—  SYMPOSIUM #11  —

New Developments in Renal Disease
Moderators: Jan A. Bruijn and J. Charles Jennette

Section 2 - Genetic and Proteomic Insights into Renal Diseases

Jan A. Bruijn
Leiden University Medical Center
Leiden, The Netherlands


Introduction
Worldwide, more than one million people suffer from end-stage renal disease (ESRD) and need renal replacement therapy. ESRD occurs secondary to a broad range of renal diseases. The rate of progression to ESRD varies among patients. Recent studies that have provided evidence for a genetic component underlying susceptibility to progressive renal disease [1]. Gene mutations may result in a disturbed function of the corresponding protein, directly leading to kidney disease. Alternatively, genetic factors may become manifest only in the presence of systemic diseases, such as hypertension and diabetes mellitus, and thus modify the outcome of the renal disorder. For example, polymorphisms in genes encoding for proteins that are able to protect renal tissue against permanent damage may determine differences in susceptibility to disease progression among patients. Identification of novel genetic factors determining renal disease susceptibility may increase the understanding of the pathogenesis of ESRD. The regenerative effects of endogenous molecules in the kidney may be exploited to efficiently counteract the growing incidence of ESRD. Finally, genomic and proteomic techniques may be applied to improve diagnostic and prognostic procedures.

Genetic Factors
In some cases the relation between genetic factors and renal disease is evident. Examples are familial forms of focal and segmental glomerulosclerosis that are caused by mutations in the podocyte molecules podocin, CD2-associated protein, alpha-actinin-4 or the canonical transient receptor potential 6. Screening for mutations in the above mentioned genes in sporadic cases of nephrotic syndrome has provided new insights and is increasingly being integrated in pediatric nephrology [2]. In focal diseases like renal cell carcinomas and polycystic kidney disease (PKD), somatic gene inactivation is likely to occur at later stages, leading to en embryonic-lethal phenotype. In particular, differential regulation of genes linked to extracellular matrix metabolism may be one of the first events leading to tubule enlargement and subsequent cyst formation [3].This is now being studied with the use of transgenic mouse lines with inducible forms of conditional gene modifications in renal cells [4].

Genetic factors frequently have a less direct influence on renal disease development and become manifest only in the presence of 'permissive conditions' like diabetes mellitus and hypertension. Conversely, not all patients suffering from these conditions develop renal disease or progress to ESRD, and it is likely that genetic factors determine the time of onset and the rate of progression of the kidney disorder. Several studies of genetic linkage analyses in diabetic nephropathy have shown a susceptibility locus on chromosome 18q. A polymorphism in the DNA sequence of the CNDP1 gene, which encodes for the enzyme carnosinase-1, on chromosome 18q in diabetic patients determines susceptibility to develop diabetic nephropathy [5]. The substrate of carnosinase-1, L-carnosine, is a potent inhibitor of oxidative stress and the formation of advanced glycation end products, and may thus act as a cytoprotective factor during diabetes. It was postulated that opposing mechanisms, i.e., hyperglycemia versus the action of protective factors such as L-carnosine, determine the net outcome of diabetic nephropathy [5]. The importance of genetic predisposition to renal disease is further emphasized by the fact that individuals with a family history of ESRD have a higher risk of ESRD. Another example of a strong genetic influence on renal disease is lupus nephritis [1, 6, 7, 8]. Results from investigations on FcgammaRIIalpha IgG receptor polymorphisms indicate the existence of a heritable riskfactor for glomerulonephritis in Brazilian patients with SLE [9].

Genetic factors also appear to play a role in transplantation. The influence of donor tissue characteristics on prognosis in kidney transplantation has been investigated by comparing functionality of two kidneys from one donor in different recipients. In a large cohort of paired donor kidneys, graft function and survival of one graft could be predicted by the performance of its "mate" graft. The data may suggest that, among other factors, the repair capacity of the graft tissue has an influence on the posttransplant course. The hypothesis that the repair capacity of the graft tissue is at least partly genetically determined, is supported by observations that polymorphisms in cytokine genes of the donor are associated with long-term graft survival in the recipient.

Experimental Models
The natural genetic heterogeneity among individuals impedes the identification of genes marking a predisposition to progressive renal disease in humans. Investigation of animal models, through comparison of strains that are progressors with those that are not, may circumvent these problems. Identified candidate genes in animal models could eventually be of relevance in human populations. An animal model for human immunodeficiency virus (HIV)-associated nephropathy has been used successfully to identify disease susceptibility loci that may be of importance for human renal diseases. Linkage analysis in HIV-transgenic mouse strains unmasked a locus on chromosome 3, which was associated with renal damage. This locus corresponds to the human chromosome 3q25-27, which has been linked to various causes of ESRD.

Genetic linkage analyses in rats have led to the identification of chromosomal loci associated with the development of glomerular lesions, hypertension, albuminuria, and proteinuria [10]. Two Lewis rat substrains with small genetic differences but with considerable difference in susceptibility to develop progressive glomerulosclerosis after induction of anti-Thy-1 glomerulonephritis have been identified [11]. Kidney and bonemarrow transplantation experiments performed in our lab showed that predisposition to progressive glomerulosclerosis is governed by genes expressed in the kidney, but not by genes expressed in bone marrow-derived cells [12]. Similar experiments are being conducted in this model to localize genes that cause a predisposition to proteinuria.

Since chromosomal regions identified by linkage analysis generally contain tens to hundreds of genes, pinpointing the genes that are affected in the case of one particular disease is an elaborate task. Mutations in the DNA sequence of such genes may give rise to altered gene expression levels. Therefore, an alternative approach for the identification of genes involved in progression or remodeling of damage to the renal tissue is the application of genome-wide gene expression analysis by employing microarray techniques. Identification of genes determining disease progression will benefit from the combined application of genetic linkage analysis and gene expression profiling. Promising recent studies have shown the use of stem cell transplantation for the repair of basement membrane collagen defects in genetic kidney disease [13].

Renal Regeneration
Several studies support the concept that the kidney has a natural capacity to remodel into its original architecture after injury. In patients with type 1 diabetes and diabetic nephropathy, ten years of normoglycemia after pancreas transplantation resulted in amelioration of glomerular and tubular lesions in the kidney [14]. Administration of an angiotensin II receptor antagonist to hypertensive rats led to regression of renal vascular and glomerular fibrosis. The molecular mechanisms governing regression of renal lesions are not yet clear. Therefore, it is useful to investigate these mechanisms, since knowledge about them might conduce to the development of therapies that target the endogenous molecular pathways to prevent or even reverse renal damage.

In this respect heme oxygenase-1 (HO-1) is a promising example. This molecule displays cytoprotective activity: due to its protection against tissue damage, HO-1 upregulation is a beneficial response after acute renal injury. A polymorphism in the promoter region of the donor's HO-1 gene, which influences the level of expression, has been associated with renal graft survival [15]. Another protein that might be able to protect the kidney against permanent damage is bone morphogenic protein-7 (BMP-7). This molecule plays a central role in kidney embryogenesis and maintenance of the tubular epithelial phenotype. BMP-7 impedes myofibroblast formation and reverses chronic renal injury, which demonstrates that recombinant BMP-7 may be a novel treatment opportunity in chronic renal disease. The prolactin receptor (PRLR) may be a novel endogenous molecule capable of protecting the kidney against damage. Gene expression profiling showed that PRLR, like BMP-7, is abundantly expressed in the cortices of normal kidneys [16]. After 6 months, PRLR mRNA levels were 30 times lower in patients that would show chronic allograft nephropathy after 12 months than in patients that would have retained normal morphology after 12 months [17]. The data may suggest that PRLR is an intrinsic factor in the protection of the kidney against permanent damage. Novel data show that in kidney transplants with rejection, decreasing expression levels of PRLR are accompanied with an increase in the extent of fibrosis.

It is important to find out why protective and regenerative mechanisms function in some patients, but fail in others. The question whether gene polymorphisms determine the expression levels of potential cytoprotective proteins like BMP-7 and PRLR also requires an answer.

Clinical Use
In addition to genetic analysis, genomic and proteomic methods offer new opportunities for clinical medicine. A promising clinical application of molecular biology is the identification of mRNA expression patterns in diseased organs [18]. The development of microarray technologies and real-time PCR enables the study of gene expression networks in renal biopsies. This, in combination with the usage of laser-capture microdissection, enables gene expression analysis in a nephron segment-specific way [19]. These mRNA expression patterns may provide information regarding diagnosis, prognosis, and responsiveness to treatment [20].

Why would it be useful to analyze levels of mRNA in renal biopsies? Firstly, mRNA quantitation could be used as a diagnostic tool. In renal pathology, it is at times difficult to formulate a diagnosis on the sole basis of clinical and histological findings. Molecular tissue analysis with RNA quantitation may help improve diagnostic accuracy. Secondly, mRNA levels could be used as prognostic tools. Studies in animal models, and, more recently, in biopsy specimens from patients have shown that alterations in mRNA levels for extracellular matrix components and matrix-regulating molecules predict the extent of scarring in later phases of the disease [21, 22, 23, 24]. Furthermore, recent studies in kidney transplantation and in native kidney diseases have shown that mRNA levels of transcripts identified by microarray analysis may complement histological findings in establishing prognosis [25]. Thirdly, mRNA assessment may be used as a tool to predict response to therapy. Support for this concept has been provided by Sarwal and colleagues. They showed that a relatively high expression of CD20, a marker for B cells, during acute rejection is associated with resistance to anti-rejection therapy. In another report, Fas ligand mRNA levels predicted unresponsiveness to anti-rejection therapy. Fourthly, mRNA assessment may be used as a tool to monitor the extent of a therapy's negative side effects over time. For example, mRNA levels of TGF- b and collagens I and III have been used to compare the fibrogenic effects of different calcineurin inhibitors on kidney grafts. Usage of sequential protocol biopsies in mRNA assessment allows for accurate monitoring of profibrotic pathways under the influence of maintenance immunosuppressive medications. Fifthly, investigation of gene expression levels in renal tissue samples will elucidate the pathogenesis of kidney diseases [26].

In addition to genomic analysis of renal tissue, proteomic investigation of urinary samples has been proposed as a non-invasive method to detect renal diseases and acute rejection of renal allografts. Capillary electrophoresis coupled to mass spectometry has been used to analyze urinary samples in order to detect acute tubulointerstitial rejection in renal transplant recipients [27]. In another study it was found that cleaved beta2-microglobulin may be used as an indicator of acute tubular injury in renal allografts [28]. This method permits fast and accurate identification of polypeptide patterns also in the urine of patients with IgA nephropathy and other glomerular diseases [29, 30]. Urinary analysis may also be directed at mRNA. For example, prediction of outcome of acute rejetion of renal transplants may be improved by measurement of mRNA encoding for FOXP3, a functional factor for regulatory T lymphocytes [31].

Overall, we envision that the use of molecular markers in addition to conventional parameters including morphology will lead to more accurate prognosis and will therefore allow more individualized therapeutic regimens. The response of patients to anti-rejection medication and maintenance therapy may be predicted more accurately on the basis of molecular markers.

Conclusion
Genetic linkage analysis in patients and animal models has led to identification of chromosomal regions associated with renal disease. Genes located in such chromosomal regions may predispose to progression of kidney disease or, alternatively, induce regeneration mechanisms in damaged renal tissue. Indeed, there is convincing evidence for the existence of endogenous molecules that protect the kidney against permanent damage. Identification of genes involved in progression will lead to a better understanding of the pathophysiology of kidney diseases. Elucidation of the regulation of the expression of cytoprotective molecules might result in improved therapies that exploit endogenous protective and regenerative mechanisms.

Measurement of mRNA transcripts may be a sensitive and efficient means to predict outcome in kidney transplantation and native kidney diseases. Messenger RNA assessment may be instrumental in predicting response to therapy and in monitoring potential long-term side effects of maintenance therapy. Any of these applications in clinical practice requires optimized protocols for extraction of intact RNA from renal biopsy specimens. Research in recent years has generated such protocols. Laser-capture microdissection allows for analysis of gene expression in specific renal tissue compartments of archival biopsy tissue. The combination of molecular biological technology, including laser-capture microdissection and differential gene expression analysis, renders a powerful instrument to investigate the mechanisms and pathogenesis of renal disease.

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