Thermogenic capacity of human white-fat: the actual picture

Presented at the 9th Greek Conference of Biochemistry and Physiology of Exercise, Thessaloniki, Greece, 18–20 October 2019Cold exposure and exercise may increase thermogenic capacity of white adipose tissue (WAT), which could subsequently enhance energy expenditure and body weight loss. We aimed to identify possible alterations in uncoupling protein 1 (UCP1)—the main biomarker of thermogenic activation—in human WAT due to both cold exposure and exercise, as well as the link between environmental temperature and thermogenic capacity of human WAT. MATERIAL & METHOD: We conducted four human experimental studies and two systematic reviews and meta-analyses—PROSPERO registration CRD42019120116, CRD42019120213. RESULTS: UCP1 mRNA was higher in winter than in summer [t(30) = 2.232, p = 0.03] in human WAT and our meta-analysis showed a main effect of cold exposure on human UCP1 mRNA [standard mean difference (Std-md) = 1.81, confidence interval (CI) = 0.50–3.13, p = 0.007]. However, UCP1 mRNA/protein expressions displayed no associations with %fat mass or BMI (p > 0.05, Cohen’s f2 < 0.20). Both a 2-hour cooling and a non-cooling protocol preceding the positron emission tomography/computed tomography (PET/CT) measurements revealed no association between environmental temperature and standardised uptake value (SUVmax) of human WAT, as well as no mean differences in SUVmax-WAT-activity between winter and summer. An 8-week exercise program had no effect on UCP1 of human WAT or on body composition. Our meta-analysis also revealed: (a) no effect of chronic exercise on human UCP1 mRNA, (b) a main effect of chronic exercise on UCP1 protein concentrations (Std-md = 0.59, CI = 0.03–1.16, p = 0.04) and UCP1 mRNA (Std-md = 1.76, CI = 0.48–3.04, p = 0.007) in WAT of normal diet animals, c) a main effect of chronic exercise on UCP1 mRNA (Std-md = 2.94, CI = 0.24–5.65, p = 0.03) and UCP1 protein concentrations (Std-md = 2.06, CI = 0.07–4.05, p = 0.04) of high-fat diet animals. CONCLUSIONS: Cold exposure represents a main stimulus for increased thermogenic capacity in human white adipocytes; however, this may have no impact on body weight loss. Chronic exercise may represent no major stimulus for UCP1 induced in human white adipocytes, while in animals it increases UCP1 gene independently of their diet. Therefore, evidence from animal studies regarding UCP1 gene activation in white adipocytes may not be applicable in humans. Finally, the identification of human WAT thermogenic capacity via PET/CT examination may be optimal with both a cooling and a non-cooling protocol.Published onlin

A functional variant in NEPH3 gene confers high risk of renal failure in primary hematuric glomerulopathies. Evidence for predisposition to microalbuminuria in the general population.

BACKGROUND: Recent data emphasize that thin basement membrane nephropathy (TBMN) should not be viewed as a form of benign familial hematuria since chronic renal failure (CRF) and even end-stage renal disease (ESRD), is a possible development for a subset of patients on long-term follow-up, through the onset of focal and segmental glomerulosclerosis (FSGS). We hypothesize that genetic modifiers may explain this variability of symptoms. METHODS: We looked in silico for potentially deleterious functional SNPs, using very strict criteria, in all the genes significantly expressed in the slit diaphragm (SD). Two variants were genotyped in a cohort of well-studied adult TBMN patients from 19 Greek-Cypriot families, with a homogeneous genetic background. Patients were categorized as "Severe" or "Mild", based on the presence or not of proteinuria, CRF and ESRD. A larger pooled cohort (HEMATURIA) of 524 patients, including IgA nephropathy patients, was used for verification. Additionally, three large general population cohorts [Framingham Heart Study (FHS), KORAF4 and SAPHIR] were used to investigate if the NEPH3-V353M variant has any renal effect in the general population. RESULTS AND CONCLUSIONS: Genotyping for two high-scored variants in 103 TBMN adult patients with founder mutations who were classified as mildly or severely affected, pointed to an association with variant NEPH3-V353M (filtrin). This promising result prompted testing in the larger pooled cohort (HEMATURIA), indicating an association of the 353M variant with disease severity under the dominant model (p = 3.0x10-3, OR = 6.64 adjusting for gender/age; allelic association: p = 4.2x10-3 adjusting for patients' kinships). Subsequently, genotyping 6,531 subjects of the Framingham Heart Study (FHS) revealed an association of the homozygous 353M/M genotype with microalbuminuria (p = 1.0x10-3). Two further general population cohorts, KORAF4 and SAPHIR confirmed the association, and a meta-analysis of all three cohorts (11,258 individuals) was highly significant (p = 1.3x10-5, OR = 7.46). Functional studies showed that Neph3 homodimerization and Neph3-Nephrin heterodimerization are disturbed by variant 353M. Additionally, 353M was associated with differential activation of the unfolded protein response pathway, when overexpressed in stressed cultured undifferentiated podocyte cells, thus attesting to its functional significance. Genetics and functional studies support a "rare variant-strong effect" role for NEPH3-V353M, by exerting a negative modifier effect on primary glomerular hematuria. Additionally, genetics studies provide evidence for a role in predisposing homozygous subjects of the general population to micro-albuminuria

C3 Glomerulonephritis/CFHR5 Nephropathy Is an Endemic Disease in Cyprus: Clinical and Molecular Findings in 21 Families

<p>Microscopic haematuria is the presenting symptom of several conditions, either heritable or acquired. A well-recognized familial condition is Alport syndrome, either of X-linked or autosomal recessive inheritance, as well as thin basement membrane nephropathy (TBMN) because of heterozygous collagen IV mutations. Even though microscopic haematuria of TBMN was long considered as a benign disease with excellent prognosis, more recent data suggest that development of chronic kidney disease (CKD) and even end-stage kidney disease (ESKD) is not a rare finding, perhaps owing to the cofounding role of modifier genes and other factors. Recent investigations in London and Cyprus culminated in the identification of another autosomal dominant condition that presents with microscopic haematuria because of heterozygous mutations in the <i>CFHR5</i> gene, which apparently plays a pivotal role in the regulation of the alterative pathway of complement system, which constitutes a significant part of innate immunity in humans. Histologically, the hallmark observation is the isolated glomerular deposition of C3 complement in the absence of immune complexes. It is considered one of the C3 glomerulopathies, and it may or may not be accompanied by mild membranoproliferative glomerulonephritis. Interestingly, a single mutation has been identified so far, a duplication of exons 2–3 of the <i>CFHR5</i> gene, and it has been described in patients of Greek-Cypriot descend only, perhaps originating on the Troodos mountains of Cyprus. Thus far, no patient with a mutation in this gene has been diagnosed in any other population. In Cyprus, it has been found in clusters of families in neighbouring villages in a total of 136 patients, and it constitutes a strong founder phenomenon. About 50% of patients over 50 years have progressed to CKD, and 14% of all patients progressed to ESKD. It is not quite well understood why males run a much higher risk to progress to CKD, compared to women.</p&gt

A miR-1207-5p binding site polymorphism abolishes regulation of HBEGF and is associated with disease severity in CFHR5 nephropathy.

Heparin binding epidermal growth factor (HBEGF) is expressed in podocytes and was shown to play a role in glomerular physiology. MicroRNA binding sites on the 3'UTR of HBEGF were predicted using miRWalk algorithm and followed by DNA sequencing in 103 patients diagnosed with mild or severe glomerulopathy. A single nucleotide polymorphism, miRSNP C1936T (rs13385), was identified at the 3'UTR of HBEGF that corresponds to the second base of the hsa-miR-1207-5p seed region. When AB8/13 undifferentiated podocytes were transfected with miRNA mimics of hsa-miR-1207-5p, the HBEGF protein levels were reduced by about 50%. A DNA fragment containing the miRSNP allele-1936C was cloned into the pMIR-Report Luciferase vector and co-transfected with miRNA mimics of hsa-miR-1207-5p into AB8/13 podocytes. In agreement with western blot data, this resulted in reduced luciferase expression demonstrating the ability of hsa-miR-1207-5p to directly regulate HBEGF expression. On the contrary, in the presence of the miRSNP 1936T allele, this regulation was abolished. Collectively, these results demonstrate that variant 1936T of this miRSNP prevents hsa-miR-1207-5p from down-regulating HBEGF in podocytes. We hypothesized that this variant has a functional role as a genetic modifier. To this end, we showed that in a cohort of 78 patients diagnosed with CFHR5 nephropathy (also known as C3-glomerulopathy), inheritance of miRSNP 1936T allele was significantly increased in the group demonstrating progression to chronic renal failure on long follow-up. No similar association was detected in a cohort of patients with thin basement membrane nephropathy. This is the first report associating a miRSNP as genetic modifier to a monogenic renal disorder

COL4A5 and LAMA5 variants co-inherited in familial hematuria: digenic inheritance or genetic modifier effect?

Abstract Background About 40–50% of patients with familial microscopic hematuria (FMH) caused by thin basement membrane nephropathy (TBMN) inherit heterozygous mutations in collagen IV genes (COL4A3, COL4A4). On long follow-up, the full phenotypic spectrum of these patients varies a lot, ranging from isolated MH or MH plus low-grade proteinuria to chronic renal failure of variable degree, including end-stage renal disease (ESRD). Methods Here, we performed Whole Exome Sequencing (WES) in patients of six families, presenting with autosomal dominant FMH, with or without progression to proteinuria and loss of renal function, all previously found negative for severe collagen IV mutations. Hierarchical filtering of the WES data was performed, followed by mutation prediction analysis, Sanger sequencing and genetic segregation analysis. Results In one family with four patients, we found evidence for the contribution of two co-inherited variants in two crucial genes expressed in the glomerular basement membrane (GBM); LAMA5-p.Pro1243Leu and COL4A5-p.Asp654Tyr. Mutations in COL4A5 cause classical X-linked Alport Syndrome, while rare mutations in the LAMA5 have been reported in patients with focal segmental glomerulosclerosis. The phenotypic spectrum of the patients includes hematuria, proteinuria, focal segmental glomerulosclerosis, loss of kidney function and renal cortical cysts. Conclusions A modifier role of LAMA5 on the background of a hypomorphic Alport syndrome causing mutation is a possible explanation of our findings. Digenic inheritance is another scenario, following the concept that mutations at both loci more accurately explain the spectrum of symptoms, but further investigation is needed under this concept. This is the third report linking a LAMA5 variant with human renal disease and expanding the spectrum of genes involved in glomerular pathologies accompanied by familial hematurias. The cystic phenotype overlaps with that of a mouse model, which carried a Lama5 hypomorphic mutation that caused severely reduced Lama5 protein levels and produced kidney cysts

COL4A3/COL4A4 Mutations Producing Focal Segmental Glomerulosclerosis and Renal Failure in Thin Basement Membrane Nephropathy

<p>Mutations in the COL4A3/COL4A4 genes of type IV collagen have been found in ~40% of cases of thin basement membrane nephropathy, which is characterized by microscopic hematuria and is classically thought to cause proteinuria and chronic renal failure rarely. Here we report our observations of 116 subjects from 13 Cypriot families clinically affected with thin basement membrane nephropathy. These families first came to our attention because they segregated microscopic hematuria, mild proteinuria, and variable degrees of renal impairment, but a dual diagnosis of focal segmental glomerulosclerosis (FSGS) and thin basement membrane nephropathy was made in 20 biopsied cases. Molecular studies identified founder mutations in both COL4A3 and COL4A4 genes in 10 families. None of 82 heterozygous patients had any extrarenal manifestations, supporting the diagnosis of thin basement membrane nephropathy. During follow-up of up to three decades, 31 of these 82 patients (37.8%) developed chronic renal failure and 16 (19.5%) reached end-stage renal disease. Mutations G1334E and G871C were detected in seven and three families, respectively, and were probably introduced by founders. We conclude that these particular COL4A3/COL4A4 mutations either predispose some patients to FSGS and chronic renal failure, or that thin basement membrane nephropathy sometimes coexists with another genetic modifier that is responsible for FSGS and progressive renal failure. The findings presented here do not justify the labelling of thin basement membrane nephropathy as a benign condition with excellent prognosis.</p&gt

Clinico-pathological correlations in 127 patients in 11 large pedigrees, segregating one of three heterozygous mutations in the COL4A3/ COL4A4 genes associated with familial haematuria and significant late progression to proteinuria and chronic kidney disease from focal segmental glomerulosclerosis

<p><strong>Background.</strong> Heterozygous mutations in the <i>COL4A3/ COL4A4</i> genes are currently thought to be responsible for familial benign microscopic haematuria and maintenance of normal long-term kidney function.</p><p><strong>Methods.</strong> We report on 11 large Cypriot pedigrees with three such mutations. A total of 236 at-risk family members were genetically studied, and 127 (53.8%) carried a heterozygous mutation. Clinico-pathological correlations were available in all of these patients. Renal biopsies in 21 of these patients all showed various stages of focal, segmental glomerulosclerosis (FSGS). Thirteen of these biopsies were also studied with EM and showed thinning of the glomerular basement membrane.</p><p><strong>Results.</strong> Mutation G1334E (<i>COL4A3</i>) was found in six pedigrees, mutation G871C (<i>COL4A3</i>) in four and mutation 3854delG (<i>COL4A4</i>) in one pedigree. Clinical and laboratory correlations in all 127 mutation carriers (MC) showed that microscopic haematuria was the only urinary finding in patients under age 30. The prevalence of 'haematuria alone' fell to 66% between 31 and 50 years, to 30% between 51 and 70 and to 23% over age 71. Proteinuria with CRF developed on top of haematuria in 8% of all MC between 31 and 50 years, to 25% between 51 and 70 years and to 50% over 71 years. Altogether 18 of these 127 MC (14%) developed ESRD at a mean age of 60 years. Two members with different mutations married, and two of their children inherited both mutations and developed adolescent, autosomal recessive Alport syndrome (ATS), confirming that these mutations are pathogenic.</p><p><strong>Conclusions.</strong> Our data confirm for the first time a definite association of heterozygous <i>COL4A3/COL4A4</i> mutations with familial microscopic haematuria, thin basement membrane nephropathy and the late development of familial proteinuria, CRF, and ESRD, due to FSGS, indicating that the term 'benign familial haematuria' is a misnomer, at least in this cohort. A strong hypothesis for a causal relationship between these mutations and FSGS is also made. Benign familial haematuria may not be so benign as commonly thought.</p&gt

Results after re-sequencing of 103 samples with mutations in <i>COL4A3</i> or <i>COL4A4</i> genes and thin basement membrane nephropathy.

<p>Sequencing primers were designed to flank the predicted target sites and also include about 300 bp on either side. Prediction hits represent the number of tools that successfully predicted the miRNA-mRNA binding.</p

Expression analysis of miR-1207-5p in various cell lines.

<p>Relative expression analysis of mature hsa-miR-1207-5p levels in various cell types, as tested by miRNA specific Real-Time PCR experiments. Both AB8/13 differentiated and undifferentiated podocytes revealed significantly high expression levels of miR-1207-5p, compared to HEK293 and SHSY-5Y neuroblastoma cells. Further examination of miR-1207-5p levels in human renal epithelial cells (HREpiC), recorded 4-fold higher mature miRNA than the AB8/13 differentiated podocyte cell line. Results represent the mean of quadruplicate values ± SEM.</p

The 1936C/T <i>HBEGF</i> genotype is overrepresented in women affected with severe CFHR5.

<p>Comparison of C1936T genotypes in women manifesting CFHR5 nephropathy. Women are known to have a milder course of the disease when compared to men and this is also demonstrated when they are statistically compared with severe CFHR5 women as regards the C1936T SNP. The 1936C allele has a significantly lower representation in severe CFHR5 women, compared to mild women, thus suggesting a protective effect for this allele (p = 0.035).</p