Neonatal nephron loss during active nephrogenesis results in altered expression of renal developmental genes and markers of kidney injury.

Preterm neonates are at a high risk for nephron loss under adverse clinical conditions. Renal damage potentially collides with postnatal nephrogenesis. Recent animal studies suggest that nephron loss within this vulnerable phase leads to renal damage later in life. Nephrogenic pathways are commonly reactivated after kidney injury supporting renal regeneration. We hypothesized that nephron loss during nephrogenesis affects renal development, which, in turn, impairs tissue repair after secondary injury. Neonates prior to 36 wk of gestation show an active nephrogenesis. In rats, nephrogenesis is ongoing until day 10 after birth. Mimicking the situation of severe nephron loss during nephrogenesis, male pups were uninephrectomized at day 1 of life (UNXd1). A second group of males was uninephrectomized at postnatal day 14 (UNXd14), after terminated nephrogenesis. Age-matched controls were sham operated. Three days after uninephrectomy transcriptional changes in the right kidney were analyzed by RNA-sequencing, followed by functional pathway analysis. In UNXd1, 1,182 genes were differentially regulated, but only 143 genes showed a regulation both in UNXd1 and UNXd14. The functional groups "renal development" and "kidney injury" were among the most differentially regulated groups and revealed distinctive alterations. Reduced expression of candidate genes concerning renal development (Bmp7, Gdnf, Pdgf-B, Wt1) and injury (nephrin, podocin, Tgf-ß1) were detected. The downregulation of Bmp7 and Gdnf persisted until day 28. In UNXd14, Six2 was upregulated and Pax2 was downregulated. We conclude that nephron loss during nephrogenesis affects renal development and induces a specific regulation of genes that might hinder tissue repair after secondary kidney injury.

Responsiveness of afferent renal nerve units in renovascular hypertension in rats.

Previous data suggest that renal afferent nerve activity is increased in hypertension exerting sympathoexcitatory effects. Hence, we wanted to test the hypothesis that in renovascular hypertension, the activity of dorsal root ganglion (DRG) neurons with afferent projections from the kidneys is augmented depending on the degree of intrarenal inflammation. For comparison, a nonhypertensive model of mesangioproliferative nephritis was investigated. Renovascular hypertension (2-kidney, 1-clip [2K1C]) was induced by unilateral clipping of the left renal artery and mesangioproliferative glomerulonephritis (anti-Thy1.1) by IV injection of a 1.75-mg/kg BW OX-7 antibody. Neuronal labeling (dicarbocyanine dye [DiI]) in all rats allowed identification of renal afferent dorsal root ganglion (DRG) neurons. A current clamp was used to characterize neurons as tonic (sustained action potential [AP] firing) or phasic (1-4 AP) upon stimulation by current injection. All kidneys were investigated using standard morphological techniques. DRG neurons exhibited less often tonic response if in vivo axonal input from clipped kidneys was received (30.4% vs. 61.2% control, p < 0.05). However, if the nerves to the left clipped kidneys were cut 7 days prior to investigation, the number of tonic renal neurons completely recovered to well above control levels. Interestingly, electrophysiological properties of neurons that had in vivo axons from the right non-clipped kidneys were not distinguishable from controls. Renal DRG neurons from nephritic rats also showed less often tonic activity upon current injection (43.4% vs. 64.8% control, p < 0.05). Putative sympathoexcitatory and impaired sympathoinhibitory renal afferent nerve fibers probably contribute to increased sympathetic activity in 2K1C hypertension.

No evidence of the unfolded protein response in the placenta of two rodent models of preeclampsia and intrauterine growth restriction.

In humans, intrauterine growth restriction (IUGR) and preeclampsia (PE) are associated with induction of the unfolded protein response (UPR) and increased placental endoplasmic reticulum (ER) stress. Especially in PE, oxidative stress occurs relative to the severity of maternal vascular underperfusion (MVU) of the placental bed. On the premise that understanding the mechanisms of placental dysfunction could lead to targeted therapeutic options for human IUGR and PE, we investigated the roles of the placental UPR and oxidative stress in two rodent models of these human gestational pathologies. We employed a rat IUGR model of gestational maternal protein restriction, as well as an endothelial nitric oxide synthase knockout mouse model (eNOS-/-) of PE/IUGR. Placental expression of UPR members was analyzed via qRT-PCR (Grp78, Calnexin, Perk, Chop, Atf6, and Ern1), immunohistochemistry, and Western blotting (Calnexin, ATF6, GRP78, CHOP, phospho-eIF2α, and phospho-IRE1). Oxidative stress was determined via Western blotting (3-nitrotyrosine and 4-hydroxy-2-nonenal). Both animal models showed a significant reduction of fetal and placental weight. These effects did not induce placental UPR. In contrast to human data, results from our rodent models suggest retention of placental plasticity in the setting of ER stress under an adverse gestational environment. Oxidative stress was significantly increased only in female IUGR rat placentas, suggesting a sexually dimorphic response to maternal malnutrition. Our study advances understanding of the involvement of the placental UPR in IUGR and PE. Moreover, it emphasizes the appropriate choice of animal models researching various aspects of these pregnancy complications.

Renal Chemerin Expression is Induced in Models of Hypertensive Nephropathy and Glomerulonephritis and Correlates with Markers of Inflammation and Fibrosis.

Chemerin and its receptor, chemokine-like receptor 1 (CmklR1), are associated with chemotaxis, inflammation, and endothelial function, especially in metabolic syndrome, coronary heart disease, and hypertension. In humans, circulating chemerin levels and renal function show an inverse relation. So far, little is known about the potential role of chemerin in hypertensive nephropathy and renal inflammation. Therefore, we determined systemic and renal chemerin levels in 2-kidney-1-clip (2k1c) hypertensive and Thy1.1 nephritic rats, respectively, to explore the correlation between chemerin and markers of renal inflammation and fibrosis. Immunohistochemistry revealed a model-specific induction of chemerin expression at the corresponding site of renal damage (tubular vs. glomerular). In both models, renal expression of chemerin (RT-PCR, Western blot) was increased and correlated positively with markers of inflammation and fibrosis. In contrast, circulating chemerin levels remained unchanged. Taken together, these findings demonstrate that renal chemerin expression is associated with processes of inflammation and fibrosis-related to renal damage. However, its use as circulating biomarker of renal inflammation seems to be limited in our rat models.

Expression of Retinoid Acid Receptor-Responsive Genes in Rodent Models of Placental Pathology.

In humans, retinoic acid receptor responders (RARRES) have been shown to be altered in third trimester placentas complicated by the pathologies preeclampsia (PE) and PE with intrauterine growth restriction (IUGR). Currently, little is known about the role of placental Rarres in rodents. Therefore, we examined the localization and expression of Rarres1 and 2 in placentas obtained from a Wistar rat model of isocaloric maternal protein restriction (E18.5, IUGR-like features) and from an eNOS-knockout mouse model (E15 and E18.5, PE-like features). In both rodent models, Rarres1 and 2 were mainly localized in the placental spongiotrophoblast and giant cells. Their placental expression, as well as the expression of the Rarres2 receptor chemokine-like receptor 1 (CmklR1), was largely unaltered at the examined gestational ages in both animal models. Our results have shown that RARRES1 and 2 may have different expression and roles in human and rodent placentas, thereby underlining immanent limitations of comparative interspecies placentology. Further functional studies are required to elucidate the potential involvement of these proteins in early placentogenesis.

Hypoxia-inducible protein 2 Hig2/Hilpda mediates neutral lipid accumulation in macrophages and contributes to atherosclerosis in apolipoprotein E-deficient mice.

Recently we identified hypoxia-inducible protein 2 (HIG2)/hypoxia-inducible lipid droplet-associated (HILPDA) as lipid droplet (LD) protein. Because HILPDA is highly expressed in atherosclerotic plaques, we examined its regulation and function in murine macrophages, compared it to the LD adipose differentiation-related protein (Adrp)/perilipin 2 (Plin2), and investigated its effects on atherogenesis in apolipoprotein E-deficient (ApoE-/-) mice. Tie2-Cre-driven Hilpda conditional knockout (cKO) did not affect viability, proliferation, and ATP levels in macrophages. Hilpda proved to be a target of hypoxia-inducible factor 1 (Hif-1) and peroxisome proliferator-activated receptors. In contrast, Adrp/Plin2 was not induced by Hif-1. Hilpda localized to the endoplasmic reticulum-LD interface, the site of LD formation. Hypoxic lipid accumulation and storage of oxidized LDL, cholesteryl esters and triglycerides were abolished in Hilpda cKO macrophages, independent of the glycolytic switch, fatty acid or lipoprotein uptake. Hilpda depletion reduced resistance against lipid overload and increased production of reactive oxygen species after reoxygenation. LPS-stimulated prostaglandin-E2 production was dysregulated in macrophages, demonstrating the substrate buffer and reservoir function of LDs for eicosanoid production. In ApoE-/- Hilpda cKO mice, total aortic plaque area, plaque macrophages and vascular Vegf expression were reduced. Thus, macrophage Hilpda is crucial to foam-cell formation and lipid deposition, and to controlled prostaglandin-E2 production. By these means Hilpda promotes lesion formation and progression of atherosclerosis.-Maier, A., Wu, H., Cordasic, N., Oefner, P., Dietel, B., Thiele, C., Weidemann, A., Eckardt, K.-U., Warnecke, C. Hypoxia-inducible protein 2 Hig2/Hilpda mediates neutral lipid accumulation in macrophages and contributes to atherosclerosis in apolipoprotein E-deficient mice.

Mild Salt-Sensitive Hypertension in Genetically Determined Low Nephron Number is Associated with Chloride but Not Sodium Retention.

BACKGROUND/AIMS: One potential pathomechanism how low nephron number leads to hypertension in later life is altered salt handling. We therefore evaluated changes in electrolyte and water content in wildtype (wt) and GDNF+/- mice with a 30% reduction of nephron number. METHODS: 32 GDNF+/- and 36 wt mice were fed with low salt (LSD, 0.03%, normal drinking water) or high salt (HSD, 4%, 0.9% drinking water) diet for 4 weeks. Blood pressure was continuously measured by telemetry in a subgroup. At the end of the experiment and after standardized ashing processes electrolyte- and water contents of the skin and the total body were determined. RESULTS: We found higher blood pressure in high salt treated GDNF+/-compared to wt mice. Of interest, we could not confirm an increase in total-body sodium as predicted by prevailing explanations, but found increased total body and skin chloride that interestingly correlated with relative kidney weight. CONCLUSION: We hereby firstly report significant total body and skin chloride retention in salt sensitive hypertension of GDNF+/-mice with genetically determined lower nephron number. Thus, in contrast to the prevailing opinion our data argue for the involvement of non-volume related mechanisms.

Blunted transcriptional response to skeletal muscle ischemia in rats with chronic kidney disease: potential role for impaired ischemia-induced angiogenesis.

Chronic kidney disease (CKD) is associated with increased cardiovascular morbidity and mortality. Previous studies indicated an impairment of ischemia-induced angiogenesis in skeletal muscle of rats with CKD. We performed a systematic comparison of early gene expression in response to ischemia in rats with or without CKD to identify potential molecular mechanisms underlying impaired angiogenesis in CKD. CKD was induced in male rats by 5/6 nephrectomy (SNX); control rats were sham operated (sham). Eight weeks later, ischemia of the right limb was induced by ligation and resection of the femoral artery. Rats were killed 24 h after the onset of ischemia, and RNA was extracted from the musculus soleus of the ischemic and the nonischemic hindlimb. To identify differentially expressed transcripts, we analyzed RNA with Affymetrix GeneChip Rat Genome 230 2.0 Arrays. RT-PCR analysis of selected genes was performed to validate observed changes. Hindlimb ischemia upregulated 239 genes in CKD and 299 genes in control rats (66% overlap), whereas only a few genes were downregulated (14 in CKD and 34 in controls) compared with the nonischemic limb of the same animals. Comparison between the ischemic limbs of CKD and controls revealed downregulation of 65 genes in CKD; 37 of these genes were also among the ischemia-induced genes in controls. Analysis of functional groups (other than angiogenesis) pointed to genes involved in leukocyte recruitment and fatty acid metabolism. Transcript expression profiling points to a relatively small number of differentially expressed genes that may underlie the impaired postischemic angiogenesis in CKD.

Hypoxia inducible factor stabilization improves defective ischemia-induced angiogenesis in a rodent model of chronic kidney disease.

Chronic kidney disease (CKD) is associated with increased risk and worse prognosis of cardiovascular disease, including peripheral artery disease. An impaired angiogenic response to ischemia may contribute to poor outcomes of peripheral artery disease in patients with CKD. Hypoxia inducible factors (HIF) are master regulators of angiogenesis and therefore represent a promising target for therapeutic intervention. To test this we induced hind-limb ischemia in rats with CKD caused by 5/6 nephrectomy and administered two different treatments known to stabilize HIF protein in vivo: carbon monoxide and a pharmacological inhibitor of prolyl hydroxylation 2-(1-chloro-4- hydroxyisoquinoline-3-carboxamido) acetate (ICA). Expression levels of pro-angiogenic HIF target genes (Vegf, Vegf-r1, Vegf-r2, Ho-1) were measured by qRT-PCR. Capillary density was measured by CD31 immunofluorescence staining and HIF expression was evaluated by immunohistochemistry. Capillary density in ischemic skeletal muscle was significantly lower in CKD animals compared to sham controls. Rats with CKD showed significantly lower expression of HIF and all measured pro-angiogenic HIF target genes, including VEGF. Both HIF stabilizing treatments rescued HIF target gene expression in animals with CKD and led to significantly higher ischemia-induced capillary sprouting compared to untreated controls. ICA was effective regardless of whether it was administered before or after induction of ischemia and led to a HIF expression in skeletal muscle. Thus, impaired ischemia-induced angiogenesis in rats with CKD can be improved by HIF stabilization, even if started after onset of ischemia.

Under-expression of α8 integrin aggravates experimental atherosclerosis.

Integrins play an important role in vascular biology. The α8 integrin chain attenuates smooth muscle cell migration but its functional role in the development of atherosclerosis is unclear. Therefore, we studied the contribution of α8 integrin to atherosclerosis and vascular remodelling. We hypothesized that α8 integrin expression is reduced in atherosclerotic lesions, and that its under-expression leads to a more severe course of atherosclerosis. α8 Integrin was detected by immunohistochemistry and qPCR and α8 integrin-deficient mice were used to induce two models of atherosclerotic lesions. First, ligation of the carotid artery led to medial thickening and neointima formation, which was quantified in carotid cross-sections. Second, after crossing into ApoE-deficient mice, the formation of advanced vascular lesions with atherosclerotic plaques was quantified in aortic en face preparations stained with Sudan IV. Parameters of renal physiology and histopathology were assessed: α8 integrin was detected in the media of human and murine vascular tissue and was down-regulated in arteries with advanced atherosclerotic lesions. In α8 integrin-deficient mice (α8(-/-) ) as well as α8(+/-) and α8(+/+) littermates, carotid artery ligation increased media:lumen ratios in all genotypes, with higher values in ligated α8(-/-) and α8(+/-) compared to ligated α8(+/+) animals. Carotid artery ligation increased smooth muscle cell number in the media of α8(+/+) mice and, more prominently, of α8(-/-) or α8(+/-) mice. On an ApoE(-/-) background, α8(+/-) and α8(-/-) mice developed more atherosclerotic plaques than α8(+/+) mice. α8 Integrin expression was reduced in α8(+/-) animals. Renal damage with increased serum creatinine and glomerulosclerosis was detected in α8(-/-) mice only. Thus, under-expression of α8 integrin aggravates vascular lesions, while a complete loss of α8 integrin results in reduced renal mass and additional renal disease in the presence of generalized atherosclerosis. Our data support the hypothesis that integrin α8ß1 has a protective role in arterial remodelling and atherosclerosis.

Renal protection by low dose irbesartan in diabetic nephropathy is paralleled by a reduction of inflammation, not of endoplasmic reticulum stress.

Diabetes can disrupt endoplasmic reticulum (ER) homeostasis which leads to ER stress. ER stress-induced renal apoptosis seems to be involved in the development of diabetic nephropathy. The present study was designed to investigate the contribution of reduced ER stress to the beneficial effects of an angiotensin receptor blocker. Insulin-dependent diabetes mellitus was induced by streptozotocin injections to hypertensive mRen2-transgenic rats. After 2weeks animals were treated with 0.7mg/kg/day irbesartan. Blood glucose, blood pressure and protein excretion were assessed. Expression of ER stress markers was measured by real-time PCR. Immunohistochemistry was performed to detect markers of ER stress, renal damage and infiltrating cells. Glomerulosclerosis and apoptosis were evaluated. Diabetic mRen2-transgenic rats developed renal injury with proteinuria, tubulointerstitial cell proliferation as well as glomerulosclerosis and podocyte injury. Moreover, an increase in inflammation, podocyte ER stress and apoptosis was detected. Irbesartan somewhat lowered blood pressure and reduced proteinuria, tubulointerstitial cell proliferation and glomerulosclerosis. Podocyte damage was ameliorated but markers of ER stress (calnexin, grp78) and apoptosis were not reduced by irbesartan. On the other hand, inflammatory cell infiltration in the tubulointerstitium and the glomerulus was significantly attenuated. We conclude that irbesartan reduced renal damage even in a very low dose. The beneficial effects of low dose irbesartan were paralleled by a reduction of blood pressure and inflammation but not by a reduction of ER stress and apoptosis. Thus, sustained endoplasmic reticulum stress in the kidney does not necessarily lead to increased inflammation and tubulointerstitial or glomerular injury.

Impaired myocardial performance in a normotensive rat model of intrauterine growth restriction.

BACKGROUND: Intrauterine growth restriction (IUGR) is an important risk factor for cardiovascular disease. Previous studies revealed altered myocardial matrix composition after IUGR. We hypothesized that IUGR is accompanied by compromised myocardial performance independently from arterial hypertension. METHODS: IUGR was induced in Wistar rats by maternal protein restriction, and hearts of male offspring were studied using echocardiography, immunohistochemistry, real-time PCR, and western blot analysis. RESULTS: At day 70 of life, in the absence of arterial hypertension (mean arterial blood pressure: 101.3 ± 7.1 mmHg in IUGR vs. 105.3 ± 4.6 mmHg in controls, not significant (NS)), echocardiography showed a reduced contractility (ejection fraction: 65.4 ± 1.8% in IUGR vs. 82.2 ± 1.5% in controls, P < 0.001) of a more distensible myocardium in IUGR rats. Altered expression patterns of myosin chains and titin isoforms and increased expression levels of atrial natriuretic peptide, Na/K-ATPase, and ß-adrenergic receptor 1 were detected. A higher number of cardiac fibroblasts and vascular cross-sections were observed in IUGR rats, accompanied by elevated expression of hypoxia inducible factor 1 target genes, such as vascular endothelial growth factor and its receptors. CONCLUSION: We observed a blood pressure-independent impairment of myocardial function after IUGR, which possibly favors cardiovascular disease later in life. Some IUGR-induced myocardial changes (e.g., sarcomeric components) may partly explain the compromised cardiac performance, whereas others (e.g., elevated vascular supply) reflect compensatory mechanisms.

Effect of lowering asymmetric dimethylarginine (ADMA) on vascular pathology in atherosclerotic ApoE-deficient mice with reduced renal mass.

The purpose of the work was to study the impact of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) and its degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH1), on atherosclerosis in subtotally nephrectomized (SNX) ApoE-deficient mice. Male DDAH1 transgenic mice (TG, n=39) and C57Bl/6J wild-type littermates (WT, n=27) with or without the deletion of the ApoE gene underwent SNX at the age of eight weeks. Animals were sacrificed at 12 months of age, and blood chemistry, as well as the extent of atherosclerosis within the entire aorta were analyzed. Sham treated (no renal mass reduction) ApoE-competent DDAH1 transgenic and wild-type littermates (n=11) served as a control group. Overexpression of DDAH1 was associated with significantly lower ADMA levels in all treatment groups. Surprisingly, SNX mice did not exhibit higher ADMA levels compared to sham treated control mice. Furthermore, the degree of atherosclerosis in ApoE-deficient mice with SNX was similar in mice with or without overexpression of DDAH1. Overexpression of the ADMA degrading enzyme, DDAH1, did not ameliorate atherosclerosis in ApoE-deficient SNX mice. Furthermore, SNX in mice had no impact on ADMA levels, suggesting a minor role of this molecule in chronic kidney disease (CKD) in this mouse model.

Intrauterine growth restriction leads to a dysregulation of Wilms' tumour supressor gene 1 (WT1) and to early podocyte alterations.

BACKGROUND: Intrauterine growth restriction (IUGR) leads to low nephron number and higher incidence of renal disease. We hypothesized that IUGR induces early podocyte alterations based on a dysregulation of Wilms' tumour suppressor gene 1 (WT1), a key player of nephrogenesis and mediator of podocyte integrity. METHODS: IUGR was induced in rats by maternal protein restriction during pregnancy. Kidneys were harvested from male offspring at Days 1 and 70 of life. qRT-PCR, immunohistochemistry and electron microscopy were performed in renal tissue. Albuminuria was assessed by enzyme-linked immunosorbent assay. RESULTS: At Day 70 of life, higher albuminuria and overt alterations of podocyte ultrastructure were detected in IUGR animals in spite of normal blood pressure. Moreover, we found increased glomerular immunoreactivity and expression of desmin, while synaptopodin and nephrin were decreased. Glomerular immunoreactivity and expression of WT1 were increased in IUGR animals at this time point with an altered expressional ratio of WT1 +KTS and -KTS isoforms. These changes of WT1 expression were already present at the time of birth. CONCLUSIONS: IUGR results in early podocyte damage possibly due to a dysregulation of WT1. We suggest that an imbalance of WT1 isoforms to the disadvantage of -KTS affects nephrogenesis in IUGR rats and that persistent dysregulation of WT1 results in a reduced ability to maintain podocyte integrity, rendering IUGR rats more susceptible for renal disease.

Cardiovascular and renal effects of high salt diet in GDNF+/- mice with low nephron number.

AIMS: To test the suggested association of low nephron number and later development of renal and cardiovascular disease we investigated the effects of high sodium diet in heterozygous GDNF+/- mice. METHODS: Aged wild type and GDNF+/- mice were grouped together according to high sodium (HS, 4%) or low sodium (LS, 0.03%) diet for 4 weeks. The heart, the aorta and the kidneys were processed for morphometric and stereological evaluations and TaqMan PCR. RESULTS: On HS GDNF+/- mice showed significantly higher drinking volume and urine production than wt and mean arterial blood pressure tended to be higher. Heart weight was higher in GDNF+/- than in wt, but the difference was only significant for LS. HS significantly increased cardiac interstitial tissue in GDNF+/-, but not in wt. On LS GDNF+/- mice had significantly larger glomeruli than wt and HS led to an additional two fold increase of glomerular area compared to LS. On electron microscopy glomerular damage after HS was seen in GDNF+/-, but not in wt. Dietary salt intake modulated renal IL-10 gene expression in GDNF+/-. CONCLUSION: In the setting of 30% lower nephron number HS diet favoured maladaptive changes of the kidney as well as of the cardiovascular system.

Sex differences in long-term kidney fibrosis following neonatal nephron loss during ongoing nephrogenesis.

BACKGROUND: Clinical studies suggest that female sex plays a protective role in the development and progression of kidney disease. Recent experimental studies indicate that in male rats early nephron loss under ongoing nephrogenesis is accompanied by severe long-term sequelae. In humans, nephron formation occurs mainly in the third trimester, ceasing with 36 weeks of gestation. Due to perinatal complications, preterm infants delivered during this vulnerable period may undergo acute nephron loss. In rats nephrogenesis persists until postnatal day 10, reflecting the situation of human preterms with persisting nephrogenesis. In our animal model of neonatal uninephrectomy, female and male rats were uninephrectomized at day 1 of life. Hypothesizing sex-dependent differences, long-term renal outcome was assessed after 1 year. RESULTS: In both sexes, neonatal uninephrectomy was not followed by arterial hypertension at 1 year of age. Compensatory weight gain and glomerular hypertrophy of the remaining kidney occurred in uninephrectomized female and male animals. Selected markers of interstitial inflammation and fibrosis were regulated sex-dependently. The expression of monocyte chemoattractant protein-1 was increased in females, while tubulointerstitial infiltration by M1 macrophages was significantly higher in males after neonatal uninephrectomy. Neonatally uninephrectomized male rats had more glomerulosclerosis and podocyte damage compared to females, which was assessed by a semiquantitative score and desmin staining. RT-PCR revealed that after neonatal uninephrectomy in the remaining contralateral kidney of female rats the expression of candidate genes of renal development and function, i.e., wt-1, nephrin, synaptopodin, gdnf, and itga8 was higher than in males. CONCLUSIONS: Based on these observations we conclude that female sex is protective in the long-term response of the kidney to acute nephron loss under active nephrogenesis.

No benefit of HIF prolyl hydroxylase inhibition for hypertensive renal damage in renovascular hypertensive rats.

Introduction: We previously reported that malignant hypertension is associated with impaired capillary density of target organs. Here, we tested the hypothesis that stabilization of hypoxia-inducible factor (HIF) in a modified "preconditioning" approach prevents the development of malignant hypertension. To stabilize HIF, we employed pharmacological inhibition of HIF prolyl hydroxylases (PHD), that profoundly affect HIF metabolism. Methods: Two-kidney, one-clip renovascular hypertension (2K1C) was induced in rats; controls were sham operated. 2K1C rats received either intermittent injections of the PHD inhibitor ICA (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate) or placebo. Thirty-five days after clipping, the frequency of malignant hypertension was assessed (based on weight loss and the occurrence of characteristic vascular lesions). In addition, kidney injury was compared between all ICA treated versus all placebo treated 2K1C, regardless of the occurrence of malignant hypertension. HIF stabilization was evaluated by immunohistochemistry, and HIF target gene expression by RT-PCR. Results: Blood pressure was elevated to the same degree in ICA- and placebo-treated 2K1C compared to control rats. ICA treatment did not affect the frequency of malignant hypertension or the extent of kidney tissue fibrosis, inflammation, or capillary density. There was a trend towards higher mortality and worse kidney function in ICA-treated 2K1C rats. ICA increased the number of HIF-1α-positive renal tubular cell nuclei and induced several HIF-1 target genes. In contrast, expression of HIF-2α protein as well as HIF-2 target genes were markedly enhanced by 2K1C hypertension, irrespective of ICA treatment. Discussion: We conclude that intermittent PHD inhibition did not ameliorate severe renovascular hypertension in rats. We speculate that the unexpected strong renal accumulation of HIF-2α in renovascular hypertension, which could not be further augmented by ICA, may contribute to the lack of a benefit from PHD inhibition.

Intrauterine growth restriction promotes vascular remodelling following carotid artery ligation in rats.

Epidemiological studies revealed an association between IUGR (intrauterine growth restriction) and an increased risk of developing CVDs (cardiovascular diseases), such as atherosclerosis or hypertension, in later life. Whether or not IUGR contributes to the development of atherosclerotic lesions, however, is unclear. We tested the hypothesis that IUGR aggravates experimentally induced vascular remodelling. IUGR was induced in rats by maternal protein restriction during pregnancy (8% protein diet). To detect possible differences in the development of vascular injury, a model of carotid artery ligation to induce vascular remodelling was applied in 8-week-old intrauterine-growth-restricted and control rat offspring. Histological and immunohistochemical analyses were performed in the ligated and non-ligated carotid arteries 8 weeks after ligation. IUGR alone neither caused overt histological changes nor significant dedifferentiation of VSMCs (vascular smooth muscle cells). After carotid artery ligation, however, neointima formation, media thickness and media/lumen ratio were significantly increased in rats after IUGR compared with controls. Moreover, dedifferentiation of VSMCs and collagen deposition in the media were more prominent in ligated carotids from rats after IUGR compared with ligated carotids from control rats. We conclude that IUGR aggravates atherosclerotic vascular remodelling induced by a second injury later in life.

Overexpression of alanine-glyoxylate aminotransferase 2 protects from asymmetric dimethylarginine-induced endothelial dysfunction and aortic remodeling.

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and increase in blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency. We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

Early glomerular alterations in genetically determined low nephron number.

An association between low nephron number and subsequent development of hypertension in later life has been demonstrated. The underlying pathomechanisms are unknown, but glomerular and postglomerular changes have been discussed. We investigated whether such changes are already present in prehypertensive "glial cell line-derived neurotrophic growth factor" heterozygous mice (GDNF+/-) with lower nephron number. Twenty-six-week-old mice [22 GDNF+/-, 29 C57B6 wild-type control (wt)] were used for in vivo experiments with intra-arterial and tail cuff blood pressure measurements. After perfusion fixation, kidneys were investigated with morphological, morphometric, stereological, and immunohistochemical techniques and TaqMan PCR analysis. As expected at this age, blood pressure was comparable between GDNF+/- and wt. Nephron number per kidney was significantly lower in GDNF+/- than in wt (-32.8%, P < 0.005), and mean glomerular volume was significantly higher (+49.5%, P < 0.001). Renal damage scores, glomerular and tubular proliferation, analysis of intrarenal arteries and peritubular capillaries, expression of relevant tubular transporter proteins, as well as gene expression of profibrotic, proinflammatory, or prohypertensive markers were not significantly different between GDNF+/- and wt. Compensatory glomerular hypertrophy in GDNF+/- was accompanied by higher numbers of endothelial and mesangial cells as well as PCNA-positive glomerular cells, whereas podocyte density was significantly reduced. Further electron microscopic analysis showed marked thickening of glomerular basement membrane. In conclusion, lower nephron number is associated with marked early glomerular structural changes, in particular lower capillary supply, reduced podocyte density, and thickened glomerular basement membrane, that may predispose to glomerular sclerosis.