Strategies of gene transfer to the kidney

Strategies of gene transfer to the kidney. Kidney targeted gene transfer has been a realistic goal for many researchers since 1991, but unfortunately, to date there is no reliable gene transfer technique for gene therapy of renal diseases. However, at the experimental level, several in vivo gene transfer methods have attempted to target certain renal structures, for example, the HVJ-liposome method and renal perfusion of adenovirus for glomerular cells, intravenous injection of oligonucleotides (ODNs) for proximal tubule, intra-arterial injection of adenovirus followed by cold incubation with a vasodilator for interstitial vasculature of the outer medulla, and adenoviral injection into the renal pelvis for the inner medullary collecting duct. As an ex vivo gene transfer method targeting the glomerulus, the transfusion of genetically-modified mesangial cells has been attempted. Implantation of genetically-modified tubular epithelial cells into the subcapsular region has been employed for ex vivo transfection to the interstitium. Gene therapy has focused particularly on the transplanted kidney, where an exogenous gene can transferred in advance. In the future, an inducible system and individual cell targeting strategy should be developed. The improvement of gene transfer techniques, especially vectors for delivering genes, is crucial. The potential application of gene transfer technologies is enormous while the therapeutic approaches have just begun to be explored. Therapeutic interventions of the process of progression of glomerulonephritis in the rat have been directed towards inhibiting the actions of growth factors. Obviously, molecular biological intervention is coming of age and there is a tremendous excitement over its potential. We believe that gene transfer techniques will become common tools for the dissection of molecular aspects of diseases and possibly for gene therapy in the field of nephrology

Immunology of membranous nephropathy [version 1; peer review: 3 approved]

Accounting for about 20 to 50% of cases of primary nephrotic syndrome, membranous nephropathy (MN) is the leading cause of nephrotic syndrome in adults. A rat model created nearly 60 years ago to research the primary MN disorder, Heymann nephritis, has provided us with a plethora of important information. Recently, our knowledge about MN has dramatically progressed. Heymann nephritis and human MN are now known to share a high degree of similarity in pathogenesis. This review summarizes our current understanding of MN pathogenesis while focusing particularly on the immunological aspects

An enhancer unit of L-type pyruvate kinase gene is responsible for transcriptional stimulation by dietary fructose as well as glucose in transgenic mice

AbstractWe produced three lines of transgenic mice containing the 5' flanking region of the L-type pyruvate kinase gene from nucleotides −189 to +37, which includes an enhancer unit and TATA box as functional elements, linked to the chloramphenicol acetyltransferase gene. Since transgene expression was stimulated by both dietary fructose and glucose in a tissue-dependent manner, we suggest that this unit is responsive to both stimuli

Role of intron 1 in smooth muscle α-actin transcriptional regulation in activated mesangial cells in vivo

Role of intron 1 in smooth muscle α-actin transcriptional regulation in activated mesangial cells in vivo.BackgroundThe activation of glomerular mesangial cells is one of the early, important features of progressive glomerular disease. Smooth muscle α-actin (SMαA) is an excellent marker of activated mesangial cells. However, the mechanisms of SMαA regulation are only available from in vitro investigation.MethodsWe examined in vivo promoter analysis of the SMαA gene-utilizing transgenic mice harboring different promoter regions of the SMαA gene fused to chloramphenicol acetyl transferase (CAT). CAT activities were tested in primary cultured mesangial cells and in glomerular legions of Habu venom glomerulonephritis.ResultsThe DNA sequence -891 to +3828, which contains exon 1, intron 1, and the first 14bp of exon 2 in addition to the 5′-flanking sequence of the SMαA gene, induced high levels of transcription in activated mesangial cells in in vivo habu venom glomerulonephritis and in cultured mesangial cells derived from transgenic mice. The DNA region -891 to -124 was a positive element in mesangial cells derived from transgenic mice. Deletions (3316 or 137bp) in intron 1 reduced transcription to undetectable levels. The 137bp sequence is highly conserved among several species, containing one CArG box element, which is one of the key motifs for transcriptional activation of contractile-related proteins. In vitro transfection analysis failed to demonstrate these positive effects of intron 1 and region -891 to -124.ConclusionsIn vivo promoter analysis of the SMαA gene provided new information about the transcriptional regulation of SMαA in activated mesangial cells. The DNA region -891 to -124 has a positive effect on SMαA transcription in cultured mesangial cells. The intron 1 region (+1088 to +1224) plays a pivotal role in SMαA transcription in activated mesangial cells in vivo. Further analysis of this conserved region in intron 1, including the CArG motif, will be of great value in understanding the molecular mechanisms of mesangial activation

Transforming growth factor-β1 antisense oligodeoxynucleotides block interstitial fibrosis in unilateral ureteral obstruction

Transforming growth factor-β1 antisense oligodeoxynucleotides block interstitial fibrosis in unilateral ureteral obstruction.BackgroundInterstitial expression of transforming growth factor-β1 (TGF-β1) is important in tubulointerstitial fibrosis, a common process in most progressive renal diseases. However, no effective therapy for progressive interstitial fibrosis is known. Recently, we developed an artificial viral envelope (AVE)-type hemagglutinating virus of Japan (HVJ) liposome-mediated retrograde ureteral gene transfer method, which allowed us to introduce the genetic material selectively into renal interstitial fibroblasts.MethodWe introduced antisense or scrambled oligodeoxynucleotides (ODNs) for TGF-β1 into interstitial fibroblasts in rats with unilateral ureteral obstruction, a model of interstitial fibrosis, to block interstitial fibrosis by retrograde ureteral injection of AVE-type HVJ liposomes.ResultsTGF-β1 and type I collagen mRNA increased markedly in the interstitium of untreated obstructed kidneys, and those were not affected by scrambled ODN transfection. Northern analysis and in situ hybridization revealed that the levels of TGF-β1 and type I collagen mRNA were dramatically decreased in antisense ODN-transfected obstructed kidneys. Consequently, the interstitial fibrotic area of the obstructed kidneys treated with antisense ODN was significantly less than that of the obstructed kidneys untreated or treated with scrambled ODN.ConclusionThe introduction of TGF-β1 antisense ODN into interstitial fibroblasts may be a potential therapeutic maneuver for interstitial fibrosis

Gene expression profile of renal proximal tubules regulated by proteinuria

Gene expression profile of renal proximal tubules regulated by proteinuria.BackgroundProximal tubules activated by reabsorption of protein are thought to play significant roles in the progression of kidney diseases. Thus, identification of genes related to proteinuria should provide insights into the pathological process of tubulointerstitial fibrosis.MethodGene expression profiles were constructed by means of direct sequencing procedures to identify genes induced in the mouse kidney proximal tubules (PT) exposed to proteinuria.ResultsBy comparing the gene expression of control PT to that of disease model PT, the abundantly expressed genes in control PT were down-regulated presumably because of potentially toxic effects of proteinuria. From the more than 1000 up-regulated genes, an immunity related gene, thymic shared antigen-1 (TSA-1), and a novel gene, GS188, were selected for further characterization. The increased expression of TSA-1, a member of the Ly-6 family, and of GS188 in response to proteinuria was confirmed by Northern analysis, immunohistochemistry, in situ hybridization and laser microdissection along with real-time PCR analysis. Full length cloning of GS188 identified it as a family member of LR8 that was reported to express predominantly in fibroblasts.ConclusionsThe gene expression profiles showed that the expression patterns in PT were changed dramatically by proteinuria. The profiles include novel genes that should be further characterized to aid the understanding of the pathophysiology of progressive kidney diseases

Transcriptional activation of a hybrid promoter composed of cytomegalovirus enhancer and β-actin/β-globin gene in glomerular epithelial cells in vivo

Transcriptional activation of a hybrid promoter composed of cytomegalovirus enhancer and β-actin/β-globin gene in glomerular epithelial cells in vivo. The aim of this study was to seek a promoter, transactivated selectively in renal cells in vivo by using transgenic (tg) mouse technology. We generated two kinds of tg mouse lines carrying a green fluorescence protein (GFP) cDNA driven either by cytomegalovirus enhancer and β-actin/β-globin promoter (CX-GFP) or by elongation factor la promoter (EF-GFP), and investigated the expression of GFP in the kidney. Microscopic examination of the renal tissues in CX-GFP-tg mice revealed that GFP was expressed only in glomeruli, mainly epithelial cells, but not in tubules, arteries and interstitium. Moreover, in situ hybridization demonstrated that GFP mRNA expression was localized in the glomerular cells. In contrast, GFP was not detectable in the kidney in any of the lines of EF-GFP-tg mouse. To exclude the possible involvement of the GFP cDNA as an enhancer, we constructed tg mice carrying the CX promoter driving a human CD4 cDNA. It was confirmed that the expression patterns of human CD4 in the kidney were quite similar to those of GFP in the kidney of CX-GFP-tg mice. These results strongly suggest that CX promoter could be transactivated in glomerular epithelial cells in vivo