81]. A previous study by Bruno et al. [82] reported significantly elevated RRI in patients with T2DM (0.65 ± 0.06) compared to non-diabetic individuals (0.59 ± 0.05) and patients with essential hypertension (0.58 ± 0.05). Interestingly, increased RRI in the diabetic subjects was associated with elevated albuminuria. In fact, the utility of RRI as an indicator of kidney disease was recently validated in high fat diet fed mice [83]. We recently reported increased RRI in db/db mouse, a more severe model of DN, compared to ZO, and this was associated with aortic stiffness and a reduction in renal 3-NTY [34]. In the present study, we observed elevated RRI in ZOC compared to ZLC (0.67 ± 0.02 versus 0.53 ± 0.03; p < 0.05), however, sac/val and val alone did not significantly suppress RRI. In contrast, hydralazine increased RRI, compared to ZOC (0.86 ± 0.03 versus 0.67 ± 0.02; p < 0.05), and this was associated with greater proteinuria in the ZOH group (Table 2 and Fig. 2a), suggesting a possible context-dependent association for hydralazine in contrast to sac/val and val.

In summary, our study provides insights into the efficacy of sac/val combination therapy compared to val monotherapy in a preclinical model of early stage DN. Our data suggest that sac/val is slightly more renoprotective and the principal mechanism involves rescue from hyperfiltration and subsequent improvement in glomerular structure and function. However, future preclinical and clinical investigations are warranted to test whether sac/val affords renoprotection specifically in the setting of DN associated with diastolic dysfunction or HFpEF. In this regard, results of the ongoing PARAGON-HF trial (Prospective Comparison of ARNI with ARB Global Outcomes in HF with Preserved Ejection Fraction) should be forthcoming in the summer of 2019 and may offer insight into whether Entresto (sac/val) also affords renoprotection in individuals with DN associated with HFpEF. Since newer classes of antihyperglycemia drugs, including the sodium glucose cotransporter 2 (SGLT2) inhibitors, glucagon-like peptide 1 receptor (GLP1R) agonists and dipeptidyl peptidase 4 (DPP4) inhibitors, slow progression of DN [84], it remains to be determined whether combining Entresto with these newer classes of drugs could provide additional renoprotection.