Kruppel‐like Factor 4 is Critical to the Maintenance of Glomerular Endothelial Integrity

Abstract

BackgroundThrombotic microangiopathy (TMA) represents the most severe manifestation of glomerular endothelial injury consisting of endothelial cell (EC) swelling, subendothelial expansion, inflammation and microthrombi. TMA occurs subsequent to various etiologies and is associated with dysregulation of thrombotic and inflammatory transcripts and complement activation. Expression of membrane bound (CD55, CD59, CD46) complement regulators, is altered in many subtypes of TMA and it is unclear if this is a driver or consequence of EC injury. To investigate transcriptional mediators involved in TMA, we reviewed expression arrays of both global and single cell RNA‐seq from human kidney biopsies with complement mediated renal microvascular injury and observed that Krüppel‐Like Factor 4 (KLF4), a zinc finger transcription factor, is both differentially expressed in ECs and predicted to regulate other differentially expressed genes. Previous studies demonstrate that KLF4 is a critical mediator of anti‐thrombotic and anti‐inflammatory phenotype in systemic vascular beds. We aimed to investigate its role in modulating renal microvascular injury and complement activation in TMA.MethodsEndothelial‐specific Klf4 knockout mice (Klf4ΔEC) were generated by crossing Klf4fl/fl mice withCdh5‐Cre mice. As two separate models of endothelial dysfunction, Klf4ΔEC mice were both aged to 1 year and crossed with Nos3−/− mice to generate double knockout mice (DKO). Periodic acid‐Schiff, electron microscopy, immunofluorescence, ELISA and RT‐PCR were performed to investigate the effects of endothelial Klf4 on the renal microvasculature. Renal histology and ultrastructure were evaluated, blinded, by renal pathology (MPR).ResultsAt 12 weeks of age, while no overt histologic changes were observed, Klf4ΔEC mice exhibited increased glomerular Vcam‐1 and Pai‐1 expression and complement activation (increased C3 and C5b‐9 deposition) as compared to Klf4fl/fl mice. To investigate the basis for this increased complement activation, we measured the expression of the membrane‐bound complement regulators and noted reduced glomerular Cd55 and Cd59 expression in Klf4ΔEC as compared with Klf4fl/fl mice. Interestingly, 1‐year‐old Klf4ΔEC mice exhibited pathology consistent with subacute TMA (glomerular capillary congestion, EC swelling, subendothelial expansion and mesangiolysis as well as increased intraglomerular von Willebrand Factor and albuminuria) as compared to age‐matched Klf4fl/fl mice. In addition, Klf4ΔEC mice crossed with nitric oxide synthase knockout (Nos3−/−) mice also exhibited increased glomerular complement activation and histological and ultrastructural features consistent with subacute TMA (loss of fenestrations, glomerular capillary congestion, tuft collapse with basement membrane wrinkling) as compared to all other groups (12 weeks). Finally, knockdown of KLF4 in cultured human microvascular endothelial cells reduced the expression of CD55, whereas induction of KLF4 increased CD55 expression in HMEC‐1s.ConclusionsLoss of endothelial Klf4 in both aged mice and in Nos3−/− mice recapitulates many pathologic features of renal‐TMA, including complement activation and is potentially mediated through its regulation of Cd55.Support or Funding InformationNIDDK