The NIDDK is seeking services to perform estimates of subpodocyte space (SPS) using serial block face scanning electron microscopy (SBFSEM) in tissue samples from research kidney biopsies in 13 Pima Indians with type 2 diabetes. SPS is a recently described glomerular structure which theoretically can induce up to 2.5-fold resistance against glomerular filtration. Hence, changes in SPS parameters are crucial in regulating single nephron glomerular filtration rate (SNGFR) and GFR. Diabetic kidney disease (DKD) is associated with a wide range of changes in GFR that are thought to be partly responsible for the development and progression of the disease. Very few studies have been performed to measure SPS and none have been performed in DKD. One major obstacle is that currently SPS studies requires three-dimensional (3D) electron microscopy, which is time consuming and expensive. The procedure is highly technically demanding, and requires very expensive specialized electron microscopes that are available in few labs in the U.S. Moreover, there are virtually no well-defined methodologies for quantification of the geometrical properties of SPS. Based on this unmet need, Dr. Behzad Najafian recently received an award from the Diabetes Complications Consortium (DiaComp) to do pilot studies for 3D modeling of SPS on biopsies from normal human and diabetic tissues (provided by our lab). Dr. Najafian has access to a Zeiss 3View-SEM machine at the University of Washington for doing these studies. In conjunction with these pilot studies he is developing much more time-efficient unbiased morphometric approaches to estimate key geometrical properties of SPS (such as volume, inflow surface area, and outflow surface area) that are directly related to flow resistance, on 2D images obtained by transmission electron microscopy (TEM). These approaches will be transformative in facilitating quantitative studies of SPS in various kidney diseases, including DKD. However, the complexity of the SPS shape requires validation and optimization of these morphometric methods. Furthermore, the DiaComp award only supports the initial pilot studies and does not support these critical validation and optimization process. Therefore, we are asking Dr. Najafian to perform these validation and optimization studies of unbiased morphometric approaches for estimation of the geometrical properties of the subpodocyte space through correlative studies of TEM and SBFSEM on tissue from kidney biopsies obtained from Pima Indians with type 2 diabetes.
Tasks to be performed
Estimation of % surface of peripheral and mesangial glomerular basement membrane (GBM) that cover the bases of SPS, interpodocyte space (IPS) and peripheral urinary space (PUS) using a line intercept method.
Estimation of slit filtration length density at bases of SPS, IPS and PUS and from there calculation of the in-flow surface area of SPS, IPS and PUS.
Estimation of volume fraction of SPS, IPS and PUS using point counting method.
Characterization of SPS exit pores to IPS and PUS and development of methods to estimate the surface area of these exit pores (SPS outflow surface area).
Correlation between paired TEM and SBFSEM data.
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