Study finds alternative pathway is main driver of aHUS
Results challenge suggestion that disease pathway requires antibodies
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Researchers reported that atypical hemolytic uremic syndrome (aHUS) is driven by abnormal activation of the alternative complement pathway, an immune response that’s independent of antibody activity, according to a study using endothelial cells that line blood vessels.
These results challenge a recent report suggesting that aHUS may be caused by abnormal activation of the classical complement pathway, which requires antibodies as mediators.
“The main finding of this study is that [alternative pathway] inhibition fully prevented complement activation and [blood clot] formation on endothelium exposed to aHUS [blood],” the researchers wrote.
The cell-based study, “Complement Alternative Pathway Dominance in Atypical Hemolytic Uremic Syndrome Revealed by Endothelial Bioassays,” was published in Kidney International Reports.
The complement system is a network of more than 30 immune proteins that, upon activation, help the body clear disease-causing microbes. It operates through two primary pathways: the classical pathway, initiated when antibodies bind to microbes, and the alternative pathway, which functions independently of antibodies. In aHUS, dysregulation of the alternative complement pathway leads to red blood cell damage (hemolytic anemia), reduced platelet counts, and blood clot formation within small blood vessels (thrombotic microangiopathy, or TMA), with the kidneys particularly affected.
Investigating cells
A recent study contested the view that the alternative pathway drives aHUS. It found that classical pathway inhibitors, but not alternative pathway inhibitors, prevented the destruction of a kidney cell line (HEK293) when mixed with blood from aHUS patients. The researchers also identified an antibody, immunoglobulin M (IgM), that drove classical pathway activation in aHUS.
HEK293 cells, however, are more similar to epithelial cells that cover organ surfaces than endothelial cells, which line blood vessels and are the primary targets of the complement system in aHUS.
Researchers in Italy used two endothelial cell lines to further investigate the role of the alternative complement pathway in aHUS.
The team first exposed HMEC-1 cells, specialized endothelial cells that line blood vessels under the skin, to blood from aHUS patients in clinical remission. This stimulated the deposition or formation of two complement proteins, C3 and C5b-9, on HMEC-1 cells, confirming a complement-mediated attack.
Adding pegcetacoplan, a molecule that blocks all complement pathways and is approved (as Empaveli) to treat adults with paroxysmal nocturnal hemoglobinuria (PNH), completely prevented blood-induced C3 deposition and C5b-9 formation on HMEC-1 cells.
Adding iptacopan, another PNH therapy (sold as Fabhalta) that inhibits factor B — a protein in the alternative complement pathway — yielded the same protective outcome as pegcetacoplan. This occurred using blood from patients with aHUS, both with and without identified genetic abnormalities.
Exposing HMEC-1 cells to aHUS blood also induced a marked increase in blood clot formation compared with cells incubated with healthy control blood. Adding either pegcetacoplan or iptacopan to patients’ blood prevented platelet aggregation on HMEC-1.
“These findings underscore the prominent role of the [alternative pathway] in driving complement activation in aHUS, even in patients in which current screening fails to detect a genetic driver,” the researchers wrote.
The team then conducted similar experiments using glomerular endothelial cells (GEnCs), which line the tiny blood vessels in the kidneys.
Complement activation in GEnCs closely matched patterns seen using HMEC-1 cells. In particular, both pegcetacoplan and iptacopan blocked aHUS blood-induced C3 deposition, C5b-9 formation, and platelet aggregation on GEnCs, restoring complement activity to normal levels.
“Finding that all the above abnormalities in GEnC were fully corrected by iptacopan, reinforces the central role of [alternative pathway] dysregulation in aHUS pathogenesis [disease processes],” the team wrote. “These results challenge a recent report that proposed an IgM-driven classical pathway stimulus as the primary driver of complement activation in aHUS.”
