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Histopathological findings on kidney sections of rge chickens.Glomeruli from mutant kidneys (Fig. 7a) are considerably larger than their wild type counterparts (Fig. 7b); whereas tubules are of approximately the same diameter. Red blood cells in large numbers are seen in the lumen of tubules and where present are associated with flattening injury to the tubular epithelium (Fig. 7c and d). These red cells are not seen in wild type kidneys (Fig. 7b, d, & f). There is a focus of severe tubulo-interstitial inflammation (lower arrow) comprising lymphocytes and macrophages infiltrating tubules in rge that are not seen in wt kidneys. Magnification 66×.
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pone-0021156-g007: Histopathological findings on kidney sections of rge chickens.Glomeruli from mutant kidneys (Fig. 7a) are considerably larger than their wild type counterparts (Fig. 7b); whereas tubules are of approximately the same diameter. Red blood cells in large numbers are seen in the lumen of tubules and where present are associated with flattening injury to the tubular epithelium (Fig. 7c and d). These red cells are not seen in wild type kidneys (Fig. 7b, d, & f). There is a focus of severe tubulo-interstitial inflammation (lower arrow) comprising lymphocytes and macrophages infiltrating tubules in rge that are not seen in wt kidneys. Magnification 66×.

Mentions: Histological examination of haematoxylin and eosin stained sections showed significant renal pathology in mutant birds compared to wild type controls. For example it is clearly evident that rge birds suffer from glomerulopathy as the sections show enhanced glomerular size (Fig. 7a) at the same magnification (60×) in comparison to wt kidney sections (Fig. 7b). Red blood cells were noted in the urinary filtration space of glomeruli (Fig. 7c). Abundant red blood cells were also seen in tubular lumina, and tubular injury with flattening of the tubular epithelium was associated with red blood cells in rge kidney sections (Fig. 7c) but not in wt (Fig. 7d). There was a marked focal tubulo-interstitial inflammatory infiltrate, with lymphocytes and macrophages infiltrating tubules in rge kidney sections (Fig. 7e). In some, but not all, foci neutrophils were present in rge sections (Fig. 7e) but not in wt (Fig. 7f). GNB3 immunohisto reactivity was totally diminished in the renal cells of the rge birds compared with an overt expression pattern of GNB3 in proximal convoluted tubule (PCT) and glomerulus (GL) in wt sections (Fig. 8a). CoxIV, a mitochondrial protein, was present in both wt and rge sections confirming the reliability of the IHC technique (Fig. 8b). The GNB3 immuno reactivity results are consistent with our previous findings that the D153del mutation affected GNB3 protein structure [30], stability, and cellular localisation (Fig. 1) with much shorter half-life than the normal GNB3 protein, as shown in our present degradation studies (Fig. 2). Given the prominent GNB3 expression in rge kidney sections the related pathological finding observed in rge chickens are considered to be a primary genetic defect causing loss of function of a specific renal transport protein or signaling molecule. Consequently, the functional disturbances of certain tubule segments lead to defects in tubular reabsorption (Fig. 7c & e). The changes of GNB3 protein expression in whole kidney observed by Western blot likely reflect GNB3 expression, as observed in the IHC.

The D153del Mutation in GNB3 Gene Causes Tissue Specific Signalling Patterns and an Abnormal Renal Morphology in Rge Chickens

Tummala H, Fleming S, Hocking PM, Wehner D, Naseem Z, Ali M, Inglehearn CF, Zhelev N, Lester DH - PLoS ONE (2011)

Bottom Line: As expected, the relative levels of cGMP and cAMP secondary messengers and their activated kinases such as MAPK, AKT and GRK2 were also found to be altered significantly in a tissue specific manner in rge chickens.These findings confirm that the D153del mutation in GNB3 gene targets GNB3 protein to early degradation.Lack of GNB3 signalling causes reduced phosphorylation activity of ERK2 and AKT leading to severe pathological phenotypes such as blindness and renal abnormalities in rge chickens.

Affiliation: School of Contemporary Sciences, University of Abertay Dundee, Dundee, Scotland, United Kingdom.

ABSTRACT

Background: The GNB3 gene is expressed in cone but not rod photoreceptors of vertebrates, where it acts as the β transducin subunit in the colour visual transduction process. A naturally occurring mutation 'D153del' in the GNB3 gene causes the recessively inherited blinding phenotype retinopathy globe enlarged (rge) disease in chickens. GNB3 is however also expressed in most other vertebrate tissues suggesting that the D153del mutation may exert pathological effects that outlie from eye.

Principal findings: Recombinant studies in COS-7 cells that were transfected with normal and mutant recombinant GNB3 constructs and subjected to cycloheximide chase showed that the mutant GNB3d protein had a much shorter half life compared to normal GNB3. GNB3 codes for the Gβ3 protein subunit that, together with different Gγ and Gα subunits, activates and regulates phosphorylation cascades in different tissues. As expected, the relative levels of cGMP and cAMP secondary messengers and their activated kinases such as MAPK, AKT and GRK2 were also found to be altered significantly in a tissue specific manner in rge chickens. Histochemical analysis on kidney tissue sections, from rge homozygous affected chickens, showed the chickens had enlargement of the glomerular capsule, causing glomerulomegaly and tubulointerstitial inflammation whereas other tissues (brain, heart, liver, pancreas) were unaffected.

Significance: These findings confirm that the D153del mutation in GNB3 gene targets GNB3 protein to early degradation. Lack of GNB3 signalling causes reduced phosphorylation activity of ERK2 and AKT leading to severe pathological phenotypes such as blindness and renal abnormalities in rge chickens.

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