Sequencing of LRP2 Reveals Multiple Rare Variants Associated with Urinary Trefoil Factor-3

Novel biomarkers are being investigated to identify patients with kidney disease. We measured a panel of 13 urinary biomarkers in participants from the Offspring Cohort of the Framingham Heart Study. Using an Affymetrix chip with imputation to 2.5 M single-nucleotide polymorphisms (SNPs), we conducted a GWAS of these biomarkers (n=2640) followed by exonic sequencing and genotyping. Functional studies in zebrafish were used to investigate histologic correlation with renal function. Across all 13 biomarkers, there were 97 significant SNPs at three loci. Lead SNPs at each locus were rs6555820 (P=6.7×10⁻⁴⁹; minor allele frequency [MAF]=0.49) in HAVCR1 (associated with kidney injury molecule-1), rs7565788 (P=2.15×10⁻¹⁶; MAF=0.22) in LRP2 (associated with trefoil factor 3 [TFF3]), and rs11048230 (P=4.77×10⁻⁸; MAF=0.10) in an intergenic region near RASSF8 (associated with vascular endothelial growth factor). Validation in the CKDGen Consortium (n=67,093) showed that only rs7565788 at LRP2, which encodes megalin, was associated with eGFR (P=0.003). Sequencing of exons 16–72 of LRP2 in 200 unrelated individuals at extremes of urinary TFF3 levels identified 197 variants (152 rare; MAF<0.05), 31 of which (27 rare) were nonsynonymous. In aggregate testing, rare variants were associated with urinary TFF3 levels (P=0.003), and the lead GWAS signal was not explained by these variants. Knockdown of LRP2 in zebrafish did not alter the renal phenotype in static or kidney injury models. In conclusion, this study revealed common variants associated with urinary levels of TFF3, kidney injury molecule-1, and vascular endothelial growth factor and identified a cluster of rare variants independently associated with TFF3.Serum creatinine, as used in most GFR estimating equations, is the primary biomarker of CKD.¹ Creatinine has significant limitations as a biomarker. It is insensitive to early declines in kidney function, and there are important extrarenal factors that influence its concentration, thus increasing the potential for misclassification when it is used to diagnose renal disease.² As a result, novel biomarkers are being investigated that could allow earlier and more accurate diagnosis of CKD.³ It is likely, however, that these biomarkers also have non-GFR determinants, including genetic factors. For example, levels of cystatin C, a novel GFR biomarker, are known to be influenced by both nongenetic factors, such as adiposity and the metabolic syndrome,⁴ and the presence of specific genetic variants in the cystatin C-gene cluster.⁵Genome-wide association studies (GWAS) allow for the evaluation of genetic associations of biomarkers in an unbiased manner. These studies could illuminate previously unrecognized pathways for CKD pathogenesis, thus identifying new potential molecular targets for therapeutic intervention. Multiple variants have been identified in association with kidney function through GWAS,⁶ whereas a variant in CUBN encoding cubilin, a proximal tubular transport protein, has been associated with albuminuria.⁷ Recently, a GWAS identified a locus at PTGDS in association with another kidney biomarker, β-trace protein.⁸To gain additional insight into biologic pathways of renal function and kidney injury, we measured a panel of 13 urinary biomarkers in participants from the Framingham Heart Study (FHS) and performed a GWAS of their levels. Here, we report the primary results from these studies as well as follow-up work to identify whether rare variants in LRP2 are associated with levels of trefoil factor 3 (TFF3).     

Glucose regulation and pain in older people—The Helsinki Birth Cohort Study

AimsTo assess if individuals with diabetes or prediabetes report more pain or have increased use of pain medication compared to normoglycaemic individuals.MethodsUsing cross-sectional data, we studied 928 men and 1075 women from the Helsinki Birth Cohort Study in 2001–2004 at a mean age of 61.5 years. Glucose regulation was assessed with a 2-h 75 g oral glucose tolerance test, and applying World Health Organization criteria, participants were defined as having normoglycaemia, prediabetes (impaired fasting glucose or impaired glucose tolerance), newly diagnosed diabetes or previously diagnosed diabetes. Self-reported pain intensity and interference during the previous 4 weeks was estimated using the RAND 36-Item Health Survey 1.0. Information on use of pain medication during the past 12 months was obtained from the Social Insurance Institution of Finland.ResultsThere was no difference in pain intensity or interference between glucose regulation groups for neither men nor women after adjusting for covariates (age, body mass index, education years, Beck Depression Inventory and physical activity). In addition, use of pain medication was similar between glucose regulation groups.ConclusionsAlthough pain is a common symptom in the general population, impairments in glucose regulation alone does not seem to increase pain among older individuals.     

Human Protoparvovirus DNA and IgG in Children and Adults with and without Respiratory or Gastrointestinal Infections

Three human protoparvoviruses, bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV), have recently been discovered in diarrheal stool. BuV has been associated with diarrhea and CuV with cutaneous T-cell lymphoma, but there are hardly any data for TuV or CuV in stool or respiratory samples. Hence, using qPCR and IgG enzyme immunoassays, we analyzed 1072 stool, 316 respiratory and 445 serum or plasma samples from 1098 patients with and without gastroenteritis (GE) or respiratory-tract infections (RTI) from Finland, Latvia and Malawi. The overall CuV-DNA prevalences in stool samples ranged between 0–6.1% among our six patient cohorts. In Finland, CuV DNA was significantly more prevalent in GE patients above rather than below 60 years of age (5.1% vs 0.2%). CuV DNA was more prevalent in stools among Latvian and Malawian children compared with Finnish children. In 10/11 CuV DNA-positive adults and 4/6 CuV DNA-positive children with GE, no known causal pathogens were detected. Interestingly, for the first time, CuV DNA was observed in two nasopharyngeal aspirates from children with RTI and the rare TuV in diarrheal stools of two adults. Our results provide new insights on the occurrence of human protoparvoviruses in GE and RTI in different countries.     

Cervical spine computed tomography utilization in pediatric trauma patients

Background

Guidelines for evaluating the cervical spine in pediatric trauma patients recommend cervical spine CT (CSCT) when plain radiographs suggest an injury. Our objective was to compare usage of CSCT between a pediatric trauma center (PTC) and referral general emergency departments (GEDs).

Methods

Patient data from a pediatric trauma registry from 2002 to 2011 were analyzed. Rates of CSI and CSCT of patients presenting to the PTC and GED were compared. Factors associated with use of CSCT were assessed using multivariate logistic regression.

Results

5148 patients were evaluated, 2142 (41.6%) at the PTC and 3006 (58.4%) at the GED. Groups were similar with regard to age, gender, GCS, and triage category. GED patients had a higher median ISS (14 vs. 9, p < 0.05) and more frequent ICU admissions (44.3% vs. 26.1% p < 0.05). CSI rate was 2.1% (107/5148) and remained stable. CSCT use increased from 3.5% to 16.1% over time at the PTC (mean 9.6% 95% CI = 8.3, 10.9) and increased from 6.8% to 42.0% (mean 26.9%, CI = 25.4, 28.4) at the GED. Initial care at a GED remained strongly associated with CSCT.

Conclusions

Despite a stable rate of CSI, rate of CSCT increased significantly over time, especially among patients initially evaluated at a GED.     

Altered mitochondrial metabolism in the insulin‐resistant heart

Obesity‐induced insulin resistance and type 2 diabetes mellitus can ultimately result in various complications, including diabetic cardiomyopathy. In this case, cardiac dysfunction is characterized by metabolic disturbances such as impaired glucose oxidation and an increased reliance on fatty acid (FA) oxidation. Mitochondrial dysfunction has often been associated with the altered metabolic function in the diabetic heart, and may result from FA‐induced lipotoxicity and uncoupling of oxidative phosphorylation. In this review, we address the metabolic changes in the diabetic heart, focusing on the loss of metabolic flexibility and cardiac mitochondrial function. We consider the alterations observed in mitochondrial substrate utilization, bioenergetics and dynamics, and highlight new areas of research which may improve our understanding of the cause and effect of cardiac mitochondrial dysfunction in diabetes. Finally, we explore how lifestyle (nutrition and exercise) and pharmacological interventions can prevent and treat metabolic and mitochondrial dysfunction in diabetes.     

Development of a realistic in vivo bone metastasis model of human renal cell carcinoma

About one-third of patients with advanced renal cell carcinoma (RCC) have bone metastases. The incidence of RCC is increasing and bone metastatic RCC merits greater focus. Realistic preclinical bone metastasis models of RCC are lacking, hampering the development of effective therapies. We developed a realistic in vivo bone metastasis model of human RCC by implanting precision-cut tissue slices under the renal capsule of immunodeficient mice. The presence of disseminated cells in bone marrow of tissue slice graft (TSG)-bearing mice was screened by human-specific polymerase chain reaction and confirmed by immunohistology using human-specific antibody. Disseminated tumor cells in bone marrow of TSG-bearing mice derived from three of seven RCC patients were detected as early as 1 month after tissue implantation at a high frequency with close resemblance to parent tumors (e.g., CAIX expression and high vascularity). The metastatic patterns of TSGs correlated with disease progression in patients. In addition, TSGs retained capacity to metastasize to bone at high frequency after serial passaging and cryopreservation. Moreover, bone metastases in mice responded to Temsirolimus treatment. Intratibial injections of single cells generated from TSGs showed 100 % engraftment and produced X-ray-visible tumors as early as 3 weeks after cancer cell inoculation. Micro-computed tomography (μCT) and histological analysis revealed osteolytic characteristics of these lesions. Our results demonstrated that orthotopic RCC TSGs have potential to develop bone metastases that respond to standard therapy. This first reported primary RCC bone metastasis model provides a realistic setting to test therapeutics to prevent or treat bone metastases in RCC.