Mechanisms of Glomerular Hypercellularity in Diabetic Nephropathy
There is a critical need for a better understanding of the pathophysiology of diabetic nephropathy (DN). Continued existence of this fundamental knowledge gap presents an important clinical problem because specific therapeutic interventions to treat or slow disease progression cannot be fully realized until this gap is filled. The long term goal of this research is to characterize key cellular and molecular pathways regulating DN and its progression in order to identify novel markers that assess disease progression, and to develop specific therapeutic interventions targeting these pathways. Accumulating evidence points to the importance of tubular injury in the pathogenesis of DN and other glomerular diseases. In addition, there is evidence in animal models that proximal tubule cells can affect the glomerular compartment. We hypothesize that growth factors derived from proximal tubules affect glomerular cellularity. Consequently, the objectives of this application are to characterize the increased cellularity found in glomeruli of human diabetic biopsies and examine the transcriptome of proximal tubules and glomeruli to quantify trophic factors and other pathways that account for the increased glomerular cellularity. The central hypothesis of this application is that the transcriptome of the proximal tubule predicts the glomerular cellularity which is a unique indicator of DN. This hypothesis has been formulated on the basis of existing literature and strong preliminary data from the applicant’s laboratory. The rationale for the proposed research is that once the unique molecular and cellular identifiers of human DN are determined, they can be used to monitor efficacy of pharmacologic interventions, identify animal models that best represent human DN for translational research, and reveal novel pharmacological approaches towards treating human DN. The central hypothesis will be tested by pursuing two specific aims: 1) Characterize the cellularity in glomeruli from human diabetic biopsies and 2) Define the transcriptome of the same glomeruli and the adjacent S1/S2 proximal tubule segments in the same set of patient biopsies. The transcriptomes will be correlated with the cell types found in aim 1. In aim 1, advanced three-dimensional tissue cytometry will be performed on bio-banked kidney biopsies from patients with diabetic nephropathy in order to quantify the cellular composition of the glomeruli. In aim 2, laser microdissection of the same glomeruli and proximal tubule sub segments on the DN biopsies. Gene expression analysis will be performed on RNA isolated from the proximal tubules and glomeruli utilizing solid-state transcriptome array chips to discern the transcriptomic signature of the isolated renal compartments. The transcriptomes will be correlated with the cellularity and specific cell types in DN glomeruli. The approach is innovative, because it represents a new and substantive advancement in the molecular and morphological interrogation of human kidney biopsies, namely a unique 3D cytometric analysis of diabetic glomeruli as well as characterization and of the transcriptomic signature of proximal tubules and glomeruli in individual human kidney biopsies. The proposed research is significant, because it is the next step in a continuum of research that is expected to identify critically needed biomarkers of disease progression, optimize preclinical studies, and develop specific and targeted therapeutic interventions in the vast clinical problem of DN.

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