Molecular and pathological signature of the human kidney tubule in progression of diabetic nephropathy.
There is a fundamental gap in understanding the mechanisms that determine disease progression in human diabetic nephropathy (DN). Continued existence of this 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 is to characterize key cellular and molecular pathways regulating progression of DN, to identify novel markers that assess disease progression, and to develop specific therapeutic interventions targeting these pathways. The cross-talk between tubular subsegments and immune cells in the kidney is an important determinant of fibrosis and disease progression in DN. Consequently, the objective of this application is to selectively examine the transcriptome of tubular subsegments and to quantify and localize immune cell subtypes in relation to tubular subsegments in patients with DN. The central hypothesis of this application is that the transcriptome of kidney tubular subsegments and the abundance and distribution of immune cell subtypes are unique and complimentary identifiers of disease progression in human DN. This hypothesis has been formulated on the basis of existing literature and strong preliminary data from the applicants’ laboratories. The rationale for the proposed research is that once the unique molecular and cellular identifiers that correlate with disease progression and long term outcomes in 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) Define the transcriptome expressed by the tubular subsegments from biopsies of patients with diabetic nephropathy and different rates of progression and 2) Determine the abundance and distribution of immune cell subtypes in the same set of patient biopsies. Under the first aim, laser microdissection of tubular subsegments will be performed on biobanked kidney biopsies from case-matched patients with DN that rapidly progressed (decrease in eGFR > 5 mL/min/1.73m2/year) and those that did not rapidly progress (decrease in eGFR < 5 mL/min/1.73m2/year). Gene expression analysis will be performed on RNA isolated from the tubular subsegments utilizing solid-state transcriptome array chips to discern the transcriptomic signature of the isolated tubular subsegments. Under the second aim, advanced three-dimensional (3-D) tissue cytometry will be performed on the biobanked kidney biopsies from the same case-matched patients described in the first aim to quantify the immune cell composition, examine spatial cellular organization, and delineate detailed morphologic differences in patients with DN that rapidly progressed and those that did not rapidly progress. The approach is innovative, because it represents a new and substantive advancement in the molecular and morphological interrogation of human kidney biopsies, namely characterization of the transcriptomic signature of tubular subsegments in individual human kidney biopsies combined with unique 3D cytometric analysis of the same 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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