- Email: [email protected]
Mast cell population in the development of diabetic nephropathy: Effects of renin angiotensin system inhibition
Biomedicine & Pharmacotherapy 107 (2018) 1115–1118
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Mast cell population in the development of diabetic nephropathy: Effects of renin angiotensin system inhibition
T
Richarlisson Borges de Moraisa,e, Victor Pereira do Couto Muniza, Emerson Nunes Costaa,f, Sebastião Rodrigues Ferreira Filhod, Karen Renata Nakamura Hirakib, ⁎ Luiz Borges Bispo-da-Silvac, Ana Paula Coelho Balbia, a
Laboratory of Physiology, Institute of Biomedical Sciences, Federal University of Uberlândia, ICBIM-UFU, Brazil Laboratory of Histology, Institute of Biomedical Sciences, Federal University of Uberlândia, ICBIM-UFU, Brazil c Laboratory of Pharmacology, Institute of Biomedical Sciences, Federal University of Uberlândia, ICBIM-UFU, Brazil d Medical Clinic Department, School of Medicine, Federal University of Uberlândia, FAMED-UFU, Brazil e Technical School of Heath, Federal University of Uberlândia, ESTES-UFU, Brazil f Hemodialysis Sector, Clinical Hospital, Federal University of Uberlândia, HC-UFU, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Diabetes Nephropathy Fibrosis Aliskiren Mast cells
Considering the importance of the renin-angiotensin system (RAS) in diabetic nephropathy (DN) and the link between mast cells (MC) and the RAS, this study evaluated the effects of RAS blockade on the MC cell population in the kidneys from rats with experimental diabetes. Wistar rats were divided into six groups: control nondiabetic (C); sham (S); diabetic (D); and D treated with enalapril (EN), losartan (LO), or aliskiren (AL). Ninety days after diabetes induction, glomerular filtration rate (GFR) and urinary albumin excretion (UAE) were determined. Kidneys were collected for MC counting. RAS blockers minimized changes in morphometrical parameters (EN), cortical collagen (LO, AL), GFR (AL) and UAE (EN, LO). An increased number of MC was observed in the kidneys from D animals. Only AL treatment prevented this increase. MC may be involved in some aspects of DN pathogenesis and the possible protective effects of AL on the kidneys might involve MC modulation.
1. Introduction
2. Materials and methods
Hyperglycemia stimulates renin release in renal tissue, resulting in high levels of angiotensin II (Ang II), an important mediator of diabetic nephropathy (DN) physiopathology [1]. Human and animal studies have used drugs capable of interfering with the renin-angiotensin system (RAS) in order to preserve renal function and decrease proteinuria [2]. In addition to the increased RAS activity in DN, renal mast cell (MC) accumulation is also a common feature of DN [3] and other kidney disorders, such as primary or secondary glomerulonephritis [4], progressive glomerular nephritis [5] and hypertensive nephropathy [6]. It has been suggested that mast cells are sources of renin, and they appear to be an important component of a local RAS [7]. Therefore, considering the role of RAS in DN development and the link between MC and RAS, this study evaluated the effects of RAS blockade on the MC population in the kidneys of rats with experimental DN, as well as on the structural and functional alterations of the kidney induced by DM.
2.1. Ethical considerations This study was approved by the Ethics Committee on Animal Use of the Federal University of Uberlândia (122/11). 2.2. Animals Male Wistar rats (200–300 g) were kept in the animal housing facility in climatized chambers, at a temperature of 22 °C, and on a 12 h light-dark cycle. 2.3. DM induction DM induction was performed by intravenous administration (through penile vein) of alloxan (50 mg/kg) (Sigma-Aldrich). Blood glucose was measured 24 h after induction using a glucometer (Accu
⁎ Corresponding author at: Department of Physiology, Institute of Biomedical Sciences, Federal University of Uberlândia, Av. Pará, 1720, Bloco 2A, Campus Umuarama, Uberlândia, MG, 38400-902, Brazil. E-mail address: [email protected] (A.P. Coelho Balbi).
https://doi.org/10.1016/j.biopha.2018.08.066 Received 8 May 2018; Received in revised form 25 July 2018; Accepted 15 August 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 107 (2018) 1115–1118
R.B. de Morais et al.
Fig. 1. Effects of renin-angiotensin system blockage on the alterations of kidney structure and function associated with diabetes in rats. A. Renal Corpuscle Area; B. Tuft Glomerular Area; C. Capsular Area; D. Cortical Collagen; E. Glomerular Filtration Rate (GFR); F. Urinary Albumin Excretion (UAE). Values are expressed as median with percentiles 25 and 75 (A, D and E) or mean ± SEM (B, C and F). The level of significance was set at p < 0.05. ***C: p < 0.001 vs. C; *C: p < 0.05 vs. C; ***S: p < 0.001 vs. S; **S: p < 0.01 vs. S; *S: p < 0.05 vs. S; **DC: p < 0.01 vs. DC; *DC: p < 005 vs. DC; **EN: p < 0.01 vs. EN; *EN: p < 005 vs. EN. Kruskal-Wallis test with Dunn's Post-test (A, D and E) and one way-ANOVA with Tukey post-test (B, C and F). C: control, S: sham, DC: diabetic control, EN: enalapril, LO: losartan, AL: aliskiren.
2.7. Statistical analysis
Check Active, Roche) and animals with blood glucose greater than 200 mg/dL were considered diabetic.
GraphPad Prism software Version 5.00 was used for all analyses. When appropriate, data are reported as mean ± SEM or median with percentiles 25 and 75. Comparisons were made by one way-ANOVA followed by the Tukey’s in the case of normally distributed variables, or by the Kruskall-Wallis test with Dunn´s post-test for nonnormally distributed variables. The data were considered to be statistically significant when p < 0.05.
2.4. Groups The animals were randomly distributed into six groups: 1) Control (C) – without DM; 2) Sham (S) – without DM with simulation of the proposed treatment; 3) Diabetic Control (DC) – DM without treatment; 4) Enalapril (EN) – DM treated with angiotensin converting enzyme inhibitor (20 mg/L, diluted in drinking water); 5) Losartan (LO) – DM treated with the AT1 receptor blocker (50 mg/L, diluted in drinking water); 6) Aliskiren (AL) – DM treated with renin inhibitor (50 mg/Kg/ day, by gavage). All diabetic animal groups (DC, EN, LO, and AL) were treated alternate days with NPH insulin (1IU/rat) (Humulin N®, Lilly) to avoid death. Drug treatments (EN, LO and AL) were started immediately after the confirmation of the diabetic state. After 90 days (d) of treatment, animals were placed in metabolic cages for 24 h for urine collection and, later, blood collection for biochemical analysis. Then the animals were anesthetized with halothane (Cristália) and their kidneys were removed for morphological analysis.
3. Results Twenty-four hours after alloxan administration (post-induction period) or 90d after DM induction animals of all experimental groups presented hyperglycemia (post-induction - C: 97.33 ± 4.62; S: 102.40 ± 2.81; CD: 453.90 ± 47.60**C**S; EN: 545.40 ± 27.10***C ***S ; LO: 481.50 ± 58.65**C**S; AL: 519.30 ± 46.45***C***S; final glycemia C: 98.83 ± 4.13; S: 109.70 ± 2.71; CD: 483.10 ± 39.74***C***S; EN: 544.70 ± 24.96***C***S; LO: 543.80 ± 26.65***C***S; AL: 418.40 ± 57.85**C**S). None of the treatments altered the effects of alloxan treatment on plasma glucose levels. The absolute weight of kidneys from diabetic animals was increased compared to that from control or sham groups (C: 1.29 ± 0.07; S: 1.21 ± 0.04; DC: 1.85 ± 0.20*C**S; EN: 1.83 ± 0.09*C**S; LO: 1.93 ± 0.19*C**S; AL: 1.42 ± 0.08), while the final body weight after 90 days was not different between the groups (data not shown). The kidney weight/body weight ratio from all diabetic animal groups was higher (DC: 0.55 ± 0.06*C*S; EN: 0.61 ± 0.06**C**S; LO: 0.71 ± 0.07***C***S; AL: 0.50 ± 0.05) than that in non-diabetic animals (C: 0.32 ± 0.01; S: 0.33 ± 0.02). There were no significant differences between groups for the sodium and potassium plasma levels; however, the AL group showed a tendency to hyperkalemia compared to other groups (C: 6.06 ± 0.37; S: 6.78 ± 0.21; CD: 6.63 ± 0.36; EN: 6.32 ± 0.34; LO: 6.40 ± 0.22; AL: 8.21 ± 0.51). Plasmatic urea from all diabetic animal groups (DC: 88.35 ± 23.10*C*S; EN: 104.00 ± 9.75*C**S; LO: 122.50 ± 25.27**C**S; AL: *C*S 88.50 ± 8.44 ) was higher than that in non-diabetic animals (C: 49.17 ± 3.60; S: 42.96 ± 2.74). Diabetes increased renal corpuscle area and only EN treatment
2.5. Functional studies Renal function was evaluated by creatinine clearance. Urinary albumin excretion (UAE) and plasma urea was determined by colorimetry (Labtest Diagnostica), while plasma sodium and potassium levels were determined by the ion-selective electrode technique, using the selective ion analyzer AVL Roche 9180.
2.6. Histological and histomorphometric analysis Renal sections (4 μm) were subjected to I) toluidine blue staining for identification of MC; II) periodic acid-reactive Schiff staining (PAS) for histomorphometric analysis (areas of the renal corpuscle, renal glomerulus and capsular space), and III) Picrosirius red for collagen visualization (analyzed using Image J software, version 1.4) 1116
Biomedicine & Pharmacotherapy 107 (2018) 1115–1118
R.B. de Morais et al.
Fig. 2. Effects of renin-angiotensin system blockage on kidney mast cell population in diabetic rats. A. Intact mast cells; B. Degranulated mast cells; C. Renal section stained with Toluidine blue evidencing degranulated mast cell (arrow) and intact mast cell (arrowhead). Light microscopy images, 1000 × . Values are expressed as median with percentiles 25 and 75. The level of significance was set at p < 0.05. ***C: p < 0.001 vs. C; **C: p < 0.01 vs. C; *C: p < 0.05 vs. C; ***S: p < 0.001 vs. S; **S: p < 0.01 vs. S; *S: p < 005 vs. S; *DC: p < 0.05 vs. DC; **EN: p < 0.01 vs. EN; **LO: p < 0.01 vs. LO. Kruskal Wallis test with Dunn's Post-test. C: control, S: sham, DC: diabetic control, EN: enalapril, LO: losartan, AL: aliskiren.
Moreover, AT2 receptor stimulation by Ang II generated by alternative pathways cannot be ruled out. These possibilities still require further investigation. The effects of the different RAS blockers were variable concerning glomerular and UAE changes induced by diabetes. Indeed, EN decreased renal corpuscle and glomerular areas, while AL and LO did not. These observations suggest that AT1 receptor stimulation is not involved in the early glomerular changes induced by diabetes and that ACE-independent effects of EN are involved in these protective actions. It is important to mention that ACE inhibitors can decrease bradykinin breakdown and that the kallikrein–kinin system has been shown to possess protective effects on the kidneys of diabetic animals [12]. The observation that EN partially decreases UAE in diabetic rats suggests that AT1 receptor stimulation is involved in the early kidney dysfunction related to this disease. However, EN but not AL partially altered UAE, pointing to an ACE-independent effect of EN in this respect and strongly suggesting that alternative pathways for Ang II generation are indeed actively present in the kidneys of diabetic animals. Diabetic rats, except for those in the AL group, presented renal hypertrophy; these data suggest that Ang I processing products from alternative ACE enzymes appear to have trophic effects in renal tissue of diabetic rats and these products could be more important in this early stage of diabetic kidney disease than Ang II, a well-known trophic peptide in the kidney tissue [10]. Despite the alterations in plasma urea levels observed in diabetic animals suggest that they could be dehydrated, the increase in kidney weight/body weight ratio in diabetic groups does not appear to reflect a dehydration status, since the absolute weight of kidneys from diabetic animals was also increased while final body weight was not different between groups. In addition, increased plasma levels of urea may be the result of reduced GFR observed in the diabetic animals.
prevented this alteration (Fig. 1A). The tuft glomerular area was also increased by diabetes and only EN or LO treatments totally or partially reversed this alteration, respectively (Fig. 1B). Diabetes increased capsular space area and none of the treatments affected this alteration (Fig. 1C). Diabetic animals showed increased cortical collagen, which was partially decreased by LO and AL treatments (Fig. 1D). GFR was decreased by diabetes and none of the treatments altered this impairment (Fig. 1E). UAE was increased by diabetes and only EN and LO partially reversed this alteration (Fig. 1 F). The total MC density and the density of active (degranulating) MC were increased by diabetes; only AL treatment reversed this alteration (Fig. 2). The experimental manipulation of the animals did not alter any of the parameters analyzed (Figs. 1 and 2; C vs. S p > 0.05).
4. Discussion Alloxan treatment was effective at inducing DM1 in rats, which was characterized by hyperglycemia (> 400 mg/dL), polydipsia, weight loss (data not shown) and polyuria. Initial diabetic renal injury was also observed 3 months after diabetes induction, as suggested by increases in kidney collagen expression, renal corpuscular area, glomerular tuft, and capsular space, all these alterations are seen in the early stages of DM [8]. Ang II appears to increase kidney collagen deposition through AT1 receptor stimulation, as this alteration was sensitive to both LO and AL treatments [9]. However, angiotensin-converting enzyme (ACE)independent pathways to Ang II generation seem to be involved, since EN did not alter collagen expression under our experimental conditions. Alternative pathways for Ang II generation from Ang I have been recognized in humans and other species: in rats, elastase-2 appears to be an important alternative Ang II-generating pathway, as rat chymases act mainly as Ang II-degrading enzymes [10]. Renal MC have been shown to release active renin and induce kidney fibrosis in a unilateral ureteral obstruction model of renal injury [7]. Interestingly, renal MC number and activity (degranulation) were increased in diabetic animals and they appear to participate in the stimulation of kidney fibrosis in alloxan-treated animals. This suggestion is based on the observation that AL not only avoided the alterations in the renal MC population associated with diabetes, but it also decreased renal fibrosis in our model. It is unlikely that Ang II generated by ACE and/or AT1 receptor stimulation are involved in kidney MC proliferation and/or activation, because neither EN nor LO treatments altered the renal MC population in diabetic rats. Therefore, the pivotal role of renin concerning the alteration of the MC population is unlinked to the main downstream effectors of the classical RAS cascade (i.e., ACE and AT1 receptors) and probably involves peptides from Ang I processing by non-ACE enzymes such as Ang1-7, which has been shown to accelerate DN progression in an animal model of diabetic disease [11].
5. Summary The main finding of this report is that AL can modulate (decrease) MC number and activity in the renal parenchyma of diabetic rats, an effect associated with decreased cortical collagen deposition; these data suggest not only that mast cells may be involved in some aspects of DN pathogenesis but also that the possible protective effects of AL on the kidneys involve MC modulation. Moreover, depending on the agent administered, treatment with RAS blockers ameliorates kidney structural and/or functional alterations induced by diabetes.
Conflict of interest The authors declare that they have no conflict of interest. 1117
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Acknowledgement
[6] P. Welker, S. Krämer, D.A. Groneberg, H.H. Neumayer, S. Bachmann, K. Amann, et al., Increased mast cell number in human hypertensive nephropathy, Am. J. Physiol. Renal Physiol. 295 (4) (2008) F1103–9. [7] A. Veerappan, A.C. Reid, N. O’Connor, R. Mora, J.A. Brazin, R. Estephan, et al., Mast cells are required for the development of renal fibrosis in the rodent unilateral ureteral obstruction model, Am. J. Physiol. Renal Physiol. 302 (1) (2012) F192–204. [8] G. Wolf, F.N. Ziyadeh, Molecular mechanisms of diabetic renal hypertrophy, Kidney Int. 56 (2) (1999) 393–405. [9] S.A. Mezzano, M. Ruiz-Ortega, J. Egido, Angiotensin II and renal fibrosis, Hypertension 38 (3 Pt 2) (2001) 635–638. [10] C. Becari, E.B. Oliveira, M.C. Salgado, Alternative pathways for angiotensin II generation in the cardiovascular system, Braz. J. Med. Biol. Res. 44 (9) (2011) 914–919. [11] Y. Shao, M. He, L. Zhou, T. Yao, Y. Huang, L.M. Lu, Chronic angiotensin (1-7) injection accelerates STZ-induced diabetic renal injury, Acta Pharmacol. Sin. 29 (7) (2008) 829–837. [12] A. Riad, J.L. Zhuo, H.P. Schultheiss, C. Tschöpe, The role of the renal kallikreinkinin system in diabetic nephropathy, Curr. Opin. Nephrol. Hypertens 16 (1) (2007) 22–26.
The paper has been prepared without any financial supports. References [1] D.B. Vidotti, D.E. Casarini, P.C. Cristovam, C.A. Leite, N. Schor, M.A. Boim, High glucose concentration stimulates intracellular renin activity and angiotensin II generation in rat mesangial cells, Am. J. Physiol. Renal Physiol. 286 (6) (2004) F1039–45. [2] R.M. Carey, Antihypertensive and renoprotective mechanisms of Renin inhibition in diabetic rats, Hypertension 52 (1) (2008) 63–64. [3] B.M. Rüger, Q. Hasan, N.S. Greenhill, P.F. Davis, P.R. Dunbar, T.J. Neale, Mast cells and type VIII collagen in human diabetic nephropathy, Diabetologia 39 (10) (1996) 1215–1222. [4] K. Hiromura, M. Kurosawa, S. Yano, T. Naruse, Tubulointerstitial mast cell infiltration in glomerulonephritis, Am. J. Kidney Dis. 32 (4) (1998) 593–599. [5] T. Tóth, R. Tóth-Jakatics, S. Jimi, M. Ihara, H. Urata, S. Takebayashi, Mast cells in rapidly progressive glomerulonephritis, J. Am. Soc. Nephrol. 10 (7) (1999) 1498–1505.
1118
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Mast cell population in the development of diabetic nephropathy: Effects of renin angiotensin system inhibition
T
Richarlisson Borges de Moraisa,e, Victor Pereira do Couto Muniza, Emerson Nunes Costaa,f, Sebastião Rodrigues Ferreira Filhod, Karen Renata Nakamura Hirakib, ⁎ Luiz Borges Bispo-da-Silvac, Ana Paula Coelho Balbia, a
Laboratory of Physiology, Institute of Biomedical Sciences, Federal University of Uberlândia, ICBIM-UFU, Brazil Laboratory of Histology, Institute of Biomedical Sciences, Federal University of Uberlândia, ICBIM-UFU, Brazil c Laboratory of Pharmacology, Institute of Biomedical Sciences, Federal University of Uberlândia, ICBIM-UFU, Brazil d Medical Clinic Department, School of Medicine, Federal University of Uberlândia, FAMED-UFU, Brazil e Technical School of Heath, Federal University of Uberlândia, ESTES-UFU, Brazil f Hemodialysis Sector, Clinical Hospital, Federal University of Uberlândia, HC-UFU, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Diabetes Nephropathy Fibrosis Aliskiren Mast cells
Considering the importance of the renin-angiotensin system (RAS) in diabetic nephropathy (DN) and the link between mast cells (MC) and the RAS, this study evaluated the effects of RAS blockade on the MC cell population in the kidneys from rats with experimental diabetes. Wistar rats were divided into six groups: control nondiabetic (C); sham (S); diabetic (D); and D treated with enalapril (EN), losartan (LO), or aliskiren (AL). Ninety days after diabetes induction, glomerular filtration rate (GFR) and urinary albumin excretion (UAE) were determined. Kidneys were collected for MC counting. RAS blockers minimized changes in morphometrical parameters (EN), cortical collagen (LO, AL), GFR (AL) and UAE (EN, LO). An increased number of MC was observed in the kidneys from D animals. Only AL treatment prevented this increase. MC may be involved in some aspects of DN pathogenesis and the possible protective effects of AL on the kidneys might involve MC modulation.
1. Introduction
2. Materials and methods
Hyperglycemia stimulates renin release in renal tissue, resulting in high levels of angiotensin II (Ang II), an important mediator of diabetic nephropathy (DN) physiopathology [1]. Human and animal studies have used drugs capable of interfering with the renin-angiotensin system (RAS) in order to preserve renal function and decrease proteinuria [2]. In addition to the increased RAS activity in DN, renal mast cell (MC) accumulation is also a common feature of DN [3] and other kidney disorders, such as primary or secondary glomerulonephritis [4], progressive glomerular nephritis [5] and hypertensive nephropathy [6]. It has been suggested that mast cells are sources of renin, and they appear to be an important component of a local RAS [7]. Therefore, considering the role of RAS in DN development and the link between MC and RAS, this study evaluated the effects of RAS blockade on the MC population in the kidneys of rats with experimental DN, as well as on the structural and functional alterations of the kidney induced by DM.
2.1. Ethical considerations This study was approved by the Ethics Committee on Animal Use of the Federal University of Uberlândia (122/11). 2.2. Animals Male Wistar rats (200–300 g) were kept in the animal housing facility in climatized chambers, at a temperature of 22 °C, and on a 12 h light-dark cycle. 2.3. DM induction DM induction was performed by intravenous administration (through penile vein) of alloxan (50 mg/kg) (Sigma-Aldrich). Blood glucose was measured 24 h after induction using a glucometer (Accu
⁎ Corresponding author at: Department of Physiology, Institute of Biomedical Sciences, Federal University of Uberlândia, Av. Pará, 1720, Bloco 2A, Campus Umuarama, Uberlândia, MG, 38400-902, Brazil. E-mail address: [email protected] (A.P. Coelho Balbi).
https://doi.org/10.1016/j.biopha.2018.08.066 Received 8 May 2018; Received in revised form 25 July 2018; Accepted 15 August 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 107 (2018) 1115–1118
R.B. de Morais et al.
Fig. 1. Effects of renin-angiotensin system blockage on the alterations of kidney structure and function associated with diabetes in rats. A. Renal Corpuscle Area; B. Tuft Glomerular Area; C. Capsular Area; D. Cortical Collagen; E. Glomerular Filtration Rate (GFR); F. Urinary Albumin Excretion (UAE). Values are expressed as median with percentiles 25 and 75 (A, D and E) or mean ± SEM (B, C and F). The level of significance was set at p < 0.05. ***C: p < 0.001 vs. C; *C: p < 0.05 vs. C; ***S: p < 0.001 vs. S; **S: p < 0.01 vs. S; *S: p < 0.05 vs. S; **DC: p < 0.01 vs. DC; *DC: p < 005 vs. DC; **EN: p < 0.01 vs. EN; *EN: p < 005 vs. EN. Kruskal-Wallis test with Dunn's Post-test (A, D and E) and one way-ANOVA with Tukey post-test (B, C and F). C: control, S: sham, DC: diabetic control, EN: enalapril, LO: losartan, AL: aliskiren.
2.7. Statistical analysis
Check Active, Roche) and animals with blood glucose greater than 200 mg/dL were considered diabetic.
GraphPad Prism software Version 5.00 was used for all analyses. When appropriate, data are reported as mean ± SEM or median with percentiles 25 and 75. Comparisons were made by one way-ANOVA followed by the Tukey’s in the case of normally distributed variables, or by the Kruskall-Wallis test with Dunn´s post-test for nonnormally distributed variables. The data were considered to be statistically significant when p < 0.05.
2.4. Groups The animals were randomly distributed into six groups: 1) Control (C) – without DM; 2) Sham (S) – without DM with simulation of the proposed treatment; 3) Diabetic Control (DC) – DM without treatment; 4) Enalapril (EN) – DM treated with angiotensin converting enzyme inhibitor (20 mg/L, diluted in drinking water); 5) Losartan (LO) – DM treated with the AT1 receptor blocker (50 mg/L, diluted in drinking water); 6) Aliskiren (AL) – DM treated with renin inhibitor (50 mg/Kg/ day, by gavage). All diabetic animal groups (DC, EN, LO, and AL) were treated alternate days with NPH insulin (1IU/rat) (Humulin N®, Lilly) to avoid death. Drug treatments (EN, LO and AL) were started immediately after the confirmation of the diabetic state. After 90 days (d) of treatment, animals were placed in metabolic cages for 24 h for urine collection and, later, blood collection for biochemical analysis. Then the animals were anesthetized with halothane (Cristália) and their kidneys were removed for morphological analysis.
3. Results Twenty-four hours after alloxan administration (post-induction period) or 90d after DM induction animals of all experimental groups presented hyperglycemia (post-induction - C: 97.33 ± 4.62; S: 102.40 ± 2.81; CD: 453.90 ± 47.60**C**S; EN: 545.40 ± 27.10***C ***S ; LO: 481.50 ± 58.65**C**S; AL: 519.30 ± 46.45***C***S; final glycemia C: 98.83 ± 4.13; S: 109.70 ± 2.71; CD: 483.10 ± 39.74***C***S; EN: 544.70 ± 24.96***C***S; LO: 543.80 ± 26.65***C***S; AL: 418.40 ± 57.85**C**S). None of the treatments altered the effects of alloxan treatment on plasma glucose levels. The absolute weight of kidneys from diabetic animals was increased compared to that from control or sham groups (C: 1.29 ± 0.07; S: 1.21 ± 0.04; DC: 1.85 ± 0.20*C**S; EN: 1.83 ± 0.09*C**S; LO: 1.93 ± 0.19*C**S; AL: 1.42 ± 0.08), while the final body weight after 90 days was not different between the groups (data not shown). The kidney weight/body weight ratio from all diabetic animal groups was higher (DC: 0.55 ± 0.06*C*S; EN: 0.61 ± 0.06**C**S; LO: 0.71 ± 0.07***C***S; AL: 0.50 ± 0.05) than that in non-diabetic animals (C: 0.32 ± 0.01; S: 0.33 ± 0.02). There were no significant differences between groups for the sodium and potassium plasma levels; however, the AL group showed a tendency to hyperkalemia compared to other groups (C: 6.06 ± 0.37; S: 6.78 ± 0.21; CD: 6.63 ± 0.36; EN: 6.32 ± 0.34; LO: 6.40 ± 0.22; AL: 8.21 ± 0.51). Plasmatic urea from all diabetic animal groups (DC: 88.35 ± 23.10*C*S; EN: 104.00 ± 9.75*C**S; LO: 122.50 ± 25.27**C**S; AL: *C*S 88.50 ± 8.44 ) was higher than that in non-diabetic animals (C: 49.17 ± 3.60; S: 42.96 ± 2.74). Diabetes increased renal corpuscle area and only EN treatment
2.5. Functional studies Renal function was evaluated by creatinine clearance. Urinary albumin excretion (UAE) and plasma urea was determined by colorimetry (Labtest Diagnostica), while plasma sodium and potassium levels were determined by the ion-selective electrode technique, using the selective ion analyzer AVL Roche 9180.
2.6. Histological and histomorphometric analysis Renal sections (4 μm) were subjected to I) toluidine blue staining for identification of MC; II) periodic acid-reactive Schiff staining (PAS) for histomorphometric analysis (areas of the renal corpuscle, renal glomerulus and capsular space), and III) Picrosirius red for collagen visualization (analyzed using Image J software, version 1.4) 1116
Biomedicine & Pharmacotherapy 107 (2018) 1115–1118
R.B. de Morais et al.
Fig. 2. Effects of renin-angiotensin system blockage on kidney mast cell population in diabetic rats. A. Intact mast cells; B. Degranulated mast cells; C. Renal section stained with Toluidine blue evidencing degranulated mast cell (arrow) and intact mast cell (arrowhead). Light microscopy images, 1000 × . Values are expressed as median with percentiles 25 and 75. The level of significance was set at p < 0.05. ***C: p < 0.001 vs. C; **C: p < 0.01 vs. C; *C: p < 0.05 vs. C; ***S: p < 0.001 vs. S; **S: p < 0.01 vs. S; *S: p < 005 vs. S; *DC: p < 0.05 vs. DC; **EN: p < 0.01 vs. EN; **LO: p < 0.01 vs. LO. Kruskal Wallis test with Dunn's Post-test. C: control, S: sham, DC: diabetic control, EN: enalapril, LO: losartan, AL: aliskiren.
Moreover, AT2 receptor stimulation by Ang II generated by alternative pathways cannot be ruled out. These possibilities still require further investigation. The effects of the different RAS blockers were variable concerning glomerular and UAE changes induced by diabetes. Indeed, EN decreased renal corpuscle and glomerular areas, while AL and LO did not. These observations suggest that AT1 receptor stimulation is not involved in the early glomerular changes induced by diabetes and that ACE-independent effects of EN are involved in these protective actions. It is important to mention that ACE inhibitors can decrease bradykinin breakdown and that the kallikrein–kinin system has been shown to possess protective effects on the kidneys of diabetic animals [12]. The observation that EN partially decreases UAE in diabetic rats suggests that AT1 receptor stimulation is involved in the early kidney dysfunction related to this disease. However, EN but not AL partially altered UAE, pointing to an ACE-independent effect of EN in this respect and strongly suggesting that alternative pathways for Ang II generation are indeed actively present in the kidneys of diabetic animals. Diabetic rats, except for those in the AL group, presented renal hypertrophy; these data suggest that Ang I processing products from alternative ACE enzymes appear to have trophic effects in renal tissue of diabetic rats and these products could be more important in this early stage of diabetic kidney disease than Ang II, a well-known trophic peptide in the kidney tissue [10]. Despite the alterations in plasma urea levels observed in diabetic animals suggest that they could be dehydrated, the increase in kidney weight/body weight ratio in diabetic groups does not appear to reflect a dehydration status, since the absolute weight of kidneys from diabetic animals was also increased while final body weight was not different between groups. In addition, increased plasma levels of urea may be the result of reduced GFR observed in the diabetic animals.
prevented this alteration (Fig. 1A). The tuft glomerular area was also increased by diabetes and only EN or LO treatments totally or partially reversed this alteration, respectively (Fig. 1B). Diabetes increased capsular space area and none of the treatments affected this alteration (Fig. 1C). Diabetic animals showed increased cortical collagen, which was partially decreased by LO and AL treatments (Fig. 1D). GFR was decreased by diabetes and none of the treatments altered this impairment (Fig. 1E). UAE was increased by diabetes and only EN and LO partially reversed this alteration (Fig. 1 F). The total MC density and the density of active (degranulating) MC were increased by diabetes; only AL treatment reversed this alteration (Fig. 2). The experimental manipulation of the animals did not alter any of the parameters analyzed (Figs. 1 and 2; C vs. S p > 0.05).
4. Discussion Alloxan treatment was effective at inducing DM1 in rats, which was characterized by hyperglycemia (> 400 mg/dL), polydipsia, weight loss (data not shown) and polyuria. Initial diabetic renal injury was also observed 3 months after diabetes induction, as suggested by increases in kidney collagen expression, renal corpuscular area, glomerular tuft, and capsular space, all these alterations are seen in the early stages of DM [8]. Ang II appears to increase kidney collagen deposition through AT1 receptor stimulation, as this alteration was sensitive to both LO and AL treatments [9]. However, angiotensin-converting enzyme (ACE)independent pathways to Ang II generation seem to be involved, since EN did not alter collagen expression under our experimental conditions. Alternative pathways for Ang II generation from Ang I have been recognized in humans and other species: in rats, elastase-2 appears to be an important alternative Ang II-generating pathway, as rat chymases act mainly as Ang II-degrading enzymes [10]. Renal MC have been shown to release active renin and induce kidney fibrosis in a unilateral ureteral obstruction model of renal injury [7]. Interestingly, renal MC number and activity (degranulation) were increased in diabetic animals and they appear to participate in the stimulation of kidney fibrosis in alloxan-treated animals. This suggestion is based on the observation that AL not only avoided the alterations in the renal MC population associated with diabetes, but it also decreased renal fibrosis in our model. It is unlikely that Ang II generated by ACE and/or AT1 receptor stimulation are involved in kidney MC proliferation and/or activation, because neither EN nor LO treatments altered the renal MC population in diabetic rats. Therefore, the pivotal role of renin concerning the alteration of the MC population is unlinked to the main downstream effectors of the classical RAS cascade (i.e., ACE and AT1 receptors) and probably involves peptides from Ang I processing by non-ACE enzymes such as Ang1-7, which has been shown to accelerate DN progression in an animal model of diabetic disease [11].
5. Summary The main finding of this report is that AL can modulate (decrease) MC number and activity in the renal parenchyma of diabetic rats, an effect associated with decreased cortical collagen deposition; these data suggest not only that mast cells may be involved in some aspects of DN pathogenesis but also that the possible protective effects of AL on the kidneys involve MC modulation. Moreover, depending on the agent administered, treatment with RAS blockers ameliorates kidney structural and/or functional alterations induced by diabetes.
Conflict of interest The authors declare that they have no conflict of interest. 1117
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Acknowledgement
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