Metabolic Bone Disease of Prematurity: Diagnosis and Management.

Metabolic Bone Disease (MBD) of prematurity is a multifactorial disorder commonly observed in very low birth weight (VLBW, <1,500 g) newborns, with a greater incidence in those extremely low birth weight (ELBW, <1,000 g). MBD is characterized by biochemical and radiological findings related to bone demineralization. Several antenatal and postnatal risk factors have been associated to MBD of prematurity, although the main pathogenetic mechanism is represented by the reduced placental transfer of calcium and phosphate related to preterm birth. The diagnosis of MBD of prematurity requires the assessment of several biochemical markers, radiological, and ultrasonographic findings. However, the best approach is the prevention of the symptomatic disease, based on the screening of subjects exposed to the risks of developing MBD. Regarding the subjects who need to be screened, there is a substantial agreement on the potential risk factors for MBD. On the contrary, different recommendations exist on the diagnosis, management and treatment of this disorder of bone metabolism. This review was aimed at: (1) identifying the subjects at risk for MBD of prematurity; (2) indicating the biochemical findings to take in consideration for the prevention of MBD of prematurity; (3) suggesting practical recommendations on nutritional intake and supplementation in these subjects. We searched for papers which report the current recommendations for biochemical assessment of MBD of prematurity and for its prevention and treatment. The majority of the authors suggest that MBD of prematurity is a disease which tends to normalize overtime, thus it is not mandatory to mimic the rate of mineral fetal accretion through parenteral or enteral supplementation. The optimization of total parenteral nutrition (TPN) and the early achievement of a full enteral feeding are important goals for the prevention and management of MBD of prematurity.

Toll-like receptor 4 signalling mediates inflammation in skeletal muscle of patients with chronic kidney disease.

BACKGROUND: Inflammation in skeletal muscle is implicated in the pathogenesis of insulin resistance and cachexia but why uremia up-regulates pro-inflammatory cytokines is unknown. Toll-like receptors (TLRs) regulate locally the innate immune responses, but it is unknown whether in chronic kidney disease (CKD) TLR4 muscle signalling is altered. The aim of the study is to investigate whether in CKD muscle, TLRs had abnormal function and may be involved in transcription of pro-inflammatory cytokine. METHODS: TLR4, phospho-p65, phospho-ikBα, tumour necrosis factor (TNF)-α, phospho p38, Murf 1, and atrogin were studied in skeletal muscle from nondiabetic CKD stage 5 patients (n = 29) and controls (n = 14) by immunohistochemistry, western blot, and RT-PCR. Muscle cell cultures (C2C12) exposed to uremic serum were employed to study TLR4 expression (western blot and RT-PCR) and TLR-driven signalling. TLR4 signalling was abrogated by a small molecule chemical inhibitor or TLR4 siRNA. Phospho AKT and phospho p38 were evaluated by western blot. RESULTS: CKD subjects had elevated TLR4 gene and protein expression. Also expression of NFkB, p38 MAPK and the NFkB-regulated gene TNF-α was increased. At multivariate analysis, TLR4 protein content was predicted by eGFR and Subjective Global Assessment, suggesting that the progressive decline in renal function and wasting mediate TLR4 activation. In C2C12, uremic serum increased TLR4 as well as TNF-α and down-regulated pAkt. These effects were prevented by blockade of TLR4. CONCLUSIONS: CKD promotes muscle inflammation through an up-regulation of TLR4, which may activate downward inflammatory signals such as TNF-α and NFkB-regulated genes.

Enhanced glomerular Toll-like receptor 4 expression and signaling in patients with type 2 diabetic nephropathy and microalbuminuria.

Toll-like receptor 4 (TLR4), a component of the innate immune system, is recognized to promote tubulointerstitial inflammation in overt diabetic nephropathy (DN). However, there is no information on immune activation in resident renal cells at an early stage of human DN. In order to investigate this, we studied TLR4 gene and protein expression and TLR4 downward signaling in kidney biopsies of 12 patients with type 2 diabetes and microalbuminuria, and compared them with 11 patients with overt DN, 10 with minimal change disease (MCD), and control kidneys from 13 patients undergoing surgery for a small renal mass. Both in microalbuminuria and in overt DN, TLR4 mRNA and protein were overexpressed 4- to 10-fold in glomeruli and tubules compared with the control kidney and in MCD. In addition, NF-κB signaling was about fourfold higher in the glomeruli. TNF-α, IL6, CCR2, CCL5, and CCR5 mRNAs were markedly (about three- to fivefold) upregulated in microdissected glomeruli. While IL6, CCL2 and CCR5-mRNA, and CD68 were overexpressed in the tubulointerstitial compartment in clinical DN, they were not expressed in microalbuminuria. In a 6-year follow-up of microalbuminuric patients, glomerular TLR4 gene expression was associated with the subsequent loss of kidney function. Thus, innate immunity is activated in the glomeruli of patients with diabetic microalbuminuria. Enhanced TLR4 signaling may contribute to the progression occurring after the incipient, microalbuminuric form of nephropathy evolves to overt disease.

Lysine triggers apoptosis through a NADPH oxidase-dependent mechanism in human renal tubular cells.

Progressive chronic kidney disease (CKD) is common in lysinuric protein intolerance (LPI), a primary inherited aminoaciduria characterized by massive Lysine excretion in urine. However, by which mechanisms Lysine may cause kidney damage to tubule cells is still not understood. This study determined whether Lysine overloading of human proximal tubular cells (HK-2) in culture enhances apoptotic cell loss and its associated mechanisms. Overloading HK-2 with Lysine levels reproducing those observed in urine of patients affected by LPI (10 mM) increased apoptosis (+30%; p < 0.01 vs.C), as well as Bax and Apaf-1 expressions (+30-50% p < 0.05), while downregulated Bcl-2 (-40% p < 0.05). Apoptosis induced by high Lysine was no longer observed after addition of caspase-9 and caspase-3 inhibitors while caspase-8 inhibitor had no protective effect. High Lysine induced elevations in ROS generation and NADPH oxidase subunits mRNAs (p22 (phox) +106 ± 23%, p67 (phox) +108 ± 22% and gp91 (phox) +75 ± 4% p < 0.05-0.01). In addition, the NADPH oxidase inhibitor DPI prevented both ROS production and apoptosis. Treating HK-2 with antioxidants, such as Cysteine and its analog, N-acetyl-L-cysteine (NAC), rescued the HK-2 from apoptosis induced by Lysine. In summary, our data show that high Lysine in vitro increases the permissiveness of proximal tubule kidney cells to apoptosis by triggering a pathway involving NADPH oxidase signaling. This event may represent a key cellular effect in the increasing the susceptibility of human tubular cells to apoptosis when the tubules cope with a high Lysine load. This effect is instrumental to renal damage and disease progression in patients with LPI.

Hyperglycemia in celiac disease: not always pretype 1 diabetes?

Celiac disease (CD) is a T-cell-mediated enteropathy, triggered in genetically susceptible individuals by the ingestion of wheat gluten or related rye and barley proteins, whose clinical picture disease is considerably heterologous. Patients with CD are at high risk of autoimmune disorders; similarly, CD is frequent in patients with type 1 diabetes mellitus (T1DM), a disorder characterized by the immune-mediated beta-cell destruction, with the cooperation of environmental factors in genetically susceptible individuals. The immunological markers of beta-cell destruction are the autoantibodies to insulin, glutamic acid decarboxylase, and the protein tyrosine phosphatase. In absence of these markers, incidental hyperglycemia in children and adolescents appears unlikely to be associated with the progression to T1DM. We report a girl with CD and incidental hyperglycemia, without immunological markers of T1DM, with a family history for hyperglycemia, and diagnosed as maturity-onset diabetes of the young. We present this case as evidence that the possibility of monogenic forms of diabetes must be suspected in children with incidental hyperglycemia, a family history for mild hyperglycemia or diabetes, and absence of markers of beta-cell destruction, even if the patients are affected by an autoimmune disease.

Wolfram syndrome (diabetes insipidus, diabetes, optic atrophy, and deafness): clinical and genetic study.

OBJECTIVE: Wolfram syndrome is an autosomal recessive neurodegenerative disorder characterized by diabetes insipidus, diabetes (nonautoimmune), optic atrophy, and deafness (a set of conditions referred to as DIDMOAD). The WFS1 gene is located on the short arm of chromosome 4. Wolfram syndrome prevalence is 1 in 770,000 live births, with a 1 in 354 carrier frequency. RESEARCH DESIGN AND METHODS: We evaluated six Italian children from five unrelated families. Genetic analysis for Wolfram syndrome was performed by PCR amplification and direct sequencing. RESULTS: Mutation screening revealed five distinct variants, one novel mutation (c.1346C>T; p.T449I) and four previously described, all located in exon 8. CONCLUSIONS: Phenotype-genotype correlation is difficult, and the same mutation gives very different phenotypes. Severely inactivating mutations result in a more severe phenotype than mildly inactivating ones. Clinical follow-up showed the progressive syndrome's seriousness.

Horseshoe kidney malformation in Turner syndrome is not associated with HNF-1beta gene mutations.

Mutations in hepatocyte nuclear factor-1beta (HNF-1beta) gene cause a subtype of maturity-onset diabetes of the young (MODY5), whose clinical features are pancreatic beta-cell dysfunction, renal malformations, and in some females, internal genital malformations. Recently, we reported the first case of MODY5 and horseshoe kidney. The patient was the only male in a three-generation family with five affected females carrying renal cysts or dysplastic kidney. Diabetes mellitus, horseshoe kidney, and X chromosome monosomy or mosaicism can be observed in Turner syndrome (TS). In particular, diabetes mellitus affects about 50% and horseshoe kidney occurs in approximately 16% of patients. To investigate whether mutations/polymorphisms of HNF-1beta and X monosomy influence horseshoe kidney development, we evaluated HNF-1beta gene sequence in 13 patients with TS and several kidney abnormalities. Analysis of the nine exons including intron-exon boundaries of HNF-1beta revealed the presence in two subjects (15%) of a known intronic polymorphism, IV8+48insC. No specific variants were found. We conclude there is no direct relationship between horseshoe kidney in TS and mutation or polymorphism of HNF-1beta gene, but we speculate that target gene(s) of HNF-1beta, likely mapped on the X chromosome, is/are responsible of the horseshoe kidney formation in TS.

Reduced serotonin and 3-hydroxyanthranilic acid levels in serum of cystatin B-deficient mice, a model system for progressive myoclonus epilepsy.

PURPOSE: To evaluate the levels of tryptophan and its metabolites along serotonin (5-HT) and kynurenine (KYN) pathways in serum of progressive myoclonus epilepsy (EPM1) patients and cystatin B (CSTB)-deficient mice, a model system for EPM1. METHODS: Tryptophan and its metabolites along serotonin (5-HT) and KYN pathways were determined in serum of EPM1 patients and CSTB-deficient mice by reverse-phase high-pressure liquid chromatography (HPLC) with electrochemical detection. RESULTS: Reduced levels of 5-HT and KYN intermediate metabolite 3-hydroxyanthranilic acid were found in serum of CSTB-deficient mice. A similar trend was found in EPM1 patients. Although tryptophan concentration was reduced in serum of EPM1 patients, no such decrease was observed in CSTB-deficient mice. CONCLUSIONS: The present study demonstrates that tryptophan metabolism along 5-HT and KYN pathways are disrupted in EPM1. Further studies are needed to elucidate the role of KYN pathway in pathogenesis of EPM1.

KCNJ11 activating mutations in Italian patients with permanent neonatal diabetes.

Permanent neonatal diabetes mellitus (PNDM) is a rare condition characterized by severe hyperglycemia constantly requiring insulin treatment from its onset. Complete deficiency of glucokinase (GCK) can cause PNDM; however, the genetic etiology is unknown in most PNDM patients. Recently, heterozygous activating mutations of KCNJ11, encoding Kir6.2, the pore forming subunit of the ATP-dependent potassium (K(ATP)) channel of the pancreatic beta-cell, were found in patients with PNDM. Closure of the K(ATP) channel exerts a pivotal role in insulin secretion by modifying the resting membrane potential that leads to insulin exocytosis. We screened the KCNJ11 gene in 12 Italian patients with PNDM (onset within 3 months from birth) and in six patients with non-autoimmune, insulin-requiring diabetes diagnosed during the first year of life. Five different heterozygous mutations were identified: c.149G>C (p.R50P), c.175G>A (p.V59M), c.509A>G (p.K170R), c.510G>C (p.K170N), and c.601C>T (p.R201C) in eight patients with diabetes diagnosed between day 3 and 182. Mutations at Arg50 and Lys170 residues are novel. Four patients also presented with motor and/or developmental delay as previously reported. We conclude that KCNJ11 mutations are a common cause of PNDM either in isolation or associated with developmental delay. Permanent diabetes of non autoimmune origin can present up to 6 months from birth in individuals with KCNJ11 and EIF2AK3 mutations. Therefore, we suggest that the acronym PNDM be replaced with the more comprehensive permanent diabetes mellitus of infancy (PDMI), linking it to the gene product (e.g., GCK-PDMI, KCNJ11-PDMI) to avoid confusion between patients with early-onset, autoimmune type 1 diabetes.