Mouad Hamzaoui

Zoubir Djerada

Valery Brunel

Paul Mulder

Vincent Richard

Jérémy Bellien

Dominique Guerrot
email: dominique.guerrot@chu-rouen.fr

nephrectomy induces different renal, cardiac and vascular consequences in 129/Sv and C57BL/6JRj mice

Experimental models of cardiovascular diseases largely depend on the genetic background. Subtotal 5/6 nephrectomy (5/6 Nx) is the most frequently used model of chronic kidney disease (CKD) in rodents. However, in mice, cardiovascular consequences of 5/6 Nx are rarely reported in details and comparative results between strains are scarce. The present study detailed and compared the outcomes of 5/6 Nx in the 2 main strains of mice used in cardiovascular and kidney research, 129/Sv and C57BL/6JRj. Twelve weeks after 5/6 Nx, CKD was demonstrated by a significant increase in plasma creatinine in both 129/ Sv and C57BL/6JRj male mice. Polyuria and kidney histological lesions were more pronounced in 129/Sv than in C57BL/6JRj mice. Increase in albuminuria was significant in 129/Sv but not in C57BL/6JRj mice. Both strains exhibited an increase in systolic blood pressure after 8 weeks associated with decreases in cardiac systolic and diastolic function. Heart weight increased significantly only in 129/Sv mice. Endothelium-dependent mesenteric artery relaxation to acetylcholine was altered after 5/6 Nx in C57BL/6JRj mice. Marked reduction of endothelium-dependent vasodilation to increased intraluminal flow was demonstrated in both strains after 5/6 Nx. Cardiovascular and kidney consequences of 5/6 Nx were more pronounced in 129/Sv than in C57BL/6JRj mice.

 . This is of critical importance in particular because transgenic mice, most commonly developed on a C57BL/6JRj background, currently represent a key experimental tool. In the literature reporting results of 5/6 Nx in mice, strain-dependent differences appear regarding hypertension, albuminuria, kidney function, and kidney histological lesions 17 . Irrespective of the genetic background, renal, cardiac and vascular consequences of 5/6 Nx in mice are generally the subject of focused studies, and their occurrence and severity are therefore poorly characterized together.

In this context, the aim of this study is to comparatively describe the consequences of 5/6 Nx in the two commonly used mouse strains C57BL/6JRj and 129/Sv, with a specific focus on structural and functional disorders of the cardiovascular system.

Methods

Animal models. All experiments were carried out in C57BL/6JRj and 129/Sv mice (n = 20 per strain), aged 8 weeks and weighing between 20 and 26 g (directly bought from Janvier laboratory, Le Genest Ste Isle, France without breeding within our facility), in accordance with the standards and ethical rules, and with approval by the national animal ethics committee (CENOMEXA C2EA-54). To limit the number of animals used, and variability related to hormonal issues which play an important role in CV disorders beyond the scope of strain-dependent differences, only male mice were studied. The surgical procedures were performed by a single experienced operator in order to ensure reproducibility. Briefly, mice were anaesthetized by intraperitoneal injection of ketamine (100 mg/kg) (Ketamine ® 1000, Virbac France) + Xylazine (10 mg/kg) (Rompun ® 2%, Bayer France). After shav- ing and disinfection with povidone (10% Betadine ® dermal, Meda), a left laparotomy was performed to expose the left kidney. First, in order to preserve the adrenal gland, it was separated from the left kidney without harming its vascularization, followed by dissection of the perihilar adipose tissue. After detecting the bifurcation of the kidney artery at the hilum, using a non-absorbable polypropylene 8-0 thread, 6.5-mm needle (Prolene ® 8-0, Ethicon), the upper limb of the kidney artery was ligated and verified by the subsequent homogeneous discoloration of the upper half of the left kidney. The identification and ligation of the upper branch of the kidney artery was easier in C56BL/6JRj than in 129/Sv mice, since the division of the artery was more proximal in C56BL/6JRj as shown in Suppl Fig. 1.

Then, using a cauterizer (Thermal Cautery Unit ® , Geiger), the lower kidney pole was cauterized with an esti- mated depth of 1 mm, avoiding the hilum (Fig. 1), and then sutured using a non-absorbable 4-0 polyamide thread (Ethilon ® 4-0, Ethicon). The entire surgical procedure was performed under a binocular magnifying glass (mag- nification x2). One week after the left partial nephrectomy, a total right nephrectomy was performed following the same steps described above, excepted that the hilum which was fully ligated and the right kidney removed. Control groups were subjected to a sham operation, undergoing a simple laparotomy. The animals received post-operative analgesia consisting in a subcutaneous injection of buprenorphine (0.05 mg/kg) (Buprecare ® , Axience). Mice were sacrificed 12 weeks (W12) after total right nephrectomy.

Blood pressure. Non-invasive measurements of systolic blood pressure (SBP) were performed by tail cuff plethysmography (CODA, Kent Scientific Corporation), before (W0), 4 (W4) and 8 (W0) weeks after surgery. These measurements were performed in conscious and trained mice, and consisted in 2 series of 10 cycles of measurements.

Urine parameters.

Urine was collected over 24 hours in metabolic cages at W0, W4 and W8 for evaluation of urine output. In addition, urine albumin was determined at W8 using an ELISA kit (Albuwell M, Exocell). Furthermore, urine aldosterone concentration was evaluated at W8 by ELISA (Aldosterone ELISA Kit, Enzo) and expressed as pg/g body weight/24 h. Plasma creatinine. At W12, mice were anesthetized (Xylazine + Ketamine as described previously), blood samples were collected and centrifuged. Plasma creatinine concentration was monitored by enzymatic method (CREP2, Roche Diagnostics).

Cardiac function and dimensions.

Ten weeks after the right nephrectomy mice were anesthetized (isoflurane 1 to 2%), LV size and function were assessed with a Vivid 7 ultrasound device (GE medical) [START_REF] Maupoint | Selective Vascular Endothelial Protection Reduces Cardiac Dysfunction in Chronic Heart Failure[END_REF] . The heart was imaged in 2-D mode (parasternal short-axis view). Ejection fraction (EF) was calculated as EF (%) = ((LVDA -LVSA)/LVDA) × 100, where LVDA and LVSA represent LV diastolic and systolic areas, estimated from end-diastolic and end-systolic diameter (EDD and ESD). Doppler measurements were also performed at the tip of mitral leaflets to determine diastolic filling profiles (apical 4-chamber view), to determine peak early (E) and late (A) mitral inflow velocities, and calculate E/A ratio, as an index of LV diastolic function.

Vascular function.

Endothelial function was first assessed on small vessel myographs and arteriographs as described previously [START_REF] Gomez | Reduction of heart failure by pharmacological inhibition or gene deletion of protein tyrosine phosphatase 1B[END_REF][START_REF] Vercauteren | Improvement of peripheral endothelial dysfunction by protein tyrosine phosphatase inhibitors in heart failure[END_REF] . After blood sampling, the mesentery was first removed and then placed in oxygenated Krebs buffer at 4 °C. A 1.5-2.0 mm segment of first order of mesenteric resistance artery segment was mounted on a myograph (DMT, Aarhus, Denmark). After normalization, endothelium-dependent relaxation to acetylcholine (10 -9 to 3.10 -5 mol/L) and endothelium-independent relaxation to sodium nitroprusside (SNP) (10 -9 to 3.10 -5 mol/L) were performed in segments pre-contracted with Phenylephrine (Phe: 10 -5 mol/L). A 2-3 mm segment of third mesenteric resistance artery segment was mounted on an arteriograph (DMT, Denmark). The dilatory response to stepwise increases in intraluminal flow (from 3 to 100 µL/min) was assessed in vessels pre-constricted with Phe (10 -5 mol/L). Only vessels which increased their constriction by more than 40% with Phe were included in the experiments.

Kidney and cardiac histology. In 5/6 Nx mice, the remnant kidney was removed at sacrifice. Kidneys were harvested and decapsulated. Kidney histological lesions were analyzed after Masson's staining. Briefly, the kidneys were fixed for 24 hours in 4% formalin and embedded in paraffin after conventional processing. Three-µm slices were thereafter stained with Masson's trichrome solution. The slides were independently examined on a blinded basis, using a 0 to 4 injury scale for the level of interstitial inflammation, interstitial fibrosis and glomerulosclerosis at magnification x20 (0: no damage; 1: < 25% of kidney damaged; 2: 25-50% of kidney damaged; 3: 50-75% of kidney damaged; 4: 75-100% of kidney damaged). Tubular lesions were analyzed at magnification x10. Vascular thickening and vascular fibrosis were analyzed at magnification x40. The upper half of the kidney was not analyzed since it was ischemic in 5/6 Nx mice. The heart was harvested and weighed. A standardized section of the left ventricle (LV) was frozen for determination of LV fibrosis, using 8-µm thick histological slices, previously stained with Sirius Red. For each heart, 12-15 consecutive images were taken at x10 magnification. Fibrosis area was determined by blinded automated morphometric analysis and expressed as a mean percentage of the total area analyzed (Image J software).

Statistical analyses.

Statistical analysis was performed with Prism software (GraphPad Prism 5.00.288).

Data were expressed as mean values ± SEM. The normality of the data and homogeneity of variances were respectively assessed by Shapiro-Wilk and Bartlett tests. Differences between groups were analyzed by one-way ANOVA test, followed by Bonferroni correction, or Kruskal-Wallis test, followed by a Dunn multiple comparison post-test, as appropriate. Differences between groups for quantitative variables measured over time were analyzed by two-way ANOVA test with group and time as factors. Bonferroni correction was applied to post-hoc t-tests when ANOVA was significant. Survival curves were analyzed using Log-rank (Mantel-Cox) test. All p values were two-tailed with statistical significance indicated by a value of p < 0.05.

Results

Body weight and survival. Body weight increased with time in sham-operated mice (Fig. 1A,B) but this increase was higher in C57BL/6JRj mice compared to 129/Sv (∆W0-W12: 7.9 ± 0.9 vs 6.1 ± 1.1 g, p < 0.01). After 5/6 Nx, body weight gradually increased in C57BL/6JRj mice but not in 129/Sv mice (∆W0-W12: 4.7 ± 0.9 vs -1.9 ± 0.8 g, p < 0.001) (Supplementary Fig. 1). After 12 weeks, survival was not significantly affected by 5/6 Nx neither in C57BL/6JRj nor in 129/Sv mice. Kidney parameters. The onset of CKD was confirmed by the significant increase in plasma creatinine 12 weeks after 5/6 Nx in both strains. There was no significant interstrain difference regarding the increase in C57BL/6JRj vs 129/Sv mice (∆W0-W12: +14 vs +28 µmol/L) (Fig. 1E). To further describe the kidney consequences of 5/6 Nx, we analyzed urine collections. As opposed to C57BL/6JRJRj mice, 129/Sv mice presented significant 2.3 and 2.8-fold increases in urine volume at week 4 and week 8 respectively (Fig. 1C,D). An increase in urinary aldosterone was observed in 129/Sv and C57BL/6JRj mice after 5/6 Nx, without difference between strains (Fig. 1F). In addition, urinary albumin/creatinine ratio was markedly increased in 129/Sv mice (4588 ± 821 vs 219 ± 52 mg/g, p < 0.001) but not in C57BL/6JRj mice (121 ± 29 vs 153 ± 14 mg/g, p = NS). Kidney histology analyses showed the development of glomerulosclerosis, tubular injury, interstitial fibrosis and inflammation, perivascular fibrosis and vascular thickening in 129/Sv and C57BL/6JRj mice after 5/6 Nx (Fig. 2A-F). All these lesions were significantly higher in 129/Sv compared to C57BL/6JRj mice.

Blood pressure. Systolic blood pressure was significantly increased in 129/Sv and C57BL/6JRj mice 8 weeks after surgery (Fig. 3A,B). 1. There was no significant difference between sham-operated and 5/6 Nx mice in both C57BL/6JRj and 129/Sv strains regarding heart rate, LV end-diastolic diameter and wall thicknesses. However, 5/6 Nx induced a similar decrease in fractional shortening and LVEF in both strains, showing a decrease in systolic function. In addition, the E/A ratio similarly decreased after 5/6 Nx in C57BL/6JRj and 129/Sv, indicating the development of diastolic dysfunction. At sacrifice, no difference in cardiac weight was observed between 5/6 Nx and sham-operated mice in the C57BL/6JRj strain, while a slight but significant increase was noticed in 129/Sv 5/6 Nx mice (Fig. 3A). In addition, there was an increase in heart fibrosis after 5/6 Nx in 129/Sv mice, but not in C57BL/6JRj (Fig. 3B).

Cardiac parameters. Echocardiographic measurements are presented in Table

Vascular function.

Relative to baseline values, there was a decrease of mesenteric artery relaxation to acetylcholine in C57BL/6JRj mice compared to sham-operated mice at week 12, without change in the relaxation response to sodium nitroprusside (SNP), demonstrating endothelial dysfunction (Fig. 4A,C). No difference was shown regarding mesenteric artery relaxation to acetylcholine and SNP in 129/Sv mice compared to sham-operated mice (Fig. 4B,D). Importantly, the relaxation to acetylcholine was altered in sham-operated 129/Sv mice (Fig. 4B), demonstrating that 129/Sv mice do not present the "normal" response to acetylcholine (acetylcholine concentration-dependent relaxation of the artery related to the release of endothelium-derived vasodilators).

Regarding arteriography, basal external diameter of the mesenteric artery increased after 5/6 Nx in C57BL/6JRj (Sham-5/6 Nx: 301 ± 10 vs 348 ± 4 µm, p = 0.001) but not in 129/Sv mice (Sham-5/6 Nx: 277 ± 14 vs 291 ± 13 µm, p = NS). Wall thickness increased significantly after 5/6 Nx in both C57BL/6JRj (Sham-5/6 Nx: 21 ± 1 vs 32 ± 2 µm, p < 0.001) and 129/Sv (Sham-5/6 Nx: 22 ± 1 vs 31 ± 1 µm, p < 0.001) strains. In addition, in precontracted mesenteric arteries we observed a marked decrease of the endothelium-dependent vasodilation in response to a gradual increase in intraluminal flow in both C57BL/6 JRj (Fig. 4E) and 129/Sv (Fig) mice, demonstrating severe endothelial dysfunction in both strains.

Discussion

The precise cardiovascular consequences of the 5/6 Nx model are poorly defined in mice, because of methodological differences between studies (Table 2). The present study demonstrates that 5/6 Nx is a relevant tool to study the cardiovascular consequences of CKD in both C57BL/6JRj and 129/Sv mice. Importantly, it highlights striking differences in terms of kidney, cardiac and vascular consequences of this model between strains, with important implications for the design and interpretation of experimental procedures.

In line with results of the present study, Leelahavanichkul et al. previously reported that C57BL/6JRj mice presented milder CKD than 129/Sv and CD-1 mice after subtotal nephrectomy [START_REF] Leelahavanichkul | Angiotensin II overcomes strain-dependent resistance of rapid CKD progression in a new remnant kidney mouse model[END_REF] . Accordingly, we found that in the remnant kidney of C57BL/6JRj mice tubular damage, interstitial fibrosis and inflammation were less severe compared to 129/Sv mice. C57BL/6JRj did not develop albuminuria while 129/Sv did, which is consistent with previous findings by Ma LJ et al. [START_REF] Ma | Model of robust induction of glomerulosclerosis in mice: importance of genetic background[END_REF] . This was associated with increased glomerulosclerosis in 129/Sv mice in both studies. Concerning cardiac consequences of CKD, in our study both strains presented diastolic dysfunction with a mild decrease in systolic function. This can be related to cardiac hypertrophy and remodelling, leading to heart wall stiffness, impairing relaxation. As opposed to C57BL/6JRj, 129/Sv exhibited hypertrophy, and presented increased cardiac fibrosis. Siedlecki et al. [START_REF] Siedlecki | Uremic cardiac hypertrophy is reversed by rapamycin but not by lowering of blood pressure[END_REF] observed cardiac hypertrophy in 129/Sv mice with modified nephrectomy (induced by cauterization of one kidney and total nephrectomy of the other one). To our knowledge, no cardiac hypertrophy after 5/6 Nx has been reported in C57BL/6JRj to date. In C57BL/6JRj, 8 weeks after 5/6 Nx Han et al. [START_REF] Han | p-Cresyl sulfate aggravates cardiac dysfunction associated with chronic kidney disease by enhancing apoptosis of cardiomyocytes[END_REF] showed diastolic dysfunction and cardiac fibrosis, and 12 weeks after 5/6 Nx Li et al. [START_REF] Li | Angiotensin (1-7) prevent heart dysfunction and left ventricular remodeling caused by renal dysfunction in 5/6 nephrectomy mice[END_REF] described systolic and diastolic dysfunction, with increased cardiac fibrosis. In 129/Sv, 16 weeks after 5/6 Nx Winterberg et al. [START_REF] Winterberg | Myocardial dysfunction occurs prior to changes in ventricular geometry in mice with chronic kidney disease (CKD)[END_REF] showed cardiac fibrosis, diastolic dysfunction without systolic dysfunction.

The pathophysiology of the cardiac consequences of CKD involves multiple factors, including hypertension, uremic toxins, FGF-23, and activation of the renin angiotensin aldosterone system [START_REF] Wanner | The heart and vascular system in dialysis[END_REF][START_REF] Sato | Relation of plasma indoxyl sulfate levels and estimated glomerular filtration rate to left ventricular diastolic dysfunction[END_REF][START_REF] Gueret | Vascular Smooth Muscle Mineralocorticoid Receptor Contributes to Coronary and Left Ventricular Dysfunction After Myocardial Infarction[END_REF] . We found an increase of urinary aldosterone excretion after 5/6 Nx, which was similar between strains. Kobayashi et al. [START_REF] Kobayashi | An angiotensin II type 1 receptor binding molecule has a critical role in hypertension in a chronic kidney disease model[END_REF] compared 5/6 Nx in C57BL/6JRj and 129/Sv mice and found no difference in plasma aldosterone between strains after 5/6 Nx. Differential expression of angiotensin II type 1 receptor-associated protein may explain the differential activity of the renin angiotensin aldosterone system, independently of plasma and urine aldosterone concentration. As is frequently the case in human, cardiac hypertrophy was associated with albuminuria in 129/Sv mice, while no albuminuria was observed in C57BL/6JRj [START_REF] Gori | Association between renal function and cardiovascular structure and function in heart failure with preserved ejection fraction[END_REF][START_REF] Wachtell | Urine albumin/creatinine ratio and echocardiographic left ventricular structure and function in hypertensive patients with electrocardiographic left ventricular hypertrophy: the LIFE study. Losartan Intervention for Endpoint Reduction[END_REF][START_REF] Chong | Renal albumin excretion in healthy young adults and its association with mortality risk in the US population[END_REF] .

Hypertension is frequently encountered in kidney diseases. As is also the case in experimental models of diabetes, strain differences have an impact in murine models of hypertension [START_REF] Gurley | Influence of genetic background on albuminuria and kidney injury in Ins2(+/C96Y) (Akita) mice[END_REF] . Although the gold standard method to measure blood pressure in mice is telemetry, controversy exists and the tail-cuff method, which we report here, provides valuable information within normal to high-normal values of blood pressure [START_REF] Lerman | Animal Models of Hypertension: A Scientific Statement From the American Heart Association[END_REF][START_REF] Luther | Under pressure-how to assess blood pressure in rodents: tail-cuff?[END_REF] . Previous studies have reported hypertension in 129/Sv after 5/6 Nx, both by plethysmography with tail cuff [START_REF] Jung | Inhibition of the soluble epoxide hydrolase promotes albuminuria in mice with progressive renal disease[END_REF] and by telemetry [START_REF] Leelahavanichkul | Angiotensin II overcomes strain-dependent resistance of rapid CKD progression in a new remnant kidney mouse model[END_REF] , contrasting with the C57BL/6JRj strain in which blood pressure most frequently does not increase after 5/6 Nx [START_REF] Ma | Model of robust induction of glomerulosclerosis in mice: importance of genetic background[END_REF] . The increase in SBP we describe in C57BL/6JRj with our model of 5/6 Nx therefore contrasts with existing literature. Wei et al. compared two models of 5/6 Nx in C57BL/6JRj mice, one with subtotal surgical removal and the other, as in our study, using the ligature of the upper branch of kidney artery, without removing the ischemic kidney parenchyma [START_REF] Wei | A new low-nephron CKD model with hypertension, progressive decline of renal function, and enhanced inflammation in C57BL/6 mice[END_REF] . Hypertension was described only in the latter model. Wei et al. found that this model upregulates kidney and systemic inflammatory response, which likely promotes the development of hypertension.

Impaired endothelial function has been extensively studied in patients with CKD, and is considered as a major player in the detrimental interactions between the kidney and the cardiovascular system in CKD [START_REF] Stam | Endothelial dysfunction contributes to renal function-associated cardiovascular mortality in a population with mild renal insufficiency: the Hoorn study[END_REF] . Endothelial dysfunction after 5/6 Nx has previously been shown in rat models of CKD [START_REF] Li | Increased circulating trimethylamine N-oxide contributes to endothelial dysfunction in a rat model of chronic kidney disease[END_REF] . In mice models of CKD impaired flow-mediated vasodilation in a resistance vessel had not been reported previously, while this is generally considered as a gold-standard method to demonstrate and study endothelial dysfunction. We demonstrated endothelial dysfunction after 5/6 Nx in both strains. Previous studies have shown features of impaired endothelial function in CKD models by means of dysregulated biomarkers. D' Apolito et al. [START_REF] D' Apolito | Urea-induced ROS cause endothelial dysfunction in chronic renal failure[END_REF] have shown impaired dilation of the thoracic aorta to acetylcholine in C57BL/6JRj mice after 5/6 Nx, with a myograph-based analysis.

129/Sv mice are more prone to CV consequences of CKD than C57BL/6JRj mice. It is therefore important to highlight that in 129/Sv mice the ex vivo analysis of resistance arteries can only be performed by arteriograph evaluation of flow-mediated dilatation and not myograph evaluation of the response to acetylcholine, since this response was incomplete in control 129/Sv mice when compared to other strains. Although data in the literature are scarce, Liu et al. [START_REF] Liu | Vascular function of the mesenteric artery isolated from thyroid hormone receptor-α knockout mice[END_REF] had already showed incomplete relaxation to acetylcholine in mesenteric artery in 129/Sv mice. Ryan et al. [START_REF] Ryan | Endothelial dysfunction and blood pressure variability in selected inbred mouse strains[END_REF] found similar results on aorta in 129/Sv mice but complete relaxation to acetylcholine when testing carotid artery. One of the explanations is a possible decrease of acetylcholine receptor in abdominal arteries, especially since the use of other endothelium-dependent agonists on the aorta induced similar relaxation as compared to C57BL/6JRj. No smooth muscle cell dysfunction was found after 5/6 Nx in our study, contrasting with increased wall thickness in both strains. The increase in wall thickness is mainly due to media remodeling, with hypertrophy and increased fibrogenesis of smooth muscle cells. Results in our study suggest that the remodeling in this model is not sufficient not to alter smooth muscle cell reactivity (precontraction and response to SNP).

The genome and the genetic differences between C57BL/6J and 129/Sv mice are available in large databases (http://bioit2.irc.ugent.be/prx/mousepost/Home.php), (http://www.informatics.jax.org/home/strain) [START_REF] Timmermans | Complete overview of protein-inactivating sequence variations in 36 sequenced mouse inbred strains[END_REF] . Over 8800 significant genetic differences (insertions, deletions, mutations, SNPs) with a potential effect on protein function exist between the 2 strains compared in our study. The database published by Timmermans et al. identifies several candidate genes playing a role in vascular physiology (Ren1, Angpt4, Notch1) or inflammation and fibrogenesis (Ddr1, Mmp9, Col6A5, Smad7) which may contribute to the differences observed in the present study [START_REF] Timmermans | Complete overview of protein-inactivating sequence variations in 36 sequenced mouse inbred strains[END_REF] . Further studies on candidate genes responsible for the variable susceptibility of these 2 strains to 5/6 Nx could suggest targets to limit the CV consequences of CKD. In conclusion this study comparatively details kidney and cardiovascular consequences of the 5/6 Nx model of CKD in two of the most frequently studied strains: C57BL/6JRj and 129/Sv male mice. 5/6 Nx induced higher kidney injury, albuminuria, hypertension, cardiac hypertrophy and fibrosis, diastolic and systolic dysfunction and endothelial dysfunction in 129/Sv. In C57BL/6JRj while mild kidney injury was observed, no albuminuria, cardiac hypertrophy or fibrosis were present. These results are of importance when comparing conclusions of the literature in different strains. They will also be particularly useful for scientists to provide arguments defining whether C57BL/6JRj or 129/Sv mice should be used to investigate specific research issues on the CV and kidney consequences of CKD.

Figure 1 .

 1 Figure 1. Evolution of body weight (A,B) and urine output (C,D) from week 0 (W0; 5/6 Nx) to W12 (sacrifice) in sham-operated and 5/6 Nx C57BL/6JRj (A,C) and 129/Sv (B,D) mice (n = 10 per group). *p < 0.05: sham vs. 5/6 Nx, **p < 0.01: sham vs. 5/6 Nx, ***p < 0.001: vs. 5/6 Nx. Plasma creatinine at week 12 (E) and aldosteronuria (F) in sham-operated and 5/6 Nx C57BL/6JRj and 129/SV mice (n = 8-9 per group). *p < 0.05: sham vs 5/6 Nx, ***p < 0.001: sham vs 5/6 Nx.

Figure 2 .

 2 Figure 2. Scoring of kidney lesions 12 weeks after surgery in sham-operated and 5/6 Nx C57BL/6JRj and 129/Sv mice (n = 6-10 per group). (A) Glomerular sclerosis (GS) lesions, (B) Tubular damage (TD) lesions, (C) Interstitial fibrosis (IF) lesions, (D) Interstitial inflammation (II) lesions, (E) Vascular thickening (VT) lesions, (F) Vascular fibrosis (VF) lesions. ***p < 0.001: sham vs 5/6 Nx, $ p < 0.05: 5/6 Nx (129/Sv) vs. 5/6 Nx (C57BL/6JRj), $$$ p < 0.001: 5/6 Nx (129/Sv) vs. 5/6 Nx (C57BL/6JRj).

Figure 3 .

 3 Figure 3. Evolution of systolic blood pressure from week 0 (W0; 5/6 Nx) to W12 (sacrifice) in sham-operated and 5/6 Nx C57BL/6JRj (A) and 129/SV (B) mice (n = 10 per group). *p < 0.05: sham vs. 5/6 Nx, ***p < 0.001: vs. 5/6 Nx. Heart weight (w) to tibia length ratio (C) and morphometric quantification of left ventricular cardiac fibrosis determined 12 weeks after surgery (D) in sham-operated and 5/6 Nx C57BL/6JRj and 129/SV mice (B) (n = 7-9 per group). **p < 0.01: sham vs. 5/6 Nx, $$ p < 0.01: 5/6 Nx (129/Sv) vs. 5/6 Nx (C57BL/6JRj), ***p < 0.001: sham vs. 5/6 Nx, $$$ p < 0.001: 5/6 Nx (129/Sv) vs. 5/6 Nx (C57BL/6JRj).

Figure 4 .

 4 Figure 4. Relaxing responses of pre-contracted mesenteric arteries in response to acetylcholine (A,B) and sodium nitroprusside (C,D) in sham-operated and 5/6 Nx C57BL/6JRj (A,C) and 129/Sv (B,D) mice (n = 7-8 per group). Dilatory responses of pre-constricted mesenteric arteries in response to stepwise increase in intraluminal flow in sham-operated and 5/6 Nx C57BL/6JRj (E) and 129/Sv (F) mice (n = 7-8 per group). *p < 0.05: sham vs. 5/6 Nx, **p < 0.01: sham vs. 5/6 Nx, ***p < 0.001: sham vs. 5/6 Nx.

Table 1 .

 1 ). Echocardiographic measurements in sham-operated and 5/6 Nx C57BL/6JRj and 129/Sv mice.

	Mice	C57BL/6JRj		Sham vs 5/6 Nx 129/Sv		Sham vs 5/6 Nx
	Parameters	Sham	5/6 Nx	p	Sham	5/6 Nx	p
	HR (bpm)	404 ± 15	437 ± 12	NS	434 ± 9	429 ± 8	NS
	LVEDD (mm)	4.0 ± 0.1	3.8 ± 0.1	NS	3.6 ± 0.1	3.8 ± 0.2	NS
	FS (%)	39 ± 4	27 ± 3	<0.05	44 ± 3	31 ± 3	<0.05
	LVEF (%)	75 ± 4	59 ± 5	<0.05	80 ± 3	64 ± 4	<0.05
	E/A	1.29 ± 0.04	0.95 ± 0.03	<0.0001	1.35 ± 0.04	0.95 ± 0.03	<0.0001

Values are expressed as mean ±SEM. (n = 8-9 per group). E/A: early to late mitral inflow peak ratio, LVEF: left ventricular ejection fraction, FS: fractional shortening, LVEDD: Left ventricular end-diastolic diameter, HR: Heart rate, NS: non significant.

Table 2 .

 2 Studies investigating kidney, cardiac and vascular consequences of 5/6 Nx in C57BL/6JRj and 129/ Sv mice. (-) no significant difference or (+) significant difference between 5/6 Nx and control mice; (ND) not determined.

		Studies	Methods	Polyuria	Urine albumin	Plasma aldosterone HTN	Cardiac hypertrophy	Cardiac fibrosis	Systolic function	Diastolic function	Endothelial function
		Leelahavanichkul A et al. 2010	surgical removal	ND	-	ND	-	ND	-	ND	ND	ND
		Ma L-J et al. 2003 surgical removal	ND	-	ND	-	ND	ND	ND	ND	ND
		Li Y et al. 2009	surgical removal	ND	ND	ND	+	+	+	+	+	ND
		Kobayashi R et al. 2017	surgical removal	+	ND	-	-	ND	ND	ND	ND	ND
		Wei J et al. 2018	surgical removal	ND	-	-	-	ND	ND	ND	ND	ND
	C57BL/6JRj	Wei J et al. 2018 Gava AL et al. 2012	artery ligation surgical removal	ND +	+ +(proteinuria) ND -	+ +	ND ND	ND ND	ND ND	ND ND	ND ND
		Lehners A et al. 2014	surgical removal	ND	+	ND	-	-	ND	ND	ND	ND
		Jin J et al. 2017	surgical removal	ND	ND	ND	-	ND	ND	ND	ND	+kidney artery
		Thomsen MB et al. 2018	surgical removal	ND	ND	ND	ND	+	ND	ND	ND	ND
		Madsen M et al. 2017	surgical removal	ND	ND	ND	ND	ND	ND	ND	ND	-aortic ring
		Leelahavanichkul A et al. 2010	surgical removal	ND	+	ND	+	ND	+	ND	ND	ND
		Ma L-J et al. 2003 surgical removal	ND	+	ND	+	ND	ND	ND	ND	ND
	129/Sv	Winterberg PD et al. 2016	surgical removal	ND	ND	ND	+	+	+	-	+	ND
		Kobayashi R et al. 2017	surgical removal	+	ND	ND	+	ND	ND	ND	ND	ND
		Jung O et al. 2010 artery ligation	ND	+	ND	+	ND	ND	ND	ND	ND

SCIENTIFIC REPORTS |(2020) 10:1524 | https://doi.org/10.1038/s41598-020-58393-w

Author contributions

M.H., D.G. and J.B. developed the idea, conducted the research and interpreted the finding. Z.D. investigate cardiac echography. V.B. conducted biochemical analysis. P.M. and V.R. helped to interpreted the finding. All authors have reviewed the manuscript.

Competing interests

The authors declare no competing interests.


Tristan De Nattes
email: denattes.tristan@gmail.com

Lucile Moreau-Grangé

Delphine Vezzosi

Julien Haddoux

Miguel Hie

Dominique Guerrot

Steven Grangé

Adrenocortical carcinoma complicated by renal thrombotic microangiopathy, a caseseries

Keywords: Thrombotic microangiopathy, Hemolytic uremic syndrome, Acute kidney injury, Adrenocortical carcinoma

Background: Cancer-related thrombotic microangiopathy (CR-TMA) is a rare entity associated with a dismal prognosis. Usually, CR-TMA is associated with mucin-producing carcinomas among which stomach, breast, prostate, lung and pancreas tumours are the most frequent. Cases presentation: We describe for the first time three cases of CR-TMA due to adrenocortical carcinoma (ACC). All of them had mechanical hemolytic anemia and thrombocytopenia without any other identifiable cause. Bicytopenia was diagnosed either simultaneously with ACC or at the time of metastatic evolution. Two patients had acute kidney injury (AKI) with severe pathological findings on kidney biopsy. Despite total adrenalectomy, chemotherapy, and specific treatment of TMA with plasma-exchanges, renal failure and hemolytic anemia remained. The only manifestation of CR-TMA in the third patient was hemolytic anemia, which resolved after surgical removal of ACC. The evolutions in these patients suggests ACC-related TMA may be related to a circulating factor. Conclusions: CR-TMAs are rare. Here we describe the first case series of ACC-related TMA, among which two had renal involvement. This entity is associated with dismal renal prognosis despite specific treatment of TMA. According to patients' evolution, the persistence of TMA may reflect an uncontrolled malignancy.

Background

Thrombotic microangiopathy (TMA) is a rare entity due to a wide variety of diseases, all characterized by mechanical hemolytic anemia, thrombocytopenia and microvascular occlusions. Causes of TMA are separated between thrombotic thrombocytopenic purpura, typical Hemolytic uremic syndrome (HUS), primary atypical HUS in patients with an abnormality of the alternative complement pathway, and HUS secondary to a heterogeneous group of causes: infections, drugs, genetic disorders, systemic diseases and cancers [START_REF] Goodship | Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "kidney disease: improving global outcomes" (KDIGO) controversies conference[END_REF]. Adrenocortical carcinoma (ACC) is a rare tumour, with an unfavourable prognosis. Hormonal complications often occur, but paraneoplastic syndromes are uncommon in the literature [START_REF] Else | Adrenocortical carcinoma[END_REF]. Here, we describe a case-series of ACCassociated TMA.

Cases presentation

Case 1

A 41-year-old woman was investigated in July 2016 for fatigue and loss of 10 kg over a few weeks. She was not taking any medication at this time and had no history of kidney disease. An abdominal computed tomography scan was performed in July, revealing a 13x13x10cm left adrenal mass of heterogeneous density with heterogeneous and prolonged enhancement after contrast agent administration. This mass was suggestive of ACC not associated with significant hormonal hypersecretion. Other lab results revealed plasma creatinine = 97 μmol/L, proteinuria = 0.7 g/g of urine creatinine, haemoglobin 9.9 g/ dL, platelet 143 G/L. Surgical procedure was planned in early-August, and no treatment was introduced.

At admission, she had stage 3 KDIGO AKI with plasma creatinine at 439 μmol/L and 0.99 g/24 h proteinuria. Urine sediment was normal. Blood pressure was 119/70 mmHg, there was no clinical sign of other organ involvement. She presented thrombocytopenia (67G/L) and mechanical hemolytic anemia: haemoglobin = 7.9 g/dL, reticulocyte = 175G/L, presence of schistocytes, Lactate Dehydrogenase (LDH) = 1058 UI/L and haptoglobin < 0.1 g/L. Extensive laboratory investigations were performed for the differential diagnosis of cytopenias. Antinuclear factors were negative, as were antibodies for antiphospholipid syndrome and scleroderma. ADAMTS 13 activity was 93%, complement investigations were normal (C4, C4, CH50, Factor I and H, anti-Factor H antibody).

The specific treatment of TMA consisted in daily plasma-exchange therapy of 60 mL/kg with plasma as replacement fluid. Intermittent hemodialysis was started on the same day. Left adrenalectomy was performed with splenectomy because of peroperative splenic decapsulation. A kidney biopsy was also performed.

ACC was confirmed by pathology, with a Weiss score at 6/9, Ki67 = 45% and 25 mitotic figures per 50 highpower field. The resection was total, and mesenteric lymphadenopathy was not metastatic, leading to stage III according to ENSAT classification. Renal pathology confirmed TMA. Twenty-six glomeruli were observed, of which 11 were ischemic with fibrin thrombi within glomerular capillary loops. Thrombi were present in interlobular arteries, arterioles and glomerular capillaries with fibrinoid deposit. No duplication of the glomerular basement membranes was found. There was moderate acute tubular necrosis with mild interstitial fibrosis and tubular atrophy (Fig. 1).

Fourteen plasma-exchanges lead to correction of hemolysis, but the patient remained dialysis-dependent, and biological TMA reappeared a few days after plasmaexchange discontinuation. Owing to the isolated renal TMA, unfavourable renal prognosis according to pathology, kidney function evolution, and the lack of efficiency of terminal complement blockade in cancerrelated TMA (CR-TMA), we decided not to escalate treatments and to undergo chronic hemodialysis.

Mitotane was started despite lack of pharmacokinetic knowledge on this treatment in hemodialysis patients, with a target plasma concentration between 14 and 20 mg/L. Eighteen months later, metastatic lesions occurred, leading to treatment by etoposide, doxorubicin and cisplatin. During all the follow-up, the patient remained anuric in hemodialysis and had persistent hemolysis with schistocytes, high LDH's value and decreased haptoglobin. Twenty-four months after diagnosis, she died in a context of severe pneumonia.

Case 2

A 23-year-old woman was diagnosed with a right ACC in 2014. She first had nephrectomy and adrenalectomy. Despite Mitotane, cerebral, hepatic, pulmonary and bone metastasis occurred, leading to several chemotherapies: Cisplatin, Lenvatinib and Gemcitabine. In October 2018, she presented cardiac tamponade related to influenza-B virus. All investigations at this time did not find any argument for a paraneoplastic aetiology of the pericardial effusion. There was no evidence of TMA and renal function was normal with plasma creatinine 99 μmol/L. Two weeks later, biological data found a KDIGO 3 AKI with plasma creatinine = 200 μmol/L. Hemoglobin was 8.0 g/dL and platelet count 25 G/L. Two weeks later, she had a new cardiac tamponade which required a pericardium-to-pleural space window. Cytopenias were also investigated at this time and revealed TMA with haptoglobin < 0.1 g/L, schistocytes, LDH = 703 UI/L and plasma creatinine = 429 μmol/L. Investigations for this TMA with AKI did not find any other cause of TMA, and metastasis were stable. Due to cytopenias and high suspicious of renal TMA, it was decided not to perform a kidney biopsy.

Daily plasma-exchanges were started for a suspicion of gemcitabine-related TMA. There were no hemodialysis criteria. After 14 plasma-exchanges, platelet count improved (145 G/L), and LDH decreased (422 UI/L) but schistocytes and undetectable haptoglobin persisted. There was no amelioration of kidney function (plasma creatinine 357 μmol/L). After discontinuation of plasmaexchanges, thrombocytopenia recurred. Due to the lack of efficiency of plasma-exchanges and the suspicion of gemcitabine-related TMA, eculizumab was started in December 2018. After second administration of eculizumab, systematic thoracic CT scan revealed new pulmonary metastasis. ACC related-TMA was diagnosed because (1) TMA and kidney function were not ameliorated by discontinuation of gemcitabine and treatment with plasma-exchanges and eculizumab, and (2) recurrent pericarditis with development of new metastatic lesions. Owing to the lack of efficiency of eculizumab in this particular issue, treatment was discontinued and chemotherapy was restarted.

Case 3

An 81-year-old woman with a medical history of polymyalgia rheumatica and ischemic cardiomyopathy had been hospitalized for the investigation of a bicytopenia which had appeared and worsened over a month. Laboratory results revealed hemolytic anemia (Hb = 9.8 g/dL, haptoglobin < 0.1 g/L and schistocytes) and thrombocytopenia (platelet count 99 G/L). The patient had no organ involvement. Bone marrow morphology confirmed the peripheral origin of the bicytopenia and Coomb's test was negative, leading to the diagnosis of TMA. Investigations were performed, and an abdominal CT scan revealed a 14 cm partially calcified left adrenal mass, which was not associated with significant hormonal hypersecretion. Left adrenalectomy was performed, with concomitant nephrectomy and splenectomy. Histopathology confirmed ACC with Weiss score at 5/9 and Ki 67 = 40%, ENSAT stage II. The next day, bicytopenia improved and haemolysis resolved, confirming CR-TMA. There was no recurrence of cytopenia during the 2 years follow-up.

Discussion and conclusion

ACC is a rare tumour, with an incidence of less than 2 cases per million per year and unfavourable prognosis with a 5-year survival rate of 13% in patients with stage-3 ACC [START_REF] Fassnacht | Limited prognostic value of the 2004 international union against cancer staging classification for adrenocortical carcinoma[END_REF]. When ACC is suspected, hormonal hypersecretion has to be investigated, especially cortisol, aldosterone and sex steroids. Complete surgical resection has to be performed for all tumours without metastasis [START_REF] Puglisi | Decision-making for adrenocortical carcinoma: surgical, systemic, and endocrine management options[END_REF]. An adjuvant treatment with mitotane is recommended. In patients with advanced disease, systemic chemotherapy with etoposide, doxorubicin and cisplatin is added [START_REF] Fassnacht | European society of endocrinology clinical practice guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the European network for the study of adrenal tumors[END_REF].

In an extensive review, most CR-TMAs were attributed to metastatic adenocarcinomas of gastric, breast, prostate, and lung origin (decreasing frequency). Pathophysiological explanations of CR-TMA remain unclear. One hypothesis is that tumour cell emboli lead to microvascular obstruction, coagulation activation, and vessel wall proliferation. The direct invasion of bone marrow vasculature which results in release of Von Willebrand Factor multimer has also been described [START_REF] Babu | Cancer-associated thrombotic microangiopathy[END_REF]. In other cases, activation of the coagulation cascade by mucin released from adenocarcinomas and immune mechanisms has been suggested.

TMA secondary to endocrine malignancies are extremely rare and mainly reported with pheochromocytoma [START_REF] Lechner | Cancer-related microangiopathic hemolytic anemia: clinical and laboratory features in 168 reported cases[END_REF]. In those cases, severe hypertension was the most probable explanation of TMA reported by the authors [START_REF] Sakai | Malignant pheochromocytoma accompanied by microangiopathic hemolytic anemia: a case report[END_REF][START_REF] Gillett | Diagnostic challenges associated with a complex case of cystic phaeochromocytoma presenting with malignant hypertension, microangiopathic haemolysis and acute renal failure[END_REF][START_REF] Schweizer | Phaeochromocytoma and thrombotic microangiopathy: favourable outcome despite advanced renal failure[END_REF].

In the first case of ACC-related TMA reported in the present case-series, TMA occurred before any medication, and extensive investigations were negative. In the second case, CR-TMA was treated as gemcitabineassociated TMA which did not improve either cytopenias or renal function. The paraneoplastic aetiology was affirmed by chronology and evolution of illness. In the third case, CR-TMA rapidly improved after total removal of tumor.

The latter case suggests that TMA in this particular situation could be explained by the secretion of a circulating factor by the ACC. This is substantiated by cases 1 and 2, in which plasma-exchange therapy lead to a transient improvement of hemolytic anemia, resuming at plasma-exchange discontinuation. The initial response to plasma-exchange therapy supports the role of a high molecular weight pathogenic factor with endothelial toxicity in the pathophysiology of ACC-related TMA. Considering the improvement of hemolytic anemia with plasma-exchange therapy, the recurrence of cytopenias at plasma-exchange discontinuation despite surgery and chemotherapy may suggest insufficient treatment of the tumor.

Therefore, we suggest that patients with TMA in a context of ACC be treated by plasma-exchange therapy until specific treatment of tumor is performed, with total surgery if possible. Due to the lack of evidence for complement involvement in ACC-related TMA, and since eculizumab efficiency in CR-TMA is not established, we believe complement blockade should not be recommended as a suitable option in this setting.

According to the first two cases, and as it is known for CR-TMA, renal prognosis in ACC-related TMA seems to be dismal. Indeed, despite plasma-exchange therapy, there was no improvement in renal function at the end of the follow-up.

CR-TMAs are rare and associated with dismal prognosis. Here we describe the first case series of ACCrelated TMA in 3 different patients. The evolutions in these patients suggests ACC-related TMA may be related to a circulating factor. We therefore suggest plasma-exchange therapy in this context until specific treatment of ACC is initiated.

Fig. 1

 1 Fig. 1 Histologic examination (H&E, × 40). Arrows: fibrin thrombi within glomerular capillary loops

Acknowledgements

Not applicable.

Funding

This study was not funded.

Availability of data and materials

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Abbreviations AAC: Adrenocortical carcinoma; AKI: Acute Kidney Injury; CR-TMA: Cancerrelated Thrombotic Microangiopathy; ENSAT: European Network for the Study of Adrenal Tumours; HUS: Hemolytic uremic syndrome; LDH: Lactate Dehydrogenase Authors' contributions TdN, LM-G, DV, JH and MH made substantial contributions to acquisition of data. TdN, DG and SG made substantial contribution to conception and design. TdN: data interpretation and analysis. TdN, JH, MH and LM-G: drafting manuscript. DV, DG, SG revised the manuscript. DG, SG: mentorship. All authors approved final manuscript. All the authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate Not applicable.

Consent for publication

Written informed consent for case 2 and 3 were obtained from the patient for publication of this case report. Written informed consent for case 1 was obtained from the patient's husband for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

Competing interests

The authors declare that they have no competing interests.

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S R Wong

C Raynaud
email: raynaud@cegely.insa-lyon.fr

D Planson

M Lazar

J.-P Chante

OBIC measurements on 1.3 kV 6H-SiC bipolar diodes protected by Junction Lateral Extension

Introduction -Due to its good electrical properties, mainly a high critical electric field and a large bandgap, silicon carbide has demonstrated potentialities for high power devices. We have designed and realised bipolar diodes to sustain a reverse voltage of about 1.3 kV. We present below the experimental breakdown voltage we achieved, and results of OBIC measurements. Samples details -6H-SiC n-type epitaxial layers from Cree Research have been used for this study. With values of thickness of 10 µm, and of doping level ND = 10 15 cm -3 , the theoretical breakdown voltage is about 1.5 kV. The p + emitter has been realised by room temperature (RT) aluminium implantations with energies varying between 25 and 300 keV, with a total dose of 1.75´10 15 cm -2 . To avoid surface premature breakdown, lateral protections have also been realised by RT Al implantations with a total dose of 1.3´10 13 cm -2 . These protections are know as Junction Terminal Extension (JTE). Then samples are annealed in a rf-induction heating furnace at 1700°C during 30 min. [1].On the half of the wafers, an additional ring, named stop-channel, have been realised by a RT nitrogen implantation. Then diodes have been metallized with Ni in backside and Al-Ti in the front side. Different kinds of diodes are available, with varying emitter diameters and JTE lengths. No passivation layers have been deposited after metallisation. Current-voltage measurements -IV measurements have been performed with a Keithley 237 Source Measurement Unit, or a picoammeter K485. Reverse biases are delivered by a 12.5 kV-100 mA supplier. In forward biases, current densities as high as 220 A cm -2 at 5V have been achieved (Figure 1), and about 50% of diodes achieved 150 A cm -2 . In reverse biases, several diodes protected with JTE have been able to block up to 1200 V (Figure 2), which is very closed to the theoretical value (given by numerical 2D simulators). The majority of non protected diodes have shown breakdown voltages at bout 500 V. This is significant of the benefit role of JTE. We have used Optical Beam Induced Current (OBIC) measurements in order to study the distribution of equipotential lines at the surface of the samples. OBIC measurements -Details about this technique have been described elsewhere [2]: a UV beam is focused at the surface of the samples, and therefore generates free carrier, up to a determined depth, which depends of the wavelength. If these carriers are generated in a space charge region, electrons and holes are separated by the electric field and a current is measured. By translating the beam, we can measure the generated current as a function of the position on the wafer. Figure 3 shows the photocurrent measured along a diameter of the diodes, the width of the current peak is relevant of the spreading out of the equipotential, and its value agrees well with the JTE length and it means that JTE are depleted even at very low voltage. The structure has been drawn on the figure for easier comparison. Figure 4 is similar analyses on a diode without JTE. It is clear that on diodes without JTE, the OBIC signal increases very fast at the edge of the emitter, which is due to the high electric field present in this corner region. We can also notice that at 800 V, a peak also appears at the emitter edge for protected diodes. This could mean that the doping level of JTE is close to the optimised value but a little lower.

Numerical simulations of the photocurrent generated by such a UV beam are currently under investigations, to correlate the OBIC signal with the distribution of the electric field. Detailed discussions and comparison with numerical simulations will be done in the full article. Conclusion -We have fabricated p + n bipolar diodes, which shows good rectifying properties, high current density in forward bias, and breakdown voltages closed to the theoretical value. OBIC analyses, correlated with numerical simulations, shows that the periphery protection has a good efficiency, and is close to the optimised value.

References -

[1] M. Lazar, L. Ottaviani, M.L. Locatelli, C. Raynaud, D. Planson, E.Morvan, P.Godignon, W. Skorupa, J.P. Chante. Mat. Sci. Forum, 353-356 (2001) p.571-574.

[2] K. Isoird, M. Lazar, L. Ottaviani, M.L. Locatelli, C. Raynaud, D. Planson, J.P.Chante.

Appl. Surf. Sci., 184 (2001) p. 477-482.

Figure 1 :

 1 Figure 1 : IV characteristics of some diodes with JTE, in forward bias, measured with a SMU K237.

Figure 2 :

 2 Figure 2 : IV characteristics of 3 diodes with JTE, in reverse bias, measured with a picoammeter K483, by step of 10 V. The lowest significant current is 10 -11 A. The last datapoints which are drawn are considered as the breakdown voltage (next point is more than 1 µA).

Figure 3 :

 3 Figure 3 : OBIC measurements performed on a p + n diode protected by JTE, for increasing reverse voltage. Wavelength is 351.1 nm, which corresponds to a penetration depth of about 11 µm in 6H-SiC.

Figure 4 :

 4 Figure 4 : OBIC measurements performed on a p + n diode without JTE, for increasing reverse voltage. Wavelength is 351.1 nm.