Basic and clinical studies of nephropathy and the progression of renal failure
(Croker, Agarwal, Madsen, Morel, Gluck, Srinivas, Reeves, Segal).

 The top four causes of chronic renal failure (CRF) leading to end stage renal disease (ESRD) are diabetes, hypertension, all causes of glomerulonephritis and chronic renal allograft failure. A variety of evidence indicates the balance between healing or resistance to kidney damage and progression to ESRD is related to the severity of the original injury and genetically determined characteristics of the kidney and progresses to a final common pathway of interstitial fibrosis. There is also evidence that autocrine and paracrine hormones form a complex network of signaling between intrinsic renal cells and infiltrating cells with the production of reactive oxygen species. Regardless, the key pathways or genes that initiate progression of renal injury through the point of no return to CRF are not known. Gene expression profiling should be helpful in identifying these genes and pathways. As part of the functional and genetic characterization of murine Systemic Lupus Erythematosus (SLE) susceptibility loci, we have defined functional pathways leading to autoimmunity. Our collection of congenic mice containing SLE-susceptibility loci, singly or in combination produced using congenic dissection, as well as the functional data that we have accumulated on those strains, constitutes an excellent system to apply new functional genomic technologies. By selecting the appropriate susceptibility loci, we can produce graded levels of lupus nephritis from normal through subclinical non-progressive disease to progressive lupus nephritis with the constellation of severe (diffuse and generalized) glomerulonephritis, proteinuria and mortality. We will examine kidneys of control C57Bl/6 (B6) mice, double congenics with morphologic but clinically silent disease (B6.NZMc4|c7) or moderately penetrant (50%) proliferative glomerulonephritis (B6.NZMc1|c7) and the triple congenic with fully penetrant (95%) glomerulonephritis (B6.NZMc1|c4|c7). Initial groups of animals will be examined at three phases of development: no histopathology (6-8 wks), developed lesions (16 wks) and progressive lesions (24 wks). We will examine five individuals in each group (4 strains - 3 time intervals). We expect to see clusters of genes which represent early events in the initiation of lupus nephritis. Clusters of genes associated with stabilization will be identified in non-progressor animals whereas progressor animals will express genes involved in progression and compensatory responses. Depending on these results, B6.NZM congenics with other promoting or suppressor loci or knockouts or transgenics involving specific genes will be evaluated. These gene expression patterns will interface with parallel studies to determine the cellular pathways involved in injury including determination of temporal and spatial sequence of specific cell types using in situ hybridization or antibody directed localization of gene products. All these findings will support the other on-going projects to identify the specific susceptibility and resistance genes in the congenic intervals by positional cloning. In addition to the studies in mice, proteinuria is also well documented as an independent risk factor in clinical studies of diabetes and SLE. These studies establish proteinuria is a prognostic marker for CRF but they do not establish a causative relationship. Therefore, we have used the protein overload model to evaluate causation. Severity of glomerular lesions in this model using diabetes related and congenic mice ranged from mild (stalk glomerulopathy) to nodular intercapillary glomerulosclerosis (Kimmelsteil Wilson lesion, severe) in a strain specific manner. Strains rank in degree of severity as follows: B6, DBA/2 << NOD.B10H2b, NON < NOD (males). NOD females were not studied because they die of diabetes in the acute phase. This model will compliment the lupus nephropathy studies. The experimental paradigm is the same as outlined in the SLE model but we will add the diabetes related strains. The time intervals will parallel disease progression. This model will help identify subsets of genes related to lupus nephritis but also patterns of gene expression more broadly related to proteinuria diabetic nephropathy and other non-inflammatory forms of glomerular disease. The congenic dissection approach has been successful in delineating pathways involved in the initiation of diabetes and lupus nephropathy. Congenic dissection coupled with expression profiling is an even more powerful approach because congenic dissection reduces the genetic differences in resistant and susceptible strains to 2 - 6% percent of the genome. This reduces the differences detected by the microarray and permits the more rapid and precise identification of significant pathogenetic pathways and candidate genes. The third facet of our approach is the confirmation of candidate genes and interactive pathways in transgenic and knockout mice. Clinical Studies - Over 50 years ago Brun and others pioneered the use of the core needle biopsy to study renal disease. This technique was soon coupled with advances in fluorescent antibody and electron microscopy technology to lead a revolution in understanding and treatment of kidney disease. Microarray technology affords a tremendous opportunity for advances in precision of diagnosis, monitoring therapy and intervention in disease processes through translational research. There are current limitations in core biopsy technology because of risk and cost. Two decades ago, fine needle aspiration (FNA) was studied as a more readily performed alternative to core biopsy. However, using currently available techniques FNA does not provide sufficient information. FNA does provide several million cells which yields sufficient RNA for microarray analysis. Once validated, this testing could be performed up to several times a week on an out patient basis to provide a level of monitoring for inflammation, toxicity and therapeutic response never before possible. Secondly it is directly adaptable to evaluation and monitoring native diseases which are often only evaluated by one core biopsy (SLE or primary glomerulonephritis) or generally not at all (type I diabetes or hypertension). This coupling of technology is expected to advance our understanding and clinical practice to greater extent than the introduction of the core biopsy fifty years ago. The initial phases of the patient biopsy protocols will focus on three conditions which are biopsied as part of accepted standard of care and where we have strengths in basic and clinical research: 1) adult diabetes, 2) lupus nephritis, and 3) renal allograft nephropathy. Microarray analysis performed on 3 - 5 mm of cortex of these groups of patient biopsies will be correlated with known clinical prognostic indicators and defined biopsy parameters (Banff, CADI, and NIH grading, WHO classification and macrophage index), in groups of stable or progressive nephropathy patients and donor kidneys. This represents two groups of patients with each disease and one set of controls. Because of the greater variability in patient disease we expect to evaluate 10 biopsies in each group. Similarities and differences between these diseases of diverse pathogenetic mechanisms should be readily apparent in stable and progressor patients. Cross reference with the parallel basic research studies is expected to optimize interpretation and opportunities for experimental evaluation of significant findings.

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