LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

The potential role of liver kinase B1 (LKB1) in the altered activation of the master metabolic and epigenetic regulator adenosine monophosphate-activated protein kinase (AMPK) in Duchenne muscular dystrophy has not been investigated so far. Hence, we analyzed both gene and protein levels of LKB1 and its related targets in gastrocnemius muscles of adult C57BL/10 mdx mice and D2 mdx mice, a model with a more severe dystrophic phenotype, as well as the sensitivity of the LKB1-AMPK pathway to AMPK activators, such as chronic exercise. Our data show, for the first time, a reduction in the levels of LKB1 and accessory proteins, MO25 and STRADα, in both mdx strains versus the respective wild type, which was further impaired by exercise, in parallel with a lack of further phosphorylation of AMPK. The AMPK-like kinase salt-inducible kinase (SIK) and class II histone deacetylases, along with expression of the HDAC target gene Mef2c, were also altered, supporting an impairment of LKB1-SIK-class II histone deacetylase signaling. Our results demonstrate that LKB1 may be involved in dystrophic progression, paving the way for future preclinical studies.

TGFbeta1 induces epithelial-mesenchymal transition, but not myofibroblast transdifferentiation of human kidney tubular epithelial cells in primary culture.

The origin and fate of renal interstitial myofibroblasts (MFs), the effector cells of renal fibrosis, are still debated. Experimental evidence suggests that renal MFs derive from tubular epithelial cells throughout the epithelial-mesenchymal transition (EMT) process. Primary human tubular epithelial cells (HUTECs) were cultured for 4 and 6 days on plastic or type I collagen-coated plates with 1, 5, 10 and 50 ng/ml of transforming growth factor beta1 (TGFbeta1). The EMT process was monitored by morphology and immunophenotyping for alphaSMA, cytokeratin 8-18, E-cadherin, vimentin and collagen III. Quantitative comparative RT/PCR and real-time PCR were used to evaluate the expression of collagen III and IV, fibronectin, tenascin, MMP-2, CTGF, E-cadherin and cadherin 11 genes, as well as those of the Smad signalling pathway. TGFbeta1 was found capable of reactivating the mesenchymal programme switched off during tubulogenesis, but it induced no de novo expression of alphaSMA gene or myofibroblast phenotype. We demonstrate that the EMT process is conditioned by the extracellular matrix and characterized by TGFbeta1-driven Smad3 downregulation. Our study results suggest that TGFbeta1 could function as a classic embryonal inducer, initiating a cascade of de-differentiating events that might be further controlled by other factors in the cellular environment.

Identification of a novel splice site mutation of CLCN5 gene and characterization of a new alternative 5' UTR end of ClC-5 mRNA in human renal tissue and leukocytes.

Mutations in the CLCN5 gene have been detected in Dent's disease and its phenotypic variants (X-linked recessive nephrolithiasis, X-linked recessive hypophosphatemic rickets, and idiopathic low-molecular-weight proteinuria of Japanese children). Dent's disease is a tubular disorder characterized by low-molecular-weight proteinuria, and nephrolithiasis associated with nephrocalcinosis and hypercalciuria. ClC-5 is the first chloride channel for which a definitive role in the trafficking and acidification-dependent recycling of apical membrane proteins has been established. In the course of CLCN5 SSCP analysis in patients with hypercalciuric nephrolithiasis, we detected a novel mutation at intron 2 of the CLCN5 gene, a T-to-G substitution, located 17 bp upstream of the AG acceptor site. To determine the effect of IVS2-17 T>G mutation on the correct splicing of intron 2, we studied ClC-5 transcripts in a patient's peripheral blood leukocytes by means of quantitative comparative RT/PCR, and found a new ClC-5 5' UTR isoform characterized by the untranslated exon 1b and by retention of intron 1b. This new isoform--isoform B1--was not correlated with mutation since it was detected also in control leukocytes and in renal tissues of kidney donors, thus confirming its physiological role. By RACE analysis we determined the putative transcriptional start site which is located at intron 1a, 251 nt upstream of the first nucleotide of the untranslated exon 1b. ORF analysis revealed that intron 1b retention in isoform B1 stabilizes the initiation of translation to the AGT at position 297 of the ClC-5 cDNA coding region.

Glycosaminoglycan therapy prevents TGF-beta1 overexpression and pathologic changes in renal tissue of long-term diabetic rats.

Chronic induction of the prosclerotic cytokine transforming growth factor beta (TGF-beta) has been implicated in the pathogenesis of diabetic nephropathy. In a rat model of diabetes mellitus-induced glomerulosclerosis, daily administration of a modified heparin (mH) glycosaminoglycan (GAG) preparation with low anticoagulant activity prevented glomerular and tubular matrix accumulation, as well as overexpression of TGF-beta1 mRNA and albuminuria, without obvious side effects. To elucidate the molecular mechanisms of GAG/mH inhibitory actions on TGF-beta1, studies using cultured mesangial cells were also performed. In these cells, high glucose-induced, dose-dependent increases in TGF-beta1 mRNA and bioactive TGF-beta protein expression were inhibited by GAG/mH treatment, whereas basal TGF-beta1 expression was not affected. Both the heparin-derived GAG and dermatan sulfate were effective, indicating that the heparin chemical structure is not necessary for inhibitory activity. Coincubation of GAG with active TGF-beta1 demonstrated no inhibitory effect on TGF-beta1 bioactivity, excluding a neutralizing effect of GAG on TGF-beta1 a the protein level. Furthermore, it was demonstrated that GAG inhibited phorbol myristate acetate-induced translocation of protein kinase C-alpha (PKC-alpha) and -beta1 and activation of PKC-alpha, as well as high glucose-induced activation of PKC-alpha. These results suggest that GAG inhibit TGF-beta1 overexpression at the transcriptional level, possibly via inhibition of high glucose-activated PKC. The findings indicate the potential of GAG therapy for the prevention of diabetic glomerulosclerosis by the inhibition of chronic disease-induced TGF-beta1 mRNA overexpression.