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Murine nephrotoxic nephritis is a widely used model of immune-complex-mediated glomerulonephritis. In this model, mice are immunized with normal sheep IgG, followed by administration of a sheep antiserum (nephrotoxic serum or NTS) raised against mouse glomerular components. This leads to glomerular injury, concomitant albuminuria and an increase in serum creatinine level. The Src-family kinases Hck, Fgr, and Lyn are critical for various disease models. However, their role in kidney diseases is poorly understood. Here we aimed to test the role of Hck, Fgr, and Lyn in accelerated nephrotoxic nephritis.
We analyzed the Nephroseq database to assess gene expression alterations in human lupus nephritis. Wild-type, Hck−/−Fgr−/−Lyn−/− triple knockout and Hck−/−, Fgr−/−, or Lyn−/− single knockout mice were preimmunized with sheep IgG followed by intravenous injection of NTS or normal sheep serum. On the fourteenth day, urine was collected over a 24-hour period. After an additional day, blood was collected, mice were perfused transcardially, then kidneys were removed, and leukocyte infiltration, glomerular injury and antibody depositions were tested with flow cytometry and light microscopy. Hck−/−Fgr−/−Lyn−/− and wild-type bone marrow chimeras were generated to narrow down the cell types where these kinases are important.
In human lupus nephritis, the expression of Hck, Fgr, and Lyn showed consistent increases, while other Src-family kinases remained unchanged. Wild-type NTS-treated mice developed severe albuminuria (approximately 7 mg/day) and hematuria (approximately 150 cells/μl) on the fourteenth day. Crescents were present in approximately 20% of the glomeruli of these mice, with additional histological signs of glomerulosclerosis and tubular injury. Flow cytometric analysis has revealed massive leukocyte infiltration of the kidneys with myeloid cell dominance. Serum creatinine levels were more than two times higher in the NTS-treated group than in control mice. In contrast to wild-type mice, NTS-treated Hck−/−Fgr−/−Lyn−/− showed normal serum creatinine levels, dramatically reduced albuminuria, and no substantial histological abnormalities and leukocyte infiltration in kidneys. The Hck–/–Fgr–/–Lyn–/– mutation did not affect glomerular antibody or complement deposition. Analysis of Hck−/−, Fgr−/−, and Lyn−/− single knockouts have revealed substantial protection of Hck−/− mice from disease development, whereas the Fgr−/− and Lyn−/− were still susceptible to induction of nephrotoxic nephritis. NTS-induced albuminuria and crescent formation were partially reduced in chimeric mice generated by transplantation of Hck−/−Fgr−/−Lyn−/− bone marrow cells into wild-type recipients.
Our results indicate a crucial role for myeloid Src-family kinases, in particular the Hck tyrosine kinase, in the development of immune complex-mediated glomerulonephritis. Src-family kinases are likely important, at least in part, in a radiosensitive hematopoietic compartment. These kinases, especially Hck, can become new therapeutic targets in immune-complex-mediated glomerulonephritis.