Novel approaches to unravel the genesis of glomerulosclerosis by new methodologies in molecular biology and molecular genetics

Jun Wada, Kenich Shikata and Hirofumi Makino

Okayama University Medical School, Department of Medicine III, Okayama, Japan

Correspondence and offprint requests to: Hirofumi Makino, MD, Professor and Chairman, Department of Medicine III, Okayama University Medical School, 2–5-1 Shikata-cho, Okayama 700– 8558, Japan.

Introduction

Glomerulosclerosis is the common pathological feature in most immunological and non-immunological renal diseases and tightly related to the progression of renal failure. Although numerous attempts have been made to elucidate its mechanism, the precise pathogenesis of glomerulosclerosis still remains to be investigated. In morphological studies, glomerulosclerosis is characterized by the depletion of glomerular cells and the accumulation of extracellular matrix, including type I, III and IV collagen, fibronectin, laminin and proteoglycans [1]. Since transforming growth factor-ß (TGF-ß) is implicated in the regulation of production and degradation of matrix glycoproteins, the role of TGF-ß was investigated extensively and has been well documented. These studies indicated that the mesangial cell-derived TGF-ß is stimulated by immunological and non-immunological insults and is responsible for glomerulosclerosis by directly stimulating the synthesis of extracellular matrix (ECM) glycoproteins and reducing collagenase production. In glomerulonephritis, the pathological immune reactions induce the activation of complement, the production of immflammatory cytokines and chemokines, i.e. interleukin 1 (IL-1), tumour necrosis factor-{alpha} (TNF-{alpha}), monocyte chemotactic protein 1 (MCP-1) and IL-8, the up-regulation of adhesion molecules and the infiltration of inflammatory cells into glomeruli. During this process, a phenotypic change of mesangial cells is induced. This provokes the production of TGF-ß and other growth factors, such as platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and IL-6, which also enhance the phenotypic changes, and vice versa. Besides glomerulonephritis, metabolic disturbances such as hyperglycaemia may induce phenotypic changes of mesangial cells [2] via activation of protein kinase C (PKC) and accumulation of advanced glycosylation end-products (AGEs) [3]. In addition, haemodynamic changes, including glomerular hyperfiltration or glomerular hypertension, also promote glomerulosclerosis. In its genesis, angiotensin II, nitric oxide [4] and adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) play an important role [5]. For the depletion of the glomerular cells in sclerotic lesions, the emerging role of apoptosis has also been recognized [6,7].

The current estimate suggests that the human genome encodes 80000 transcripts, and only 10% of the genes that exist in the human genome have been cloned and identified so far. Thus, most molecular biology studies on the mechanisms of glomerulosclerosis addressed only known and functionally characterized gene products. Recently, new techniques in molecular biology and molecular genetics have been introduced which promise to throw new light on this issue. These studies deal with the isolation of glomerulosclerosis-related novel genes by screening cDNA libraries or by whole-genome scanning without information on the structure or function of the gene products. In this editorial comment, we attempt to survey such new approaches to the glomerulosclerosis-related genes and provide perspectives concerning future trends in the research of kidney diseases.

Gene expression profile in normal, developmental and pathological states of the kidney

In mammalian tissues, mRNA expression is altered dynamically in various developmental and pathophysiological states. The isolation of these genes promises to unravel the molecular mechanism operative in the genesis of diverse diseases. Kidney-specific genes or genes involved in renal development and in high glucose- or ischaemia-induced kidney injuries have been searched, since they may be involved in renal pathology. The subtractive screening method has been used for the isolation of novel genes, but it requires vast amounts of mRNA to drive the subtraction process. It is also time consuming. To overcome these problems, a PCR-based differential cloning method, i.e. differential display reverse transcription–PCR (DDRT–PCR), was introduced. It is a new powerful technique for the isolation of novel genes. Various genes have been isolated by DDRT–PCR and it has been shown that they are up-regulated when mesangial cells are cultured in high glucose media [811], or when kidneys are subjected to ischaemic injury [12,13].

Another PCR-based subtraction method, i.e. representational difference analysis of cDNA, has also been applied successfully for the isolation of developmentally regulated kidney genes [14], or genes up-regulated in the kidney of STZ-induced diabetic mice [15], the reperfused kidney after ischaemic injury [16] and in subtotal nephrectomy remnants [17]. Diabetes and high glucose induced a number of genes, including lactate dehydrogenase, amiloride-sensitive sodium channel, ubiquitin-like protein [8], munc13s [9], prolyl 4-hydroxylase {alpha}, thrombospondin-1, a novel zinc-finger protein [10], fibronectin, caldesmon, PAI-1, connective tissue growth factor [11], Na+, K+-ATPase and a fusion protein of ubiquitin and ribosomal protein L40 [15]. Ischaemic kidney injury also induced up-regulation of the receptor for activated C kinase (RACK1) [12], calcyclin belonging to the S100 family of calcium-binding proteins [13], and kidney injury molecule-1 (KIM-1) [16]. It is noteworthy that the mitochondrial genome-encoded genes, such as cytochrome c oxidase I and NADH dehydrogenase, were up-regulated in the diabetic rat [8] and subtotally resected mouse kidney [17]. They were in turn down-regulated in the nephrotic human kidney [18]. The discovery of the translocase of inner mitochondrial membrane-44 (Tim44) in the diabetic kidney, which facilitates protein import from the cytosol to mitochondria [15], also suggests that stimulation of the function of mitochondria, i.e. oxidative phosphorylation, is involved in the initial events in diabetic nephropathy. These novel genes, which are up- or down-regulated in pathological states of the kidney, were cloned and identified. Nevertheless, the functional link between altered gene expression and the final result, i.e. glomerulosclerosis, remains to be explored.

By these subtractive cloning methods, it is difficult to survey the complete gene expression pattern unless more than thousands of clones are screened. Each report revealed only a partial picture of the alteration of gene expression. We may overcome this problem by constructing a database of gene expression profiles by sequencing the large-scale cDNA libraries in various tissues (body mapping) [19,20] or 9–10 base cDNA tags, i.e. serial analysis of gene expression (SAGE) [21]. The database of randomly cloned and sequenced 5' and 3' cDNA fragments (EST: expressed sequence tag) is expanding as information accumulates in rat, mouse and human genome projects. Since the EST database is available from on-line servers, ESTs have become invaluable tools for gene discovery. They can be utilized for the investigation of differential gene expression. By blotting thousands of EST clones on nylon filters (DNA macroarray) or DNA chips (DNA microarray) and hybridization with radiolabelled or fluorescence-labelled cDNAs, one can survey the transcriptional activities of tens of thousands of genes in various pathological conditions at one time. This method would facilitate the `Fast Track' gene discovery and define candidate genes potentially involved in the pathogenesis of glomerulosclerosis.

Functional studies using knockout and transgenic animals

Although the efficient isolation of known and unknown genes which are differentially expressed in various pathological states is now progressing, altered expression does not necessarily imply that they are involved directly in the genesis of glomerulosclerosis. In contrast, gene disruption or transgenic experiments in mice provide unambiguous information regarding the role of certain genes in the genesis of glomerulosclerosis, even if the pathophysiological function of the respective gene product is unknown. For instance, adult mice, homozygous for a disruption of Mvp17 by the recombinant retrovirus technique, developed nephrotic syndrome and progressive glomerular sclerosis [22]. Subsequent studies revealed that cDNA of Mvp17 encodes a putative peroxisomal membrane protein, which stimulates intracellular production of reactive oxygen species and the expression of MMP-2 [22]. Another example is the uteroglobin knockout mouse. Uteroglobin is a steroid-inducible secreted protein of unknown function. Interestingly, massive accumulation of fibronectin is observed in the kidney of uteroglobin knockout mice [23]. Uteroglobin seems to prevent the deposition of fibronectin and collagen by the formation of fibronectin–uteroglobin heterodimers in normal mice. Since glomerulosclerosis is characterized by the accumulation of various ECM proteins, this finding will prompt studies to explore the mechanisms which control remodelling of the ECM.

Approach to glomerulosclerosis-related genes using molecular genetics

Molecular genetics is another powerful method to search for the susceptible genes for glomerulosclerosis. Although many patients and extensive screening procedures are required, one can identify the disease-related loci and genes without any knowledge of the structural and functional features of gene products, when genome-wide linkage analysis is used. Epidemiologic studies in humans provided increasing evidence for genetic susceptibility to glomerulosclerosis following various insults, such as hypertension, diabetes and glomerulonephritis. Similarly, in experimental animal models, marked species or strain differences with respect to the severity of glomerulosclerosis are noted after induction of diabetes or subtotal renal ablation [24]. Using genome-wide linkage analysis to identify genetic loci responsible for glomerulosclerosis, several loci have been identified in experimental animals and humans. In the fawn-hooded rat, a model of hypertension complicated by chronic renal failure, Brown et al. [25] were able to identify the localization of two genes Rf-1 and Rf-2, which are responsible for renal impairment, independently of blood pressure. In glomerulosclerosis-prone ROP-Os/+ mice, the presence of at least 8–10 loci was suggested by the analysis of backcrossed animal with non-sclerotic CH3, i.e. [(ROP-Os/+ xCH3)F1x ROP-Os/+ ] [26]. Furthermore, two genetic loci linked to glomerulosclerosis have been identified by linkage analysis in backcrossed NOD and Mus spretus mice, i.e. [(NODxMus spretus)F1xNOD] (K. Shikata and M. Hattori, personal communication). In humans, a locus for the gene responsible for focal glomerulosclerosis with autosomal dominant inheritance was identified and located on chromosome 19q13 [27]. Recently, susceptibility genes for microvascular diabetic complications have also been identified by sib-pair linkage analysis in Pima Indians [28]. The task of future studies will be the cloning of cDNA, the characterization of these genes and clarification of the genesis of glomerulosclerosis.

Conclusions

Rapid advances in modern molecular biology and genetics have enabled us to explore the greatest enigma for the nephrologist, i.e. the molecular mechanism of glomerulosclerosis. Although the above approaches to clarify the mechanism of glomerulosclerosis by molecular biology and molecular genetics are progressing independently, we are convinced that they ultimately will converge and provide new understanding of the pathobiology of glomerulosclerosis in the near future. The expected rich harvest of data will not only help to understand the disease process, but also will provide novel targets for molecular intervention in such diverse renal diseases as diabetes, hypertension and glomerulonephritis.

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