Neutrophil gelatinase–associated lipocalin is instrumental in the pathogenesis of antibody‐mediated nephritis in mice

Abstract

Objective The mechanism by which anti-DNA antibodies mediate lupus nephritis has yet to be conclusively determined. Previously, we found that treatment of mesangial cells with anti-DNA antibodies induced high expression of Neutrophil Gelatinase Associated Lipocalin (NGAL), an iron-binding protein upregulated in response to kidney injury. However, whether NGAL is instrumental in pathogenesis, induced as part of repair, or irrelevant to damage/repair pathways, is not known. Methods To investigate the role of NGAL in antibody-mediated nephritis, we induced nephrotoxic nephritis by passive antibody transfer to B6 or 129 mice. To determine if NGAL upregulation is instrumental, we compared the severity of renal damage in NGAL wild-type and knock-out mice following induction of nephrotoxic nephritis. Results We found that kidney NGAL expression, as well as urinary NGAL levels, were significantly increased in nephrotoxic nephritis as compared to control injected mice. Tight correlations were observed between NGAL expression, renal histopathology, and urinary NGAL excretion. NGAL knock-out mice had attenuated proteinuria and improved renal histopathology as compared to wild-type mice. Similarly, following nephritis induction, NGAL injection significantly exacerbated nephritis and decreased survival. NGAL induces apoptosis via caspase-3 activation, and upregulates inflammatory gene expression in kidney cells in vitro and when injected in vivo. Conclusion We conclude that kidney binding of pathogenic antibodies stimulates local expression of NGAL, which plays a crucial role in the pathogenesis of nephritis via promotion of inflammation and apoptosis. NGAL blockade may be a novel therapeutic approach for the treatment of nephritis mediated by pathogenic antibodies, including anti-GBM disease and lupus nephritis.

Serum autoantibodies in pristane induced lupus are regulated by neutrophil gelatinase associated lipocalin

“…Thorsten Berger and Tak M. Mak, The Campbell Family Institute for Breast Cancer Research and the Ontario Cancer Institute, University Health Network (Toronto, CA), and bred at the Einstein Institute for Animal Studies. Generation of NGAL−/− mice on the B6 background has been described; these mice appear normal and display no gross phenotype, as previously reported [8,17,26]. We confirmed that the D1Mit105 lupus susceptibility locus on chromosome 1 in the B6.NGAL−/− strain was of B6 (non-risk), not 129/Sv origin (data not shown).…”

“…For cDNA synthesis, reverse transcription was performed from 2 μg of total RNA using the SuperScript II system from Invitrogen. Real time PCR was performed in duplicate or triplicate using the SYBR green PCR mastermix (Power SyBr, Applied Biosystems, Warrington, UK) and the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems), using the ΔCt method as described previously [17]. GAPDH was used as a housekeeping gene for normalization of the CT values for the gene of interest.…”

“…In human disease, we found that NGAL was upregulated in the serum and urine of patients with lupus nephritis as compared to normal controls [16]. Furthermore, we determined that NGAL is detrimental in antibody-mediated nephritis, as NGAL−/− mice were protected against renal injury induced by challenge with nephrotoxic serum [17]. …”

Deficiency of Fibroblast Growth Factor-Inducible 14 (Fn14) Preserves the Filtration Barrier and Ameliorates Lupus Nephritis

“…CellTiter 96 Solution (Promega) was used for the detection of cell viability. 62 In some of the permeability and viability experiments, the caspase inhibitor z-VAD-fmk (G-Biosciences) was added to the media at a concentration of 20 mM during Fc-TWEAK stimulation.…”

Temporal Regulation of Glomerular and Cortical Tubulointerstitial Genes Involved in the Development of Nephrotoxic Serum Nephritis

“…Likewise, the expression of Slc40a2, also known as the iron exporter Ferroportin-1, was downregulated in the cortical tubulointerstitium at all time points. In contrast, the gene Lcn2, encoding the protein NGAL, which possesses iron-binding properties and plays a key role in iron metabolism, was significantly upregulated in NTN mice in cortical tubulointerstitial at all time points peaking on day 21 (31.9-fold change, p < 0.0001) [11]. The gene expression of Transferrin was significantly downregulated in whole kidney samples but unchanged in the cortical tubulointerstitium (Table 4).…”