Urinary tract obstruction during kidney development causes tubular apoptosis, tubular necrosis, and interstitial inflammation. Necroptosis is a subtype of programmed necrosis mediated by the receptor-interacting serine/threonine-protein kinase-3 (RIPK3) and the pseudokinase mixed lineage kinase domain-like (MLKL). Necrosis induces inflammation and stimulates cell death in an autoamplification loop named necroinflammation. Here, we studied necroptosis and necroinflammation in obstructive nephropathy induced by unilateral ureteral obstruction (UUO) in neonatal C57Bl/6J mice. Ureteral obstruction induced tubular dilatation, tubular basement membrane thickening, cast formation, and increased expression of kidney injury molecule-1 (KIM-1). Morphological investigations showed either apoptotic or necrotic cells in the tubular compartment. Biochemical analysis revealed increased caspase-8 activity and upregulation of RIPK3 as well as phosphorylated-MLKL in UUO-kidneys. Pro-inflammatory cytokines (IL-1α, INF-γ, TNF-α) were upregulated following UUO. Taken together we show that necroptosis and necroinflammation are accompanied phenomena in neonatal kidneys with obstruction. These findings may help to develop novel strategies to treat congenital obstructive nephropathy.

Urinary tract obstruction during nephron development causes tubular apoptosis, tubular atrophy, and interstitial fibrosis. Leukocyte recruitment is critical in the development of obstructive nephropathy leading to interstitial inflammation and renal fibrosis. RAGE, the receptor of advanced glycation end products, is implicated in chronic inflammation and has been recently identified as a novel receptor for the β2-integrin Mac-1, cooperating with ICAM-1 and thereby directly mediating leukocyte recruitment in vivo. Here, we studied the role of RAGE and ICAM-1 in a model of unilateral ureteral obstruction in neonatal mice. Interestingly, the number of infiltrating leukocytes was independent of RAGE and ICAM-1 in the ureteral obstructed neonatal kidney. By contrast, galectin-3, a marker for profibrogenic M2 macrophages, was strongly reduced in ureteral obstructed RAGE and RAGE-Icam1 knockout mice. Snail expression and loss of E-cadherin but not NF-κB activation were attenuated in both knockout models. Epithelial cell cycle arrest at G2/M, which mediates kidney fibrosis, and transforming growth factor-β expression were reduced in ureteral obstructed RAGE knockout mice. Thus, RAGE and ICAM-1 promote renal fibrosis in the developing kidney upon ureteral obstruction. Combined RAGE- and ICAM-1-blocking strategies may prove beneficial in neonatal obstructive nephropathy.

BackgroundCongenital obstructive nephropathy is the main cause of end-stage renal disease in infants and children. Renal insufficiency is due to impaired growth and maturation in the developing kidney with obstruction. Congenital obstructive nephropathy leads to cytokine mediated inflammation and the development of interstitial fibrosis. The Janus kinase-2 (JAK-2) and Signal Transducer and Activator of Transcription’-3 (STAT3) are involved in cytokine production, inflammation, and interstitial fibrosis.MethodsWe studied the role of JAK2/STAT3 in a model of congenital obstructive nephropathy using unilateral ureteral obstruction (UUO) in neonatal mice at the second day of life. Cytokine production, inflammation, and interstitial fibrosis were analyzed in obstructed and sham operated kidneys of neonatal mice treated with or without JAK2/STAT3 inhibitor Tyrphostin AG490. To mimic obstruction and distension, proximal tubular cells were stretched in vitro.ResultsWe show that STAT3 is highly activated in the developing kidney with obstruction and in proximal tubular cells following stretch. JAK2/STAT3 activation mediates cytokine release and leukocyte recruitment into neonatal kidneys after UUO. Pharmacological blockade of JAK2/STAT3 by Tyrphostin AG490 reduced inflammation, tubular apoptosis, and interstitial fibrosis. JAK2/STAT3 blockade decreased pro-inflammatory and profibrotic mediators in tubular cells.ConclusionOur findings provide evidence that JAK2/STAT3 mediates inflammation and fibrosis in the developing kidney with obstruction. Blocking JAK2/STAT3 may prove beneficial in congenital obstructive nephropathy in children.

Congenital obstructive nephropathy hinders normal kidney development. The severity and the duration of obstruction determine the compensatory growth of the contralateral, intact opposite kidney. We investigated the regulation of renal developmental genes, that are relevant in congenital anomalies of the kidney and urinary tract (CAKUT) in obstructed and contralateral (intact opposite) kidneys after unilateral ureteral obstruction (UUO) in neonatal and adult mice. Newborn and adult mice were subjected to complete UUO or sham-operation, and were sacrificed 1, 5, 12 and 19 days later. Quantitative RT-PCR was performed in obstructed, intact opposite kidneys and sham controls for Gdnf, Pax2, Six4, Six2, Dach1, Eya1, Bmp4, and Hnf-1β. Neonatal UUO induced an early and strong upregulation of all genes. In contrast, adult UUO kidneys showed a delayed and less pronounced upregulation. Intact opposite kidneys of neonatal mice revealed a strong upregulation of all developmental genes, whereas intact opposite kidneys of adult mice demonstrated only a weak response. Only neonatal mice exhibited an increase in BMP4 protein expression whereas adult kidneys strongly upregulated phosphatidylinositol 3 kinase class III, essential for compensatory hypertrophy. In conclusion, gene regulation differs in neonatal and adult mice with UUO. Repair and compensatory hypertrophy involve different genetic programs in developing and adult obstructed kidneys.