Over the last five years, we have demonstrated that the activation of NOD-like receptor protein with pryin domain containing 3 (NLRP3) inflammasomes in podocytes triggers and promotes glomerular dysfunction and sclerosis during hyperhomocysteinemia (hHcy). Given that the NLRP3 inflammasomes are activated primarily in the cytosol, their products such as IL-1?, IL-18 or high mobility group protein B1 (HMGB1) may not be secreted out of cells via a classical and Golgi apparatus-mediated delivery pathway. It is now imperative to address how NLRP3 activation-derived products are released out of podocytes to trigger the inflammatory response leading to injury in glomeruli. Our preliminary studies have shown that homocysteine (Hcy) stimulation or hHcy not only resulted in NLRP3 inflammasome activation within podocytes, but also increased the release of exosomes that are enriched with NLRP3 inflammasome products. Lysosomal acid sphingomyelinase (ASM) activation was importantly involved in hHcy-induced lysosome dysfunction, which reduced the degradation of multivesicular body (MVB) resulting in robust exosome secretion. We therefore hypothesize that an exosome secretory mechanism mediated by lysosomal ASM-ceramide signaling pathway is concurrently activated with NLRP3 inflammasomes in podocytes during hHcy and both work in concert to trigger or promote local inflammatory response, leading to glomerular injury and sclerosis. To test this hypothesis, three Specific Aims are proposed.
Specific Aim 1 will determine whether lysosomal ASM-ceramide signaling pathway contributes to NLRP3 inflammasome activation and enhanced release of inflammatory exosomes in podocytes during hHcy, which together trigger local glomerular inflammation, injury and even sclerosis using Smpd1-/- mice, podocyte-specific Smpd1 transgenic mice (Smpd1trg/PodoCre), and their wild type littermates (WT/WT and Smpd1trg/Podowt).
Specific Aim 2 will determine whether Hcy in vitro or hHcy in vivo stimulates the formation of MVBs containing NRLP3 inflammasome products in podocytes to release inflammatory exosomes and whether these inflamed exosomes induce glomerular dysfunction and injury.
Specific Aim 3 will explore the molecular mechanisms by which lysosome trafficking and interactions with MVBs are altered upon Hcy stimulation to prolong the fate of MVBs, thereby promoting robust release of inflammatory exosomes in primary cultures of podocytes or glomeruli from Smpd1-/- and Smpd1trg/PodoCre mice and their wild type littermates. These proposed studies represent the first effort in the field to link the activation of NLRP3 inflammasomes in podocytes to the instigation of glomerular inflammatory response and injury. The findings may lead to a paradigm shift in how we understand the molecular mechanisms of glomerular inflammation during hHcy and in how to develop new therapeutic strategies to mechanistically counteract glomerular injury or end-stage renal disease during hHcy.

Public Health Relevance

In this research program, we will test how elevations of blood homocysteine (Hcy), a toxic amino acid, cause kidney inflammation which results in end stage renal disease (ESRD). We will demonstrate how activation of a protein complex named as inflammasomes in podocytes (a type of cells that manage renal urine formation) triggers renal glomerular inflammation with a focus on the mechanisms responsible for secretion of small particles ? exosomes, which contain various inflamed factors. The findings from our proposed studies will help develop new therapy for prevention and treatment of ESRD.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK054927-21A1
Application #
10072247
Study Section
Hypertension and Microcirculation Study Section (HM)
Program Officer
Sadusky, Anna Burkart
Project Start
1998-06-01
Project End
2024-05-31
Budget Start
2020-06-04
Budget End
2021-05-31
Support Year
21
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
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