isolation-conditionally-immortalized-mouse-glomerular-endothelial

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative ...

The Icahn School of Medicine at Mount Sinai

The anticancer and immunosuppressant thiopurines cause 6-thioguanine (6-TG) to accumulate in nuclear DNA. We report that 6-TG is also readily incorporated into mitochondrial DNA (mtDNA) where it is rapidly oxidized. The oxidized forms of mtDNA 6-TG inhibit replication by DNA Pol-γ. Accumulation of oxidized 6-TG is associated with reduced mtDNA transcription, a decline in mitochondrial protein levels, and loss of mitochondrial function. Ultraviolet A radiation (UVA) also oxidizes mtDNA 6-TG. Cells without mtDNA are less sensitive to killing by a combination of 6-TG and UVA than their mtDNA-containing counterparts, indicating that photochemical mtDNA 6-TG oxidation contributes to 6-TG-mediated UVA photosensitization.

6-Thioguanine damages mitochondrial DNA and causes mitochondrial dysfunction in human cells.

Microvascular endothelial cells poised to take center stage in experimental renal fibrosis.

The molecular signaling mechanisms between glomerular cell types during initiation/progression of diabetic kidney disease (DKD) remain poorly understood. We compared the early transcriptome profile between DKD-resistant C57BL/6J and DKD-susceptible DBA/2J (D2) glomeruli and demonstrated a significant downregulation of essential mitochondrial genes in glomeruli from diabetic D2 mice, but not in C57BL/6J, with comparable hyperglycemia. Diabetic D2 mice manifested increased mitochondrial DNA lesions (8-oxoguanine) exclusively localized to glomerular endothelial cells after 3 weeks of diabetes, and these accumulated over time in addition to increased urine secretion of 8-oxo-deoxyguanosine. Detailed assessment of glomerular capillaries from diabetic D2 mice demonstrated early signs of endothelial injury and loss of fenestrae. Glomerular endothelial mitochondrial dysfunction was associated with increased glomerular endothelin-1 receptor type A (Ednra) expression and increased circulating endothelin-1 (Edn1). Selective Ednra blockade or mitochondrial-targeted reactive oxygen species scavenging prevented mitochondrial oxidative stress of endothelial cells and ameliorated diabetes-induced endothelial injury, podocyte loss, albuminuria, and glomerulosclerosis. In human DKD, increased urine 8-oxo-deoxyguanosine was associated with rapid DKD progression, and biopsies from patients with DKD showed increased mitochondrial DNA damage associated with glomerular endothelial EDNRA expression. Our studies show that DKD susceptibility was linked to mitochondrial dysfunction, mediated largely by Edn1-Ednra in glomerular endothelial cells representing an early event in DKD progression, and suggest that cross talk between glomerular endothelial injury and podocytes leads to defects and depletion, albuminuria, and glomerulosclerosis.

Glomerular Endothelial Mitochondrial Dysfunction Is Essential and Characteristic of Diabetic Kidney Disease Susceptibility.

Mitochondria Matter: A Critical Role of  in Stabilizing the CoQ Complex in Podocytes in Steroid-Resistant Nephrotic Syndrome.

In the setting of diabetes mellitus, mitochondrial dysfunction and oxidative stress are important pathogenic mechanisms causing end organ damage, including diabetic kidney disease (DKD), but mechanistic understanding at a cellular level remains obscure. In mouse models of DKD, glomerular endothelial cell (GEC) dysfunction precedes albuminuria and contributes to neighboring podocyte dysfunction, implicating GECs in breakdown of the glomerular filtration barrier. In the following studies we wished to explore the cellular mechanisms by which GECs become dysfunctional in the diabetic milieu, and the impact to neighboring podocytes.

The diabetic microenvironment causes mitochondrial oxidative stress in glomerular endothelial cells and pathological crosstalk with podocytes.

Loss of normal kidney function affects more than 10% of the population and contributes to morbidity and mortality. Kidney diseases are currently treated with immunosuppressive agents, antihypertensives and diuretics with partial but limited success. Most kidney disease is characterized by breakdown of the glomerular filtration barrier (GFB). Specialized podocyte cells maintain the GFB, and structure-function experiments and studies of intercellular communication between the podocytes and other GFB cells, combined with advances from genetics and genomics, have laid the groundwork for a new generation of therapies that directly intervene at the GFB. These include inhibitors of apolipoprotein L1 (APOL1), short transient receptor potential channels (TRPCs), soluble fms-like tyrosine kinase 1 (sFLT1; also known as soluble vascular endothelial growth factor receptor 1), roundabout homologue 2 (ROBO2), endothelin receptor A, soluble urokinase plasminogen activator surface receptor (suPAR) and substrate intermediates for coenzyme Q10 (CoQ). These molecular targets converge on two key components of GFB biology: mitochondrial function and the actin-myosin contractile machinery. This Review discusses therapies and developments focused on maintaining GFB integrity, and the emerging questions in this evolving field.

The glomerular filtration barrier: a structural target for novel kidney therapies.

Menon et al. report cell-specific transcriptional changes in podocytes and glomerular endothelial cells that indicate cell stress and increased bidirectional crosstalk among these cells in apparently healthy human allografts. They identified common and independent podocytes and glomerular endothelial cell-specific responses in nondiabetic and diabetic transplant recipients, as well as parallels in genes related to podocyte and glomerular endothelial cell stress in experimental focal segmental glomerular sclerosis. These findings could explain hypertrophy-associated glomerular disease progression associated with podocyte detachment after transplantation.

Article	Year
6-Thioguanine damages mitochondrial DNA and causes mitochondrial dysfunction in human cells. FEBS letters\| PubMed ID: 22062154	2011
Microvascular endothelial cells poised to take center stage in experimental renal fibrosis. Journal of the American Society of Nephrology : JASN\| PubMed ID: 25535302	2015
Glomerular Endothelial Mitochondrial Dysfunction Is Essential and Characteristic of Diabetic Kidney Disease Susceptibility. Diabetes\| PubMed ID: 27899487	2017
Mitochondria Matter: A Critical Role of in Stabilizing the CoQ Complex in Podocytes in Steroid-Resistant Nephrotic Syndrome. Journal of the American Society of Nephrology : JASN\| PubMed ID: 32381602	2020
The diabetic microenvironment causes mitochondrial oxidative stress in glomerular endothelial cells and pathological crosstalk with podocytes. Cell communication and signaling : CCS\| PubMed ID: 32641054	2020
The glomerular filtration barrier: a structural target for novel kidney therapies. Nature reviews. Drug discovery\| PubMed ID: 34262140	2021
Hypertrophy and glomerular cell adaptation through crosstalk leads to glomerular injury after kidney transplantation. Kidney international\| PubMed ID: 35314050	2022

Ilse S. Daehn

Biography

JoVE Journal Publications

Other Publications