Anubhuti Gupta

PhD Student

Berend Isermann

Project Leader

Diabetic nephropathy (dNP) is a chronic kidney disease observed in patients with diabetes mellitus (DM). An altered hemostatic system and an important role of coagulation proteases in the pathophysiology of dNP is well established. Patients with DM have platelet-hyperreactivity and enhanced platelet activation. However, studies evaluating a mechanistic role of platelets in dNP are lacking. In this regard, when activated platelets come in contact with the injured glomerular filtration barrier, it can result in glomerular endothelial injury and production of pro-inflammatory cytokines. This results in glomerular endothelial dysfunction and loss of barrier function. Loss of NF-E2, which is required for the completion of megakaryocyte maturation and platelet production, results in a quantitative platelet deficiency in mice, making it a suitable model to study the role of platelets in diseases. Interestingly, a platelet independent and trophoblast specific role of NF-E2 for placental development has been shown. Mechanistically, these placental effects are regulated through epigenetic modifications such as acetylation and sumoylation. However, the role of platelet dependent as well platelet independent (renal resident cell dependent) role of p45 NF-E2 and associated mechanisms insights in dNP remains unknown. Therefore, within the current study we will dissect the role of platelets as well as transcription factor p45-NFE2 in dNP. In order to specifically address the role of platelets, we will inhibit platelet activation using aspirin (pharmaceutical inhibition) or using platelet specific Gaq knockout mice (Gaq^LoxP x PF4^cre, genetic inhibition). We will further study the effect of platelets on glomerular endothelial barrier function by evaluating markers of endothelial dysfunction (eNOS, tight junction proteins). Markers for cell death (TUNEL) and inflammation (IL 1β and NLRP3) will be evaluated using immunoblotting and staining in glomerular endothelial cells. Furthermore, we will perform trans-endothelial barrier assays using microfluidics based approach on co-cultures of knockout GENC and podocytes. To study the role of p45 NFE-2 in renal resident cells, we will first conduct in vitro studies to identify the renal cell types which express NFE2 and are relevant for our model. We will then study the effect of high glucose in NFE2 deficient cells which will be generated using CRISPR or shRNA knockdown approaches. Mechanistically, we will study molecular markers of inflammation (IL 1β, HMGB1), kidney injury (KIM-1, Nephrin), cell death (TUNEL) and senescence (p21, p16, β-Gal) using immunoblotting and staining. Furthermore, we will use NFE2^LoxP mice to delete the expression of NFE2 in relevant renal resident cells or platelets. Mechanisms of cellular proliferation, senescence and epigenetic modifications will be evaluated in NFE-2 deficient cells in cooperation with Project 5 and Project 7. Taken together, these studies will establish the contribution of platelets in the pathogenesis of dNP and distinguish it from the role of p45 NF-E2 in renal resident cells.

Disturbances in the endothelial function and coagulation pathway may cause initial platelet activation. Intrinsic changes in the platelet metabolism and changes in intra-platelet signaling pathways due to diabetes contribute to the increased platelet hyperactivity in T2DM. This results in glomerular endothelial dysfunction which is characterized by loss of eNOS and fenestrations. Danger signals released from platelets can activate inflammasome and promote cell death disrupting the barrier. On the other hand, a renal cell specific loss of NFE2, a transcription factor regulating platelet formation, can result in impaired cellular proliferation and kidney regeneration. Furthermore, cell intrinsic NFE2 can modulate renal cell metabolism by regulating epigenetic modifications.