Targeted addition of mini-dystrophin into rDNA locus of Duchenne muscular dystrophy patient-derived iPSCs.

Duchenne muscular dystrophy (DMD), the most common lethal muscular disorder, affects 1 in 5000 male births. It is caused by mutations in the X-linked dystrophin gene (DMD), and there is no effective treatment currently. Gene addition is a promising strategy owing to its universality for patients with all gene mutations types. In this study, we describe a site-specific gene addition strategy in induced pluripotent stem cells (iPSCs) derived from a DMD patient with exon 50 deletion. By using transcription activator-like effector nickases (TALENickases), the mini-dystrophin cassette was precisely targeted at the ribosomal RNA gene (rDNA) locus via homologous recombination with high targeting efficiency. The targeted clone retained the main pluripotent properties and was differentiated into cardiomyocytes. Significantly, the dystrophin expression and membrane localization were restored in the genetic corrected iPSCs and their derived cardiomyocytes. More importantly, the enhanced spontaneous contraction was observed in modified cardiomyocytes. These results provide a proof of principle for an efficient targeted gene addition for DMD gene therapy and represents a significant step toward precisely therapeutic for DMD.

Identification of marker proteins of muscular dystrophy in the urine proteome from the mdx-4cv model of dystrophinopathy.

Since the protein constituents of urine present a dynamic proteome that can reflect a variety of disease-related alterations in the body, the mass spectrometric survey of proteome-wide changes in urine promises new insights into pathogenic mechanisms. Urine can be investigated in a completely non-invasive way and provides valuable biomedical information on body-wide changes. In this report, we have focused on the urine proteome in X-linked muscular dystrophy using the established mdx-4cv mouse model of dystrophinopathy. In order to avoid potential artefacts due to the manipulation of the biofluid proteome prior to mass spectrometry, crude urine specimens were analyzed without the prior usage of centrifugation steps or concentration procedures. Comparative proteomics revealed 21 increased and 8 decreased proteins out of 870 identified urinary proteoforms using 50 µl of biofluid per investigated sample, i.e. 14 wild type versus 14 mdx-4cv specimens. Promising marker proteins that were almost exclusively found in mdx-4cv urine included nidogen, parvalbumin and titin. Interestingly, the mass spectrometric identification of urine-associated titin revealed a wide spread of peptides over the sequence of this giant muscle protein. The newly established urinomic signature of dystrophinopathy might be helpful for the design of non-invasive assays to improve diagnosis, prognosis, therapy-monitoring and evaluation of potential harmful side effects of novel treatments in the field of muscular dystrophy research.

Discovery of potential urine-accessible metabolite biomarkers associated with muscle disease and corticosteroid response in the mdx mouse model for Duchenne.

Urine is increasingly being considered as a source of biomarker development in Duchenne Muscular Dystrophy (DMD), a severe, life-limiting disorder that affects approximately 1 in 4500 boys. In this study, we considered the mdx mice-a murine model of DMD-to discover biomarkers of disease, as well as pharmacodynamic biomarkers responsive to prednisolone, a corticosteroid commonly used to treat DMD. Longitudinal urine samples were analyzed from male age-matched mdx and wild-type mice randomized to prednisolone or vehicle control via liquid chromatography tandem mass spectrometry. A large number of metabolites (869 out of 6,334) were found to be significantly different between mdx and wild-type mice at baseline (Bonferroni-adjusted p-value < 0.05), thus being associated with disease status. These included a metabolite with m/z = 357 and creatine, which were also reported in a previous human study looking at serum. Novel observations in this study included peaks identified as biliverdin and hypusine. These four metabolites were significantly higher at baseline in the urine of mdx mice compared to wild-type, and significantly changed their levels over time after baseline. Creatine and biliverdin levels were also different between treated and control groups, but for creatine this may have been driven by an imbalance at baseline. In conclusion, our study reports a number of biomarkers, both known and novel, which may be related to either the mechanisms of muscle injury in DMD or prednisolone treatment.

Phase 1 Study of Edasalonexent (CAT-1004), an Oral NF-κB Inhibitor, in Pediatric Patients with Duchenne Muscular Dystrophy.

BACKGROUND: Edasalonexent is an orally administered small molecule designed to inhibit NF-κB, which is activated from infancy in Duchenne muscular dystrophy and is central to causing muscle damage and preventing muscle regeneration. OBJECTIVE: Evaluate the safety, tolerability, pharmacokinetics and exploratory pharmacodynamics of three doses of edasalonexent in ambulatory males ≥4 to <8 years of age with genetically confirmed Duchenne muscular dystrophy. METHODS: This was a 1-week, open-label, multiple-dose study with 3 sequential ascending doses (33, 67 and 100 mg/kg/day) of edasalonexent administered under different dietary conditions to 17 males with a mean age of 5.5 years. RESULTS: All doses of edasalonexent were well tolerated, with no serious adverse events, no drug discontinuations and no dose reductions. The majority of adverse events were mild, and the most common adverse events were gastrointestinal (primarily diarrhea). Edasalonexent was rapidly absorbed with peak levels observed 2-6 hours after dosing and exposures appeared to increase nearly proportionally to dose for the 2 lower and all 3 doses under low-fat and high-fat meal conditions, respectively. Only minor plasma accumulation of edasalonexent was observed with 7 days of dosing. After treatment with edasalonexent for 7 days, levels of NF-κB-regulated genes and serum proteins were decreased. CONCLUSIONS: This first report of edasalonexent oral administration for one week in male pediatric patients with Duchenne muscular dystrophy showed that treatment was well tolerated and inhibited NF-kB pathways.

Analysis of extracellular mRNA in human urine reveals splice variant biomarkers of muscular dystrophies.

Urine contains extracellular RNA (exRNA) markers of urogenital cancers. However, the capacity of genetic material in urine to identify systemic diseases is unknown. Here we describe exRNA splice products in human urine as a source of biomarkers for the two most common forms of muscular dystrophies, myotonic dystrophy (DM) and Duchenne muscular dystrophy (DMD). Using a training set, RT-PCR, droplet digital PCR, and principal component regression, we identify ten transcripts that are spliced differently in urine exRNA from patients with DM type 1 (DM1) as compared to unaffected or disease controls, form a composite biomarker, and develop a predictive model that is 100% accurate in our independent validation set. Urine also contains mutation-specific DMD mRNAs that confirm exon-skipping activity of the antisense oligonucleotide drug eteplirsen. Our results establish that urine mRNA splice variants can be used to monitor systemic diseases with minimal or no clinical effect on the urinary tract.

Urinary prostaglandin metabolites as Duchenne muscular dystrophy progression markers.

BACKGROUND: Patients with Duchenne muscular dystrophy (DMD) exhibit increased prostaglandin D2 (PGD2) expression in necrotic muscle and increased PGD2 metabolites in their urine. In mouse models, inhibiting PGD2 production suppresses muscle necrosis, suggesting a possible intervention through PGD2-mediated activities. OBJECTIVE: We investigated the involvement of PGD2 and its potential use as a marker of pathological progression in DMD. METHODS: Sixty-one male children with DMD and thirty-five age-matched controls were enrolled in the study. DMD patients were divided into "ambulant" and "non-ambulant" groups, which were further subdivided into "steroid" and "non-steroid" therapy groups. Levels of the PGD2 metabolite tetranor-PGDM (t-PGDM) and creatinine were measured in both spot and 24-hour urine samples, with comparisons between groups made according to geometric mean values. RESULTS: DMD patients had significantly higher levels of creatinine-corrected t-PGDM in spot urine samples as compared with the control group. Additionally, both ambulant and non-ambulant DMD groups had significantly higher levels of t-PGDM as compared with controls, with no significant difference in t-PGDM levels observed between steroid and non-steroid groups. Moreover, total creatinine excretion in 24-hour urine samples was significantly lower in DMD patients as compared with controls, and although DMD patients had lower muscle mass than controls, their overall levels of t-PGDM did not differ significantly from those in the non-ambulant and control groups. CONCLUSION: PGD2 might help explain the progression and symptomatic presentations (e.g., ambulatory difficulty) associated with DMD, suggesting it as a useful pathological marker and use of a selective PGD2 inhibitor as a potential treatment modality.

Receiver operating curve analyses of urinary titin of healthy 3-y-old children may be a noninvasive screening method for Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive, fatal muscle wasting disease. Early detection of DMD by mass screening may enable the early treatment of these patients. We have reported that urinary titin concentration, an indicator of severe muscle wasting, is a diagnostic biomarker for DMD. METHODS: Urinary titin concentrations were measured in healthy 3-y-old children and, by comparison with concentrations in 4 DMD patients, and validated as a screening biomarker for DMD. Urine samples were obtained from 100 healthy Japanese children, 52 boys and 48 girls, and their urinary titin concentrations measured by ELISA. RESULTS: The mean ±â€¯SD urinary titin concentration was 1.5 ±â€¯2.5 nmol/l, and the mean urinary titin concentration normalized to creatinine was 2.2 ±â€¯4.1 pmol/mg creatinine, with no differences between boys and girls. Histograms and box-and-whisker plots showed that almost all titin and normalized titin concentrations were in narrow ranges, with one outlier in common. Receiver operating characteristic curve analysis showed that titin and normalized-titin concentrations from healthy 3-y-olds were completely separate from those of 3-y-old DMD patients. CONCLUSIONS: These findings indicate that urinary titin may be an excellent non-invasive biomarker to screen for DMD.

Neopterin/7,8-dihydroneopterin is elevated in Duchenne muscular dystrophy patients and protects mdx skeletal muscle function.

NEW FINDINGS: What is the central question of this study? We examined whether the macrophage-synthesized antioxidant 7,8-dihydroneopterin was elevated in Duchenne muscular dystrophy (DMD) patients. We then examined whether 7,8-dihydroneopterin could protect dystrophic skeletal mouse muscle from eccentric contraction-induced force loss and improve recovery. What is the main finding and its importance? Urinary neopterin/creatinine and 7,8-dihydroneopterin/creatinine were elevated in DMD patients. 7,8-Dihydroneopterin attenuated eccentric contraction-induced force loss of dystrophic skeletal mouse muscle and accelerated recovery of force. These results suggest that eccentric contraction-induced force loss is mediated, in part, by an oxidative component and provides a potential protective role for 7,8-dihydroneopterin in DMD. ABSTRACT: Macrophage infiltration is a hallmark of dystrophin-deficient muscle. We tested the hypothesis that Duchenne muscular dystrophy (DMD) patients would have elevated levels of the macrophage-synthesized pterins, neopterin and 7,8-dihydroneopterin, compared with unaffected age-matched control subjects. Urinary neopterin/creatinine and 7,8-dihydroneopterin/creatinine were elevated in DMD patients, and 7,8-dihydroneopterin/creatinine was associated with patient age and ambulation. Urinary 7,8-dihydroneopterin corrected for specific gravity was also elevated in DMD patients. Given that 7,8-dihydroneopterin is an antioxidant, we then identified a potential role for 7,8-dihydroneopterin in disease pathology. We assessed whether 7,8-dihydroneopterin could: (i) protect against isometric force loss in wild-type skeletal muscle exposed to various pro-oxidants; and (ii) protect wild-type and mdx muscle from eccentric contraction-induced force loss, which has an oxidative component. Force loss was elicited in isolated extensor digitorum longus (EDL) muscles by 10 eccentric contractions, and recovery of force after the contractions was measured in the presence of exogenous 7,8-dihydroneopterin. 7,8-Dihydroneopterin attenuated isometric force loss by wild-type EDL muscles when challenged by H2 O2 and HOCl, but exacerbated force loss when challenged by SIN-1 (NO• , O2• , ONOO- ). 7,8-Dihydroneopterin attenuated eccentric contraction-induced force loss in mdx muscle. Isometric force production by EDL muscles of mdx mice also recovered to a greater degree after eccentric contractions in the presence of 7,8-dihydroneopterin. The results corroborate macrophage activation in DMD patients, provide a potential protective role for 7,8-dihydroneopterin in the susceptibility of dystrophic muscle to eccentric contractions and indicate that oxidative stress contributes to eccentric contraction-induced force loss in mdx skeletal muscle.

High urinary ferritin reflects myoglobin iron evacuation in DMD patients.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in the dystrophin gene leading to the absence of the normal dystrophin protein. The efforts of many laboratories brought new treatments of DMD to the reality, but ongoing and forthcoming clinical trials suffer from absence of valuable biomarkers permitting to follow the outcome of the treatment day by day and to adjust the treatment if needed. In the present study the levels of 128 urinary proteins including growth factors, cytokines and chemokines were compared in urine of DMD patients and age related control subjects by antibody array approach. Surprisingly, statistically significant difference was observed only for urinary ferritin whose level was 50 times higher in young DMD patients. To explain the observed high urinary ferritin content we analysed the levels of iron, iron containing proteins and proteins involved in regulation of iron metabolism in serum and urine of DMD patients and their age-matched healthy controls. Obtained data strongly suggest that elevated level of urinary ferritin is functionally linked to the renal management of myoglobin iron derived from leaky muscles of DMD patients. This first observation of the high level of ferritin in urine of DMD patients permits to consider this protein as a new urinary biomarker in muscular dystrophies and sheds light on the mechanisms of iron metabolism and kidney functioning in DMD.

Results, meta-analysis and a first evaluation of UNOxR, the urinary nitrate-to-nitrite molar ratio, as a measure of nitrite reabsorption in experimental and clinical settings.

We recently found that renal carbonic anhydrase (CA) is involved in the reabsorption of inorganic nitrite (NO2-), an abundant reservoir of nitric oxide (NO) in tissues and cells. Impaired NO synthesis in the endothelium and decreased NO bioavailability in the circulation are considered major contributors to the development and progression of renal and cardiovascular diseases in different conditions including diabetes. Isolated human and bovine erythrocytic CAII and CAIV can convert nitrite to nitrous acid (HONO) and its anhydride N2O3 which, in the presence of thiols (RSH), are further converted to S-nitrosothiols (RSNO) and NO. Thus, CA may be responsible both for the homeostasis of nitrite and for its bioactivation to RSNO/NO. We hypothesized that enhanced excretion of nitrite in the urine may contribute to NO-related dysfunctions in the renal and cardiovascular systems, and proposed the urinary nitrate-to-nitrite molar ratio, i.e., UNOxR, as a measure of renal CA-dependent excretion of nitrite. Based on results from clinical and experimental animal studies, here, we report on a first evaluation of UNOxR. We determined UNOxR values in preterm neonates, healthy children, and adults, in children suffering from type 1 diabetes mellitus (T1DM) or Duchenne muscular dystrophy (DMD), in elderly subjects suffering from chronic rheumatic diseases, type 2 diabetes mellitus (T2DM), coronary artery disease (CAD), or peripheral arterial occlusive disease (PAOD). We also determined UNOxR values in healthy young men who ingested isosorbide dinitrate (ISDN), pentaerythrityl tetranitrate (PETN), or inorganic nitrate. In addition, we tested the utility of UNOxR in two animal models, i.e., the LEW.1AR1-iddm rat, an animal model of human T1DM, and the APOE*3-Leiden.CETP mice, a model of human dyslipidemia. Mean UNOxR values were lower in adult patients with rheumatic diseases (187) and in T2DM patients of the DALI study (74) as compared to healthy elderly adults (660) and healthy young men (1500). The intra- and inter-variabilities of UNOxR were of the order of 50% in young and elderly healthy subjects. UNOxR values were lower in black compared to white boys (314 vs. 483, P = 0.007), which is in line with reported lower NO bioavailability in black ethnicity. Mean UNOxR values were lower in DMD (424) compared to healthy (730) children, but they were higher in T1DM children (1192). ISDN (3 × 30 mg) decreased stronger UNOxR compared to PETN (3 × 80 mg) after 1 day (P = 0.046) and after 5 days (P = 0.0016) of oral administration of therapeutically equivalent doses. In healthy young men who ingested NaNO3 (0.1 mmol/kg/d), UNOxR was higher than in those who ingested the same dose of NaCl (1709 vs. 369). In LEW.1AR1-iddm rats, mean UNOxR values were lower than in healthy rats (198 vs. 308) and comparable to those in APOE*3-Leiden.CETP mice (151).

Downregulation of miRNA-29, -23 and -21 in urine of Duchenne muscular dystrophy patients.

AIM: To study the signature of 87 urinary miRNAs in Duchenne muscular dystrophy (DMD) patients, select the most dysregulated and determine statistically significant differences in their expression between controls, ambulant (A) and nonambulant (NA) DMD patients, and patients on different corticosteroid regimens. Patients/materials & methods: Urine was collected from control (n = 20), A (n = 31) and NA (n = 23) DMD patients. miRNA expression was measured by reverse transcription-quantitative PCR. RESULTS: miR-29c-3p was significantly downregulated in A DMD patients while miR-23b-3p and miR-21-5p were significantly downregulated in NA DMD patients compared with age-matched controls. CONCLUSION: miR-29c-3p, miR-23b-3p and miR-21-5p are promising novel noninvasive biomarkers for DMD, and miR-29c-3p levels are differentially affected by different steroid regimens, supporting the antifibrotic effect of steroid therapy.

Diagnostic and clinical significance of the titin fragment in urine of Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is a fatal progressive muscle wasting disease of childhood. Titin in sarcomere is digested by calcium dependent protease. To explore muscle damage in DMD, the urinary concentrations of the N-terminal fragment of titin were determined using a newly developed enzyme linked immune sorbent assay kit. The urinary titin concentrations were normalized to creatinine (Cr). A total of 145 urine samples were obtained at a single Japanese hospital from 113 DMD patients aged 3-29years. Normalized urinary titin concentration was 965.8±1011.9 (Mean±SD) pmol/mg Cr in patients with DMD. This was nearly 700-fold higher than healthy children (1.4±0.8pmol/mg Cr). The concentration was significantly higher in DMD than in BMD patients who had significantly higher urinary titin than normal. Urinary titin in DMD patients tended to decrease with age. The median concentration of urinary titin in the youngest (aged 3-7years) and oldest (aged ≥16years) groups was 1468.3 and 411.3pmol/mg Cr, respectively, with significant difference. Urinary concentration of titin correlated significantly with serum creatine kinase concentration, the best-known biomarker of DMD. The N-terminal fragment of titin in urine has potential as a diagnostic and clinical biomarker for DMD.

Dramatic elevation in urinary amino terminal titin fragment excretion quantified by immunoassay in Duchenne muscular dystrophy patients and in dystrophin deficient rodents.

Enzyme-linked and electrochemiluminescence immunoassays were developed for quantification of amino (N-) terminal fragments of the skeletal muscle protein titin (N-ter titin) and qualified for use in detection of urinary N-ter titin excretion. Urine from normal subjects contained a small but measurable level of N-ter titin (1.0 ± 0.4 ng/ml). A 365-fold increase (365.4 ± 65.0, P = 0.0001) in urinary N-ter titin excretion was seen in Duchene muscular dystrophy (DMD) patients. Urinary N-ter titin was also evaluated in dystrophin deficient rodent models. Mdx mice exhibited low urinary N-ter titin levels at 2 weeks of age followed by a robust and sustained elevation starting at 3 weeks of age, coincident with the development of systemic skeletal muscle damage in this model; fold elevation could not be determined because urinary N-ter titin was not detected in age-matched wild type mice. Levels of serum creatine kinase and serum skeletal muscle troponin I (TnI) were also low at 2 weeks, elevated at later time points and were significantly correlated with urinary N-ter titin excretion in mdx mice. Corticosteroid treatment of mdx mice resulted in improved exercise performance and lowering of both urinary N-ter titin and serum skeletal muscle TnI concentrations. Low urinary N-ter titin levels were detected in wild type rats (3.0 ± 0.6 ng/ml), while Dmdmdx rats exhibited a 556-fold increase (1652.5 ± 405.7 ng/ml, P = 0.002) (both at 5 months of age). These results suggest that urinary N-ter titin is present at low basal concentrations in normal urine and increases dramatically coincident with muscle damage produced by dystrophin deficiency. Urinary N-ter titin has potential as a facile, non-invasive and translational biomarker for DMD.

Establishment of a highly sensitive sandwich ELISA for the N-terminal fragment of titin in urine.

Muscle damage and loss of muscle mass are triggered by immobilization, loss of appetite, dystrophies and chronic wasting diseases. In addition, physical exercise causes muscle damage. In damaged muscle, the N-terminal and C-terminal regions of titin, a giant sarcomere protein, are cleaved by calpain-3, and the resulting fragments are excreted into the urine via glomerular filtration. Therefore, we considered titin fragments as promising candidates for reliable and non-invasive biomarkers of muscle injury. Here, we established a sandwich ELISA that can measure the titin N-terminal fragment over a biologically relevant range of concentrations, including those in urine samples from older, non-ambulatory Duchenne muscular dystrophy patients and from healthy donors under everyday life conditions and after exercise. Our results indicate that the established ELISA could be a useful tool for the screening of muscular dystrophies and also for monitoring the progression of muscle disease, evaluating the efficacy of therapeutic approaches, and investigating exercise-related sarcomeric disruption and repair processes.

The L-arginine/NO pathway and homoarginine are altered in Duchenne muscular dystrophy and improved by glucocorticoids.

The L-arginine/nitric oxide (L-Arg/NO) pathway regulates endothelial function and may play an important role in the pathogenesis of Duchenne muscular dystrophy (DMD). Yet, this pathway is poorly investigated in children suffering from DMD. Endothelial dysfunction can affect the perfusion of contracting muscles, thus leading to ischemia and hypoxia. In the present study, we tested the hypothesis that reduced NO production due to elevated synthesis of N (G),N (G)-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of NO synthesis, is a possible pathophysiological mechanism for progressive intramuscular muscle ischemia and disturbed endothelial function in children with DMD. Given the possible antagonistic action of homoarginine (hArg) on ADMA, we also analyzed this amino acid. We investigated 55 male patients with DMD and 54 healthy male controls (HC; aged 11.9 ± 4.8 vs. 11.1 ± 4.9 years, mean ± SD). Urinary creatinine and metabolites of the L-Arg/NO pathway were measured in plasma and urine by GC-MS or GC-MS/MS. Urine levels of ADMA and its major urinary metabolite dimethylamine (DMA), nitrite and nitrate (P < 0.001 for all) and hArg (P = 0.002) were significantly higher in DMD patients compared to HC, while the urinary DMA/ADMA molar ratio was lower (P = 0.002). In plasma, nitrate (P < 0.001), hArg (P = 0.002) and the hArg/ADMA ratio (P < 0.001) were lower in DMD than in HC. In plasma, ADMA (631 ± 119 vs. 595 ± 129 nM, P = 0.149), arginine and nitrite did not differ between DMD and HC. In DMD, positive correlations between ADMA, DMA or nitrate excretion and the stage of disease (according to Vignos and Thompson) were found. In DMD patients on steroid medication, lower concentrations of ADMA in plasma, and of DMA, ADMA, nitrate and hArg in urine were observed compared to non-treated patients. The L-Arg/NO pathway is impaired in DMD patients, with the disease progression being clinically negatively correlated with the extent of impairment. One of the underlying mechanisms in DMD may involve insufficient antagonism of ADMA by hArg. Steroids, but not creatine supplementation, seems to improve the L-Arg/NO pathway in DMD.

Renal function in children and adolescents with Duchenne muscular dystrophy.

Improved life expectancy and the need for robust tools to monitor renal safety of emerging new therapies have fueled the interest in renal function in Duchenne muscular dystrophy (DMD) patients. We aimed to establish a methodology to accurately assess their renal function. Twenty DMD patients (5-22 years) were included in this prospective study. After obtaining medical history, all patients underwent a clinical examination, 24-hour ambulatory blood pressure monitoring, ultrasound of the kidneys, direct GFR measurement ((51)Cr-EDTA, mGFR), complete blood and urine analysis. Seventeen of 20 patients were treated with corticosteroids and 5/20 with angiotensin converting enzyme inhibitor (lisinopril). No patient suffered from urinary tract infections or other renal diseases. Hypertension (systolic or diastolic blood pressure >P95) was found in 9/20 patients (8/9 patients were on steroid treatment) and a non-dipping blood pressure profile in 13/20 subjects (10/13 patients were on steroid treatment). Urinary protein to creatinine ratio was elevated in 17/18 patients, whereas 24-hour urine protein excretion was normal in all subjects. Median interquartile range (IQR) mGFR was 130.4 (29.1) mL/min/1.73 m(2). Hyperfiltration (mGFR >150 mL/min/1.73 m(2)) was found in 5/20 patients. Inverse correlation between mGFR and age was observed (R(2) = 0.45, p = 0.001). Serum creatinine based estimated GFR (eGFR) equations overestimated mGFR up to 300%. eGFR based on cystatin C Filler equation was closest to the mGFR (median eGFR (IQR) of 129.5 (39.7) mL/min/1.73 m(2)). Our study demonstrates a high prevalence of hyperfiltration and hypertension in children and adolescents with DMD. Because the majority of hypertensive patients were under corticosteroid treatment, the iatrogenic cause of hypertension cannot be excluded. Serum or urine creatinine measurements are of no value to evaluate renal function in DMD patients due to the reduced skeletal muscle mass.

Proteomics profiling of urine reveals specific titin fragments as biomarkers of Duchenne muscular dystrophy.

Diagnosis of muscular dystrophies is currently based on invasive methods requiring muscle biopsies or blood tests. The aim of the present study was to identify urinary biomarkers as a diagnostic tool for muscular dystrophies. Here, the urinary proteomes of Duchenne muscular dystrophy (DMD) patients and healthy donors were compared with a bottom-up proteomic approach. Label-free analysis of more than 1100 identified proteins revealed that 32 of them were differentially expressed between healthy controls and DMD patients. Among these 32 proteins, titin showed the highest fold change between healthy subjects and DMD patients. Interestingly, most of the sequenced peptides belong to the N-terminal and C-terminal parts of titin, and the presence of the corresponding fragments in the urine of DMD patients was confirmed by Western blot analysis. Analysis of a large cohort of DMD patients and age-matched controls (a total of 104 individuals aged from 3 to 20 years) confirmed presence of the N-ter fragment in all but two patients. In two DMD patients aged 16 and 20 years this fragment was undetectable and two healthy controls of 16 and 19 years with serum CK >800 IU/L demonstrated a low level of the fragment. N- and C-terminal titin fragments were also detected in urine from patients with other muscular dystrophies such as Becker muscular dystrophy and Limb-girdle muscular dystrophy (type 1D, 2D and 2J) but not in neurogenic spinal muscular atrophy. They were also present in urine of dystrophin-deficient animal models (GRMD dogs and mdx mice). Titin is the first urinary biomarker that offers the possibility to develop a simple, non-invasive and easy-to-use test for pre-screening of muscular dystrophies, and may also prove to be useful for the non-invasive follow up of DMD patients under treatment.

Dystrophin-deficient cardiomyocytes derived from human urine: new biologic reagents for drug discovery.

The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.