Both authors contributed equally to this work.
Laser Therapy in Metabolic Syndrome-Related Kidney Injury
Article first published online: 4 MAR 2013
© 2013 The Authors Photochemistry and Photobiology © 2013 The American Society of Photobiology
Photochemistry and Photobiology
Special Issue: Symposium in Print on Upper-Room Ultraviolet Germicidal Irradiation for Air Disinfection
Volume 89, Issue 4, pages 953–960, July/August 2013
How to Cite
Ucero, A. C., Sabban, B., Benito-Martin, A., Carrasco, S., Joeken, S. and Ortiz, A. (2013), Laser Therapy in Metabolic Syndrome-Related Kidney Injury. Photochemistry and Photobiology, 89: 953–960. doi: 10.1111/php.12055
- Issue published online: 2 JUL 2013
- Article first published online: 4 MAR 2013
- Accepted manuscript online: 29 JAN 2013 10:38AM EST
- Manuscript Accepted: 21 JAN 2013
- Manuscript Received: 9 OCT 2012
- CERES GmbH and BMWi
- CERES GmbH, Livetec GmbH, and Fundación Conchita Rábago
- Programa Intensificación Actividad Investigadora. Grant Numbers: CAM S2010/BMD-2378, REDINREN RETIC 06/0016, RD12/0021/0001
Metabolic syndrome is characterized by hyperglycemia, hypertension, dyslipidemia and obesity. Diabetes and hypertension are the main causes of chronic end-stage kidney disease in humans. Chronic kidney disease is characterized by kidney inflammation and eventual development of kidney fibrosis. Low-level laser (or light) therapy (LLLT) can be used to relieve pain associated with some inflammatory diseases due to photochemical effects. Despite the known contribution of inflammation to metabolic syndrome and kidney disease, there is scarce information on the potential therapeutic use of LLLT in renal disease. The aim of this randomized, placebo-controlled study was to test the hypothesis that LLLT could modulate chronic kidney injury. Rats with nephropathy, hypertension, hyperlipidemia and type II diabetes (strain ZSF1) were subjected to three different conditions of LLLT or sham treatment for 8 weeks, and then sacrificed 10 weeks later. The main findings of this study are that the LLLT-treated rats had lower blood pressure after treatment and a better preserved glomerular filtration rate with less interstitial fibrosis upon euthanasia at the end of follow-up. This initial proof-of-concept study suggests that LLLT may modulate chronic kidney disease progression, providing a painless, noninvasive, therapeutic strategy, which should be further evaluated.
Experimental and clinical studies have identified the metabolic syndrome as an important contributor to chronic kidney disease (CKD) [1, 2]. The metabolic syndrome includes insulin resistance, impaired glucose tolerance, hyperinsulinemia, high levels of very low-density lipoprotein (VLDL) triglycerides, low levels of high-density lipoprotein (HDL) cholesterol, central obesity and hypertension . The World Health Organization definition of metabolic syndrome requires at least one of three major features namely type 2 diabetes, impaired glucose tolerance and insulin resistance, plus at least two so-called minor features, comprising hypertension, obesity, hypertriglyceridemia or microalbuminuria [4, 5]. It is estimated that 34% of U.S. adults have metabolic syndrome , and the prevalence of diabetes increases with increasing body weight, from 8% for normal weight individuals to 43% for individuals with obesity class 3 .
CKD defined as a glomerular filtration rate (GFR) under 60 mL min−1 per 1.73 m2 is estimated to be present in one of eight adults . CKD is usually a progressive disease leading to end-stage renal disease requiring replacement of renal function or death from CKD-related cardiovascular injury. Interstitial fibrosis gradually replacing functional nephrons with a scar composed of extracellular matrix proteins is a key feature of progressive CKD . Inflammation is a key contributor to nephron loss and fibrosis . Metabolic changes, toxic drugs or proteinuria activate the proinflammatory transcription factor NF-κB and recruit inflammatory cytokines and chemokines that recruit inflammatory cells in the interstitium, activate fibroblasts and contribute to progression of fibrosis [11-20]. Key inflammatory mediators of kidney disease include the angiotensin system, tumor necrosis factor (TNF-α) superfamily cytokines (TNF-α, Fas ligand TRAIL, TWEAK), interleukin-1β (IL-1β), transforming growth factor-beta1 (TGF-β1), Connective tissue growth factor (CTGF) and chemokines [11-20]. Furthermore, inflammation also contributes to CKD mortality . While several drugs, mainly drugs targeting angiotensin II (AngII), slow progression of CKD, the therapeutic armamentarium is still incomplete and the prevalence of CKD around the world is increasing. Indeed, despite available therapy diabetes and hypertension are still the leading causes of end-stage renal disease in the United States .
The biological effects of LLLT were first identified in 1967 by Hungarian physician Endre Mester , when he observed that LLLT accelerated hair growth in mice. LLLT has been used in medicine since at least 1974, when the USSR Ministry of Health gave permission for clinical application for the first helium–neon laser-based device for laser therapy, and it was introduced as an alternative noninvasive treatment for rheumatoid arthritis . In LLLT, cells or tissues are exposed to low energy density, generally between 1 and 20 wavelength, of extremely pure single-wavelength red or near-infrared (630–1000 nm) light generated from a low power laser or LED light source. The power range of a low-level laser is between 5 and 500 mW, thus the therapeutic effect is not thermal, but rather relates to photochemical reactions that change cellular functions in radiated cells. Laser irradiation to treat acute inflammatory pain resulting from soft-tissue injury can modulate inflammatory pain in a dose-dependent manner .
The main purpose of this study was to evaluate whether noninvasive kidney irradiation with LLLT could preserve renal function or structure in a rat model of metabolic syndrome-induced CKD.
Material and Methods
Transcutaneous therapeutic LLLT of kidney tissue was designed based on different physiological interactions of laser light with kidney tissue with the aim of preserving renal function and structure.
Both transmission depth and degree of absorption highly influence the effect of laser irradiation. This is also influenced by the effective device power, dosage, wavelength, tissue type, optical barrier (reflection, refraction), as well as by the optical, and the directive penetration depth. The highest penetration depth is provided by a wavelength range between 630 and 850 nm, termed the optical window of the skin. This means the absorption level is low and the light can permeate the deeper skin layers well . Based on these facts, a semiconductor laser with a wavelength of 785 nm was chosen. The therapeutic effect is based on photon biomodulation, which begins with photon absorption. In turn, some molecules, like the cellular chromoproteins (flavoproteins, catalases, and peroxidases), absorb photon radiation.
Based on the physical dimensions of the rat kidney, a customized laser was designed and built by Livetec GmbH Germany. The wavelength of the “nephro-laser” LLLT is 785 nm (min and max wavelength: 770 and 795 nm), between visible red and invisible near-infrared light. On the basis of a rat model, we estimated a penetration depth of approximately 1 cm for a 700–800 nm wavelength that should reach the kidneys . The heterogeneous composition of tissue of the kidney absorbs and scatters parts of the light, so the dosage was chosen in consideration of this loss and dissipation of energy. The laser electrode included five laser diodes that were the energy sources (Light-Emitting Diodes or LEDs), and the power of each LED determined the applied dosage. The continuous wave mode was used, with different duration of treatment time. The dosage was calculated using these equations:
To experimentally determine the correct dosage, various test dosages were selected, in relation to the minimal responder dose of 1.0 J cm−².
Studies were conducted according with the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Testing, and Education issued by the New York Academy of Sciences Adhoc Committee on Animal Research. Pathogen-free ZSF1 rats were from Charles River (Wilmington, MA). The ZSF1 strain is a first-generation (F1) hybrid rat derived from two well-characterized parental strains: the Zucker diabetic fat (ZDF, fa/fa) rat and the spontaneous hypertensive heart failure rat (SHHF) . Rats were maintained from 6 week of age to the end of the study at 45 week of age. LLLT was applied five times per week, from 26 to 34 week of age (Fig. 1). Four groups (each n = 12) received different fluences of LLLT (Table 1). The aim of the study was to compare sham treatment with high-dose LLLT. In addition, intermediate dose LLLT groups were included to better understand the dose kinetics. Group L4 received 1.5 J cm−2 (five diodes, 3 mW each, for 4.25 min). Group L11 received 4 J cm−2 (five diodes, 3 mW each, for 11.33 min). Group L20 received 12 J cm−2 (five diodes, 5 mW each, for 20.4 min). Finally, group C (control) received the treatment similar to group L20 but with device switched off (0 J cm−2). Electrodes were programmed depending on parameters like power density, power of diodes and treatment time. For a homogenous irradiation, the optimized arrangement of diodes was calculated beforehand. One diode was centered and the other four were arranged circularly with a distance of 1.05 cm to the middle point. The light source was situated right over the skin. The spot area is determined by the arrangement of the five diodes and the divergence angle of the radiation. In a depth of 1 cm, this results in a spot area for the five laser diodes which is a circular disk with a diameter of 1.5 cm. Both kidneys were irradiated simultaneously (fluence parameters are calculated for each light source, so each kidney was exposed to those conditions).
|Dosage/Treatment (J cm−2)||Number of diodes||Power/Diode (mW)||Treatment time (min)||Mode||Power/5 Diodes (mW)|
Rats were fed Purina diet 5008 ad libitum and were housed in steel cages and acclimatized to a 12:12-h light-dark (7 A.M.–7 P.M.) cycle. Body weights and blood metabolic profiles were monitored throughout the experiment. Blood samples were obtained from the tail vein from fasting animals. No differences in activity condition were observed in treated groups versus control group. At the end of treatment period, systolic blood pressure (SBP) was measured in conscious, restrained rats by tail-cuff sphygmomanometer (NARCO; Biosystems, CO).
At the end the study, rats were euthanized, blood samples obtained and kidneys perfused in situ with cold saline before removal. One kidney was fixed in buffered formalin, embedded in paraffin and used for optical microscopy.
Blood and urine analysis
Blood was extracted from the tail vein and collected in EDTA tubes. Then, blood was centrifuged at 1000 g for 10 min to obtain the plasma. Twenty-four hour urine was collected from each rat in metabolic cages. Plasma levels of triglycerides, total cholesterol, creatinine, and urea and urine levels of creatinine, protein and urea were measured on a Beckman CX4CE Clinical System. Glomerular filtration rate (GFR) was calculated as the mean of creatinine clearance (CCr) and urea clearance (CUrea) as creatinine clearance overestimates and urea clearance infraestimates GFR (GFR = (CCr + CUrea)/2). CCr and CUrea were calculated as ([molecule] urine × diuresis/[molecule]plasma)/1440 min and adjusted per weight.
Rat kidneys were localized by sonography to determine the site of laser diode placement in rats with shaved loins (Fig. 2). Siemens ×300 sonographer and a compact linear probe VF13-5 (Siemens, Munich, Germany) were used to localize the kidneys (frequency 11.4 MHz). Once the kidneys were located, we corroborated that the size of both kidney and laser diode were compatible.
Histological studies were carried out in 5 μm-thick paraffin-embedded tissue sections . For sirius red staining, tissue sections were deparaffinized with xylene and graded concentrations of ethanol up to 70% ethanol where slides remained for 5 days at 4°C. Direct Red 80 (Sigma) was dissolved in Picro-sirius acid and incubated with tissue section for 30 min at room temperature. Samples were dehydrated with a 100% ethanol wash and xylene. Slides were mounted in DPX medium (Merck, Darmstadt, Germany).
Image quantification was carried out with ImageProPlus software (MediaCybernetics, Bethesda, MD). This software allows selecting and calculating the area of pixels with similar color. Results are shown as percentage of interstitial sirius red positive stained area in 10 fields per kidney (×200 magnification). Glomeruli were excluded from the quantification .
Statistical analysis was performed using SPSS software (SPSS Inc., Chicago, IL). A parametric test (Student's t-test) was used for analysis of statistic differences between the high-dose LLLT and the sham groups. A P value <0.05 was considered significant. Results are expressed as mean ± SEM.
ZSF1 rats develop metabolic syndrome
Control sham-treated rats displayed hyperglycemia, hyperlipidemia, obesity (Table 2) and high SBP (164 ± 2 mmHg). Values for age-matched Wistar Kyoto rats are glycemia 126 ± 2 mg dL−1, total cholesterol 45 ± 12 mg dL−1, weight 225 ± 49 g (ZSF1 rats weight at baseline 588 ± 10 g) and SBP 138 ± 7 mmHg [11, 18]. In addition, untreated control rats displayed a progressive increase in serum creatinine and urea indicative of renal insufficiency, as well as abnormal urinary protein excretion (Table 3). Thus, a model of CKD secondary to metabolic syndrome characterized by type II diabetes, hypertension and obesity was reproduced.
|Group||Basal (week 0)||End of treatment (week 8)||End of study (week 18)|
|Δ weight (%)||100 ± 2||100 ± 2||100 ± 2||100 ± 1||108 ± 2||106 ± 2||107 ± 2||109 ± 1||118 ± 2||117 ± 2||118 ± 2||122 ± 1|
|Glucose (mg dL−1)||278 ± 21||262 ± 11||261 ± 9||257 ± 10||298 ± 22||361 ± 30||299 ± 18||320 ± 17||260 ± 19||316 ± 26||319 ± 31||*342 ± 30|
|Cholesterol (mg dL−1)||225 ± 5||233 ± 3||221 ± 5||*241 ± 2||338 ± 59||386 ± 60||271 ± 7||340 ± 43||590 ± 55||524 ± 40||723 ± 53||574 ± 40|
|Triglycerides (mg dL−1)||208 ± 14||192 ± 10||193 ± 10||174 ± 9||255 ± 10||208 ± 9||228 ± 13||*205 ± 16||115 ± 16||103 ± 14||88 ± 5||156 ± 30|
|Group||Basal (week 0)||End of treatment (week 8)||End of study (week 18)|
|Cr (mg dL−1)||0.26 ± 0.01||0.25 ± 0.02||0.26 ± 0.01||0.23 ± 0.01||0.40 ± 0.02||0.40 ± 0.01||0.36 ± 0.01||0.35 ± 0.02||0.66 ± 0.04||0.62 ± 0.03||0.75 ± 0.03||0.74 ± 0.04|
|Urea (mg dL−1)||48.7 ± 1.9||50.6 ± 1.5||48.1 ± 2.8||52.8 ± 2.3||47.7 ± 1.9||47.9 ± 1.5||43.8 ± 1.1||47.8 ± 2.0||59.6 ± 4.8||55.9 ± 1.8||52.2 ± 3.5||48.6 ± 3.6|
|CCr (mL min−1 kg−1)||2.45 ± 0.16||2.85 ± 0.29||2.77 ± 0.14||2.73 ± 0.13||2.65 ± 0.27||2.32 ± 0.33||3.53 ± 0.31||2.88 ± 0.39||0.80 ± 0.05||0.86 ± 0.06||0.98 ± 0.10||1.07 ± 0.07|
|CUrea (mL min−1 kg−1)||1.15 ± 0.20||1.00 ± 0.27||1.30 ± 0.32||0.95 ± 0.25||1.14 ± 0.45||1.07 ± 0.38||1.15 ± 0.18||1.38 ± 0.32||0.46 ± 0.12||0.49 ± 0.12||0.45 ± 0.12||0.48 ± 0.11|
|GFR (mL min−1 kg−1)||1.80 ± 0.10||1.93 ± 0.17||2.04 ± 0.11||1.81 ± 0.14||1.89 ± 0.2||1.70 ± 0.22||2.34 ± 0.18||2.12 ± 0.22||0.63 ± 0.04||0.68 ± 0.05||0.71 ± 0.07||*0.79 ± 0.04|
|UPro/UCr||11.2 ± 1.7||7.6 ± 0.9||8.8 ± 1.1||9.1 ± 1.3||9.3 ± 1.0||8.3 ± 1.0||9.5 ± 1.0||9.6 ± 1.8||12.3 ± 1.3||10.4 ± 1.2||10 ± 1.6||9.8 ± 1.3|
Effect of LLLT to the kidneys on metabolic parameters
High-dose LLLT (L20) was associated with a transient decrement in serum triglyceride levels at the end of therapy that was no longer observed at the end of follow-up. In addition, the high-dose group rats displayed higher glucose levels at the end of follow-up (Table 2). The cause for this observation is not readily apparent, but we speculate that it may be related to increased appetite as there was a trend toward a greater weight gain in this group. We must emphasize that given this observation, any changes in renal function or histology cannot be attributed to improved metabolic control.
Effect of LLLT to the kidneys on renal function
No significant influence of LLLT was observed on plasma creatinine or urea or on urinary protein excretion either at the end of treatment or at the end of follow-up (Table 3). However, a trend toward increased CCr, a measure of GFR, was observed in the high-dose LLLT L20 group both at the end of the treatment period and at the end of follow-up. When the GFR was calculated more precisely as the mean of CCr and CUrea, a trend toward better preserved GFR was observed at the end of treatment in the high-dose LLLT L20 group that translated into a significantly better preserved GFR in L20 rats at the end of the study (Fig. 3). Furthermore, a LLLT dose–response trend was observed toward better preserved Ccr, better preserved GFR and lower plasma urea at the end of follow-up when all groups of rats were considered (Table 2 and Fig. 3).
At the end of the treatment, all groups showed a high SBP (>140 mmHg). However, SBP was significantly lower in L20 than in sham untreated control rats and a trend toward a LLLT dose–response was observed (Fig. 4).
Interstitial collagen fiber accumulation is reduced by kidney laser treatment
Interstitial fibrosis is the hallmark of irreversible CKD. Collagen is the main extracellular matrix component of interstitial fibrosis. Sirius red staining allowed the quantification of interstitial collagen deposition. Images of sirius red-stained sham untreated kidneys showed the common features of CKD including tubular atrophy and dilatation and increased collagen fiber staining indicative of fibrosis in the interstitium and glomeruli (Fig. 5A). Collagen deposition was evaluated in the tubulointerstitial space as percentage of sirius red positive area, as there is a better correlation between tubulointerstitial fibrosis and renal function outcome than between glomerular injury and kidney function outcome even in primarily glomerular disorders [28-30]. LLLT resulted in a dose-related decrease in tubulointerstitial fibrosis that reached statistical significance in the high-dose group (L20) (L20 14.1 ± 0.45 versus C 17.4 ± 0.96%, P = 0.022) (Fig. 5B).
The aim of this study was to evaluate the potential therapeutic effect of LLLT L20 in experimental CKD. In addition, a dose-ranging exploration of lower LLLT doses was performed. For this purpose, a rat model of CKD representative of the main human causes of CKD, obesity-related type 2 diabetes and arterial hypertension was chosen. The ZSF1 rat strain shows obesity, hyperglycemia, dyslipidemia and hypertension as well as progressive renal failure. The main finding of the study is that LLLT better preserves GFR and results in less fibrosis than control sham-treated rats. However, protection of renal function was partial and renal function at the end of the study was worse than at baseline in all groups.
LLLT is used to treat arthritis rheumatoid, an autoimmune inflammatory disease, and may modulate inflammation and wound healing [24, 25]. The mechanisms by which LLLT exerts its therapeutic effects are not completely known. In animal models of acute lung inflammation, LLLT reduced the levels of cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-8 (IL–8), and reduced the neutrophil influx [31-33]. Some of these LLLT effects were also observed in cultured synoviocytes . CKD is an inflammatory disease  and TNF superfamily members, mainly TNF-α, are key mediators in acute and chronic renal injury . Inflammation and inflammatory cytokines contribute to CKD progression by promoting parenchymal cell death and fibrosis [12, 14, 36]. Therefore, we hypothesized that the potential LLLT-induced decrease in cytokines such as TNF-α might reduce CKD-associated kidney inflammation and preserve renal function. However, there are few studies which address the effect of LLLT on renal function or renal fibrosis in experimental CKD. In rabbits with renal colic, low intensive laser irradiation prevented some ultrastructural changes in proximal tubules as assessed by electron microscopy . However, no assessment of fibrosis or renal function was provided. More recently, LLLT was shown to modulate kidney oxidation-related pathways in experimental type I diabetes and a nonsignificant trend toward lower blood glucose, serum creatinine and blood urea nitrogen (BUN) was observed . However, neither GFR nor fibrosis was assessed. There are critical differences between this study and the one presented here. Thus, the rat model did not display obesity, hypertension or progressive renal failure. Furthermore, the wavelength of the laser (670 nm) differed.
The combination of diabetes, obesity and hypertension is the main cause of end-stage renal disease in Western societies. We now report the effects of LLLT in a rat model of progressive renal failure induced by a combined metabolic and hypertensive disorder. LLLT had persistent positive effects on both renal functional and structural parameters that persisted for several weeks beyond the end of treatment. Thus, GFR was better preserved at the end of follow-up and this was associated with a decreased renal fibrosis. This beneficial effect was observed despite the persistence of hyperglycemia. Indeed, glycemia was higher in the rats from the L20 group. As these rats tended to gain more weight, we hypothesize that a better health may have resulted in increased weight gain and hyperglycemia. These parameters may be related to better appetite or to a better preservation of renal function. In this regard, the control of glycemia improves with worsening renal function in diabetic subjects (“Burnt-out” diabetes), as a consequence of the combination of lower appetite, increased insulin half-life and other factors . A better general well-being may also explain their improved GFR despite lack of changes in plasma creatinine between groups. GFR is considered a better indicator of kidney function than serum creatinine, serum urea or their respective clearances. Muscle mass is the main source of creatinine and preservation of muscle mass is associated with increased creatinine values . In this regard, kidney failure leads to sarcopenia, which limits the increase in serum creatinine. Protein intake and kidney function are key determinants of serum urea. Decreased kidney function promotes anorexia, which limits protein intake and the rise of serum urea. Finally, CCr overestimates and CUrea infraestimates kidney function.
In addition, in our model, better SBP control was observed at the end of treatment in groups L11 and L20. We hypothesize that this may be directly related to a direct effect of LLLT on the kidneys. Indeed, LLLT is known to have anti-inflammatory properties and Bernardo Rodriguez-Iturbe et al. have extensively documented a hypertension promoting effect of kidney inflammation [41-45].
Laser treatment accelerates the wound-healing process . This may be due, at least in part, to proliferative effects of photoirradiation [47-52], including on stem cells . In the kidney, healing, proliferative actions and anti-inflammatory actions of laser may contribute to the repair process and the observed beneficial effect on kidney function and structure. Low-level lasers have nonthermal effects, but the molecular mechanisms are poorly understood. The main accepted hypothesis is that most of the laser effects occur due to the stimulation of photoreactive proteins like cytochrome C in the mitochondrial respiratory chain [54-58]. This may improve ATP availability in the cell and modulate reactive oxygen species (ROS) [59-62]. Laser therapies could increase collagen deposition and fibroblasts proliferation to accelerate wound-healing process both in vitro and in vivo . Otherwise, there are studies indicating a reduction in total collagen deposition in organs other than skin, such as oral mucosa or muscle, measured by picro-sirius acid staining or hydroxyproline content [64, 65]. In these publications, the antifibrotic effect is associated with a decreased inflammation (neutrophil influx and NF-κB blockade respectively) after tissue damage, suggesting that the prolonged inflammatory process is leading the higher deposition of collagen. Heterogeneity of the fibroblast populations depending on the tissue, or even the individual, could also explain the different effect of LLLTs regarding collagen production after tissue injury [63, 66, 67]. An additional element to be considered is the possibility of biphasic dose responses. In this regard, dose responses may not be necessarily linear. This concept is important for any potential extrapolation to humans. In this regard, the ultimate aim of experimental studies is to pave the way for human intervention. As LLLT has already been applied to humans, key safety issues have already been addressed. As potential advantages for the treatment of human CKD we find the noninvasive nature of the procedure. As a potential disadvantage the need for almost daily application for long periods of time due to the long time course of human CKD. In this regard, advances in renal imaging that allow the early, noninvasive detection and monitoring of kidney fibrosis may facilitate human trials and the definition of realistic end points. In addition, inflammation is also a feature of acute kidney injury and the procedure may also be useful in this context.
In conclusion, we now report significant beneficial effects of LLLT on kidney function and structure in a rat model of renal progressive failure induced by a metabolic and hypertensive disorder representative of the main cause of end-stage renal disease in humans. A preliminary dose-ranging analysis suggests that the full potential of this therapy might be achieved with higher dose of those tested. This dose dependence seems to be a main characteristic of laser therapy and different doses may induce different cell type responses . In this regard, further investigations on dosage should be done. In addition, further study on the molecular and cellular mechanisms of the beneficial effects should be performed. On the basis of these results, we propose that LLLT is a promising therapeutic strategy for CKD.
This study was supported by a grant from CERES GmbH and BMWi (Germany). A.C.U was supported by CERES GmbH, Livetec GmbH, and Fundación Conchita Rábago. A.O. was supported by Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM), CAM S2010/BMD-2378 and REDINREN RETIC 06/0016 and RD12/0021/0001. EDNSG. We thank Dr. Olga Sanchez Pernaute for helping with the sonography.
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