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ADMA elevation does not exacerbate development of diabetic nephropathy in mice with streptozotocin-induced diabetes mellitus
Atherosclerosis Supplements 40 (2019) 100e105
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ADMA elevation does not exacerbate development of diabetic nephropathy in mice with streptozotocin-induced diabetes mellitus Roman N. Rodionov a, *, 1, Natalia Jarzebska a, b, 1, Alfred Schneider c, Annett Rexin d, € ger e, Jan Sradnick d, Silke Brilloff a, Jens Martens-Lobenhoffer e, Stefanie M. Bode-Bo d d a d Vladimir Todorov , Christian Hugo , Norbert Weiss , Bernd Hohenstein €t Dresden, University Center for Vascular Medicine, Department of Internal Medicine III, University Hospital Carl Gustav Carus, Technische Universita Fetscherstrasse 74, 01307, Dresden, Germany €t Dresden, Germany Department of Anesthesiology and Critical Care Medicine, University Hospital Dresden, Technische Universita c €t Dresden, Fetscherstrasse 74, 01307, Dresden, Germany Department of Visceral Surgery, University Hospital Carl Gustav Carus, Technische Universita d €t Dresden, Fetscherstrasse 74, Division of Nephrology, Department of Internal Medicine III, University Hospital Carl Gustav Carus, Technische Universita 01307, Dresden, Germany e Institute of Clinical Pharmacology, Otto-von-Guericke University, Leipziger Str.44, 39120, Magdeburg, Germany a
b
a b s t r a c t Keywords: Asymmetric dimethylarginine Diabetic nephropathy Dimethylarginine dimethylaminohydrolase 1 Mouse model Streptozotocin-induced diabetes mellitus
Background and aims: Cardiovascular disease is nowadays the major cause of mortality and morbidity worldwide. The risk of developing cardiovascular disease is significantly increased in patients with diabetic nephropathy. It has been suggested that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthases (NOS), may play an important role in the pathogenesis of diabetic nephropathy. ADMA is mainly metabolized by dimethylarginine dimethylaminohydrolase 1 (DDAH1). The goal of this study was to test the hypothesis that elevation of systemic ADMA levels by knocking out DDAH1 would exacerbate functional and structural glomerular abnormalities in a murine model of diabetic nephropathy. Methods: Streptozotocin (STZ) was used to induce diabetes in adult DDAH1 knock-out and wild type mice. Healthy mice served as controls. Mice were sacrificed after 20 weeks of diabetes. Plasma ADMA levels were assessed by isotope-dilution tandem mass spectrometry and albumin by ELISA. Kidneys were used for FACS analysis and were also stained for markers of inflammation, cell proliferation, glomerular cells and cell matrix. Results: STZ led to development of diabetes mellitus in all injected animals. Deficiency of DDAH1 led to a significant increase in plasma ADMA levels in healthy and diabetic mice. The diabetic state itself did not influence systemic ADMA levels. Diabetic mice of both genotypes developed albuminuria and had increased glomerulosclerosis index. There were no changes in desmin expression, glomerular cell proliferation rate, matrix expansion and expression of Mac-2 antigen in the diabetic mice of both genotypes as compared to the healthy ones. Conclusions: In summary, STZ-induced diabetes led to the development of early features of diabetic nephropathy. Deficiency of DDAH1 and subsequent increase in systemic ADMA levels did not exacerbate these changes, indicating that ADMA is not the major mediator of diabetic nephropathy in this experiment model. © 2019 Elsevier B.V. All rights reserved.
1. Introducion * Corresponding author. University Center for Vascular Medicine and Division of Angiology, Department of Internal Medicine III, University Hospital Carl Gustav Carus at the Technische Universit€ at Dresden, Fetscherstraße 74, 01307, Dresden, Germany. E-mail address: [email protected] (R.N. Rodionov). 1 Both Authors contributed equally to this work. https://doi.org/10.1016/j.atherosclerosissup.2019.08.040 1567-5688/© 2019 Elsevier B.V. All rights reserved.
In the Western countries end stage renal failure is most commonly caused by diabetic nephropathy [1]. One third of patients suffering from diabetes will develop diabetic nephropathy at some stage of the disease, which puts them at high risk for end stage renal disease and, in parallel, for severe micro- and
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macrovascular complications [2]. The earliest clinically detectable consequences of diabetic nephropathy include glomerular hyperfiltration, infiltration of inflammatory cells, and development of capillary leakage leading to microalbuminuria [3]. Nitric oxide (NO) is believed to be involved in the early and late alterations of glomerular hemodynamics due to diabetes [4]. It is hypothesized that increased levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, can be at least to some extent responsible for the decreased bioavailability of NO in diabetic nephropathy. ADMA is an endogenous dimethylated derivative of L-arginine, which was shown to compete with L-arginine for the active site of NOS and inhibit NO production in vitro and in vivo. [5] ADMA is constantly produced from the proteolysis of proteins that are methylated on arginine residues and hydrolysed to citrulline and methylamines by dimethylarginine dimethylaminohydrolase (DDAH). DDAH1 is the major isoform responsible for this reaction and it is widely expressed in vascular and nonvascular tissues. ADMA was demonstrated to be elevated in hypertension, congestive heart failure, atherosclerosis, stroke, hypercholesterolemia, hyperhomocysteinemia, peripheral arterial disease, preeclampsia, chronic kidney disease (CKD), and diabetes mellitus [5]. Elevation of plasma ADMA levels in animal models leads to endothelial dysfunction, increased systemic vascular resistance, and elevated blood pressure [6]. The association between diabetes, diabetic complications and ADMA has been shown in many reports. Plasma ADMA levels are elevated in patients with insulin resistance [7], diabetes mellitus type 1 [8] and type 2 [9], ADMA levels predict cardiovascular events in patients with diabetes mellitus [10]. Plasma ADMA was also shown to be associated with retinopathy in both type 1 and type 2 diabetes [11,12]. Elevation of ADMA has been shown to be a potent predictor of the progression of nephropathy in adult Japanese type 2 diabetic patients [13]. Plasma concentration of ADMA predicts cardiovascular morbidity and mortality in type 1 diabetic patients with diabetic nephropathy [14,15]. The kidney is a major organ regulating plasma ADMA levels [5], while CKD is a major cause of ADMA elevation [16]. ADMA accumulates in the plasma of patients with end-stage renal disease (ESRD), while dialysis decreases ADMA levels and improves endothelial function [17]. Plasma ADMA is a strong and independent predictor of progression of CKD into ESRD [18]. Furthermore, plasma ADMA is independently associated with carotid intimamedia thickness (IMT) in patients with CKD [19] and is a strong and independent predictor of overall mortality and cardiovascular outcome in ESRD [20]. Infusion of ADMA in healthy volunteers decreases renal blood flow and increases renovascular resistance, sodium retention and systemic blood pressure [21], suggesting potential mechanisms for ADMA to accelerate progression of renal failure and promote cardiovascular complications. The aim of the current study was to determine whether elevation of the plasma levels of ADMA by deficiency of dimethylarginine dimethylaminohydrolase 1 (DDAH1) exacerbates progression of diabetes-induced impairment of glomerular morphology and function. 2. Materials and methods 2.1. Animals Eight to ten week-old male DDAH1 gene-deficient (DDAH1 KO) mice and their gender- and age-matched wild type littermates (wt) controls were used for the experiments. All animals had the same, C57BL/6J genetic background. The DDAH1 KO mice were a kind gift from Prof. Yingjie Chen from University of Minnesota, USA and they
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were bred from heterozygous parents and the offspring was genotyped with the following primer pairs: wild type allele forward 50 AAT CTG CAC AGA AGG CCC TCA A-30 , reverse 50 -GGA GGA TCC ATT GTT ACA AGC CCT TAA CGC-3’; knock-out allele forward 50 -TGC AGG TCG AGG GAC CTA ATA ACT-30 , reverse 50 -AAC CAC ACT GCT AGA TGA AGT TCC-3’. All animals were housed at constant humidity (60 ± 5%), temperature (24 ± 1 C), and a 12 h light/dark cycle (6 a.m. to 6 p.m. light). Mice had unlimited access to water and food. All protocols for animal experiments were approved by the ethical committee of the Technische Universit€ at Dresden. 2.2. Induction of diabetes mellitus Diabetes mellitus was induced with a single intraperitoneal injection of streptozotocin in the dose of 180 mg/kg body weight. The animals were considered diabetic if the blood glucose levels reached 350 mg/ml or more. Mice were divided into four groups: healthy wild type (n ¼ 6), diabetic wild type (n ¼ 15), healthy DDAH1 KO (n ¼ 5), diabetic DDAH1 KO (n ¼ 15). All animals were sacrificed after 20 weeks of the experiment. Two diabetic DDAH1 KO and three diabetic wild type mice died in the course of the disease and were excluded from the analysis. 2.3. Collection of urine, plasma and tissue samples 24-h urine was collected in metabolic cages at the last day of the experiment and stored at 80 C. Mice were subjected to isofluorane anesthesia and blood was collected by cardiac puncture into EDTA containing tubes (final concentration 5 mmol/L). Mice were subsequently perfused with 0.9% (w/v) NaCl-solution. Left and right kidneys were harvested, left ones were fixed in zinc solution for 24 h and embedded in paraffin. Plasma was separated by centrifugation and stored at 80 C. Kidney samples were collected and flash-frozen immediately after the sacrifice of the animals and stored at 80 C until further analysis. 2.4. Measurement of blood glucose, ADMA and SDMA in plasma, creatinine and albumin The glucose levels were measured in a small drop of blood from the tail vein using the Accu-Chek® Aviva Plus device according to manufacturer's instructions. Plasma levels of ADMA and SDMA were measured by isotope-dilution tandem mass spectrometry (LC-MS/MS) as previously described [22]. Urinary and plasma creatinine was measured in the Clinical Chemistry Department at the University Hospital Dresden using standard procedures. Urinary albumin concentrations were determined using a commercially available ELISA kit (Bethyl Laboratories; Montgomery, TX; USA) according to manufacturer's instructions. 2.5. Immunohistochemical staining Harvested kidneys were fixed in Zn fixative overnight and were further processed as described elsewhere [23]. Paraffin-embedded kidneys were cut in 2 mm-thick sections. Sections were stained with the periodic acid-Schiff reagent (PAS). The following scoring system was used to quantify PAS-positivity in the glomerulus: 0 e healthy glomerulus; 1 e less than 25% of glomerulus shows increased PAS positivity; 2 - less than 50% of glomerulus shows increased PAS positivity; 3 - less than 75% of glomerulus shows increased PAS positivity; 4 - more than 75% of glomerulus shows increased PAS positivity. For immunoperoxidase staining sections were incubated with the following primary and secondary antibodies: desmin, a mouse monoclonal antibody (Dako); PCNA, a goat polyclonal antibody to detect actively proliferating cells (Santa Cruz); Mac-2, a
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rat monoclonal antibody to detect monocytes (Cedarlane). The antibodies were diluted in sterile PBS containing 1% (w/v) BSA. Negative controls were performed by deletion of the primary antibody. All tissue sections were incubated with the primary antibodies at 4 C over night in a wet chamber. All slides were incubated with the secondary antibodies, according to the species in which the primary antibodies were generated: biotinylated antigoat (Vector Laboratories); biotinylated anti-rat (Vector Laboratories); biotinylated anti-mouse (Biolegend) for 1 h in darkness in room temperature. All tissue stainings were developed with the DAB HRP (3,3 e diaminobenzidine horseradish peroxidase) Substrate Kit (Vector Laboratories) according to manufacturer's instructions. 2.6. Silver staining Paraffin-embedded tissues were cut into 2 mm sections, deparaffinized, rehydrated, washed in deionized water and incubated in 1% periodic acid for 10 min. Next the slides were washed 3 30 s in deionized water, immersed for 45 min in silver nitrate/ methenamine-borate solution at 65 C, washed 3 30 s in deionized water and stained with 0.25% gold chloride solution for 1 min. Afterwards the slides were washed 3 30 s in deionized water, placed for 2 min in 2% nitric acid sodium thiosulphate and put for 3 min under running tap water. In a next step the slides were washed 3 30 s in deionized water, immersed for 3 min in light green SF solution, washed 30 s in deionized water, dehydrated in increasing concentrations of alcohol, cleared with xylene and mounted with Moviol. 2.7. Flow cytometry Flow cytometry was performed using FACSCanto II from BD with subsequent analysis using the FlowJo data analysis software (FlowJo, Ashland, OR). Renal tissues were processed in culture dishes (Greiner Bio-One, Frickenhausen, Germany) and cut into small pieces (1e3 mm) immediately after washing with PBS containing 5% FCS. To digest renal tissue, collagenase type IA (1 mg/ml; Darmstadt, Life Technologies) was used in combination with DNAse (Sigma-Aldrich; Taufkirchen, Germany) at 37 C in 5% CO2 for 45 min to prevent clumping of cells due to DNA release form dead cells. After dissociation, cells suspensions were filtered through 40 mm nylon mesh (BD Bioscience, Heilderberg, Germany). Before incubation with antibody non-specific stainings were prevented using anti-mouse CD16/CD32 (FC-block) and apoptotic cells were excluded using 7-AAD staining. Macrophages, DCs, B cells and T cells were identified after stainings for 30 min at room temperature with antibodies against CD11c PE-Cy7, CD11b BV 421, GR1 PerCP Cy5.5, F4/80 APC, CD4 PE-Cy7, CD8 APC, CD19 APC-Cy7, IgM PE-Cy7 and CD45R PE Cy5.5 (B220). IgG2a antibodies served as isotype controls for each procedure. All antibodies were obtained from BD Biosciences or ebioscience (Frankfurt, Germany). 2.8. Measurement of glomerular filtration rate The glomerular filtration rate (GFR) was measured in conscious and free-moving mice. Hair was removed from the back from an area of app. 2 cm in diameter and sterile FITC-S (fluorescein-isothiocyanate-labelled sinistrin) was injected via the tail vain (35 mg pro ml in 0.9% NaCl, 4 ml per kg body weigh). Fluorescence was measured for 1 h and GFR was calculated using the formula: GFR [ml/min/100 g b.w.] ¼ 1416.8/ t
1/2
2.9. Image analysis Images were taken with a digital microscope (BIOREVO BZ9000, Keyence GmbH, Neu-Isenburg, Germany) under 200 or 400-fold magnification in a minimum 30 glomeruli per mouse. ImageJ software (National Institute of Health, http://rsb.info.nih.gov/ij/) was used for computer assisted image analysis. 2.10. Statistical analysis Statistical analysis was performed using SigmaPlot 12.0. Normal distribution of the data was tested using the D'Agostino and Pearson omnibus normality test. Comparisons between the groups were performed using unpaired two-tailed Student's t-test (two groups) or one-way analysis of variance (more than two groups). Statistical significance was defined as a P value < 0.05. Values are reported as mean ± SEM. 3. Results 3.1. Successful induction of diabetes mellitus All mice used for final analysis became diabetic within 2e3 weeks, defined as reaching blood glucose levels above 350 mg/dl after injection of STZ. After 20 weeks of diabetes there was no difference in the body weight or blood glucose between the wild type and DDAH1 KO mice (Fig. 1 A and B). 3.2. STZ-induced diabetes does not alter plasma ADMA levels Consistent with the role of DDAH1 as an ADMA-metabolizing enzyme, the mice with global deficiency of DDAH1 had app. twice higher plasma ADMA levels (Fig. 2, p < 0.001). Contrary to our expectations, the plasma ADMA levels were not higher in the diabetic animals than in the healthy controls (Fig. 2). 3.3. DDAH1 gene-deficiency did not influence albuminuria and renal function In a next step we wanted to determine whether deficiency of DDAH1 influences the development of renal impairment in our experimental model. As expected, after 20 weeks of diabetes we observed a significant rise in the total urinary albumin levels in the diabetic wild type and diabetic DDAH1 KO animals as compared to the healthy controls. However, there was no influence of DDAH1 gene deficiency on albuminuria (Fig. 3 A). Our experimental model did not result in changes in glomerular filtration rate (Fig. 3 B) nor in plasma SDMA levels (data not shown). 3.4. DDAH1 gene-deficiency did not influence infiltration of renal tissue with inflammatory cells We investigated infiltration of renal tissue with inflammatory cells by flow cytometry. After 20 weeks of diabetes we observed increased amounts of CD8 positive T cells, CD4 positive T cells and dendritic cells. There was no change in the number of macrophages or neutrophils. However, the global deficiency of DDAH1 did not affect the amount of any of the cell type investigated (Table 1). 3.5. DDAH1 deficiency did not influence degree of glomerulosclerosis, glomerular matrix expansion, cell proliferation or podocyte injury
FITC-S In the following step we investigated whether DDAH1 deficiency influences diabetic changes by histology. The
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Fig. 1. Body weight (A) and blood glucose levels (B) in wild type (Wt) and DDAH1 knock-out (KO) mice. - STZ: without induction of diabetes; þ STZ: with induction of diabetes. Mice were weighted and blood glucose was measured at 20 weeks of the disease, before sacrifice.
Fig. 2. Plasma ADMA levels in the wild type (Wt) and DDAH1 knock-out (KO) mice. - STZ: without induction of diabetes; þ STZ: with induction of diabetes. Plasma was collected at 20 weeks of the disease, before sacrificing the mice. ***p < 0.001.
glomerulosclerosis index as assessed by PAS staining, was significantly increased by the diabetic state (8.5-fold for the Wt mice and 3.0 for the DDAH1 KO mice, p < 0.001 in both cases), but there was no difference in DDAH1 KO mice (Fig. 4). We also quantified glomerular matrix expansion with silver staining and saw no difference between the groups (Supplementary Data, Fig. 5 A). Additional staining for collagen IV in the glomeruli and interstitium also did not reveal any effect of the diabetic state or the absence of DDAH1 (Supplementary Data, Fig. 5B and C). As diabetic nephropathy also leads to mesangial cells activation and proliferation, we assessed glomerular cell proliferation in all mice, but could not detect any difference between the groups (Supplementary Data, Fig. 6 A). The same was true for infiltration of the glomeruli with macrophages e an indicator of initiation of the inflammatory response (Supplementary Data, Fig. 6 B). There was also no difference in the degree of podocyte injury as assessed by staining for
Fig. 3. Renal function in wild type (Wt) and DDAH1 knock-out (KO) mice. - STZ: without induction of diabetes; þ STZ: with induction of diabetes. Urine was collected at 20 weeks of the disease, before sacrificing the mice. **p < 0.001 vs. eSTZ wt; ##p < 0.01 vs. eSTZ KO.
Table 1 Infiltration of renal tissue with inflammatory cells.
DDAH1 WT -STZ DDAH1 WT þ STZ DDAH1 KO -STZ DDAH1 KO þ STZ
CD8 positive T cells
CD4 positive T cells
Dendritic cells
macrophages
neutrophils
0.47 ± 0.06 0.89 ± 0.09 ** 0.35 ± 0.01 0.91 ± 0.12 #
0.84 ± 0.06 1.51 ± 0.15 ** 0.79 ± 0.03 1.53 ± 0.23
1.18 ± 0.18 1.96 ± 0.14 ** 1.13 ± 0.05 1.81 ± 0.32
0.09 ± 0.14 0.09 ± 0.02
0.14 ± 0.05 0.21 ± 0.03
0.07 ± 0.01 0.11 ± 0.02
0.17 ± 0.04 0.22 ± 0.07
**p < 0.01 vs DDAH1 WT eSTZ; #p < 0.05 vs DDAH1 KO eSTZ.
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Fig. 4. Glomerulosclerosis index in wild type (Wt) and DDAH1 knock-out (KO) mice. - STZ: without induction of diabetes; þ STZ: with induction of diabetes. Kidneys were harvested after 20 weeks of the disease, before sacrifice. ***p < 0.001 vs. eSTZ wt; ###p < 0.01 vs. eSTZ KO.
desmin, among all the groups of mice (Supplementary Data, Fig. 6 C). 4. Discussion In the current study we aimed to investigate the effect of DDAH1 deficiency on the development of STZ-induced diabetes mellitus. Both the wild type and the DDAH1 KO mice developed diabetes type 1 after STZ injection. As expected, deficiency of DDAH1 caused a significant increase in plasma ADMA (asymmetric dimethylarginine) levels in both the healthy and diabetic animals. Quite surprisingly though STZ-induced diabetes did not increase plasma ADMA levels in the wild type nor in the DDAH1 deficient mice. In diabetic patients kidney disease starts from microalbuminuria (>30 mg protein in urine/day) [24]. On cellular level, glomerular membrane thickening is believed to be the earliest electron microscopical lesion [25] and mesangial expansion to be the most common histopathological change [24]. The other important contributors to epithelial injury in diabetic kidney disease are dysfunctional endothelium and loss of both glomerular and tubulointerstitial capillaries [24]. What is more, it is reported that the number of viable and functional podocytes correlates with proteinuria and is currently established as one of the best predictors of the disease progression [26]. The factors triggering loss of podocytes are not fully understood, but it is believed that hyperglycemia-induced reactive oxygen species generation causing podocyte apoptosis or detachment could be one of the reasons [27]. One of the limitations of mouse models of diabetic kidney disease is that the animals do not manifest progressive decline of renal function, which is so characteristic for humans [28]. Our experimental protocol used in the current study led to the development of albuminuria. However, global deletion of DDAH1 and subsequent increase in systemic ADMA levels did not exacerbate these changes, indicating that ADMA is not the major mediator of the early diabetic changes in this experimental model of diabetes type 1. Even though the mice had significantly elevated blood glucose levels over a period of 5 months, they did not develop profound histological renal changes, characteristic for diabetic kidney disease in humans. In the histological analysis we focused on changes in glomerulus, which are known as the initiating step in diabetic nephropathy, as reflected by the increase in albuminuria. However, we were not able to detect significant differences between the wild type and DDAH1 KO diabetic mice. The design of this experiment included an
assumption that at least 35% change in glomerular matrix expansion would occur, which was clearly not the case. The experimental model of diabetes mellitus type 1 used in this study did not result in elevation of plasma ADMA levels. To the best of our knowledge, there is no data published on plasma ADMA levels in mice with STZ-induced diabetes mellitus type 1. The available studies with rats show that STZ injection leads to plasma ADMA elevation as compared to the healthy animals [29e31]. However, some groups reported only a mild increase or no difference at all [32e34]. The lack of significant changes in plasma ADMA levels which we observed does not necessarily exclude the role of ADMA in local pathological processes, since it has been shown that intracellular ADMA concentration could be 5e10 folds higher than ADMA levels in plasma [35]. In human studies it was reported that adult patients with diabetes mellitus type 1 have increased circulating ADMA levels even before development of vascular complications [8]. The group of Tarnow and coworkers has also described elevation of plasma ADMA levels in adult patients with diabetes mellitus type 1 suffering from early diabetic nephropathy [15]. However, the data on systemic ADMA levels in children and adolescents with diabetes type 1 are contradictory. Some studies showed decreased plasma ADMA levels [36e38], other report no difference comparing to the healthy control subjects [39e41]. One limitation of the current study results from the fact that the STZ (streptozotocin)-induced model of diabetes mellitus type 1 is known to cause a rather mild course of disease, especially in animals with the C57BL/6 genetic background [28,42], which is the background of DDAH1 knock-out mice. However, since there is no other commonly acceptable inducible model of diabetes mellitus type 1, which could be used to directly study the effects of DDAH1 deficiency in mice, we had to use the STZ model in order to answer our study question and accept the model's limitations. In summary, in the present study we successfully induced diabetes mellitus type 1 in both wild type and DDAH1 knock-out mice. However, contrary to our expectations the diabetic animals did not have increased systemic ADMA levels and the absence of the DDAH1 transgene did not exacerbate diabetic nephropathy changes indicating that ADMA is not the major mediator of the early diabetic nephropathy in this experimental model. Subsequent experiment with introducing the DDAH1 global deletion into diabetes prone mouse strains will be necessary to further evaluate the direct relevance of ADMA in more advanced stages of diabetic nephropathy. Conflicts of interest disclosures None to declare. Financial Support € ner-FreseThis work was supported by the grant from Else Kro nius-Stiftung 2011_A168 to R.R. and B.H. Authors contributions R.N.R., S.M.BeB., V.T.T., Ch.P.M.H., N.W., B.H. e research design, data analysis, manuscript preparation; J.M-L. e research design, experimental work, data analysis, manuscript preparation; A.H., A.S., S.B. e experimental work, data analysis, manuscript preparation; N.J. e data analysis, manuscript preparation. Acknowledgements This article is part of a supplement entitled ‘Therapeutic Apheresis e Current advances for the treatment of metabolic,
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cardiovascular and autoimmune diseases. Based on the contributions to the 2nd Congress of the European Group e International Society for Apheresis, March 22-24, 2018, Vienna, Austria’, published with support of the European Group e International Society for Apheresis e E-ISFA office. E-ISFA gratefully acknowledges support of this supplement by B. Braun, DIAMED Medizintechnik, Fresenius Medical Care, and Kaneka.
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.atherosclerosissup.2019.08.040.
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Reidy K, Kang HM, Hostetter T, Susztak K. Molecular mechanisms of diabetic kidney disease. J Clin Investig 2014;124:2333e40. Tervaert TW, Mooyaart AL, Amann K, Cohen AH, Cook HT, Drachenberg CB, Ferrario F, Fogo AB, Haas M, de Heer E, Joh K, Noel LH, Radhakrishnan J, Seshan SV, Bajema IM, Bruijn JA. Pathologic classification of diabetic nephropathy. J Am Soc Nephrol 2010;21:556e63. White KE, Bilous RW, Marshall SM, El Nahas M, Remuzzi G, Piras G, De Cosmo S, Viberti G. Podocyte number in normotensive type 1 diabetic patients with albuminuria. Diabetes 2002;51:3083e9. Susztak K, Raff AC, Schiffer M, Bottinger EP. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes 2006;55:225e33. Brosius 3rd FC, Alpers CE, Bottinger EP, Breyer MD, Coffman TM, Gurley SB, Harris RC, Kakoki M, Kretzler M, Leiter EH, Levi M, McIndoe RA, Sharma K, Smithies O, Susztak K, Takahashi N, Takahashi T. Mouse models of diabetic nephropathy. J Am Soc Nephrol 2009;20:2503e12. Chen Y, Xu X, Sheng M, Zhang X, Gu Q, Zheng Z. Prmt-1 and ddahs-induced adma upregulation is involved in ros- and ras-mediated diabetic retinopathy. Exp Eye Res 2009;89:1028e34. Wang S, Jiang JL, Hu CP, Zhang XJ, Yang DL, Li YJ. Relationship between protective effects of rosiglitazone on endothelium and endogenous nitric oxide synthase inhibitor in streptozotocin-induced diabetic rats and cultured endothelial cells. Diabetes Metab Res Rev 2007;23:157e64. Xiong Y, Lei M, Fu S, Fu Y. Effect of diabetic duration on serum concentrations of endogenous inhibitor of nitric oxide synthase in patients and rats with diabetes. Life Sci 2005;77:149e59. Shibata R, Ueda S, Yamagishi S, Kaida Y, Matsumoto Y, Fukami K, Hayashida A, Matsuoka H, Kato S, Kimoto M, Okuda S. Involvement of asymmetric dimethylarginine (adma) in tubulointerstitial ischaemia in the early phase of diabetic nephropathy. Nephrol Dial Transplant 2009;24:1162e9. Onozato ML, Tojo A, Leiper J, Fujita T, Palm F, Wilcox CS. Expression of ng,ngdimethylarginine dimethylaminohydrolase and protein arginine n-methyltransferase isoforms in diabetic rat kidney: effects of angiotensin ii receptor blockers. Diabetes 2008;57:172e80. Palm F, Buerk DG, Carlsson PO, Hansell P, Liss P. Reduced nitric oxide concentration in the renal cortex of streptozotocin-induced diabetic rats: effects on renal oxygenation and microcirculation. Diabetes 2005;54:3282e7. Masuda H, Goto M, Tamaoki S, Azuma H. Accelerated intimal hyperplasia and increased endogenous inhibitors for no synthesis in rabbits with alloxaninduced hyperglycaemia. Br J Pharmacol 1999;126:211e8. Chrysis D, Efthymiadou A, Mermigka A, Kritikou D, Spiliotis BE. Osteoprotegerin, rankl, adma, and fetuin-a serum levels in children with type i diabetes mellitus. Pediatr Diabetes 2017 Jun;18(4):277e82. https://doi.org/10.1111/ pedi.12384. Epub 2016 Mar 29. Takaya J, Tanabe Y, Kuroyanagi Y, Kaneko K. Asymmetric dimethylarginine is negatively correlated with hyperglycemia in children. Endocr J 2015;62: 551e6. Krebs A, Doerfer J, Grunert SC, Wohrl J, Stier B, Schmidt-Trucksass A, Lichte K, Winkler K, Grulich-Henn J, Holder M, Schwab KO. Decreased levels of homoarginine and asymmetric dimethylarginine in children with type 1 diabetes: associations with cardiovascular risk factors but no effect by atorvastin. J Pediatr Endocrinol Metab 2015;28:147e52. Glowinska-Olszewska B, Luczynski W, Jablonska J, Otocka A, Florys B, Bossowski A. [asymmetric dimethylarginine (adma) in children with diabetes type 1]. Pediatr Endocrinol Diabetes Metab 2010;16:137e41. Jehlicka P, Stozicky F, Mayer Jr O, Varvarovska J, Racek J, Trefil L, Siala K. Asymmetric dimethylarginine and the effect of folate substitution in children with familial hypercholesterolemia and diabetes mellitus type 1. Physiol Res/ Academia Scientiarum Bohemoslovaca 2009;58:179e84. 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ADMA elevation does not exacerbate development of diabetic nephropathy in mice with streptozotocin-induced diabetes mellitus Roman N. Rodionov a, *, 1, Natalia Jarzebska a, b, 1, Alfred Schneider c, Annett Rexin d, € ger e, Jan Sradnick d, Silke Brilloff a, Jens Martens-Lobenhoffer e, Stefanie M. Bode-Bo d d a d Vladimir Todorov , Christian Hugo , Norbert Weiss , Bernd Hohenstein €t Dresden, University Center for Vascular Medicine, Department of Internal Medicine III, University Hospital Carl Gustav Carus, Technische Universita Fetscherstrasse 74, 01307, Dresden, Germany €t Dresden, Germany Department of Anesthesiology and Critical Care Medicine, University Hospital Dresden, Technische Universita c €t Dresden, Fetscherstrasse 74, 01307, Dresden, Germany Department of Visceral Surgery, University Hospital Carl Gustav Carus, Technische Universita d €t Dresden, Fetscherstrasse 74, Division of Nephrology, Department of Internal Medicine III, University Hospital Carl Gustav Carus, Technische Universita 01307, Dresden, Germany e Institute of Clinical Pharmacology, Otto-von-Guericke University, Leipziger Str.44, 39120, Magdeburg, Germany a
b
a b s t r a c t Keywords: Asymmetric dimethylarginine Diabetic nephropathy Dimethylarginine dimethylaminohydrolase 1 Mouse model Streptozotocin-induced diabetes mellitus
Background and aims: Cardiovascular disease is nowadays the major cause of mortality and morbidity worldwide. The risk of developing cardiovascular disease is significantly increased in patients with diabetic nephropathy. It has been suggested that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthases (NOS), may play an important role in the pathogenesis of diabetic nephropathy. ADMA is mainly metabolized by dimethylarginine dimethylaminohydrolase 1 (DDAH1). The goal of this study was to test the hypothesis that elevation of systemic ADMA levels by knocking out DDAH1 would exacerbate functional and structural glomerular abnormalities in a murine model of diabetic nephropathy. Methods: Streptozotocin (STZ) was used to induce diabetes in adult DDAH1 knock-out and wild type mice. Healthy mice served as controls. Mice were sacrificed after 20 weeks of diabetes. Plasma ADMA levels were assessed by isotope-dilution tandem mass spectrometry and albumin by ELISA. Kidneys were used for FACS analysis and were also stained for markers of inflammation, cell proliferation, glomerular cells and cell matrix. Results: STZ led to development of diabetes mellitus in all injected animals. Deficiency of DDAH1 led to a significant increase in plasma ADMA levels in healthy and diabetic mice. The diabetic state itself did not influence systemic ADMA levels. Diabetic mice of both genotypes developed albuminuria and had increased glomerulosclerosis index. There were no changes in desmin expression, glomerular cell proliferation rate, matrix expansion and expression of Mac-2 antigen in the diabetic mice of both genotypes as compared to the healthy ones. Conclusions: In summary, STZ-induced diabetes led to the development of early features of diabetic nephropathy. Deficiency of DDAH1 and subsequent increase in systemic ADMA levels did not exacerbate these changes, indicating that ADMA is not the major mediator of diabetic nephropathy in this experiment model. © 2019 Elsevier B.V. All rights reserved.
1. Introducion * Corresponding author. University Center for Vascular Medicine and Division of Angiology, Department of Internal Medicine III, University Hospital Carl Gustav Carus at the Technische Universit€ at Dresden, Fetscherstraße 74, 01307, Dresden, Germany. E-mail address: [email protected] (R.N. Rodionov). 1 Both Authors contributed equally to this work. https://doi.org/10.1016/j.atherosclerosissup.2019.08.040 1567-5688/© 2019 Elsevier B.V. All rights reserved.
In the Western countries end stage renal failure is most commonly caused by diabetic nephropathy [1]. One third of patients suffering from diabetes will develop diabetic nephropathy at some stage of the disease, which puts them at high risk for end stage renal disease and, in parallel, for severe micro- and
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macrovascular complications [2]. The earliest clinically detectable consequences of diabetic nephropathy include glomerular hyperfiltration, infiltration of inflammatory cells, and development of capillary leakage leading to microalbuminuria [3]. Nitric oxide (NO) is believed to be involved in the early and late alterations of glomerular hemodynamics due to diabetes [4]. It is hypothesized that increased levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, can be at least to some extent responsible for the decreased bioavailability of NO in diabetic nephropathy. ADMA is an endogenous dimethylated derivative of L-arginine, which was shown to compete with L-arginine for the active site of NOS and inhibit NO production in vitro and in vivo. [5] ADMA is constantly produced from the proteolysis of proteins that are methylated on arginine residues and hydrolysed to citrulline and methylamines by dimethylarginine dimethylaminohydrolase (DDAH). DDAH1 is the major isoform responsible for this reaction and it is widely expressed in vascular and nonvascular tissues. ADMA was demonstrated to be elevated in hypertension, congestive heart failure, atherosclerosis, stroke, hypercholesterolemia, hyperhomocysteinemia, peripheral arterial disease, preeclampsia, chronic kidney disease (CKD), and diabetes mellitus [5]. Elevation of plasma ADMA levels in animal models leads to endothelial dysfunction, increased systemic vascular resistance, and elevated blood pressure [6]. The association between diabetes, diabetic complications and ADMA has been shown in many reports. Plasma ADMA levels are elevated in patients with insulin resistance [7], diabetes mellitus type 1 [8] and type 2 [9], ADMA levels predict cardiovascular events in patients with diabetes mellitus [10]. Plasma ADMA was also shown to be associated with retinopathy in both type 1 and type 2 diabetes [11,12]. Elevation of ADMA has been shown to be a potent predictor of the progression of nephropathy in adult Japanese type 2 diabetic patients [13]. Plasma concentration of ADMA predicts cardiovascular morbidity and mortality in type 1 diabetic patients with diabetic nephropathy [14,15]. The kidney is a major organ regulating plasma ADMA levels [5], while CKD is a major cause of ADMA elevation [16]. ADMA accumulates in the plasma of patients with end-stage renal disease (ESRD), while dialysis decreases ADMA levels and improves endothelial function [17]. Plasma ADMA is a strong and independent predictor of progression of CKD into ESRD [18]. Furthermore, plasma ADMA is independently associated with carotid intimamedia thickness (IMT) in patients with CKD [19] and is a strong and independent predictor of overall mortality and cardiovascular outcome in ESRD [20]. Infusion of ADMA in healthy volunteers decreases renal blood flow and increases renovascular resistance, sodium retention and systemic blood pressure [21], suggesting potential mechanisms for ADMA to accelerate progression of renal failure and promote cardiovascular complications. The aim of the current study was to determine whether elevation of the plasma levels of ADMA by deficiency of dimethylarginine dimethylaminohydrolase 1 (DDAH1) exacerbates progression of diabetes-induced impairment of glomerular morphology and function. 2. Materials and methods 2.1. Animals Eight to ten week-old male DDAH1 gene-deficient (DDAH1 KO) mice and their gender- and age-matched wild type littermates (wt) controls were used for the experiments. All animals had the same, C57BL/6J genetic background. The DDAH1 KO mice were a kind gift from Prof. Yingjie Chen from University of Minnesota, USA and they
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were bred from heterozygous parents and the offspring was genotyped with the following primer pairs: wild type allele forward 50 AAT CTG CAC AGA AGG CCC TCA A-30 , reverse 50 -GGA GGA TCC ATT GTT ACA AGC CCT TAA CGC-3’; knock-out allele forward 50 -TGC AGG TCG AGG GAC CTA ATA ACT-30 , reverse 50 -AAC CAC ACT GCT AGA TGA AGT TCC-3’. All animals were housed at constant humidity (60 ± 5%), temperature (24 ± 1 C), and a 12 h light/dark cycle (6 a.m. to 6 p.m. light). Mice had unlimited access to water and food. All protocols for animal experiments were approved by the ethical committee of the Technische Universit€ at Dresden. 2.2. Induction of diabetes mellitus Diabetes mellitus was induced with a single intraperitoneal injection of streptozotocin in the dose of 180 mg/kg body weight. The animals were considered diabetic if the blood glucose levels reached 350 mg/ml or more. Mice were divided into four groups: healthy wild type (n ¼ 6), diabetic wild type (n ¼ 15), healthy DDAH1 KO (n ¼ 5), diabetic DDAH1 KO (n ¼ 15). All animals were sacrificed after 20 weeks of the experiment. Two diabetic DDAH1 KO and three diabetic wild type mice died in the course of the disease and were excluded from the analysis. 2.3. Collection of urine, plasma and tissue samples 24-h urine was collected in metabolic cages at the last day of the experiment and stored at 80 C. Mice were subjected to isofluorane anesthesia and blood was collected by cardiac puncture into EDTA containing tubes (final concentration 5 mmol/L). Mice were subsequently perfused with 0.9% (w/v) NaCl-solution. Left and right kidneys were harvested, left ones were fixed in zinc solution for 24 h and embedded in paraffin. Plasma was separated by centrifugation and stored at 80 C. Kidney samples were collected and flash-frozen immediately after the sacrifice of the animals and stored at 80 C until further analysis. 2.4. Measurement of blood glucose, ADMA and SDMA in plasma, creatinine and albumin The glucose levels were measured in a small drop of blood from the tail vein using the Accu-Chek® Aviva Plus device according to manufacturer's instructions. Plasma levels of ADMA and SDMA were measured by isotope-dilution tandem mass spectrometry (LC-MS/MS) as previously described [22]. Urinary and plasma creatinine was measured in the Clinical Chemistry Department at the University Hospital Dresden using standard procedures. Urinary albumin concentrations were determined using a commercially available ELISA kit (Bethyl Laboratories; Montgomery, TX; USA) according to manufacturer's instructions. 2.5. Immunohistochemical staining Harvested kidneys were fixed in Zn fixative overnight and were further processed as described elsewhere [23]. Paraffin-embedded kidneys were cut in 2 mm-thick sections. Sections were stained with the periodic acid-Schiff reagent (PAS). The following scoring system was used to quantify PAS-positivity in the glomerulus: 0 e healthy glomerulus; 1 e less than 25% of glomerulus shows increased PAS positivity; 2 - less than 50% of glomerulus shows increased PAS positivity; 3 - less than 75% of glomerulus shows increased PAS positivity; 4 - more than 75% of glomerulus shows increased PAS positivity. For immunoperoxidase staining sections were incubated with the following primary and secondary antibodies: desmin, a mouse monoclonal antibody (Dako); PCNA, a goat polyclonal antibody to detect actively proliferating cells (Santa Cruz); Mac-2, a
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rat monoclonal antibody to detect monocytes (Cedarlane). The antibodies were diluted in sterile PBS containing 1% (w/v) BSA. Negative controls were performed by deletion of the primary antibody. All tissue sections were incubated with the primary antibodies at 4 C over night in a wet chamber. All slides were incubated with the secondary antibodies, according to the species in which the primary antibodies were generated: biotinylated antigoat (Vector Laboratories); biotinylated anti-rat (Vector Laboratories); biotinylated anti-mouse (Biolegend) for 1 h in darkness in room temperature. All tissue stainings were developed with the DAB HRP (3,3 e diaminobenzidine horseradish peroxidase) Substrate Kit (Vector Laboratories) according to manufacturer's instructions. 2.6. Silver staining Paraffin-embedded tissues were cut into 2 mm sections, deparaffinized, rehydrated, washed in deionized water and incubated in 1% periodic acid for 10 min. Next the slides were washed 3 30 s in deionized water, immersed for 45 min in silver nitrate/ methenamine-borate solution at 65 C, washed 3 30 s in deionized water and stained with 0.25% gold chloride solution for 1 min. Afterwards the slides were washed 3 30 s in deionized water, placed for 2 min in 2% nitric acid sodium thiosulphate and put for 3 min under running tap water. In a next step the slides were washed 3 30 s in deionized water, immersed for 3 min in light green SF solution, washed 30 s in deionized water, dehydrated in increasing concentrations of alcohol, cleared with xylene and mounted with Moviol. 2.7. Flow cytometry Flow cytometry was performed using FACSCanto II from BD with subsequent analysis using the FlowJo data analysis software (FlowJo, Ashland, OR). Renal tissues were processed in culture dishes (Greiner Bio-One, Frickenhausen, Germany) and cut into small pieces (1e3 mm) immediately after washing with PBS containing 5% FCS. To digest renal tissue, collagenase type IA (1 mg/ml; Darmstadt, Life Technologies) was used in combination with DNAse (Sigma-Aldrich; Taufkirchen, Germany) at 37 C in 5% CO2 for 45 min to prevent clumping of cells due to DNA release form dead cells. After dissociation, cells suspensions were filtered through 40 mm nylon mesh (BD Bioscience, Heilderberg, Germany). Before incubation with antibody non-specific stainings were prevented using anti-mouse CD16/CD32 (FC-block) and apoptotic cells were excluded using 7-AAD staining. Macrophages, DCs, B cells and T cells were identified after stainings for 30 min at room temperature with antibodies against CD11c PE-Cy7, CD11b BV 421, GR1 PerCP Cy5.5, F4/80 APC, CD4 PE-Cy7, CD8 APC, CD19 APC-Cy7, IgM PE-Cy7 and CD45R PE Cy5.5 (B220). IgG2a antibodies served as isotype controls for each procedure. All antibodies were obtained from BD Biosciences or ebioscience (Frankfurt, Germany). 2.8. Measurement of glomerular filtration rate The glomerular filtration rate (GFR) was measured in conscious and free-moving mice. Hair was removed from the back from an area of app. 2 cm in diameter and sterile FITC-S (fluorescein-isothiocyanate-labelled sinistrin) was injected via the tail vain (35 mg pro ml in 0.9% NaCl, 4 ml per kg body weigh). Fluorescence was measured for 1 h and GFR was calculated using the formula: GFR [ml/min/100 g b.w.] ¼ 1416.8/ t
1/2
2.9. Image analysis Images were taken with a digital microscope (BIOREVO BZ9000, Keyence GmbH, Neu-Isenburg, Germany) under 200 or 400-fold magnification in a minimum 30 glomeruli per mouse. ImageJ software (National Institute of Health, http://rsb.info.nih.gov/ij/) was used for computer assisted image analysis. 2.10. Statistical analysis Statistical analysis was performed using SigmaPlot 12.0. Normal distribution of the data was tested using the D'Agostino and Pearson omnibus normality test. Comparisons between the groups were performed using unpaired two-tailed Student's t-test (two groups) or one-way analysis of variance (more than two groups). Statistical significance was defined as a P value < 0.05. Values are reported as mean ± SEM. 3. Results 3.1. Successful induction of diabetes mellitus All mice used for final analysis became diabetic within 2e3 weeks, defined as reaching blood glucose levels above 350 mg/dl after injection of STZ. After 20 weeks of diabetes there was no difference in the body weight or blood glucose between the wild type and DDAH1 KO mice (Fig. 1 A and B). 3.2. STZ-induced diabetes does not alter plasma ADMA levels Consistent with the role of DDAH1 as an ADMA-metabolizing enzyme, the mice with global deficiency of DDAH1 had app. twice higher plasma ADMA levels (Fig. 2, p < 0.001). Contrary to our expectations, the plasma ADMA levels were not higher in the diabetic animals than in the healthy controls (Fig. 2). 3.3. DDAH1 gene-deficiency did not influence albuminuria and renal function In a next step we wanted to determine whether deficiency of DDAH1 influences the development of renal impairment in our experimental model. As expected, after 20 weeks of diabetes we observed a significant rise in the total urinary albumin levels in the diabetic wild type and diabetic DDAH1 KO animals as compared to the healthy controls. However, there was no influence of DDAH1 gene deficiency on albuminuria (Fig. 3 A). Our experimental model did not result in changes in glomerular filtration rate (Fig. 3 B) nor in plasma SDMA levels (data not shown). 3.4. DDAH1 gene-deficiency did not influence infiltration of renal tissue with inflammatory cells We investigated infiltration of renal tissue with inflammatory cells by flow cytometry. After 20 weeks of diabetes we observed increased amounts of CD8 positive T cells, CD4 positive T cells and dendritic cells. There was no change in the number of macrophages or neutrophils. However, the global deficiency of DDAH1 did not affect the amount of any of the cell type investigated (Table 1). 3.5. DDAH1 deficiency did not influence degree of glomerulosclerosis, glomerular matrix expansion, cell proliferation or podocyte injury
FITC-S In the following step we investigated whether DDAH1 deficiency influences diabetic changes by histology. The
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Fig. 1. Body weight (A) and blood glucose levels (B) in wild type (Wt) and DDAH1 knock-out (KO) mice. - STZ: without induction of diabetes; þ STZ: with induction of diabetes. Mice were weighted and blood glucose was measured at 20 weeks of the disease, before sacrifice.
Fig. 2. Plasma ADMA levels in the wild type (Wt) and DDAH1 knock-out (KO) mice. - STZ: without induction of diabetes; þ STZ: with induction of diabetes. Plasma was collected at 20 weeks of the disease, before sacrificing the mice. ***p < 0.001.
glomerulosclerosis index as assessed by PAS staining, was significantly increased by the diabetic state (8.5-fold for the Wt mice and 3.0 for the DDAH1 KO mice, p < 0.001 in both cases), but there was no difference in DDAH1 KO mice (Fig. 4). We also quantified glomerular matrix expansion with silver staining and saw no difference between the groups (Supplementary Data, Fig. 5 A). Additional staining for collagen IV in the glomeruli and interstitium also did not reveal any effect of the diabetic state or the absence of DDAH1 (Supplementary Data, Fig. 5B and C). As diabetic nephropathy also leads to mesangial cells activation and proliferation, we assessed glomerular cell proliferation in all mice, but could not detect any difference between the groups (Supplementary Data, Fig. 6 A). The same was true for infiltration of the glomeruli with macrophages e an indicator of initiation of the inflammatory response (Supplementary Data, Fig. 6 B). There was also no difference in the degree of podocyte injury as assessed by staining for
Fig. 3. Renal function in wild type (Wt) and DDAH1 knock-out (KO) mice. - STZ: without induction of diabetes; þ STZ: with induction of diabetes. Urine was collected at 20 weeks of the disease, before sacrificing the mice. **p < 0.001 vs. eSTZ wt; ##p < 0.01 vs. eSTZ KO.
Table 1 Infiltration of renal tissue with inflammatory cells.
DDAH1 WT -STZ DDAH1 WT þ STZ DDAH1 KO -STZ DDAH1 KO þ STZ
CD8 positive T cells
CD4 positive T cells
Dendritic cells
macrophages
neutrophils
0.47 ± 0.06 0.89 ± 0.09 ** 0.35 ± 0.01 0.91 ± 0.12 #
0.84 ± 0.06 1.51 ± 0.15 ** 0.79 ± 0.03 1.53 ± 0.23
1.18 ± 0.18 1.96 ± 0.14 ** 1.13 ± 0.05 1.81 ± 0.32
0.09 ± 0.14 0.09 ± 0.02
0.14 ± 0.05 0.21 ± 0.03
0.07 ± 0.01 0.11 ± 0.02
0.17 ± 0.04 0.22 ± 0.07
**p < 0.01 vs DDAH1 WT eSTZ; #p < 0.05 vs DDAH1 KO eSTZ.
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Fig. 4. Glomerulosclerosis index in wild type (Wt) and DDAH1 knock-out (KO) mice. - STZ: without induction of diabetes; þ STZ: with induction of diabetes. Kidneys were harvested after 20 weeks of the disease, before sacrifice. ***p < 0.001 vs. eSTZ wt; ###p < 0.01 vs. eSTZ KO.
desmin, among all the groups of mice (Supplementary Data, Fig. 6 C). 4. Discussion In the current study we aimed to investigate the effect of DDAH1 deficiency on the development of STZ-induced diabetes mellitus. Both the wild type and the DDAH1 KO mice developed diabetes type 1 after STZ injection. As expected, deficiency of DDAH1 caused a significant increase in plasma ADMA (asymmetric dimethylarginine) levels in both the healthy and diabetic animals. Quite surprisingly though STZ-induced diabetes did not increase plasma ADMA levels in the wild type nor in the DDAH1 deficient mice. In diabetic patients kidney disease starts from microalbuminuria (>30 mg protein in urine/day) [24]. On cellular level, glomerular membrane thickening is believed to be the earliest electron microscopical lesion [25] and mesangial expansion to be the most common histopathological change [24]. The other important contributors to epithelial injury in diabetic kidney disease are dysfunctional endothelium and loss of both glomerular and tubulointerstitial capillaries [24]. What is more, it is reported that the number of viable and functional podocytes correlates with proteinuria and is currently established as one of the best predictors of the disease progression [26]. The factors triggering loss of podocytes are not fully understood, but it is believed that hyperglycemia-induced reactive oxygen species generation causing podocyte apoptosis or detachment could be one of the reasons [27]. One of the limitations of mouse models of diabetic kidney disease is that the animals do not manifest progressive decline of renal function, which is so characteristic for humans [28]. Our experimental protocol used in the current study led to the development of albuminuria. However, global deletion of DDAH1 and subsequent increase in systemic ADMA levels did not exacerbate these changes, indicating that ADMA is not the major mediator of the early diabetic changes in this experimental model of diabetes type 1. Even though the mice had significantly elevated blood glucose levels over a period of 5 months, they did not develop profound histological renal changes, characteristic for diabetic kidney disease in humans. In the histological analysis we focused on changes in glomerulus, which are known as the initiating step in diabetic nephropathy, as reflected by the increase in albuminuria. However, we were not able to detect significant differences between the wild type and DDAH1 KO diabetic mice. The design of this experiment included an
assumption that at least 35% change in glomerular matrix expansion would occur, which was clearly not the case. The experimental model of diabetes mellitus type 1 used in this study did not result in elevation of plasma ADMA levels. To the best of our knowledge, there is no data published on plasma ADMA levels in mice with STZ-induced diabetes mellitus type 1. The available studies with rats show that STZ injection leads to plasma ADMA elevation as compared to the healthy animals [29e31]. However, some groups reported only a mild increase or no difference at all [32e34]. The lack of significant changes in plasma ADMA levels which we observed does not necessarily exclude the role of ADMA in local pathological processes, since it has been shown that intracellular ADMA concentration could be 5e10 folds higher than ADMA levels in plasma [35]. In human studies it was reported that adult patients with diabetes mellitus type 1 have increased circulating ADMA levels even before development of vascular complications [8]. The group of Tarnow and coworkers has also described elevation of plasma ADMA levels in adult patients with diabetes mellitus type 1 suffering from early diabetic nephropathy [15]. However, the data on systemic ADMA levels in children and adolescents with diabetes type 1 are contradictory. Some studies showed decreased plasma ADMA levels [36e38], other report no difference comparing to the healthy control subjects [39e41]. One limitation of the current study results from the fact that the STZ (streptozotocin)-induced model of diabetes mellitus type 1 is known to cause a rather mild course of disease, especially in animals with the C57BL/6 genetic background [28,42], which is the background of DDAH1 knock-out mice. However, since there is no other commonly acceptable inducible model of diabetes mellitus type 1, which could be used to directly study the effects of DDAH1 deficiency in mice, we had to use the STZ model in order to answer our study question and accept the model's limitations. In summary, in the present study we successfully induced diabetes mellitus type 1 in both wild type and DDAH1 knock-out mice. However, contrary to our expectations the diabetic animals did not have increased systemic ADMA levels and the absence of the DDAH1 transgene did not exacerbate diabetic nephropathy changes indicating that ADMA is not the major mediator of the early diabetic nephropathy in this experimental model. Subsequent experiment with introducing the DDAH1 global deletion into diabetes prone mouse strains will be necessary to further evaluate the direct relevance of ADMA in more advanced stages of diabetic nephropathy. Conflicts of interest disclosures None to declare. Financial Support € ner-FreseThis work was supported by the grant from Else Kro nius-Stiftung 2011_A168 to R.R. and B.H. Authors contributions R.N.R., S.M.BeB., V.T.T., Ch.P.M.H., N.W., B.H. e research design, data analysis, manuscript preparation; J.M-L. e research design, experimental work, data analysis, manuscript preparation; A.H., A.S., S.B. e experimental work, data analysis, manuscript preparation; N.J. e data analysis, manuscript preparation. Acknowledgements This article is part of a supplement entitled ‘Therapeutic Apheresis e Current advances for the treatment of metabolic,
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cardiovascular and autoimmune diseases. Based on the contributions to the 2nd Congress of the European Group e International Society for Apheresis, March 22-24, 2018, Vienna, Austria’, published with support of the European Group e International Society for Apheresis e E-ISFA office. E-ISFA gratefully acknowledges support of this supplement by B. Braun, DIAMED Medizintechnik, Fresenius Medical Care, and Kaneka.
[21]
[22]
Appendix A. Supplementary data
[23]
Supplementary data to this article can be found online at https://doi.org/10.1016/j.atherosclerosissup.2019.08.040.
[24]
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