Connective Tissue Growth Factor (CTGF) Expression Modulates Response to High Glucose

<div><p>Connective tissue growth factor (CTGF) is an important mediator of fibrosis; emerging evidence link changes in plasma and urinary CTGF levels to diabetic kidney disease. To further ascertain the role of CTGF in responses to high glucose, we assessed the consequence of 4 months of streptozotocin-induced diabetes in wild type (+/+) and CTGF heterozygous (+/−) mice. Subsequently, we studied the influence of glucose on gene expression and protein in mice embryonic fibroblasts (MEF) cells derived from wildtype and heterozygous mice. At study initiation, plasma glucose, creatinine, triglyceride and cholesterol levels were similar between non-diabetic CTGF+/+ and CTGF+/− mice. In the diabetic state, plasma glucose levels were increased in CTGF+/+ and CTGF+/− mice (28.2 3.3 mmol/L vs 27.0 3.1 mmol/L), plasma triglyceride levels were lower in CTGF+/− mice than in CTGF+/+ (0.7 0.2 mmol/L vs 0.5 0.1 mmol/L, p<0.05), but cholesterol was essentially unchanged in both groups. Plasma creatinine was higher in diabetic CTGF+/+ group (11.7±1.2 vs 7.9±0.6 µmol/L p<0.01), while urinary albumin excretion and mesangial expansion were reduced in diabetic CTGF+/− animals. Cortices from diabetic mice (both CTGF +/+ and CTGF +/−) manifested higher expression of CTGF and thrombospondin 1 (TSP1). Expression of nephrin was reduced in CTGF +/+ animals; this reduction was attenuated in CTGF+/− group. In cultured MEF from CTGF+/+ mice, glucose (25 mM) increased expression of pro-collagens 1, IV and XVIII as well as fibronectin and thrombospondin 1 (TSP1). In contrast, activation of these genes by high glucose was attenuated in CTGF+/− MEF. We conclude that induction of <i>Ctgf</i> mediates expression of extracellular matrix proteins in diabetic kidney. Thus, genetic variability in CTGF expression directly modulates the severity of diabetic nephropathy.</p></div

E14.5 and E10.5 embryos.

<p>A) and E) are fixed embryos at E14.5 from a cross between <i>Ctgf Lo/+</i> and <i>EIIa-Cre +/−</i> B6.129 mixed background parents. A) is a normal <i>Lo/+</i> and E) is a small atypical <i>Hi/+</i> at higher magnification. Top arrow denotes less developed eye in E) and the lower two arrows point to lateral and midline facial clefting, respectively. Embryos B) to D) and F) to H) are E10.5 embryos from a cross between <i>Ctgf Lo/Lo</i> and <i>EIIa-Cre +/−</i> 129/SvEv background parents. B), C), and F), G), are unfixed embryos. B), C) and D) are normal <i>Ctgf Lo/+</i> embryos. F), G), and H) are <i>Ctgf Hi/+</i> embryos. The top arrow in B) and F) points to the forebrain which appears smaller in F) and the lower arrow points to the mouth gape in F) also indicative of a small forebrain. The top arrow in C) and G) points to the eye and the bottom arrow points to the hindbrain which both appears abnormal in G). The pictures in D) and H) are from scanning electron microscopy. The arrows in D) and H) point to the first (top) and second (bottom) pharangeal (branchial) clefts. In D) there is normal closure of the clefts and in H) the clefts are abnormally patent (open). B) and C) are the same embryo and F) to H) are the same embryo. The bars in A), B), E), and F) represent 1000 µm and in C), D), G), and H) the bars represent 250 µm.</p

Stimulus set, averaged by category.

<p>Stimulus set, averaged by category.</p

CTGF mRNA levels in kidneys from newborn (A) and 1-month old mice (B).

<p>Total RNA was isolated and expression was determined by qRT-PCR as described in methods. Panel <b>C</b> depicts CTGF protein levels in kidney from wildtype and heterozygous adult mice. Protein was determined by Western blotting analysis. *p<0.05; n = 6.</p

Category-level classification results.

<p>All electrodes and time samples of the brain response were used together in the six-class category-level classification. An equal number of observations from each category were used. Left: Confusion matrix showing proportions of classifier output. Rows represent actual labels and columns represent predicted labels. Values along the diagonal indicate proportions of correct classifications. Mean accuracy for this classification was 40.68%, compared to chance-level accuracy of 16.67% (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135697#pone.0135697.g002" target="_blank">Fig 2</a>). Middle: Multidimensional scaling (MDS) plot derived from the confusion matrix, visualizing the non-hierarchical structure of the representational space. MDS dimensions are sorted in descending order of variance explained. Right: Dendrogram visualizing the hierarchical structure of the representation. The Human Face category is most separate from the other categories, while the two Inanimate categories form the tightest category cluster.</p

Immunofluorescence of E11.5 <i>Ctgf Lo</i> and <i>Hi</i> embryos.

<p>Panels A) – D) are stained with an anti-CTGF antibody and a Alexa 594 (Texas Red) secondary. Panels E) – H) are stained with DAPI to highlight nuclei. Panels I) – L) are a merge of the Red and the DAPI layer. Genotypes for each panel are as marked on the panel. The panels in each row are from the same section. All panels are at 5× magnification.</p

Strains in the <i>Ctgf</i> allelic series.

<p><i>Ctgf</i> expression levels are relative to WT. A single WT allele expresses 50% (2×50% = 100%). Each copy of the <i>Ctgf Lo</i> allele expresses about 30% of WT (<i>Lo/+</i> = 30%+50%≈85%). Each copy of the <i>Ctgf Hi</i> allele expresses about 300% to 860% of WT. Abbreviations: KO is knockout, del is exon 3–5 deletion, and 3′ is 3′UTR.</p

Phenotype of E13.5 embryos.

<p>Embryos are from a cross between <i>Ctgf Lo/+</i> and <i>EIIa-Cre +/-</i> parents. The number of embryos for each genotype classified as Normal, Small, or Small atypical. <i>+/+</i> n = 9, <i>Lo/+</i> n = 15, <i>Cre⁺</i> n = 11, <i>Hi/+</i> n = 7. The +/+ animals are wild type for <i>Ctgf</i> and do not have a Cre allele, <i>Lo/+</i> animals are heterozygous for the <i>Ctgf Lo</i> allele and do not have a Cre allele. <i>Cre⁺</i> are wild type for <i>Ctgf</i> and have one Cre allele. And <i>Hi/+</i> are heterozygous for the <i>Ctgf Hi</i> allele and have one Cre allele.</p

Survivors of <i>Ctgf Hi/+</i> embryonic lethality.

<p>Gross morphology of high <i>Ctgf</i>-expressing survivors at 10 months old. Males are shown in A) and B) and females are shown in C) and D). In A) and B) the <i>Ctgf Hi/+</i> mice (foreground) have small ears, shortened face and short body length. B) The short curly tail of the <i>Hi/+</i> male. D) Short kinked tail and short body of the <i>Hi/+</i> female (top) compared with a control sibling (bottom). All the animals shown are siblings from a cross between <i>Ctgf Lo/+</i> and <i>EIIa-Cre +/−</i> animals on a mixed B6.129 background. The controls (background of A) and C) and bottom of D)) are of the genotype <i>Ctgf Lo/+</i>.</p

Generation of mice with disrupted <i>Ctgf</i> gene.

<p>Depiction of wildtype allele is represented in Panel <b>A</b>. I,II, III, IV and V represents exons 1 to 5 of <i>Ctgf</i> gene. The arrow straddling exons I and II represents 5′ probe. B (for <i>Bam</i>HI); NheI and NciI represent restriction enzyme sites. Targeted allele (Panel <b>B</b>) demonstrating loss of exons III to V following recombination in ES cells. <i>Neo</i> represents neomycin resistance gene. Southern blot analysis of genomic DNA is shown in Panel <b>C</b>. The targeted allele was identified by 5′probe that hybridizes to a 5 kb fragment in the heterozygous and 12 kb fragment in the wildtype. PCR of genomic DNA from MEF is represented in Panel <b>D</b>. In panel A, <b>a</b>,<b>b</b> and <b>c</b> indicate location of primers used for PCR, whereas B denotes restriction site for <i>Bam</i>H1. Primers <b>a</b> and <b>b</b> gives a 550 bp endogenous product, while primers <b>a</b> and <b>c</b> gives a 320 bp targeted product as indicated in panel D.</p