Nephron-deficient Fvb mice develop rapidly progressive renal failure and heavy albuminuria involving excess glomerular GLUT1 and VEGF.

Reduced nephron numbers may predispose to renal failure. We hypothesized that glucose transporters (GLUTs) may contribute to progression of the renal disease, as GLUTs have been implicated in diabetic glomerulosclerosis and hypertensive renal disease with mesangial cell (MC) stretch. The Os (oligosyndactyly) allele that typically reduces nephron number by approximately 50%, was repeatedly backcrossed from ROP (Ra/+ (ragged), Os/+ (oligosyndactyly), and Pt/+ (pintail)) Os/+ mice more than six times into the Fvb mouse background to obtain Os/+ and +/+ mice with the Fvb background for study. Glomerular function, GLUT1, signaling, albumin excretion, and structural and ultrastructural changes were assessed. The FvbROP Os/+ mice (Fvb background) exhibited increased glomerular GLUT1, glucose uptake, VEGF, glomerular hypertrophy, hyperfiltration, extensive podocyte foot process effacement, marked albuminuria, severe extracellular matrix (ECM) protein deposition, and rapidly progressive renal failure leading to their early demise. Glomerular GLUT1 was increased 2.7-fold in the FvbROP Os/+ mice vs controls at 4 weeks of age, and glucose uptake was increased 2.7-fold. These changes were associated with the activation of glomerular PKCbeta1 and NF-kappaB p50 which contribute to ECM accumulation. The cyclic mechanical stretch of MCs in vitro, used as a model for increased MC stretch in vivo, reproduced increased GLUT1 at 48 h, a stimulus for increased VEGF expression which followed at 72 h. VEGF was also shown to act in a positive feedback manner on MC GLUT1, increasing GLUT1 expression, glucose uptake and fibronectin (FN) accumulation in vitro, whereas antisense suppression of GLUT1 largely blocked FN upregulation by VEGF. The FvbROP Os/+ mice exhibited an early increase in glomerular GLUT1 leading to increased glomerular glucose uptake PKCbeta1, and NF-kappaB activation, with excess ECM accumulation. A GLUT1-VEGF-GLUT1 positive feedback loop may play a key role in contributing to renal disease in this model of nondiabetic glomerulosclerosis.

Igf2r improves the survival and transmission ratio of Igf2 transgenic mice.

Mammals with excess insulin-like growth factor 2 (IGFII) during embryogenesis have developmental defects that can lead to perinatal lethality. In adults, higher levels of IGFII increase the risk of cancer and may accelerate the development of atherosclerosis. IGFII can be increased as a consequence of genetic abnormalities and polymorphisms, and through epigenetic mechanisms. Decreasing IGFII levels thus can benefit human health. Degradation of IGFII is mediated by the insulin-like growth factor type 2 receptor (IGF2R). The growth-stimulatory effects of IGFII, and their attenuation by the IGF2R, are considered important for the evolution of IGFII/IGF2R interaction and imprinting. The IGFII/IGF2R interactions during development have been previously examined in mice carrying knock-out alleles of these genes or their regulators. Here we tested the ability of the IGF2R to ameliorate the negative effects of IGFII on development and survival in crosses between Igf2 and Igf2r transgenic mice, which may be a better model for natural variations in the levels of these genes' products. A fraction of hemizygous Igf2 transgenic mice die in the perinatal period, some with cleft palates, with an ensuing reduction in the frequency of transgenic mice among the surviving offspring. The Igf2r transgene lowers the frequency of cleft palate and increases the percentage of Igf2 transgenic mice among the live offspring. These findings draw attention to the fact that Igf2-associated lethality selects for the retention of IGFII/IGF2R binding in present day mammals; it may have played a similar role in the acquisition of IGFII/IGF2R binding in ancient mammals.

Delayed onset of Igf2-induced mammary tumors in Igf2r transgenic mice.

The insulin-like growth factor-II (IGF-II) receptor (IGF2R) regulates the level or activity of numerous proteins, including factors that control growth and differentiation. Frequent loss or inactivation of this receptor in a diverse group of tumors indicates that it may act as a tumor suppressor, but it is not known which functions of this receptor are selected against in the tumors. Lysosomal targeting and degradation of the growth-promoting IGF-II has been proposed as a mechanism for the tumor suppressor effects of IGF2R. As a genetic test of this hypothesis in vivo, we have produced Igf2r transgenic mice that ubiquitously express the transgene and have crossed these mice with mice that develop mammary tumors as a consequence of Igf2 overexpression. Our findings indicate that the presence of the Igf2r transgene delays mammary tumor onset and decreases tumor multiplicity in Igf2 transgenic mice. These findings are relevant to human tumors and preneoplastic conditions accompanied by altered IGF2 expression.

Oligosyndactylism mice have an inversion of chromosome 8.

The radiation-induced mutation Oligosyndactylism (Os) is associated with limb and kidney defects in heterozygotes and with mitotic arrest and embryonic lethality in homozygotes. We reported that the cell cycle block in Os and in the 94-A/K transgene-induced mutations is due to disruption of the Anapc10 (Apc10/Doc1) gene. To understand the genetic basis of the limb and kidney abnormalities in Os mice we characterized the structural changes of chromosome 8 associated with this mutation. We demonstrate that the Os chromosome 8 has suffered two breaks that are 5 cM ( approximately 10 Mb) apart and the internal fragment delineated by the breaks is in an inverted orientation on the mutant chromosome. While sequences in proximity to the distal break are present in an abnormal Os-specific Anapc10 hybrid transcript, transcription of these sequences in normal mice is low and difficult to detect. Transfer of the Os mutation onto an FVB/N background indicated that the absence of dominant effects in 94-A/K mice is not due to strain background effects on the mutation. Further analysis of this mutation will determine if a gene interrupted by the break or a long-range effect of the rearrangement on neighboring genes is responsible for the dominant effects of Os.

Transgene instability in mice injected with an in vitro methylated Igf2 gene.

Foreign DNA injected into mouse embryos integrates into the host chromosomes and is usually transmitted stably to the progeny. Rare cases of transgene instability have been described, and these can help our understanding of the rules that govern the organization and stability of endogenous DNA. We have observed unusual inheritance in three transgenic lines produced with a partially in vitro methylated Igf2 construct. All three founders transmitted to their progeny two different transgene patterns, A and B. Pattern A was inherited in accordance with expectation, whereas pattern B was associated with several abnormal characteristics, including fewer than expected transgenic progeny, evidence for instability and loss from the somatic tissues of some of the progeny, and high incidence of runting and perinatal death that did not appear correlated with transgene retention. The absence of these features in transgenic mice produced with the unmethylated version of the same construct indicated that prior methylation played a role in the unusual behavior of these transgenes. We hypothesize that patterns A and B were formed by transgenes that differed in their methylation, and that pattern B methylation led to instability of the transgene locus. Runting and early lethality in the pattern B sublines may be the result of transgene rearrangements, which result in transgene amplification with adverse effects of increased IGFII dosage, and/or deletions, which may affect endogenous genes required for viability. These findings provide further evidence that DNA methylation plays a role in genome stability and indicate that perturbations in the normal pattern of methylation may have destabilizing effects that extend through several generations.