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Our previous work supports a major role for angiotensin-converting enzyme (ACE)-independent intrarenal angiotensin (ANG) II formation on microvascular function in type II diabetes. We tested the hypothesis that there is a switch from renal vascular ACE-dependent to chymase-dependent ANGII formation in diabetes. The in vitro juxtamedullary afferent arteriole (AA) contractile responses to the intrarenal conversion of the ACE-specific, chymase-resistant ANGI peptide ([Pro10]ANGI) to ANGII were significantly reduced in kidneys of diabetic (db/db) compared to control (db/m) mice. AA responses to the intrarenal conversion of the chymase-specific, ACE-resistant ANGI peptide ([Pro11, DAla12]ANGI) to ANGII were significantly enhanced in kidneys of diabetic compared to control mice. AA diameters were significantly reduced by 9 ± 2, 15 ± 3 and 24 ± 3% of baseline in diabetic kidneys in response to 10, 100, and 1000 nmol/L [Pro11, DAla12]ANGI, respectively and the responses were significantly attenuated by angiotensin type 1 (AT1) receptor or chymase-specific (JNJ-18054478) inhibition. [Pro11, DAla12]ANGI did not produce a significant AA vasoconstriction in control kidneys. Chymase inhibition significantly attenuated ANGI-induced AA vasoconstriction in diabetic, but not control kidneys. Renal vascular mMCP-4 (mouse mast cell protease-4; chymase)/β-actin mRNA expression was significantly augmented by 5.1 ± 1.4 fold; while ACE/β-actin mRNA expression was significantly attenuated by 0.42 ± 0.08 fold in diabetic compared to control tissues. In summary, intrarenal formation of ANGII occurs primarily via ACE in the control, but via chymase in the diabetic vasculature. In conclusion, chymase-dependent mechanisms may contribute the progression of diabetic kidney disease.
Classically, angiotensin converting enzyme (ACE) is considered the major pathway for angiotensin (ANG) II formation. ACE-independent enzymatic pathways include serine proteases, tonin, cathepsin G, trypsin, and kallikrein.1 Evidence is mounting for an important role of chymase-dependent ANGII formation in human tissues:2,3 heart4, vasculature5, and kidney.6,7 Chymases are serine proteases that have chymotrypsin-like cleavage properties for the conversion of ANGI to ANGII at a rate 20 times greater than ACE.8,9 Human chymase has been identified as an efficient ANG converting enzyme, selectively hydrolyzing ANGI at Phe8 to generate bioactive ANGII.8 Mouse mast cell protease-4 (mMCP-4) is the functional homologue to human chymase.10 ACE and mMCP-4 cleave ANGI at identical sites to generate ANGII.10 Chymase inhibitors have emerged as potential therapeutic agents for treating various inflammatory, allergic, cardiovascular, and renal disorders.11 Thus, ACE inhibitor monotherapy may allow for the continued generation of ANGII via ACE-independent pathways. The current studies were performed to investigate alternative intrarenal angiotensin ANGII forming pathways that may be enhanced in the diabetic kidney with the overall goal of identifying new targets for treatment of diabetic kidney disease.
Recently, there has been growing interest in the role of chymase in various renal pathophysiologic states. Increased chymase expression has been observed in humans with diabetic nephropathy (DN),12,13 IgA nephropathy,14,15 autosomal dominant polycystic kidney disease,16 and hypertensive nephropathy17 suggesting a central role of chymase in many forms of kidney disease in humans. Interestingly, in patients with DN, the number of renal chymase-positive mast cells is positively correlated with the severity of DN,18 suggesting that degranulation of mast cells promotes renal inflammation and fibrosis. Increased chymase expression in mesangial and vascular smooth muscle cells in human DN13 indicates that chymase is important for progression of the disease and suggests that pharmacological blockade of chymase may provide beneficial effects.
The current studies were performed in the db/db mouse (BKS.Cg-Dock7m +/+ Leprdb/J) which is an animal model of type II diabetes exhibiting features of human diabetic nephropathy 19–21. Our previous study22, as well as those of the research group of Batlle et al.23–25 have demonstrated a significant decrease in ACE protein expression and activity in the diabetic db/db compared to the control db/m kidney. Plasma and kidney ANGII levels were similar in db/db and db/m mice suggesting an augmentation of alternative ANGII forming enzymatic pathways in the db/db mice.22 In spite of reduced ACE activity, afferent arterioles (AA) of control and diabetic kidneys responded with a similar magnitude of vasoconstriction to the intrarenal conversion of bath applied ANGI to ANGII.22 In kidneys of control mice, AA vasoconstrictor responses were mediated by ACE-dependent conversion of ANGI to ANGII; in contrast, AA vasoconstrictor responses in diabetic mice were mediated by serine protease-dependent conversion of ANGI to ANGII.22 The rationale for conducting the current studies was to provide direct evidence for chymase as the specific serine protease responsible for ANGII formation in diabetic renal vascular disease.
We tested the hypothesis that there is a switch from renal ACE-dependent to chymase-dependent ANGII formation in diabetic vascular disease. Renal AA vascular responses to the intrarenal enzymatic conversion of ACE-specific and chymase-specific ANGI analogs to ANGII were determined in the absence or presence of a chymase inhibitor in order to determine the specific serine protease-dependent enzyme responsible for the intrarenal conversion of ANGI to ANGII in normal and diabetic kidneys. Quantification of vascular ACE and chymase mRNA expression was performed to provide support for the determination of the predominant intrarenal ANGII forming enzymes on vascular function in the type II diabetic kidney.
An extended Methods section is available in the online-only Data Supplement.
Experiments were performed in adult male control db/m (n=38, Dock7m Leprdb) and diabetic db/db (n=38, BKS.Cg-Dock7m +/+ Leprdb/J; #000642) mouse littermates.
Experiments were conducted using the mouse in vitro blood perfused juxtamedullary nephron technique as we have previously reported in detail.22,26,27 AA diameters were measured during the following protocols:
AA luminal diameters were measured manually and continuously using a digital image-shearing monitor22,26,27. One-way repeated-measures ANOVA, two-way ANOVA followed by Dunnett’s or Bonferroni’s test, paired t-test, or unpaired t-test were used as appropriate. p ≤ 0.05 was considered statistically significant. Valuesare means ± SEM.
Body weight was significantly higher in 18-wk-old adult male diabetic (48.7 ± 0.07 g; n=38) compared to control (32.2 ± 0.04 g; n=38) mouse littermates. Baseline AA diameters of kidneys from diabetic mice (14.6 ± 0.5 μm; n=31) were significantly larger than AAs from control (13.0 ± 0.5 μm; n=27) mice.
Figure 1 demonstrates the AA vasoconstriction to the ACE-specific, chymase-resistant ANGI peptide ([Pro10]ANGI; 0 - 1000 nmol/L). Figure 1A illustrates the average AA responses plotted in microns and Figure 1B illustrates the average AA responses plotted as the delta % of baseline to [Pro10]ANGI in kidneys from control and diabetic mice. Significant AA vasoconstriction to 10, 100, 1000 nmol/L [Pro10]ANGI was observed in kidneys of control (−12 ± 2, −18 ± 5, −16 ± 4%, n=7), but not diabetic (n=7) mice. [Pro10]ANGI produced a significantly greater response in AAs from control compared to diabetic mice.
Figure 2A demonstrates the AA vasoconstriction plotted as the delta % of baseline to the chymase-specific, ACE-resistant ANGI peptide ([Pro11,D-Ala12]ANGI; 0 - 1000 nmol/L). Significant AA vasoconstriction to 10, 100, 1000 nmol/L [Pro11,D-Ala12]ANGI was observed in kidneys of diabetic (−9 ± 2, −15 ± 3, −24 ± 3%, n=8), but not control (n=9) mice. [Pro11,D-Ala12]ANGI produced a significantly greater constrictor response in AAs from diabetic compared to control mice.
Pretreatment with an AT1 receptor blocker (100 μmol/L candesartan) did not produce a change from the baseline diameter in either group. The AA vasoconstrictor responses to [Pro11,D-Ala12]ANGI were significantly attenuated by AT1 receptor blockade in both groups (Figure 2B).
AA diameters of diabetic kidneys did not change in response to [Pro11,D-Ala12]ANGI (1000 nmol/L: 4 ± 2%, n=3) in the presence of chymase inhibition (data not shown). AA vasoconstrictor responses to [Pro11,D-Ala12]ANGI were significantly attenuated by chymase blockade compared to [Pro11,D-Ala12]ANGI alone in kidneys of diabetic mice (data not shown).
Figure 3 illustrates the average AA responses to ANGI in the presence of chymase inhibition plotted in microns (Figure 3A) and delta % of baseline (Figure 3B) in kidneys from control and diabetic mice. In the presence of chymase inhibition, significant AA vasoconstriction to 10, 100, 1000 nmol/L ANGI (−12 ± 2, −19 ± 4, −30 ± 3%, n=6) was observed in kidneys of control mice. However, chymase inhibition significantly attenuated the AA vasoconstriction to 10, 100, 1000 nmol/L ANGI (−7 ± 4, −9 ± 3, −11 ± 4%, n=6) in kidneys of diabetic mice. In the presence of chymase inhibition, ANGI produced a significantly greater response in AAs from control compared to diabetic mice.
At the conclusion of protocols 1 and 2, AA contractile responsiveness to ANGI remained intact in control (−12 ± 2%, n=11) and diabetic (−11 ± 1%, n=10) kidneys (Figure S1A). In the continued presence of AT1 receptor blockade, norepinephrine (NE) produced a rapid and significant vasoconstriction in AAs of control and diabetic kidneys (−46 ± 4, −35 ± 6%; n=5, 7 respectively) (Figure S1B). AA contractile responsiveness to ANGII remained intact in control (−24 ± 2%, n=10) and diabetic (−18 ± 2%, n=9) kidneys at the conclusion of protocols 2 and 5 (Figure S1C). The magnitude of the AA vasoconstrictions produced by ANGI, NE, and ANGII were not significantly different between kidneys of control and diabetic mice (Figure S1).
The renal vascular tissue isolation procedure yielded a significant 14.2 ± 0.5 fold enrichment of alpha-smooth muscle actin (α-SMA) protein expression compared to renal cortical tissues from control mice (Figure 4A, B). Renal cortical tissue ACE protein expression was 16.5 ± 0.7 fold enriched compared to renal vascular tissues from control mice (Figure 4C, D). Renal vascular tissues isolated from diabetic mice expressed significantly augmented mMCP-4 (chymase) mRNA expression of 5.1±1.4 fold (Figure 4E) and attenuated ACE mRNA expression of 0.42±0.08 fold (Figure 4F) compared to control mice.
Current drug therapies for the treatment of diabetic renal disease may slow the progression of the damage, but do not stop disease progression or restore normal kidney function for these patients. Despite the widespread use of inhibitors of the renin-angiotensin system and glucose-lowering medications, the incidence of diabetes-related end-stage renal disease continues to rise steadily indicating the need for the continued search for the mechanisms involved in the development and progression of DN.
Chymase has received considerable attention as an ACE-independent means to produce ANGII. It has been shown that the formation of ANGII from ANGI in coronary arteries33 and kidney microvessels22,34,35 is dependent on both ACE (captopril inhibitable) and ACE-independent (chymostatin inhibitable) pathways. Our published work22 indicates that AA vasoconstriction produced by the intrarenal conversion of ANGI to ANGII is of similar magnitude in diabetic and control kidneys. Inhibition of microvascular responses to intrarenal conversion of ANGI to ANGII by captopril indicated that ACE is the predominant pathway for ANGII formation in the normal mouse kidney. In contrast, in diabetic kidneys AA vasoconstriction produced by the intrarenal conversion of ANGI to ANGII was not attenuated by ACE inhibition, but was significantly attenuated by serine protease inhibition. Our earlier studies utilized ACE and non-specific serine protease inhibitors as a means to determine the major enzymatic pathways for intrarenal conversion of exogenously applied ANGI. Sequences for the synthesis of ANGI peptide analogs were obtained from the work of Husain and colleagues.28,36 The ANGI analogs contain specific amino acid sequences that make them substrate-specific for either ACE or chymase enzymatic activity which allowed for the quantitative assessment of renal microvascular functional responses to the intrarenal conversion of these analogs to ANGII. ANGII synthesized via ACE and/or chymase-dependent pathways within the renal endothelium, microvasculature, glomerulus, tubules, and/or interstitium may act in an autocrine/paracrine manner via binding to plasma membrane AT1 receptors located on AA vascular smooth muscle cells.
The efficiency of human heart chymase for peptides with proline in the P2′ position of ANGI is decreased by 95% compared to ANGI,37 indicating that this analog is chymase-resistance. Furthermore, the positive inotropic response of [Pro10]ANGI on hamster papillary muscle was completely suppressed by captopril pretreatment,36 indicating that the analog is specific for ACE. AA vasoconstriction to the ACE-specific, chymase-resistant [Pro10]ANGI was significantly greater in control than diabetic mice confirming significantly reduced ACE activity in the diabetic kidney. The significant AA responses to the ACE-specific, chymase-resistant ANGI analog confirm our previous work22 demonstrating that ACE is the predominant ANGII forming enzyme in the normal kidney.
It is well known that peptides with a proline in the penultimate position prevent ACE from cleaving a dipeptide from the carboxy terminus. The addition of a carboxy-terminal D-Ala prevents carboxypeptidases from making the penultimate proline into a carboxy-terminal proline. Hoit et al.38 cleverly combined these strategies to synthesize an ANGI analog that is resistant to ACE and carboxypeptidases, [Pro11,D-Ala12]ANGI, and allows for the in vitro and in vivo quantification of chymase activity. Li et al.28 demonstrated a lack of vasoconstrictor responses to [Pro11, D-Ala12]ANGI in mesenteric arteries of mast cell deficient Kitw/Kitw-v mice, while mesenteric arteries of control mice produced a vasoconstriction similar in magnitude to an equimolar dose of ANGII. AA vasoconstriction to the chymase-specific, ACE-resistant [Pro11,D-Ala12]ANGI peptide is significantly greater in diabetic than control mice which allowed for the identification of chymase as the serine protease responsible for ANGI to ANGII conversion in diabetic kidneys. The maximal AA vasoconstriction to [Pro11,D-Ala12]ANGI in the diabetic kidney was of similar magnitude as we previously reported for ANGI,22 suggesting that for the duration of the experiment the analog is converted to ANGII as effectively as ANGI. Husain and colleagues demonstrated that the cardiovascular effects of [Pro11,D-Ala12]ANGI were not impacted by ACE inhibition in the conscious baboon38 and mouse28. Additionally, the control kidney has minimal chymase activity. A significantly greater chymase and reduced ACE mRNA expression was detected in the renal vasculature of diabetic compared to control mice. The renal vascular isolation technique yielded an enhanced α-SMA and diminished ACE protein expression compared to cortical tissues demonstrating a significant enrichment of vascular tissue and minimal tubular tissue in the protein and RNA extracts. These data are consistent with the microvascular physiological functional studies for intrarenal ANGII formation and support our hypothesis that there is a switch from ACE-dependent to chymase-dependent activity in the diabetic kidney.
Our previous work demonstrated a significant reduction in the density of renal cortical tubular ACE immunohistochemical staining and cortical ACE activity in diabetic compared to control mice22 which is consistent with the work of Ye et al.23 In further studies, Ye et al. 24 reported that the percentage of glomeruli with strong endothelial ACE staining was significantly greater in 8-wk old female db/db mice compared to control mice which conflicts with our functional data in 18-wk old male db/db mice. Of interest are the findings of Soler et al.39 in which the percent of renal vessels demonstrating strong endothelial ACE immunostaining was increased in streptozotocin-induced type I diabetic compared to control mice. It is not clear if the age, gender, or type of diabetic model influences renal arterial endothelial ACE protein expression.
The vasoconstrictor responses to conversion of [Pro11, D-Ala12] to ANGII in the diabetic kidney are due to AT1 receptor activation since the responses were blocked by ANG receptor blocker. In addition, the vasoconstrictor responses to conversion of [Pro11, D-Ala12] to ANGII are due to intrarenal chymase activity since these responses were blocked by the chymase inhibitor. These data implicate the importance of chymase as the primary route of formation of ANGII from ANGI in diabetic kidneys.
Most importantly, key data suggest that the AA vasoconstriction induced by intrarenal conversion of the endogenous form of ANGI to ANGII is significantly attenuated by a specific chymase inhibitor in kidneys of diabetic mice, but not in kidneys of control mice. The potent phosphinate chymase inhibitor, JNJ-18054478, complexes with mammalian chymases and exhibits a potency of approximately 0.07 μmol/L against human and macaque chymases and 5 μmol/L for guinea pig and hamster chymases.40 The 10μM dose of the chymase inhibitor, JNJ-18054478, produced a complete inhibition of the AA vasoconstriction produced by intrarenal conversion of the chymase-specific, ACE-resistant [Pro11,D-Ala12]ANGI to ANGII in the diabetic kidney providing strong support for the efficacy of this inhibitor in the mouse kidney. This is the first study to document the efficacy of the chymase inhibitor, JNJ-18054478, to block AngII formation in the mouse renal vasculature.
At the conclusion of the [Pro10]ANGI and [Pro11, D-Ala12]ANGI protocols, bath application of ANGI or ANGII produced a significant vasoconstriction in AAs of control and diabetic kidneys indicating that the intrarenal ANGI forming enzymatic machinery and vascular smooth muscle cell AT1 receptor-mediated contractile properties were intact. In the presence of ANG receptor blockade, NE produced a potent vasoconstriction in control and diabetic kidneys indicating that although the AAs did not respond to bath applied ANGI analog, vascular smooth muscle cell vasoconstriction was not diminished. The maintenance of renal microvascular vasoconstrictor potential of AAs from both diabetic and control kidneys provides support for the significant differences observed between AAs from diabetic and control kidneys in response to the enzyme-specific ANGI analogs.
The magnitude of the AA vasoconstriction of the diabetic kidney to 1 μmol/L [Pro11, D-Ala12]ANGI (−24 ± 3%) was very similar to the magnitude of the vasoconstriction of the control kidney in response to 1 μmol/L ANGI in the presence of chymase inhibition (−30 ± 3%). These data suggest that there is a similar magnitude of ANGII formation by chymase-dependent and ACE-dependent pathways in diabetic and control kidneys, respectively. Recent studies have shown that chymase inhibition protects against renal dysfunction in type I diabetic hamsters.41 In addition, chymase (mMCP-4) deficient mice exhibit lower proteinuria, blood creatinine and urea nitrogen levels, and less severe renal damage compared to wild-type mice indicating an aggravating role of renal chymase in glomerulonephritis disease progression.42
The most significant finding of the present study is the identification of chymase as the major ACE-independent pathway for the formation of ANGII in the type II diabetic leptin-receptor deficient mouse kidney. In the diabetic kidney, AA vasoconstriction to intrarenally formed ANGII from the substrate ANGI is blocked by inhibition of chymase activity. In contrast, intrarenally formed ANGII from the substrate ANGI produces a potent AA vasoconstriction in the presence of chymase inhibition in the control kidney. Our studies may provide a potential mechanism involved in the superior renoprotective effects of combining an ACE inhibitor with an AT1 receptor antagonist relative to ACE inhibitor therapy alone in patients with DN which has been reported in some clinical studies.43,44 In addition, the presence of this ACE-independent pathway for ANGII formation may explain the continued proteinuria in some patients on maximal ACE inhibitor therapy.45 However, the ONTARGET trial indicated that in patients with cardiovascular disease or diabetes, the combination of ANG receptor blocker and ACE inhibitor provided more adverse events without an increase in benefit compared to either monotherapy.46 We suggest that ACE inhibitor monotherapy may allow for the continued generation of ANGII via chymase-dependent pathways which contributes to fibrosis, proteinuria, and reduced renal function in diabetic patients.
Despite the first-line use of ACE inhibitors and ANG receptor blockers for the treatment of DN, there is still a large need to improve therapies for the prevention of DN and dramatically reduce the rates of disease progression for these patients. Our studies support a major role for chymase-dependent ANGII formation in the db/db renal vasculature and thus provide a novel translational approach to human disease. Chymase inhibition may provide substantial renal protection in diabetic patients. Physicians may treat with an ANG receptor blocker when diabetic renal disease patients are unresponsive to ACE inhibition. Treatment with an ANG receptor blocker may provide additional benefit due to the inhibition of ANGII produced by ACE-dependent and chymase-dependent pathways. Targeting chymase as a therapeutic target for chronic kidney disease patients with normal blood pressure may provide the advantage of reducing intrarenal chymase-dependent fibrosis, proteinuria, and vasoconstriction without causing systemic hypotension that can lead to further reductions in glomerular filtration rate and renal blood flow, which is often observed with treatment with ACE inhibitors or ANG receptor blockers.
These are the first studies to indicate a significant contribution of chymase to the intrarenal formation of ANGII on afferent arteriolar function in the diabetic kidney. Future studies will test the ability of chymase inhibitors, specifically JNJ-18054478, to attenuate the in vivo disease-related changes in diabetic renal function.
The authors’ gratefully acknowledge the technical assistance of Paul M. Berner. Dr. Anders Ljunggren (Astra Hassle, Gothenburg, Sweden) generously provided the AT1 receptor antagonist, candesartan.
Sources of Funding
This work was supported by 2R56DK62003, P20RR018766, American Heart Association Grant-In-Aid 2250875 (Harrison-Bernard), T35HL105350 (Ford), and the American Physiological Society Undergraduate Summer Research Fellowship (Xu).
Conflict of Interest: The authors have declared that no conflict of interest exists.