Rescue of Defective Branching Nephrogenesis in Renal-Coloboma Syndrome by the Caspase Inhibitor, Z-VAD-fmk

Abstract

Abstract. In renal-coloboma syndrome (RCS), null mutations of the PAX2 gene cause renal hypoplasia due to a congenital deficit of nephrons; affected individuals may develop renal insufficiency in childhood. During normal kidney development, PAX2, is expressed at high levels throughout the arborizing ureteric bud (UB); recent observations suggest that one of its key roles is to suppress apoptosis in this collecting duct lineage. The authors hypothesized that increased UB cell apoptosis due to PAX2 haploinsufficiency must directly influence the rate of branching morphogenesis in developing kidney and the number of nephrons that can be formed before birth, when nephrogenesis in humans comes to an end. If so, the authors reasoned that caspase inhibitors might be used to suppress unwanted UB cell apoptosis during kidney development in Pax2 1Neu mutant mice and rescue the genetic UB branching defect. E17.5 kidneys from Pax2 1Neu mutant mice had smaller (Ϫ25%) longitudinal cross-sectional area and 3.5-fold increase in collecting duct cell apoptosis versus wild-type littermates; mutant E13.5 kidney explants allowed to arborize for 50 h in vitro had 18% fewer terminal branches than wild-types. However, exposure to the caspase inhibitor, Z-VAD-fmk (25 M), significantly increased terminal branch number in mutant explants (23%). It also increased branching in wild-type explants, apparently reflecting an effect of Z-VAD-fmk on basal apoptosis induced by ex vivo culture conditions. Similarly, when pregnant mice were injected daily with Z-VAD-fmk (10 g/g weight from E10.5 to E17.5), apoptosis of Pax2 1Neu fetal collecting duct cells was suppressed to 40% of untreated mutants; by E14, terminal branch number was increased to 152% that of untreated litters. These studies support the hypothesis that PAX2 normally optimizes the rate of branching morphogenesis in fetal kidney by suppressing UB apoptosis. Furthermore, it suggests that caspase inhibitors can rescue the branching defect caused by PAX2 mutations.During normal kidney development, interactions between the arborizing ureteric bud (UB) and the metanephric mesenchyme (MM) generate the nephrons of each kidney. This process begins at 5 to 6 wk fetal age, when the UB emerges from the nephric duct, grows laterally into the undifferentiated mesenchyme, and begins to arborize. Each terminal branch of the UB signals adjacent MM cells to form the proximal portion of a nephron, which then fuses to the common collecting system. By about 1 mo before birth, when nephrogenesis comes to a halt, final nephron number has been determined by how many UB branching events have occurred in the intervening period. This crop of fetal nephrons will constitute the individual's nephron endowment for life.Nephron number varies widely from 0.3 to 1.3 million/ kidney among normal humans (1,2). Children born at the lower end of this spectrum may have increased risk of hypertension or susceptibility to acquired renal disease later in life (3). In families with the autosomal dominant renal-coloboma syndrom...

PAX2 is reactivated in urinary tract obstruction and partially protects collecting duct cells from programmed cell death

“…However, it is conceivable that the extent of renal parenchymal loss is modulated by the PAX2 response. We recently found that the caspase inhibitor Z-VAD-fmk can substitute for the loss of PAX2 antiapoptotic function during renal development (6). It is tempting to speculate that caspase-inhibitory drugs might ameliorate renal parenchymal loss during acute urinary tract obstruction when endogenous mechanisms are overwhelmed.…”

“…Although Pax2 appears to have a number of roles during renal development, a key function is to suppress programmed cell death in UB cells, thereby optimizing the rate of branching nephrogenesis (6,10,19,22). Heterozygous Pax2 mutant mice display increased apoptosis of UB cells without any change in proliferative rate (19,23).…”

“…Constant, Quebec). Heterozygous Pax2 1Neu mutant male mice bearing a single base-pair G insertion within the DNA-binding paired domain were crossed with wild-type (C3H) females as previously described (6,19). Offspring were genotyped before weaning using specially designed PCR primers to create a unique restriction site for XcmI in the presence of the mutation as reported elsewhere (6).…”

“…As a consequence, UB branching is suboptimal and mice are born with reduced nephron number (19). Renal hypoplasia can be rescued by treating the pregnant mice with caspase-inhibitory drugs which replace the missing antiapoptotic function of PAX2 in mutant fetal kidney (6). Conversely, when endogenous PAX2 levels in cultured collecting duct cells are reduced when transfected with an antisense Pax2 cDNA, apoptosis is increased (23).…”

Multicystic Dysplastic Kidney and Variable Phenotype in a Family with a Novel Deletion Mutation of PAX2

“…The presence of increased apoptosis in Pax2 1neu mice heterozygous for PAX2 mutations may also reflect the important role of PAX2 in regulating apoptosis. In addition, increased apoptosis in homozygous congenital polycystic kidney (cpk) mice heterozygous for PAX2 mutations has been suggested to lead to the observed slowing of the progression of renal cystic disease (35,36) in mice that carry the additional PAX2 mutation compared with those without a PAX2 mutation.…”