Specific Activation of K-RasG12D Allele in the Bladder Urothelium Results in Lung Alveolar and Vascular Defects

K-ras is essential for embryogenesis and its mutations are involved in human developmental syndromes and cancer. To determine the consequences of K-ras activation in urothelium, we used uroplakin-II (UPK II) promoter driven Cre recombinase mice and generated mice with mutated KrasG12D allele in the urothelium (UPK II-Cre;LSL-K-rasG12D). The UPK II-Cre;LSL-K-rasG12D mice died neonatally due to lung morphogenesis defects consisting of simplification with enlargement of terminal air spaces and dysmorphic pulmonary vasculature. A significant alteration in epithelial and vascular basement membranes, together with fragmentation of laminin, points to extracellular matrix degradation as the causative mechanism of alveolar and vascular defects. Our data also suggest that altered protease activity in amniotic fluid might be associated with matrix defects in lung of UPK II-Cre;LSL-K-rasG12. These defects resemble those observed in early stage human neonatal bronchopulmonary dysplasia (BPD), although the relevance of this new mouse model for BPD study needs further investigation

Ketogenic essential amino acids replacement diet ameliorated hepatosteatosis with altering autophagy-associated molecules

AbstractKetogenic amino acid (KAA) replacement diet has been shown to cure hepatic steatosis, a serious liver disease associated with diverse metabolic defects. In this study, we investigated the effects of KAA replacement diet on nutrition sensing signaling pathway and analyzed whether induction of hepatic autophagy was involved. Mice are fed with high fat diet (HFD) or KAA replacement in high-fat diet (30% fat in food; HFD)-fed (HFDKAAR) and sacrificed at 8, 12, 16weeks after initiation of experimental food. Hepatic autophagy was analyzed in protein expression of several autophagy-associated molecules and in light chain-3 green fluorescent protein (LC-3 GFP) transgenic mice. HFDKAAR showed increased AMP-activated protein kinase (AMPK) phosphorylation and enhanced liver kinase B1 (LKB1) expression compared to control HFD-fed mice. The KAA-HFD-induced activation of AMPK was associated with an increased protein expression of sirtuin 1 (Sirt1), decreased forkhead box protein O3a (Foxo3a) level, and suppression of mammalian target of rapamycin (mTOR) phosphorylation compared with the HFD-fed mice. The intervention study revealed that a KAA-replacement diet also ameliorated all the established metabolic and autophagy defects in the HFD-fed mice, suggesting that a KAA-replacement diet can be used therapeutically in established diseases. These results indicate that KAA replacement in food could be a novel strategy to combat hepatic steatosis and metabolic abnormalities likely involvement of an induction of autophagy

STOX1 deficiency is associated with renin-mediated gestational hypertension and placental defects

The pathogenesis of preeclampsia and other hypertensive disorders of pregnancy remains poorly defined despite the substantial burden of maternal and neonatal morbidity associated with these conditions. In particular, the role of genetic variants as determinants of disease susceptibility is understudied. Storkhead-box protein 1 (STOX1) was first identified as a preeclampsia risk gene through family-based genetic linkage studies in which loss-of-function variants were proposed to underlie increased preeclampsia susceptibility. We generated a genetic Stox1 loss-of-function mouse model (Stox1 KO) to evaluate whether STOX1 regulates blood pressure in pregnancy. Pregnant Stox1-KO mice developed gestational hypertension evidenced by a significant increase in blood pressure compared with WT by E17.5. While severe renal, placental, or fetal growth abnormalities were not observed, the Stox1-KO phenotype was associated with placental vascular and extracellular matrix abnormalities. Mechanistically, we found that gestational hypertension in Stox1-KO mice resulted from activation of the uteroplacental renin-angiotensin system. This mechanism was supported by showing that treatment of pregnant Stox1-KO mice with an angiotensin II receptor blocker rescued the phenotype. Our study demonstrates the utility of genetic mouse models for uncovering links between genetic variants and effector pathways implicated in the pathogenesis of hypertensive disorders of pregnancy

Loss of placental growth factor ameliorates maternal hypertension and preeclampsia in mice

Preeclampsia remains a clinical challenge due to its poorly understood pathogenesis. A prevailing notion is that increased placental production of soluble fms-like tyrosine kinase-1 (sFlt-1) causes the maternal syndrome by inhibiting proangiogenic placental growth factor (PlGF) and VEGF. However, the significance of PlGF suppression in preeclampsia is uncertain. To test whether preeclampsia results from the imbalance of angiogenic factors reflected by an abnormal sFlt-1/PlGF ratio, we studied PlGF KO (Pgf-/-) mice and noted that the mice did not develop signs or sequelae of preeclampsia despite a marked elevation in circulating sFLT-1. Notably, PlGF KO mice had morphologically distinct placentas, showing an accumulation of junctional zone glycogen. We next considered the role of placental PlGF in an established model of preeclampsia (pregnant catechol-O-methyltransferase-deficient [COMT-deficient] mice) by generating mice with deletions in both the Pgf and Comt genes. Deletion of placental PlGF in the context of COMT loss resulted in a reduction in maternal blood pressure and increased placental glycogen, indicating that loss of PlGF might be protective against the development of preeclampsia. These results identify a role for PlGF in placental development and support a complex model for the pathogenesis of preeclampsia beyond an angiogenic factor imbalance

Elevation of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline: a blood pressure-independent beneficial effect of angiotensin I-converting enzyme inhibitors

Blockade of the renin-angiotensin system (RAS) is well recognized as an essential therapy in hypertensive, heart, and kidney diseases. There are several classes of drugs that block the RAS; these drugs are known to exhibit antifibrotic action. An analysis of the molecular mechanisms of action for these drugs can reveal potential differences in their antifibrotic roles. In this review, we discuss the antifibrotic action of RAS blockade with an emphasis on the potential importance of angiotensin I-converting enzyme (ACE) inhibition associated with the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP)

Oral Administration of N-Acetyl-seryl-aspartyl-lysyl-proline Ameliorates Kidney Disease in Both Type 1 and Type 2 Diabetic Mice via a Therapeutic Regimen

Kidney fibrosis is the final common pathway of progressive kidney diseases including diabetic nephropathy. Here, we report that the endogenous antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), the substrate of angiotensin-converting enzyme (ACE), is an orally available peptide drug used to cure kidney fibrosis in diabetic mice. We utilized two mouse models of diabetic nephropathy, streptozotocin- (STZ-) induced type 1 diabetic CD-1 mice and type 2 diabetic nephropathy model db/db mice. Intervention with the ACE inhibitor imidapril, oral AcSDKP, or imidapril + oral AcSDKP combination therapy increased urine AcSDKP levels. AcSDKP levels were significantly higher in the combination group compared to those of the other groups. AcSDKP oral administration, either AcSDKP alone or in addition to imidapril, ameliorated glomerulosclerosis and tubulointerstitial fibrosis. Plasma cystatin C levels were higher in both models, at euthanasia, and were restored by all the treatment groups. The levels of antifibrotic miRs, such as miR-29 or let-7, were suppressed in the kidneys of both models; all treatments, especially the combination of imidapril + oral AcSDKP, restored the antifibrotic miR levels to a normal value or even higher. AcSDKP may be an oral antifibrotic peptide drug that would be relevant to combating fibroproliferative kidney diseases such as diabetic nephropathy

Normal kidney development in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A-B: H&E stained kidney sections from day E17.5 showing normal development both in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and controls (X200).</p

Lung phenotype of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A–B: Representative H&E stained sections showing normality of lung (X200) morphology in <i>UPK II-Cre;LSL-K-ras^G12D</i> when compared with controls at E14.5 (pseudoglandular stage). <b>C–H</b>: Formation of air spaces was impaired in lungs (X100) of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice at E17.5 (<b>C,D</b>) and E19.5 (<b>E,F</b>), reflecting a defective septation process. P1 lung morphology was also different between controls and transgenic mice, which exhibited an enlargement and simplification of sacculi (<b>G,H</b>). <b>I–J</b>: Morphometric analysis of lung sections showed a decreased total air space (<b>I</b>) and mean alveolar (saccular) area (<b>J</b>) in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice at E17.5 (n = 5); the impairment of air space development led to an increased total air space area and mean alvelolar area in the early postnatal period (P1; n = 12) in transgenic mice when compared to controls; bars, SEM. <b>K–L</b>: Masson tri-chrome staining of day P1 <i>UPK II-Cre;LSL-K-ras^G12D</i> lungs (X100), showing absence of fibrosis both in cases and controls. <b>M</b>: The direct expression of K<i>-ras^G12D</i> was ruled out with specific PCR showing the absence of recombination between <i>K-ras</i> and the Lox sequence in lung and placenta.</p

Fragmentation of ECM components in lungs of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A: Total protein lysates from whole lung were analyzed by Western blot for for laminin β-1 from P1 <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and control (Cre-) mice, showing an additional low molecular weight (mw) band (35 kDa) which suggest fragmentation. <b>B</b>: Representative images of immunofluorescence for laminin β-1 (X200) showed a disorganized membrane pattern in E17.5 lung from <i>UPK II-Cre;LSL-K-ras^G12D</i> mice. <b>C</b>: WB for lung E-cadherin in P1 lung, with an increase in low mw bands. (53 and 32 KDa) also in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.</p

Urothelial hyperplasia in the <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A: Urothelial-restricted expression of K-ras^G12D. <b>B–E</b>: H&E analysis of bladders (X200) reveals a hyperplastic urothelium at E17.5 (<b>B,C</b>) and P1 (<b>D,E</b>) (black arrows). <b>F</b>: Differences in urothelial cellularity (cells/0.15 mm²) between <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and controls were significant both at E17.5 (n = 2/group; *, <i>P</i> = 0.05) and P1 (n>6/group; **, <i>P</i><0.0001); bars, SEM. <b>G–H</b>: BRDU staining of bladder (X200) showing a higher proliferation in E17.5 <i>UPK II-Cre;LSL-K-ras^G12D</i> mice (urothelium limit is marked with a blue line). The mean number of BrdU positive nuclei/200 µm of urothelium was significantly higher than in controls (n = 10; 6±2 positive nuclei/200 µm vs 1.33±0.81; <i>P</i> = 0.01).</p