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Carnosine treatment in combination with ACE inhibition in diabetic rats
Regulatory Peptides 194–195 (2014) 36–40
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Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
Carnosine treatment in combination with ACE inhibition in diabetic rats V. Peters a,⁎,1, E. Riedl b,1, M. Braunagel b, S. Höger b, S. Hauske b, F. Pfister b, J. Zschocke c, B. Lanthaler c, U. Benck b, H.-P. Hammes b, B.K. Krämer b, C.P. Schmitt a, B.A. Yard b, H. Köppel b a b c
Centre for Pediatric and Adolescent Medicine, University of Heidelberg, Heidelberg, Germany Vth Department of Medicine, Nephrology, Endocrinology, Diabetology & Rheumatology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Division of Human Genetics, Medical University Innsbruck, Austria
a r t i c l e
i n f o
Article history: Received 16 June 2014 Received in revised form 8 September 2014 Accepted 11 September 2014 Available online 16 September 2014 Keywords: Diabetic nephropathy Carnosine Diabetes mellitus Cataract Proteinuria
a b s t r a c t In humans, we reported an association of a certain allele of carnosinase gene with reduced carnosinase activity and absence of nephropathy in diabetic patients. CN1 degrades histidine dipeptides such as carnosine and anserine. Further, we and others showed that treatment with carnosine improves renal function and wound healing in diabetic mice and rats. We now investigated the effects of carnosine treatment alone and in combination with ACE inhibition, a clinically established nephroprotective drug in diabetic nephropathy. Male Sprague–Dawley rats were injected i.v. with streptozotocin (STZ) to induce diabetes. After 4 weeks, rats were unilaterally nephrectomized and randomized for 24 weeks of treatment with carnosine, lisinopril or both. Renal CN1 protein concentrations were increased under diabetic conditions which correlated with decreased anserine levels. Carnosine treatment normalized CN1 abundance and reduced glucosuria, blood concentrations of glycosylated hemoglobin (HbA1c), carboxyl-methyl lysine (CML), N-acetylglucosamine (GlcNac; all p b 0.05 vs. non-treated STZ rats), reduced cataract formation (p b 0.05) and urinary albumin excretion (p b 0.05), preserved podocyte number (p b 0.05) and normalized the increased renal tissue CN1 protein concentration. Treatment with lisinopril had no effect on HbA1C, glucosuria, cataract formation and CN1 concentration, but reduced albumin excretion rate more effectively than carnosine treatment (p b 0.05). Treatment with both carnosine and lisinopril combined the effects of single treatment, albeit without additive effect on podocyte number or albuminuria. Increased CN1 amount resulted in decreased anserine levels in the kidney. Both carnosine and lisinopril exert distinct beneficial effects in a standard model of diabetic nephropathy. Both drugs administered together combine the respective effects of single treatment, albeit without exerting additive nephroprotection. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Diabetic nephropathy (DN) is the most common cause of end stage renal disease in the Western world. Despite increasing knowledge on the pathogenesis of DN and potential therapeutic interventions, progression to end stage renal disease still occurs in a substantial number of diabetic patients [1,2]. At present, pharmacological nephroprotection is largely limited to tight glucose and blood pressure control and antiproteinuric intervention using antagonists of the renin–angiotensin system [3]. ACE inhibitors are known to reduce proteinuria and have been the first-line agents in the management of diabetic nephropathy [4,5] during the last years.
⁎ Corresponding author at: Centre for Pediatric and Adolescent Medicine, Metabolic Centre, University of Heidelberg, Im Neuenheimer Feld 669, 69120 Heidelberg, Germany. Tel.: +49 6221 5631715; fax: +49 6221 565565. E-mail address: [email protected] (V. Peters). 1 Both authors contributed equally.
http://dx.doi.org/10.1016/j.regpep.2014.09.005 0167-0115/© 2014 Elsevier B.V. All rights reserved.
A genome-wide linkage study revealed strong evidence for linkage of DN to the CNDP1 gene, encoding serum carnosinase (CN1) [6]. (CTG)5 homozygous individuals display low CN1 activity in serum [6] and have a reduced susceptibility to DN. Carnosine, the natural substrate for CN1 [7–9] has several protective functions, such as antioxidant activity [10], scavenging of reactive oxygen species [11] and glycation inhibition [12]. In both in vitro and in vivo models high carnosine levels protect against diabetic complications by improving glucose metabolism, reduction of carbonyl stress and minimizing formation of advanced glycation end products (AGEs) [13–15]. An intriguing treatment approach in patients with diabetic nephropathy should be the combination of the direct nephroprotective action of ACE inhibitors and the carnosine mediated nephroprotection which is based on a variety of protective functions, such as scavenging of reactive oxygen species [11], inhibition of angiotensin converting enzyme [12], of protein glycation [12], of cellular senescence [16] and of matrix protein synthesis [6]. To assess the potential of ACE inhibition and pharmacological doses of carnosine in DN, we treated diabetic rats for 24 weeks with lisinopril, carnosine and a combination of both. Diabetes mellitus was
V. Peters et al. / Regulatory Peptides 194–195 (2014) 36–40
induced by streptozotocin (STZ) which results in a well-defined nephropathy. The rats were moreover uninephrectomized in order to reduce the time until development of overt DN [17,18]. These animals underwent detailed analysis of glucose metabolism, carbonyl stress, renal morphology and renal function. 2. Material and methods 2.1. Animal and induction of experimental diabetes Diabetes was induced in male Sprague–Dawley rats weighing 350 to 420 g by a single intravenous injection of 50 mg/kg bodyweight streptozotocin (STZ; Sigma-Aldrich Chemical, Deisenhofen, Germany). A second STZ injection was given in case blood glucose levels were below 400 mg/dl. Blood glucose levels were monitored regularly, i.e. daily in the first week after STZ-injection and weekly thereafter. Insulin glargine (Lantus, Sanofi-Aventis, Frankfurt, Germany) was injected s.c. in individually adjusted doses to maintain a blood glucose level of 400–600 mg/dl to allow for stable diabetes mellitus. Injection of insulin was daily in the first week after STZ-injection and thrice weekly thereafter. Rats were housed in a climate-controlled animal house with a 12 h light cycle and free access to standard diet and drug supplemented tap water. 2.2. Experimental design Four weeks following STZ injection unilateral nephrectomy was performed and 52 rats were randomized to four groups, L-carnosine treatment (C, 1 g/kg bodyweight, Flamma S.p.A, Bergamo, Italy), lisinopril treatment (L, 2.5 mg/kg bodyweight, AstraZeneca GmbH, Wedel, Germany), the respective combined treatment (L + C) and untreated rats (STZ). Ten additional rats without preceding STZ injection served as age matched untreated controls (C). Treatment was administered via drinking water for 24 weeks. Blood glucose was controlled by thrice weekly insulin injections in all STZ groups. Drinking volumes were recorded daily, and bodyweight assessed twice weekly and drug dosing adjusted accordingly. Body weight and blood glucose levels and insulin doses administered did not differ between groups. Animals were placed in metabolic cages on week 28 post-STZ injection for 24 h. Following sacrifice, organs were removed immediately and either stored in liquid nitrogen or 4% w/v paraformaldehyde, respectively. Blood samples were collected for the determination of glycated hemoglobin concentration by affinity chromatography (MicromatII™; Bio-Rad Laboratories GmbH, Munich, Germany). Serum creatinine was measured by immunoassay (Dimension; Dade Behring, Germany), urinary albumin by ELISA as previously published [19] and serum carboxyl-methyl lysine was measured using a commercial ELISA (CML-ELISA Kit, Cell Biolabs, BIOCAT GmbH, Germany). All animal procedures were approved by the Regierungspräsidium Karlsruhe AZ 35-9185.81/G-5306. 2.3. Quantification of cataract formation The formation of cataract was assessed semiquantitatively by macroscopic evaluation in a blinded setting using a score of 0–4, with 0 representing no cataract formation, 1 incipient cataract, 2 incipient cataract of both eyes or moderate cataract in one eye, 3 moderate cataract of both eyes and 4 with both eyes being completely opaque. 2.4. Histological analysis Paraffin embedding of kidneys was performed using routine procedures and sections were stained with hematoxylin-eosin. Tissue sections were evaluated by two different, blinded individuals scoring a minimum of 20 microscopic fields per kidney (H. Köppel and R Waldherr). A semiquantitative score was used for grading of glomerular size, mesangial matrix expansion, interstitial fibrosis and tubular
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atrophy. The histologic grading scale ranged was from 0 to 3 (0 = not present, 1 = mild alteration, 2 = moderate alteration, and 3 = severe alteration). For determination of podocyte number, sections were stained for markers of podocytes (WT-1) and endothelial cells (CD31) according to standard protocols. Renal carnosinase abundance was assessed by immunohistochemistry, using an anti-CN1 (RYSK 173) monoclonal antibody (generated by our group; [9]). Staining intensity was classified as minute (= 1), moderate (= 2) and high (= 3), respectively. 2.5. N-Acetylglucosamine immunoblotting Shock frozen kidneys were homogenized in lysis buffer [150 mM NaCl, 10 mM Tris–HCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0 · 5% Na-deoxycholate, 1 μM dithiothreitol (DTT), 1 μg/ml aprotinin, 1 mM phenylmethylsulphonyl fluoride (PMSF)] using a Polytron homogenizer (IKA Labortechnik/Fischer Scientific, Schwerte, Germany) and incubated for 5 min on ice. Lysates were centrifuged (15 min at 16 000 g), protein concentrations in the supernatants measured using Coomassie Reagent (Pierce, Rockford, IL, USA). Proteins were separated on a 10% w/v sodium dodecyl sulfate–polyacrylamide gel by electrophoresis (SDS-PAGE) and semidry blotted onto a polyvinyl difluoride (PVDF) membrane (Roche Diagnostics, Mannheim, Germany). The membrane was incubated overnight in Tris-buffered saline (TBS) (10 mM Tris–HCl, pH 8 · 0, 150 mM NaCl) containing 5% w/v milk powder. Thereafter, the blots were incubated for 1 h with specific primary antibody for N-Acetylglucosamine-modified proteins (GlucNAc; Abcamplc, Cambridge, United Kingdom), followed by incubation with appropriate horseradish peroxidase (HRP) secondary antibody. Proteins were visualized by enhanced chemoluminescence technology according to the manufacturer's instructions (Pierce). To confirm equal protein loading, membranes were stripped with 62 · 5 mM Tris–HCl, 2% w/v SDS and 100 mM β-mercaptoethanol and incubated with antibodies against GAPDH (Abcamplc, Cambridge, United Kingdom). Intensity of specific bands was measured by chemiluminescence using the ImageJ 1.36b software and means of the absolute values of each group were expressed relative to WT. 2.6. Carnosine and anserine concentrations Kidneys were removed and immediately homogenized in cold buffer containing 20 mM HEPES, 1 mM ethylene glycol-tetra-acetic acid (EGTA), 210 mM mannitol and 70 mM sucrose per gram tissue, pH 7.2. The homogenate was centrifuged at 1500 ×g for 5 min at 4 °C. Carnosine and anserine levels were measured in the supernatant by highperformance liquid chromatography as previously described [20]. Briefly, the samples were diluted with sulfosalicylic acid to precipitate proteins. After derivatization with carbazole-9-carbonyl chloride (CFC), the samples underwent liquid chromatography and quantification by fluorescence. All samples were measured twice, and one sample was spiked with the standard to identify anserine. 2.7. Statistical analyses All values are shown as mean ± SD if not indicated otherwise. For survival, Kaplan–Meier survival estimates were calculated. Differences between treatment groups were analyzed by one-way ANOVA followed by t-test and Mann–Whitney in case of non-Gaussian distribution. Values of p b 0.05 were considered to be significant. 3. Results STZ treatment increased glucosuria, glycosylated hemoglobin (HbA1c), carboxy-methyl lysine (CML) and N-Acetylglucosamine (GlcNAc) blood levels. Significant cataract developed over the study period, kidney morphology and function declined (Table 1), and renal
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Table 1 Comparison between carnosine and lisinopril treatment in diabetic rats. Parameter
Control
STZ no treatment (STZ)
STZ + carnosinetreated (C)
STZ + lisinopriltreated (L)
STZ + carnosine + lisinopril treated (L + C)
Glucose metabolism Glucosuria (mg glucose/mg creatinine) Glycosylated hemoglobin (HbA1c) % (mmol/mol) Carboxyl-methyl lysine (CML) ng/mg protein normalized for control N-Acetylglucosamine (GlcNAc) ratio to control Cataract score 0–4, median (min, max)
0.1 ± 0.1 4.9 ± 1.0 1.0 ± 0 1.0 ± 0.1 0 (0, 0)
1096 ± 560 12.8 ± 2.4 16.3 ± 3.5 3.2 ± 1.0 1 (0, 3)
310 ± 378a 9.3 ± 2.2a 12.7 ± 2.0a 1.8 ± 0.9a 0 (0, 0)a
1214 ± 547b 13.1 ± 1.9b 15.4 ± 2.0 1.7 ± 0.9a 2 (0, 4)b
266 ± 234a, c 11.8 ± 1.2b 17.3 ± 2.2b 1.7 ± 0.5a 0 (0, 3)a, c
Renal morphology/function Kidney/body weight[%] Podocytes per endothelial cells CreaCl (ml/min/100 g bw) Urinary albumin/creatinine ratio
0.3 ± 0.0 0.31 ± 0.04 0.9 ± 0.3 0.3 ± 0.3
1.0 ± 0.1 0.18 ± 0.02 0.6 ± 0.3 34.0 ± 39.0
0.8 ± 0.1 0.23 ± 0.02a 0.8 ± 0.2 14.6 ± 21.7a
0.8 ± 0.1 0.28 ± 0.04a, b 0.9 ± 0.2 2.0 ± 2.9a, b
0.8 ± 0.1 0.25 ± 0.02a, c 0.6 ± 0.1 1.1 ± 1.0a, b
Outcome of treatment with carnosine (C), lisinopril (L) or both (C + L) of STZ rats. CreaCl = creatinine clearance. a = p b 0.05 vs. untreated STZ; b = p b 0.05 vs. STZ + C; c = p b 0.05 vs. STZ + L.
tissue CN1 abundance increased (Fig. 1). Body weight gain was significantly impaired in STZ rats after the onset of hyperglycemia and resulted in reduced body weight at the end of the study (430 ± 39 vs. 509 ± 53 g in STZ vs. non-STZ treated control rats; p b 0.05). Treatment with carnosine but not with lisinopril increased bodyweight gain of diabetic rats (body weight gain STZ controls: 40 ± 63 g; STZ + C: 77 ± 70 g; STZ + L: 11 ± 45 g; STZ + C + L: 104 ± 41 g, p b 0.001 for STZ + C + L vs. STZ controls). 3.1. Glucose metabolism Treatment with carnosine reduced glucosuria, HbA1c, CML accumulation, and GlcNAc levels. Treatment with carnosine and lisinopril reduced glucosuria and GlcNAc levels (Table 1). Lisinopril treatment did not have any effect on glucose metabolism, CML levels remained high, GlcNAc concentration declined. 3.2. Cataract formation Cataract formation could not be reduced by treatment with lisinopril, but by treatment with carnosine alone and in combination with lisinopril (p b 0.01 for STZ + C and STZ + C + L vs. non-treated STZ rats, Table 1). 3.3. Kidney morphology and function Kidney weight/bodyweight ratio was used as a surrogate parameter of hyperfiltration following uninephrectomy. Upon diabetes induction and reduction of kidney mass, relative kidney weight increased significantly in untreated, diabetic rats (1.0 ± 0.1 vs. 0.3 ± 0.0% in controls, p b 0.001, Table 1) and could not be reduced consistently by lisinopril, carnosine and the combined treatment, respectively. Lisinopril and carnosine treatment reduced albuminuria significantly, lisinopril, however, to a much greater extent (p b 0.05). Combined treatment of carnosine and lisinopril did not further reduce urinary albuminuria. Podocyte number was preserved by carnosine, lisinopril and the combined therapy and correlated with albuminuria (r = −0.83, p b 0.05). Other indices of renal damage such as glomerulosclerosis, mesangial thickening, tubular atrophy, interstitial fibrosis and the creatinine clearance remained largely unchanged (Table 1). 3.4. Renal CN1 abundance and histidine dipeptide concentrations Immunostaining of kidney sections revealed a marked increase in CN1 abundance in diabetic rats. Treatment with carnosine normalized CN1 abundance, whereas lisinopril had no effect (Fig. 1). The combined treatment of C and L also normalized CN1 abundance in the kidney. Staining was largely confined to the cytoplasm of mesangial cells and
surrounding matrix. In line with CN1 abundance, anserine, the methylated form of carnosine and one of the main substrates of CN1, was reduced in STZ rats (76.1 ± 21.6 nmol/mg in control vs. 5.9 ± 3.2 nmol/mg in STZ, p b 0.001), however carnosine was not reduced in STZ rats (23.1 ± 12.9 nmol/mg in control vs. 25.6 ± 19.3 nmol/mg in STZ, p = n.s.). Treatment with carnosine increased carnosine and anserine levels (carnosine treatment: 26.4 ± 15.5/42.3 ± 26.9 nmol anserine/ carnosine per mg protein and combined treatment: 18.6 ± 6.4/ 39.5 ± 12.5 nmol/mg; p b 0.05 for both) whereas lisinopril treatment alone had no effect on anserine and carnosine levels (7.0 ± 4.1/ 19.4 ± 12.5 nmol/mg; p = n.s. vs. untreated STZ rats). 4. Discussion Experimental evidence suggests that the histidine dipeptide metabolism and carnosine/anserine availability play an important role in diabetes associated complication [14,21,22]. A considerable nephroprotective potential of exogenous carnosine administration in diabetes animals has been demonstrated recently by our and other groups [21, 23–26]. To compare this effect to the well described and clinically proven nephroprotective action of ACE inhibition [27] and to describe the potential benefits exerted by the combination of both compounds, we treated unilaterally nephrectomised STZ rats with lisinopril and carnosine and the combination of both. STZ induced diabetes is a well-defined model of DN. The additional uninephrectomy resulted in a threefold enlargement of the remaining kidney as previously described [28]. During the first week following STZ-treatment, blood glucose levels were maintained constant at 400– 600 mg/dl by repeated insulin treatment to allow for better survival and to minimize effects exerted by different degrees of diabetes rather than drug related effects on nephropathy. Both, the treatment with carnosine and with lisinopril exerted distinct nephroprotective effects. Both drugs substantially reduced albuminuria compared to untreated diabetic rats. Both drugs preserved podocyte number relative to endothelial cell number. Carnosine furthermore improved glucose metabolism, and reduced cataract formation. The combination of both drugs combined the beneficial effects seen with single treatments but without any synergy regarding proteinuria and preservation of podocytes. An impact of carnosine on glucose metabolism has recently been shown in diabetic mice (db/db) and the reaction of carnosine with glycated proteins and inhibition of advanced glycation end product formation was demonstrated [14]. In db/db mice, Sauerhöfer et al. demonstrated a significant correlation of L-carnosine levels with β-cell mass and carnosine treatment decreased blood levels of HbA1c, of CML, a surrogate parameter of AGE formation, and of N-acetylglucosamine, a derivate of glucose [14]. Of note, the beneficial effect of carnosine on hyperglycemia and glycative stress was less pronounced when carnosine treatment was combined with lisinopril. The recently demonstrated
V. Peters et al. / Regulatory Peptides 194–195 (2014) 36–40
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Fig. 1. Glomerular staining of CN1. Representative glomeruli of sections stained with anti-CN1 antibody are given for each study group. CN1 levels were increased under diabetic conditions (STZ) and could be reduced by carnosine treatment alone (STZ + C) and in combination with lisinopril (STZ + C + L) but not by lisinopril (STZ + L) alone.
suppression of diabetic cataract formation in STZ rats by carnosine [23] could be reconfirmed in the present study. In contrast, lisinopril had no impact on cataract formation. The observed suppression of Nacetylglucosamine levels by lisinopril is readily explained by a suppressive effect on glutamine fructose-6-phosphate amidotransferase (GFAT), the rate limiting enzyme of hexosamine-pathway. It can be induced not only by hyperglycemia but also by angiotensin II [29]. Within our experimental model of severe diabetes mellitus and uninephrectomy both treatments were able to reduce podocyte loss as related to capillary surface but not to prevent major histomorphometric transformation of the kidneys 24 weeks after induction of diabetes. The semiquantitative assessment revealed no changes regarding mesangial thickening, indices of glomerulosclerosis, tubular atrophy and interstitial fibrosis with lisinopril and carnosine, respectively. In line with this, kidney function as measured by creatinine clearance was not improved in any of the treatment groups. Still, the preservation of podocyte number by lisinopril and carnosine should be of interest. The improvement of podocyte number by ACE inhibition is well described [30]. We recently demonstrated that exogenous carnosine improves glomerular podocyte number in STZ-induced diabetic rats [24]. Interestingly, the podocyte preserving effect of lisinopril and carnosine was in the same range. In humans and in experimental models podocyte loss has been
linked to the development of proteinuria and glomerulosclerosis [31] and is predictive for the progression of DN [32]. In our model, improved glomerular podocyte number was also associated with a decline in albuminuria, a finding which has consistently been demonstrated for ACEinhibitors in humans. Diabetic patients that are homozygous for a certain CN1 allele, the so-called Mannheim allele, are less often affected by DN. This genotype is associated with a low serum CN1 activity [6]. In line with our findings in diabetic mice [21], kidney tissue CN1 abundance was increased in the STZ-rats. Renal tissue CN1 concentrations correlate with reduced tissue concentrations of anserine, the methylated form of carnosine [21]. Whether the nephroprotective effect of exogenous carnosine is exerted directly, e.g. via the antioxidative or carbonyl quenching action of the dipeptides, via suppression of ACE or indirectly via downregulation of CN1 as previously described for a diabetic mouse model by our group [21] deserves further study. In conclusion, carnosine treatment improves glucose homeostasis and mitigates diabetic sequelae such as cataract formation and DN. In an experimental model of severe and long lasting diabetes mellitus the non-renal effects of exogenous carnosine can be added to the beneficial effects of lisinopril, however without further improving nephroprotection.
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Conflict of interest The authors have not declared any conflict of interest. Acknowledgments Parts of this work were supported by a grant of the Federal Ministry of Education and Research (BMBF Förderkennzeichen 01GU0621) as well as grants by the Deutsche Forschungsgemeinschaft (SFB 1118 and Ma2510/3-1 and Zs17/5-1) and the Dr. Pfleger Foundation Bamberg, Germany. We would like to thank Rüdiger Waldherr (R.W.) for his help with the histological analyses and Kristina Klingbeil and Haang Jeung for technical assistance. References [1] Harvey JN. Trends in the prevalence of diabetic nephropathy in type 1 and type 2 diabetes. Curr Opin Nephrol Hypertens 2003;3:317–22. [2] Usrds: The United States renal data system. Am J Kidney Dis 2003;6(Suppl. 5): 1–230. [3] Tylicki L, Lizakowski S, Rutkowski B. Renin-angiotensin-aldosterone system blockade for nephroprotection: Current evidence and future directions. J Nephrol 2012; 6:900–10. [4] Vivian E, Mannebach C. Therapeutic approaches to slowing the progression of diabetic nephropathy - is less best? Drugs in Context 2013:212249. [5] Klahr S, Morrissey J. Comparative effects of ace inhibition and angiotensin ii receptor blockade in the prevention of renal damage. Kidney Int Suppl 2002;82:S23–6. [6] Janssen B, Hohenadel D, Brinkkoetter P, et al. Carnosine as a protective factor in diabetic nephropathy: Association with a leucine repeat of the carnosinase gene cndp1. Diabetes 2005;8:2320–7. [7] Teufel M, Saudek V, Ledig JP, et al. Sequence identification and characterization of human carnosinase and a closely related non-specific dipeptidase. J Biol Chem 2003;8:6251–531. [8] Peters V, Jansen EE, Jakobs C, et al. Anserine inhibits carnosine degradation but in human serum carnosinase (CN1) is not correlated with histidine dipeptide concentration. Clin Chim Acta 2011;3–4:263–7. [9] Peters V, Kebbewar M, Jansen EW, et al. Relevance of allosteric conformations and homocarnosine concentration on carnosinase activity. Amino Acids 2010;5: 1607–15. [10] Boldyrev A. Does carnosine possess direct antioxidant activity? Int J Biochem 1993; 8:1101–7. [11] Mozdzan M, Szemraj J, Rysz J, et al. Antioxidant properties of carnosine re-evaluated with oxidizing systems involving iron and copper ions. Basic Clin Pharmacol Toxicol 2005;96:352–60. [12] Alhamdani M, Al-Azzawie HF, Abbas FK. Decreased formation of advanced glycation end-products in peritoneal fluid by carnosine and related peptides. Perit Dial Int 2007;1:86–9.
[13] Aldini G, Orioli M, Rossoni G, et al. The carbonyl scavenger carnosine ameliorates dyslipidemia and renal function in Zucker obese rats. J Cell Mol Med 2011;15: 1339–13354. [14] Sauerhöfer S, Yuan G, Braun GS, et al. L-carnosine, a substrate of carnosinase-1, influences glucose metabolism. Diabetes 2007;10:2425–32. [15] Hou W, Chen HJ, Lin YH. Antioxidant peptides with angiotensin converting enzyme inhibitory activities and applications for angiotensin converting enzyme purification. J Agric Food Chem 2003;6:1706–17093. [16] Hipkiss A. Carnosine, a protective, anti-ageing peptide? Int J Biochem Cell Biol 1998: 863–86. [17] Hu YY, Ye SD. Experimental models of type 2 diabetic nephropathy. Chin Med J 2013;3:574–7. [18] Kang MJ, Ingram A, Ly H, et al. Effects of diabetes and hypertension on glomerular transforming growth factor-beta receptor expression. Kidney Int 2000;4:1677–85. [19] Obermueller N, Kranzlin B, Blum WF, et al. An endocytosis defect as a possible cause of proteinuria in polycystic kidney disease. Am J Physiol Renal Physiol 2001;280: F244–53. [20] Schönherr J. Analysis of products of animal origin in feeds by determination of carnosine and related dipeptides by high-performance liquid chromatography. J Agric Food Chem 2002;7:1945–50. [21] Peters V, Schmitt CP, Zschocke J, et al. Carnosine treatment largely prevents alterations of renal carnosine metabolism in diabetic mice. Amino Acids 2012;6:2411–6. [22] Ansurudeen I, Sunkari VG, Grunler J, et al. Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes. Amino Acids 2012;1:127–34. [23] Pfister F, Riedl E, Wang Q, et al. Oral carnosine supplementation prevents vascular damage in experimental diabetic retinopathy. Cell Physiol Biochem 2011;1:125–36. [24] Riedl E, Pfister F, Braunagel M, et al. Carnosine prevents apoptosis of glomerular cells and podocyte loss in stz diabetic rats. Cell Physiol Biochem 2011;2:279–88. [25] Aldini G, Orioli M, Rossoni G, et al. The carbonyl scavenger carnosine ameliorates dyslipidaemia and renal function in zucker obese rats. J Cell Mol Med 2011;6: 1339–54. [26] Menini S, Iacobini C, Ricci C, et al. D-carnosine octylester attenuates atherosclerosis and renal disease in ApoE null mice fed a western diet through reduction of carbonyl stress and inflammation. Br J Pharmacol 2012;4:1344–56. [27] Fried LF, Emanuele N, Zhang JH, et al. Combined angiotensin inhibition for the treatment of diabetic nephropathy. N Engl J Med 2013;20:1892–903. [28] Babizhayev MA, Lankin VZ, Savel'Yeva EL, et al. Diabetes mellitus: Novel insights, analysis and interpretation of pathophysiology and complications management with imidazole-containing peptidomimetic antioxidants. Recent Pat Drug Deliv Formul 2013;3:216–56. [29] James LR, Ingram A, Ly H, et al. Angiotensin II activates the GFAT promoter in mesangial cells. Am J Physiol Renal Physiol 2001;1:F151–62. [30] Macconi D, Sangalli F, Bonomelli M, et al. Podocyte repopulation contributes to regression of glomerular injury induced by ACE inhibition. Am J Pathol 2009;3: 797–807. [31] Kriz W, Gretz N, Lemley KV. Progression of glomerular diseases: Is the podocyte the culprit? Kidney Int 1998;3:687–97. [32] Meyer TW, Bennett PH, Nelson RG. Podocyte number predicts long-term urinary albumin excretion in pima indians with type II diabetes and microalbuminuria. Diabetologia 1999;11:1341–4.
Contents lists available at ScienceDirect
Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
Carnosine treatment in combination with ACE inhibition in diabetic rats V. Peters a,⁎,1, E. Riedl b,1, M. Braunagel b, S. Höger b, S. Hauske b, F. Pfister b, J. Zschocke c, B. Lanthaler c, U. Benck b, H.-P. Hammes b, B.K. Krämer b, C.P. Schmitt a, B.A. Yard b, H. Köppel b a b c
Centre for Pediatric and Adolescent Medicine, University of Heidelberg, Heidelberg, Germany Vth Department of Medicine, Nephrology, Endocrinology, Diabetology & Rheumatology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Division of Human Genetics, Medical University Innsbruck, Austria
a r t i c l e
i n f o
Article history: Received 16 June 2014 Received in revised form 8 September 2014 Accepted 11 September 2014 Available online 16 September 2014 Keywords: Diabetic nephropathy Carnosine Diabetes mellitus Cataract Proteinuria
a b s t r a c t In humans, we reported an association of a certain allele of carnosinase gene with reduced carnosinase activity and absence of nephropathy in diabetic patients. CN1 degrades histidine dipeptides such as carnosine and anserine. Further, we and others showed that treatment with carnosine improves renal function and wound healing in diabetic mice and rats. We now investigated the effects of carnosine treatment alone and in combination with ACE inhibition, a clinically established nephroprotective drug in diabetic nephropathy. Male Sprague–Dawley rats were injected i.v. with streptozotocin (STZ) to induce diabetes. After 4 weeks, rats were unilaterally nephrectomized and randomized for 24 weeks of treatment with carnosine, lisinopril or both. Renal CN1 protein concentrations were increased under diabetic conditions which correlated with decreased anserine levels. Carnosine treatment normalized CN1 abundance and reduced glucosuria, blood concentrations of glycosylated hemoglobin (HbA1c), carboxyl-methyl lysine (CML), N-acetylglucosamine (GlcNac; all p b 0.05 vs. non-treated STZ rats), reduced cataract formation (p b 0.05) and urinary albumin excretion (p b 0.05), preserved podocyte number (p b 0.05) and normalized the increased renal tissue CN1 protein concentration. Treatment with lisinopril had no effect on HbA1C, glucosuria, cataract formation and CN1 concentration, but reduced albumin excretion rate more effectively than carnosine treatment (p b 0.05). Treatment with both carnosine and lisinopril combined the effects of single treatment, albeit without additive effect on podocyte number or albuminuria. Increased CN1 amount resulted in decreased anserine levels in the kidney. Both carnosine and lisinopril exert distinct beneficial effects in a standard model of diabetic nephropathy. Both drugs administered together combine the respective effects of single treatment, albeit without exerting additive nephroprotection. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Diabetic nephropathy (DN) is the most common cause of end stage renal disease in the Western world. Despite increasing knowledge on the pathogenesis of DN and potential therapeutic interventions, progression to end stage renal disease still occurs in a substantial number of diabetic patients [1,2]. At present, pharmacological nephroprotection is largely limited to tight glucose and blood pressure control and antiproteinuric intervention using antagonists of the renin–angiotensin system [3]. ACE inhibitors are known to reduce proteinuria and have been the first-line agents in the management of diabetic nephropathy [4,5] during the last years.
⁎ Corresponding author at: Centre for Pediatric and Adolescent Medicine, Metabolic Centre, University of Heidelberg, Im Neuenheimer Feld 669, 69120 Heidelberg, Germany. Tel.: +49 6221 5631715; fax: +49 6221 565565. E-mail address: [email protected] (V. Peters). 1 Both authors contributed equally.
http://dx.doi.org/10.1016/j.regpep.2014.09.005 0167-0115/© 2014 Elsevier B.V. All rights reserved.
A genome-wide linkage study revealed strong evidence for linkage of DN to the CNDP1 gene, encoding serum carnosinase (CN1) [6]. (CTG)5 homozygous individuals display low CN1 activity in serum [6] and have a reduced susceptibility to DN. Carnosine, the natural substrate for CN1 [7–9] has several protective functions, such as antioxidant activity [10], scavenging of reactive oxygen species [11] and glycation inhibition [12]. In both in vitro and in vivo models high carnosine levels protect against diabetic complications by improving glucose metabolism, reduction of carbonyl stress and minimizing formation of advanced glycation end products (AGEs) [13–15]. An intriguing treatment approach in patients with diabetic nephropathy should be the combination of the direct nephroprotective action of ACE inhibitors and the carnosine mediated nephroprotection which is based on a variety of protective functions, such as scavenging of reactive oxygen species [11], inhibition of angiotensin converting enzyme [12], of protein glycation [12], of cellular senescence [16] and of matrix protein synthesis [6]. To assess the potential of ACE inhibition and pharmacological doses of carnosine in DN, we treated diabetic rats for 24 weeks with lisinopril, carnosine and a combination of both. Diabetes mellitus was
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induced by streptozotocin (STZ) which results in a well-defined nephropathy. The rats were moreover uninephrectomized in order to reduce the time until development of overt DN [17,18]. These animals underwent detailed analysis of glucose metabolism, carbonyl stress, renal morphology and renal function. 2. Material and methods 2.1. Animal and induction of experimental diabetes Diabetes was induced in male Sprague–Dawley rats weighing 350 to 420 g by a single intravenous injection of 50 mg/kg bodyweight streptozotocin (STZ; Sigma-Aldrich Chemical, Deisenhofen, Germany). A second STZ injection was given in case blood glucose levels were below 400 mg/dl. Blood glucose levels were monitored regularly, i.e. daily in the first week after STZ-injection and weekly thereafter. Insulin glargine (Lantus, Sanofi-Aventis, Frankfurt, Germany) was injected s.c. in individually adjusted doses to maintain a blood glucose level of 400–600 mg/dl to allow for stable diabetes mellitus. Injection of insulin was daily in the first week after STZ-injection and thrice weekly thereafter. Rats were housed in a climate-controlled animal house with a 12 h light cycle and free access to standard diet and drug supplemented tap water. 2.2. Experimental design Four weeks following STZ injection unilateral nephrectomy was performed and 52 rats were randomized to four groups, L-carnosine treatment (C, 1 g/kg bodyweight, Flamma S.p.A, Bergamo, Italy), lisinopril treatment (L, 2.5 mg/kg bodyweight, AstraZeneca GmbH, Wedel, Germany), the respective combined treatment (L + C) and untreated rats (STZ). Ten additional rats without preceding STZ injection served as age matched untreated controls (C). Treatment was administered via drinking water for 24 weeks. Blood glucose was controlled by thrice weekly insulin injections in all STZ groups. Drinking volumes were recorded daily, and bodyweight assessed twice weekly and drug dosing adjusted accordingly. Body weight and blood glucose levels and insulin doses administered did not differ between groups. Animals were placed in metabolic cages on week 28 post-STZ injection for 24 h. Following sacrifice, organs were removed immediately and either stored in liquid nitrogen or 4% w/v paraformaldehyde, respectively. Blood samples were collected for the determination of glycated hemoglobin concentration by affinity chromatography (MicromatII™; Bio-Rad Laboratories GmbH, Munich, Germany). Serum creatinine was measured by immunoassay (Dimension; Dade Behring, Germany), urinary albumin by ELISA as previously published [19] and serum carboxyl-methyl lysine was measured using a commercial ELISA (CML-ELISA Kit, Cell Biolabs, BIOCAT GmbH, Germany). All animal procedures were approved by the Regierungspräsidium Karlsruhe AZ 35-9185.81/G-5306. 2.3. Quantification of cataract formation The formation of cataract was assessed semiquantitatively by macroscopic evaluation in a blinded setting using a score of 0–4, with 0 representing no cataract formation, 1 incipient cataract, 2 incipient cataract of both eyes or moderate cataract in one eye, 3 moderate cataract of both eyes and 4 with both eyes being completely opaque. 2.4. Histological analysis Paraffin embedding of kidneys was performed using routine procedures and sections were stained with hematoxylin-eosin. Tissue sections were evaluated by two different, blinded individuals scoring a minimum of 20 microscopic fields per kidney (H. Köppel and R Waldherr). A semiquantitative score was used for grading of glomerular size, mesangial matrix expansion, interstitial fibrosis and tubular
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atrophy. The histologic grading scale ranged was from 0 to 3 (0 = not present, 1 = mild alteration, 2 = moderate alteration, and 3 = severe alteration). For determination of podocyte number, sections were stained for markers of podocytes (WT-1) and endothelial cells (CD31) according to standard protocols. Renal carnosinase abundance was assessed by immunohistochemistry, using an anti-CN1 (RYSK 173) monoclonal antibody (generated by our group; [9]). Staining intensity was classified as minute (= 1), moderate (= 2) and high (= 3), respectively. 2.5. N-Acetylglucosamine immunoblotting Shock frozen kidneys were homogenized in lysis buffer [150 mM NaCl, 10 mM Tris–HCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0 · 5% Na-deoxycholate, 1 μM dithiothreitol (DTT), 1 μg/ml aprotinin, 1 mM phenylmethylsulphonyl fluoride (PMSF)] using a Polytron homogenizer (IKA Labortechnik/Fischer Scientific, Schwerte, Germany) and incubated for 5 min on ice. Lysates were centrifuged (15 min at 16 000 g), protein concentrations in the supernatants measured using Coomassie Reagent (Pierce, Rockford, IL, USA). Proteins were separated on a 10% w/v sodium dodecyl sulfate–polyacrylamide gel by electrophoresis (SDS-PAGE) and semidry blotted onto a polyvinyl difluoride (PVDF) membrane (Roche Diagnostics, Mannheim, Germany). The membrane was incubated overnight in Tris-buffered saline (TBS) (10 mM Tris–HCl, pH 8 · 0, 150 mM NaCl) containing 5% w/v milk powder. Thereafter, the blots were incubated for 1 h with specific primary antibody for N-Acetylglucosamine-modified proteins (GlucNAc; Abcamplc, Cambridge, United Kingdom), followed by incubation with appropriate horseradish peroxidase (HRP) secondary antibody. Proteins were visualized by enhanced chemoluminescence technology according to the manufacturer's instructions (Pierce). To confirm equal protein loading, membranes were stripped with 62 · 5 mM Tris–HCl, 2% w/v SDS and 100 mM β-mercaptoethanol and incubated with antibodies against GAPDH (Abcamplc, Cambridge, United Kingdom). Intensity of specific bands was measured by chemiluminescence using the ImageJ 1.36b software and means of the absolute values of each group were expressed relative to WT. 2.6. Carnosine and anserine concentrations Kidneys were removed and immediately homogenized in cold buffer containing 20 mM HEPES, 1 mM ethylene glycol-tetra-acetic acid (EGTA), 210 mM mannitol and 70 mM sucrose per gram tissue, pH 7.2. The homogenate was centrifuged at 1500 ×g for 5 min at 4 °C. Carnosine and anserine levels were measured in the supernatant by highperformance liquid chromatography as previously described [20]. Briefly, the samples were diluted with sulfosalicylic acid to precipitate proteins. After derivatization with carbazole-9-carbonyl chloride (CFC), the samples underwent liquid chromatography and quantification by fluorescence. All samples were measured twice, and one sample was spiked with the standard to identify anserine. 2.7. Statistical analyses All values are shown as mean ± SD if not indicated otherwise. For survival, Kaplan–Meier survival estimates were calculated. Differences between treatment groups were analyzed by one-way ANOVA followed by t-test and Mann–Whitney in case of non-Gaussian distribution. Values of p b 0.05 were considered to be significant. 3. Results STZ treatment increased glucosuria, glycosylated hemoglobin (HbA1c), carboxy-methyl lysine (CML) and N-Acetylglucosamine (GlcNAc) blood levels. Significant cataract developed over the study period, kidney morphology and function declined (Table 1), and renal
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Table 1 Comparison between carnosine and lisinopril treatment in diabetic rats. Parameter
Control
STZ no treatment (STZ)
STZ + carnosinetreated (C)
STZ + lisinopriltreated (L)
STZ + carnosine + lisinopril treated (L + C)
Glucose metabolism Glucosuria (mg glucose/mg creatinine) Glycosylated hemoglobin (HbA1c) % (mmol/mol) Carboxyl-methyl lysine (CML) ng/mg protein normalized for control N-Acetylglucosamine (GlcNAc) ratio to control Cataract score 0–4, median (min, max)
0.1 ± 0.1 4.9 ± 1.0 1.0 ± 0 1.0 ± 0.1 0 (0, 0)
1096 ± 560 12.8 ± 2.4 16.3 ± 3.5 3.2 ± 1.0 1 (0, 3)
310 ± 378a 9.3 ± 2.2a 12.7 ± 2.0a 1.8 ± 0.9a 0 (0, 0)a
1214 ± 547b 13.1 ± 1.9b 15.4 ± 2.0 1.7 ± 0.9a 2 (0, 4)b
266 ± 234a, c 11.8 ± 1.2b 17.3 ± 2.2b 1.7 ± 0.5a 0 (0, 3)a, c
Renal morphology/function Kidney/body weight[%] Podocytes per endothelial cells CreaCl (ml/min/100 g bw) Urinary albumin/creatinine ratio
0.3 ± 0.0 0.31 ± 0.04 0.9 ± 0.3 0.3 ± 0.3
1.0 ± 0.1 0.18 ± 0.02 0.6 ± 0.3 34.0 ± 39.0
0.8 ± 0.1 0.23 ± 0.02a 0.8 ± 0.2 14.6 ± 21.7a
0.8 ± 0.1 0.28 ± 0.04a, b 0.9 ± 0.2 2.0 ± 2.9a, b
0.8 ± 0.1 0.25 ± 0.02a, c 0.6 ± 0.1 1.1 ± 1.0a, b
Outcome of treatment with carnosine (C), lisinopril (L) or both (C + L) of STZ rats. CreaCl = creatinine clearance. a = p b 0.05 vs. untreated STZ; b = p b 0.05 vs. STZ + C; c = p b 0.05 vs. STZ + L.
tissue CN1 abundance increased (Fig. 1). Body weight gain was significantly impaired in STZ rats after the onset of hyperglycemia and resulted in reduced body weight at the end of the study (430 ± 39 vs. 509 ± 53 g in STZ vs. non-STZ treated control rats; p b 0.05). Treatment with carnosine but not with lisinopril increased bodyweight gain of diabetic rats (body weight gain STZ controls: 40 ± 63 g; STZ + C: 77 ± 70 g; STZ + L: 11 ± 45 g; STZ + C + L: 104 ± 41 g, p b 0.001 for STZ + C + L vs. STZ controls). 3.1. Glucose metabolism Treatment with carnosine reduced glucosuria, HbA1c, CML accumulation, and GlcNAc levels. Treatment with carnosine and lisinopril reduced glucosuria and GlcNAc levels (Table 1). Lisinopril treatment did not have any effect on glucose metabolism, CML levels remained high, GlcNAc concentration declined. 3.2. Cataract formation Cataract formation could not be reduced by treatment with lisinopril, but by treatment with carnosine alone and in combination with lisinopril (p b 0.01 for STZ + C and STZ + C + L vs. non-treated STZ rats, Table 1). 3.3. Kidney morphology and function Kidney weight/bodyweight ratio was used as a surrogate parameter of hyperfiltration following uninephrectomy. Upon diabetes induction and reduction of kidney mass, relative kidney weight increased significantly in untreated, diabetic rats (1.0 ± 0.1 vs. 0.3 ± 0.0% in controls, p b 0.001, Table 1) and could not be reduced consistently by lisinopril, carnosine and the combined treatment, respectively. Lisinopril and carnosine treatment reduced albuminuria significantly, lisinopril, however, to a much greater extent (p b 0.05). Combined treatment of carnosine and lisinopril did not further reduce urinary albuminuria. Podocyte number was preserved by carnosine, lisinopril and the combined therapy and correlated with albuminuria (r = −0.83, p b 0.05). Other indices of renal damage such as glomerulosclerosis, mesangial thickening, tubular atrophy, interstitial fibrosis and the creatinine clearance remained largely unchanged (Table 1). 3.4. Renal CN1 abundance and histidine dipeptide concentrations Immunostaining of kidney sections revealed a marked increase in CN1 abundance in diabetic rats. Treatment with carnosine normalized CN1 abundance, whereas lisinopril had no effect (Fig. 1). The combined treatment of C and L also normalized CN1 abundance in the kidney. Staining was largely confined to the cytoplasm of mesangial cells and
surrounding matrix. In line with CN1 abundance, anserine, the methylated form of carnosine and one of the main substrates of CN1, was reduced in STZ rats (76.1 ± 21.6 nmol/mg in control vs. 5.9 ± 3.2 nmol/mg in STZ, p b 0.001), however carnosine was not reduced in STZ rats (23.1 ± 12.9 nmol/mg in control vs. 25.6 ± 19.3 nmol/mg in STZ, p = n.s.). Treatment with carnosine increased carnosine and anserine levels (carnosine treatment: 26.4 ± 15.5/42.3 ± 26.9 nmol anserine/ carnosine per mg protein and combined treatment: 18.6 ± 6.4/ 39.5 ± 12.5 nmol/mg; p b 0.05 for both) whereas lisinopril treatment alone had no effect on anserine and carnosine levels (7.0 ± 4.1/ 19.4 ± 12.5 nmol/mg; p = n.s. vs. untreated STZ rats). 4. Discussion Experimental evidence suggests that the histidine dipeptide metabolism and carnosine/anserine availability play an important role in diabetes associated complication [14,21,22]. A considerable nephroprotective potential of exogenous carnosine administration in diabetes animals has been demonstrated recently by our and other groups [21, 23–26]. To compare this effect to the well described and clinically proven nephroprotective action of ACE inhibition [27] and to describe the potential benefits exerted by the combination of both compounds, we treated unilaterally nephrectomised STZ rats with lisinopril and carnosine and the combination of both. STZ induced diabetes is a well-defined model of DN. The additional uninephrectomy resulted in a threefold enlargement of the remaining kidney as previously described [28]. During the first week following STZ-treatment, blood glucose levels were maintained constant at 400– 600 mg/dl by repeated insulin treatment to allow for better survival and to minimize effects exerted by different degrees of diabetes rather than drug related effects on nephropathy. Both, the treatment with carnosine and with lisinopril exerted distinct nephroprotective effects. Both drugs substantially reduced albuminuria compared to untreated diabetic rats. Both drugs preserved podocyte number relative to endothelial cell number. Carnosine furthermore improved glucose metabolism, and reduced cataract formation. The combination of both drugs combined the beneficial effects seen with single treatments but without any synergy regarding proteinuria and preservation of podocytes. An impact of carnosine on glucose metabolism has recently been shown in diabetic mice (db/db) and the reaction of carnosine with glycated proteins and inhibition of advanced glycation end product formation was demonstrated [14]. In db/db mice, Sauerhöfer et al. demonstrated a significant correlation of L-carnosine levels with β-cell mass and carnosine treatment decreased blood levels of HbA1c, of CML, a surrogate parameter of AGE formation, and of N-acetylglucosamine, a derivate of glucose [14]. Of note, the beneficial effect of carnosine on hyperglycemia and glycative stress was less pronounced when carnosine treatment was combined with lisinopril. The recently demonstrated
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Fig. 1. Glomerular staining of CN1. Representative glomeruli of sections stained with anti-CN1 antibody are given for each study group. CN1 levels were increased under diabetic conditions (STZ) and could be reduced by carnosine treatment alone (STZ + C) and in combination with lisinopril (STZ + C + L) but not by lisinopril (STZ + L) alone.
suppression of diabetic cataract formation in STZ rats by carnosine [23] could be reconfirmed in the present study. In contrast, lisinopril had no impact on cataract formation. The observed suppression of Nacetylglucosamine levels by lisinopril is readily explained by a suppressive effect on glutamine fructose-6-phosphate amidotransferase (GFAT), the rate limiting enzyme of hexosamine-pathway. It can be induced not only by hyperglycemia but also by angiotensin II [29]. Within our experimental model of severe diabetes mellitus and uninephrectomy both treatments were able to reduce podocyte loss as related to capillary surface but not to prevent major histomorphometric transformation of the kidneys 24 weeks after induction of diabetes. The semiquantitative assessment revealed no changes regarding mesangial thickening, indices of glomerulosclerosis, tubular atrophy and interstitial fibrosis with lisinopril and carnosine, respectively. In line with this, kidney function as measured by creatinine clearance was not improved in any of the treatment groups. Still, the preservation of podocyte number by lisinopril and carnosine should be of interest. The improvement of podocyte number by ACE inhibition is well described [30]. We recently demonstrated that exogenous carnosine improves glomerular podocyte number in STZ-induced diabetic rats [24]. Interestingly, the podocyte preserving effect of lisinopril and carnosine was in the same range. In humans and in experimental models podocyte loss has been
linked to the development of proteinuria and glomerulosclerosis [31] and is predictive for the progression of DN [32]. In our model, improved glomerular podocyte number was also associated with a decline in albuminuria, a finding which has consistently been demonstrated for ACEinhibitors in humans. Diabetic patients that are homozygous for a certain CN1 allele, the so-called Mannheim allele, are less often affected by DN. This genotype is associated with a low serum CN1 activity [6]. In line with our findings in diabetic mice [21], kidney tissue CN1 abundance was increased in the STZ-rats. Renal tissue CN1 concentrations correlate with reduced tissue concentrations of anserine, the methylated form of carnosine [21]. Whether the nephroprotective effect of exogenous carnosine is exerted directly, e.g. via the antioxidative or carbonyl quenching action of the dipeptides, via suppression of ACE or indirectly via downregulation of CN1 as previously described for a diabetic mouse model by our group [21] deserves further study. In conclusion, carnosine treatment improves glucose homeostasis and mitigates diabetic sequelae such as cataract formation and DN. In an experimental model of severe and long lasting diabetes mellitus the non-renal effects of exogenous carnosine can be added to the beneficial effects of lisinopril, however without further improving nephroprotection.
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Conflict of interest The authors have not declared any conflict of interest. Acknowledgments Parts of this work were supported by a grant of the Federal Ministry of Education and Research (BMBF Förderkennzeichen 01GU0621) as well as grants by the Deutsche Forschungsgemeinschaft (SFB 1118 and Ma2510/3-1 and Zs17/5-1) and the Dr. Pfleger Foundation Bamberg, Germany. We would like to thank Rüdiger Waldherr (R.W.) for his help with the histological analyses and Kristina Klingbeil and Haang Jeung for technical assistance. References [1] Harvey JN. Trends in the prevalence of diabetic nephropathy in type 1 and type 2 diabetes. Curr Opin Nephrol Hypertens 2003;3:317–22. [2] Usrds: The United States renal data system. Am J Kidney Dis 2003;6(Suppl. 5): 1–230. [3] Tylicki L, Lizakowski S, Rutkowski B. Renin-angiotensin-aldosterone system blockade for nephroprotection: Current evidence and future directions. J Nephrol 2012; 6:900–10. [4] Vivian E, Mannebach C. Therapeutic approaches to slowing the progression of diabetic nephropathy - is less best? Drugs in Context 2013:212249. [5] Klahr S, Morrissey J. Comparative effects of ace inhibition and angiotensin ii receptor blockade in the prevention of renal damage. Kidney Int Suppl 2002;82:S23–6. [6] Janssen B, Hohenadel D, Brinkkoetter P, et al. Carnosine as a protective factor in diabetic nephropathy: Association with a leucine repeat of the carnosinase gene cndp1. Diabetes 2005;8:2320–7. [7] Teufel M, Saudek V, Ledig JP, et al. Sequence identification and characterization of human carnosinase and a closely related non-specific dipeptidase. J Biol Chem 2003;8:6251–531. [8] Peters V, Jansen EE, Jakobs C, et al. Anserine inhibits carnosine degradation but in human serum carnosinase (CN1) is not correlated with histidine dipeptide concentration. Clin Chim Acta 2011;3–4:263–7. [9] Peters V, Kebbewar M, Jansen EW, et al. Relevance of allosteric conformations and homocarnosine concentration on carnosinase activity. Amino Acids 2010;5: 1607–15. [10] Boldyrev A. Does carnosine possess direct antioxidant activity? Int J Biochem 1993; 8:1101–7. [11] Mozdzan M, Szemraj J, Rysz J, et al. Antioxidant properties of carnosine re-evaluated with oxidizing systems involving iron and copper ions. Basic Clin Pharmacol Toxicol 2005;96:352–60. [12] Alhamdani M, Al-Azzawie HF, Abbas FK. Decreased formation of advanced glycation end-products in peritoneal fluid by carnosine and related peptides. Perit Dial Int 2007;1:86–9.
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