Improving Genomic Prediction of Crossbred and Purebred Dairy Cattle.

This study assessed the accuracy and bias of genomic prediction (GP) in purebred Holstein (H) and Jersey (J) as well as crossbred (H and J) validation cows using different reference sets and prediction strategies. The reference sets were made up of different combinations of 36,695 H and J purebreds and crossbreds. Additionally, the effect of using different sets of marker genotypes on GP was studied (conventional panel: 50k, custom panel enriched with, or close to, causal mutations: XT_50k, and conventional high-density with a limited custom set: pruned HDnGBS). We also compared the use of genomic best linear unbiased prediction (GBLUP) and Bayesian (emBayesR) models, and the traits tested were milk, fat, and protein yields. On average, by including crossbred cows in the reference population, the prediction accuracies increased by 0.01-0.08 and were less biased (regression coefficient closer to 1 by 0.02-0.16), and the benefit was greater for crossbreds compared to purebreds. The accuracy of prediction increased by 0.02 using XT_50k compared to 50k genotypes without affecting the bias. Although using pruned HDnGBS instead of 50k also increased the prediction accuracy by about 0.02, it increased the bias for purebred predictions in emBayesR models. Generally, emBayesR outperformed GBLUP for prediction accuracy when using 50k or pruned HDnGBS genotypes, but the benefits diminished with XT_50k genotypes. Crossbred predictions derived from a joint pure H and J reference were similar in accuracy to crossbred predictions derived from the two separate purebred reference sets and combined proportional to breed composition. However, the latter approach was less biased by 0.13. Most interestingly, using an equalized breed reference instead of an H-dominated reference, on average, reduced the bias of prediction by 0.16-0.19 and increased the accuracy by 0.04 for crossbred and J cows, with a little change in the H accuracy. In conclusion, we observed improved genomic predictions for both crossbreds and purebreds by equalizing breed contributions in a mixed breed reference that included crossbred cows. Furthermore, we demonstrate, that compared to the conventional 50k or high-density panels, our customized set of 50k sequence markers improved or matched the prediction accuracy and reduced bias with both GBLUP and Bayesian models.

Angiotensin receptor blocker vs ACE inhibitor effects on HDL functionality in patients on maintenance hemodialysis.

BACKGROUND AND AIMS: Angiotensin receptor blockers (ARB) and angiotensin converting enzyme inhibitors (ACEI) reduce cardiovascular events in the general population. Maintenance hemodialysis (MHD) patients are at high cardiovascular risk but few studies have directly addressed the comparative efficacy of these drugs. MHD disrupts the normally atheroprotective actions of high density lipoprotein (HDL), therefore, we compared ACEI or ARB treatment on HDL functions in MHD. METHODS AND RESULTS: HDL was isolated at the starting point (pre) and 3-6 months later (post) in 30 MHD randomly assigned to placebo, ramipril or valsartan. Outcomes included cholesterol efflux, inflammatory cytokine response, effects on Toll-like receptors (TLR), superoxide production, methylarginine and serum amyloid A (SAA) levels. HDL from ARB- or ACEI-treated subjects was more effective in maintaining efflux than HDL of placebo. HDL from ARB- or ACEI-treated subjects but not placebo lessened cellular superoxide production. In contrast, neither ARB nor ACEI improved HDL anti-inflammatory effect. Indeed, HDL of ACEI-treated subjects potentiated the cytokine responses in association with activation of TLR but did not alter the HDL content of methylarginines or SAA. CONCLUSION: Both ACEI and ARB stabilized HDL cholesterol acceptor function and sustained cellular anti-oxidative effects but not anti-inflammatory effects, and ACEI-treatment instead amplified the HDL inflammatory response. The findings reveal possible utility of antagonizing angiotensin actions in MDH and suggest a possible mechanism for superiority of ARB vs ACEI in the setting of advanced kidney disease.

Effect of sevelamer hydrochloride exposure on carotid intima media thickness in hemodialysis patients.

BACKGROUND: Elevated phosphorus (P) and calcium (Ca)-P product (Ca × P) are associated with vascular calcification and cardiovascular disease (CVD) morbidity and CVD and all-cause mortality. OBJECTIVES: This study examined the effect of sevelamer hydrochloride exposure (regardless of calcium carbonate exposure) on carotid and femoral intima media thickness (IMT), reliable surrogate measures of prospective intimal thickening, in end-stage renal disease patients on maintenance hemodialysis. METHODS: The present cross-sectional study is nested in the Sevelamer hydrochloride and ultrasound-measured femoral and carotid intima media thickness progression in end-stage renal disease (SUMMER) clinical trial. Carotid and femoral arteries were visualized in B-mode ultrasonography. Log-transformed IMT was compared by sevelamer hydrochloride exposure and modeled using multiple linear regression. RESULTS: Forty-five subjects were exposed to sevelamer hydrochloride and 130 were not. Exposed subjects had significantly lower carotid IMT, an association which persisted in the multiple linear regression model even after controlling for potentially confounding variables including serum Ca, history of CVD and body weight. Exposed subjects had lower low-density lipoprotein cholesterol levels and significantly higher parathyroid hormone, but no differences in P, Ca and Ca × P. CONCLUSIONS: Sevelamer hydrochloride was associated with lower carotid IMT. This association may be mediated through reduction in Ca load, low-density lipoprotein cholesterol lowering or some other pleiotropic effect.

Update on endothelins - biology and clinical implications.

In the decade since the initial discovery and characterization of endothelin, its biology as a powerful vasoconstrictor has been dramatically demonstrated. Studies have clarified the existence of endothelin isoforms, complex mechanisms of biosynthesis, interaction with specific receptors, and pathogenic implications. We are on the brink of using endothelin antagonism as a clinical treatment for disease processes where endothelin plays an important role, including congestive heart failure and hypertension. Novel observations have been made about the unexpectedly profound contribution endothelins make to normal fetal maturation, especially in cardiac and enteric development.

Dual renin gene targeting by Cre-mediated interchromosomal recombination.

This study describes a new approach to targeting clustered genes. Our study began with the establishment of two lines of mice carrying different mutations in either Ren1 or Ren2. These two genes, both encoding renin, span over 40 kb in tandem on chromosome 1. Each gene was mutated by gene targeting to contain loxP sites. These two mutants and Cre transgenic mice were mated to produce offspring carrying the mutant Ren1 and Ren2 genes, as well as the Cre transgene concurrently. Initially, two mutant Ren genes were located on separate chromosomes. Southern analysis of mice from the second generation revealed that the mutant Ren1 and Ren2 were interchromosomally recombined at the loxP sites to produce a new dually mutated allele on the chromosome at the rate of 9.6% (7/73). Thus, interchromosomal recombination can be efficiently programmed by mating as designed using the Cre-loxP system.

The angiotensin type II receptor tonically inhibits angiotensin- converting enzyme in AT2 null mutant mice.

BACKGROUND: Pharmacologic inhibition of the angiotensin-converting enzyme (ACE) limits angiotensin II (Ang II)-induced vasoconstriction and cellular proliferation. There is emerging evidence that some of the beneficial effects of ACE inhibitors may be endogenously available through the angiotensin receptor type 2 (AT2). METHODS: To evaluate whether AT2 modulates ACE activity, we used an high-performance liquid chromatography (HPLC)-based enzymatic assay in tissues from AT2 knockout mice (Agtr2-/y) and cultured cells. These studies were complimented by physiologic studies of pharmacologic inhibition of AT2. RESULTS: Circulating (C) and tissue ACE activities in heart (H), lung (L), and kidney (K) were doubled in Agtr2-/y mice compared with wild-type mice [162.9 +/- 17.6 mU/mL (C), 97.7 +/- 20.7 (H), 6282.1 +/- 508.3 (L), and 2295.0 +/- 87.0 (K) mU/g tissue for Agtr2-/y vs. 65.3 +/- 35.4 mU/mL (C), 44.5 +/- 8.7 (H), 3392.4 +/- 495.2 (L), and 1146.1 +/- 217.3 (K) mU/g tissue for wild-type mice, P < or = 0.05, 0.025, 0.002, and 0.0001, respectively]. Acute pharmacologic inhibition of AT2 [PD123319 (PD), 50 microg/kg/min, i. v.] significantly increased ACE activity in kidneys of wild-type mice (1591.2 +/- 104.4 vs. 1233.6 +/- 88.0 mU/g tissue in saline-infused mice, P < 0.05; P < 0.01 vs. uninfused, wild-type mice). Moreover, ACE activity increased in A10 cells exposed to PD (10-6 mol/L) together with Ang II (10-7 mol/L), but not with an AT1 antagonist (losartan, 10-6 mol/L). This heightened ACE activity appears functionally relevant because infusion of angiotensin I caused more prompt hypertension in Agtr2-/y mice than in wild-type littermates. Likewise, infusion of bradykinin, also a substrate for ACE, caused significantly less hypotension in Agtr2-/y mice than controls. CONCLUSIONS: These studies indicate that AT2 functions to decrease ACE activity tonically, which may, in part, underlie AT2's increasingly recognized attenuation of AT1-mediated actions.

ACE I/D gene polymorphism predicts renal damage in congenital uropathies.

We investigated angiotensin converting enzyme gene (ACE I/D) polymorphism as a risk for progressive renal damage in congenital uropathies. The ACE I/D genotype was determined in 196 Caucasian patients with congenital uropathies and 163 individuals with no clinical or sonographic evidence of any urological malformations. The study group included patients with ureteropelvic junction obstruction (n=49), primary obstructive megaureter (n=19), primary vesicoureteral reflux (VUR) (n=67), and posterior urethral valves (n=27). Thirty-four patients were excluded because of additional diseases or insufficient follow-up. There was no difference in the ACE I/D distribution between children with uropathies and normal controls (II 16%, ID 56%. DD 28% vs. II 26%, ID 50%, DD 24%). Renal lesions were found in 99 of 162 children by ultrasonography, intravenous pyelography, and nuclear scans. In these children there was significant over-representation of the DD genotype (II 11%, ID 53%, DD 36%) compared with normals (P<0.005, X2=14.9) or with patients with uropathies but no renal lesions (II 23%, ID 62%, DD 15%, P<0.005, X2=14.9). Because ACE I/D has been linked with progressive deterioration of renal function, we evaluated a subset of patients with initially normal kidneys who developed radiographic renal lesions (n=28). Among these patients there was an even greater over-representation of the DD genotype (II 0%, ID 43%, DD 57%, P<0.001, X2=22.6) compared with patients with uropathies but no radiographic lesions. Multivariate analysis revealed that the DD genotype is a risk factor for parenchymal destruction, which was independent of time of diagnosis, surgical intervention, or urinary tract infection. This finding was particularly relevant in patients with VUR who constituted the majority with initially normal kidneys who developed radiographic damage (22/28). Indeed, the odds ratio of developing parenchymal damage with VUR was significantly increased if the individual had the DD genotype (4.2, 95% confidence interval 1.4-13.0). In conclusion the ACE I/D gene polymorphism is a risk factor for renal parenchymal damage in patients with congenital urological abnormalities and appears particularly relevant in children with VUR, where it is an independent predisposing factor.

Angiotensin type 2 receptor is important in the normal development of the ureter.

In humans, the actions of angiotensin II are transduced through the AT1 and AT2 receptors which have recently been implicated in renal organogenesis. Polymorphisms in the human angiotensin II receptor genes have been linked to cardiovascular and nephrological disorders. In this study we evaluated 35 patients with either primary obstructive megaureter or posterior urethral valves. Each was genotyped for the A1166 AT1 polymorphism and the recently described A-1332G AT2 transition. The incidence of these genetic variants was also evaluated in normal controls without any ultrasonographic urological abnormalities. Similar to our previous findings in congenital urological abnormalities, the AT1 receptor genotype distribution did not differ between patients with either primary obstructive megaureter or posterior urethral valves versus controls. In contrast, compared with normal controls, there was a dramatic increase in the occurrence of the AT2 A-1332G transition in patients with primary obstructive megaureter (75.0% vs. 41.9% in controls, P<0.025). In patients with posterior urethral valves, there was no difference in the occurrence of the transition versus controls (36.9%, P=NS). Thus, there is no correlation between the AT1 receptor gene polymorphism and urological abnormalities. However there is an increased incidence in the AT2 genetic variant in patients with primary obstructive megaureter.

Angiotensin II, type 2 receptor in the development of vesico-ureteric reflux.

OBJECTIVE: To investigate if mutation of the angiotensin II (Ang II) receptors AT2 is involved in primary vesico-ureteric reflux (VUR) in humans. PATIENTS AND METHODS: Genetic polymorphisms in the AT1 and AT2 receptors was evaluated in 23 patients having the most common congenital urological abnormality, namely primary congenital VUR. The occurrence of the A1166C transition in the AT1 receptor gene and the A-1332G transition in the AT2 receptor gene were evaluated and compared with the incidence in normal controls with no urological abnormalities. RESULT: The distribution of the AT1 receptor genotypes was no different between patients with VUR and healthy controls. Furthermore, 10 of 23 (44%) patients with VUR and seven of 19 (42%) controls carried the AT2 receptor gene variation. These results contrast with our previous finding of an association between the A-1332G transition in the AT2 receptor gene and primary obstructive megaureter, and pelvi-ureteric junction obstruction. CONCLUSIONS: We propose that while the AT2 receptor is crucial for the normal development of the ureter, it does not contribute to the processes which culminate in VUR, which is primarily an abnormality in the bladder trigone.

Renin-angiotensin genes in renal development and the occurrence and progression of renal diseases.

The function of angiotensin II has evolved from recognition of its vasoconstrictive functions and regulations of fluid balance to its pivotal role in progressive tissue destruction, particularly renal scarring. This review highlights angiotensin's newest role in normal and abnormal organogenesis as well as the impact on renal damage of subtle variations in genes that control angiotensin's actions including angiotensinogen, angiotensin converting enzyme, and its receptors.

IgA nephropathy.

Immunoglobulin A nephropathy results from the abnormal deposition of IgA immunoglobulin in the glomerulus, which leads to the characteristic presentation of painless hematuria. It is the most common glomerulonephritis worldwide. Originally described 30 years ago, it was thought to follow a benign course. We now know that IgA nephropathy leads to progressive renal destruction in about one third of affected patients. Alteration in glycosylation of circulating IgA may be an important pathophysiologic mechanism that predisposes to IgA deposition, although how this leads to parenchymal damage remains unclear. Hypertension, high-grade proteinuria, and elevated serum creatinine levels are known risk factors for progressive renal destruction. In addition to these well-recognized risk factors, there appear to be genetic variants, particularly within the angiotensin-converting enzyme gene, that portend a worse outcome.

Role of the renin-angiotensin system in disorders of the urinary tract.

PURPOSE: The role of the renin-angiotensin system in the homeostasis of fluid, electrolyte and blood pressure has been known for quite some time. Recent developments indicate that angiotensin has a profound role, not only in the developing urinary tract but also in the response of the urinary tract to injury. In this review we outline these characteristics. MATERIALS AND METHODS: We summarize the clinical approach to congenital abnormalities of the kidney and urinary tract, and report new data obtained in genetically engineered mice. Furthermore, we present the connection between the mutant mice observations and human congenital abnormalities. RESULTS: Genetically engineered mutants clearly indicate that the renin-angiotensin system is important for normal renal and urological development. As in glomerular disease, the renin-angiotensin system is involved in progressive damage due to urological disease. CONCLUSIONS: While the renin-angiotensin system is important for blood pressure regulation, it also affects the embryogenesis of the urinary tract and modulates renal injury due to specific disease processes. The importance of angiotensin and its blockade provides an exciting avenue for possible early treatment in children with congenital anomalies of the kidney and urinary tract.

Renal angiotensin converting enzyme promotes renal damage during ureteral obstruction.

PURPOSE: We and others have shown that angiotensin II has a pivotal role in renal damage in various renal injuries. Although most angiotensin II actions are associated with the angiotensin type 1 receptor, there is increasing evidence that the angiotensin type 2 receptor also transduces some important effects of angiotensin II. In this regard we recently observed that mice with genetically engineered disruption of the angiotensin type 2 receptor, termed Agtr2 mutants, are more susceptible to structural renal damage after ureteral obstruction. Recent evidence suggests that a genetically determined increase in angiotensin converting enzyme activity in humans promotes end organ damage. Therefore, we determined whether renal damage in Agtr2 mutants is associated with heightened angiotensin converting enzyme activity. MATERIALS AND METHODS: We studied 28 wild type and 19 Agtr2 mutant mice with unilateral ureteral obstruction. Seven days after obstruction was created serum samples were obtained to evaluate angiotensin converting enzyme activity. The obstructed and contralateral kidneys were harvested for histological analysis and determination of renal angiotensin converting enzyme activity by high pressure liquid chromatography. RESULTS: Renal angiotensin converting enzyme was uniformly higher than serum angiotensin converting enzyme in normal wild type and Agtr2 mutant mice. However, even at baseline Agtr2 mutant mice had strikingly higher renal angiotensin converting enzyme activity than normal wild type mice (mean plus or minus standard error 1,492+/-83 versus 450+/-60 milliunits per gm. tissue weight, p <0.0005). Histological analysis revealed more extensive parenchymal damage in the obstructed kidneys of mutant mice than in identically treated controls. Notably while unilateral ureteral obstruction decreased renal angiotensin converting enzyme activity in each group, activity remained persistently higher in the Agtr2 mutants than in normal mice (mean 742+/-146 versus 310+/-43 milliunits per gm. tissue weight, p <0.005). CONCLUSIONS: We propose that elevated renal angiotensin converting enzyme activity contributes to more severe renal parenchymal damage in ureteral obstruction by promoting the availability of growth factors, such as angiotensin II, or depleting antiproliferation factors, such as bradykinin or nitric oxide. These findings complement previous observations that angiotensin converting enzyme inhibition preserves the renal parenchyma after injury, including obstruction.

Accelerated fibrosis and collagen deposition develop in the renal interstitium of angiotensin type 2 receptor null mutant mice during ureteral obstruction.

We examined the role of angiotensin in renal remodeling that is specifically channeled through the angiotensin type 2 receptor (AT2 receptor). Previously, we observed that in mouse embryonic kidneys the AT2 mRNA is predominantly expressed in the mesenchyme. We therefore chose a model of unilateral ureteral obstruction, characterized by activation of the renin-angiotensin system, while fibrosis develops prominently within the renal interstitium. Male wild-type mice (Agtr2 -/Y) and mice null mutant for the AT2 gene (Agtr2 -/Y) were subjected to a complete unilateral ureteral ligation for 5 or 14 days. Obstructed kidneys of Agtr2 -/Y mice showed more severe interstitial fibrosis than those of Agtr2 +/Y mice, confirmed by increased collagen by point-counting on Masson trichrome stained sections, and increased alpha 1(I) collagen mRNA expression by Northern blot. Immunohistochemistry staining for PCNA (a marker of cell proliferation), F4/80 (a marker of macrophages), vimentin (a marker of fibroblasts), and alpha SMA (a marker of myofibroblasts) revealed that, while the two groups were comparable in the degree of cell proliferation and macrophage infiltration, fibroblasts/ myofibroblasts were present in a greater abundance in obstructed kidneys of Agtr2 -/Y mice than in Agtr2 +/Y at both 5 and 14 days after obstruction. Moreover, cells undergoing apoptosis were significantly less in Agtr2 -/Y than in Agtr2 +/Y. Thus, the AT2 receptor significantly impacts the remodeling process within renal interstitium, potentially by regulating the population of collagen-producing cells.

Angiotensin II type I receptor polymorphism in African Americans lower frequency of the C1166 variant.

The C1166 variant, an A to C substitution polymorphism at the 1166 position of the angiotensin II type I (AT1) receptor, has been previously associated with hypertension in Caucasians. This study determines the frequency of the C1166 variant in an African American population. Normotensive African American (n = 99) and Caucasian (n = 100) subjects were genotyped to determine the frequency of the C1166 variant. This study establishes the frequency of the C1166 variant in African Americans (0.05 +/- 0.01) and demonstrates a significantly lower frequency in African Americans compared with Caucasians (0.05 vs. 0.25, respectively, chi 2 = 30.7, p < < 0.001, 1 df).

Potential risk factors associated with progressive renal damage in childhood urological diseases: the role of angiotensin-converting enzyme gene polymorphism.

PURPOSE: Experimental as well as human studies have established an important role for the renin-angiotensin system in the progressive deterioration of renal function. Recently genetic polymorphism in components of the renin-angiotensin system has been associated with several cardiovascular diseases, particularly variations in the angiotensin-converting enzyme gene that involve insertion (I) or deletion (D) of a 287 bp fragment. The D variant has been associated with myocardial infarction and cardiac hypertrophy. MATERIALS AND METHODS: To assess whether this genetic variant is associated with worse prognosis in renal disorders we evaluated 70 children with congenital urological abnormalities, since a substantial number have progressive renal deterioration even after early corrective intervention. Renal deterioration was assessed by the presence or absence of radiographic evidence of parenchymal damage and serum creatinine. RESULTS: Among patients with no radiographic renal parenchymal damage angiotensin-converting enzyme genotype distribution of II, ID and DD was 24, 67 and 9%, respectively. In contrast, a significantly different angiotensin-converting enzyme genotype distribution was observed in patients with evidence of parenchymal damage, that is 10, 49 and 41% for II, ID and DD, respectively (p < 0.05, chi-square 5.0). Mean serum creatinine plus or minus standard error in the former group was normal at 0.6 +/- 0.1 mg./dl., while in those with scarring it was elevated at 1.1 +/- 0.1 mg./dl., as expected. In patients with the DD genotype an overwhelming frequency of parenchymal damage was observed, that is of all 22 with that genotype 20 (91%) had parenchymal damage. CONCLUSIONS: Considered together, these studies suggest that there are differences in the distribution of angiotensin-converting enzyme gene polymorphism in patients with congenital urological abnormalities who have evidence of renal parenchymal damage versus those who do not have such damage. Given that this genetic variation activates the renin-angiotensin system and this activation may be particularly robust in the kidney, we propose that the genotype of an individual independent of other factors modifies the likelihood of parenchymal loss in this setting.

A novel in vivo mechanism for angiotensin type 1 receptor regulation.

This study examined whether a regulatory mechanism exists for the angiotensin II receptor that is compatible with in vivo homeostatic need. Experiments were conducted under two different experimental stresses, (1) deletion of receptor protein and (2) chronic extracellular fluid (ECF) volume depletion. To circumvent potentially dampening intermediary feedback signals in vivo, any feedback gain was completely averted through genetic engineering. The coding exon of angiotensin type 1A (AT1A) receptor gene (Agtr1a) was targeting-replaced with a reporter gene, lacZ, so that the transcription of lacZ, instead of Agtr1a, is driven by the native Agtr1a promoter. ECF volume depletion by dietary sodium restriction enhanced Agtr1a gene expression in the adrenal gland of wild-type mice. However, although blood pressure fell in the homozygous targeted mice, Agtr1a gene expression remained unchanged in the adrenal, indicating that adrenal Agtr1a gene expression is regulated entirely through angiotensin receptor-ligand interactions. In the kidney, AT1A mRNA assessed by Northern blotting also did not change in AT1A null-mutated mice with or without sodium restriction. However, tissue examinations for lacZ mRNA and activities indicated that sodium restriction and receptor protein depletion result in dramatic up-regulation of Agtr1a gene expression within the renal arterioles, which can be nullified by an experimental normalization of blood pressure. No such change was observed in wild-type mice. This study demonstrates a presence within the resistance vessel of a blood pressure-sensitive mechanism for AT1 receptor regulation that opposes a down-regulatory influence of the ligand during ECF volume depletion.

Endothelin in ischemic acute renal failure.

The endothelin peptides comprise a family of potent and long-lasting vascoconstrictors, to which the renal microcirculation is particularly susceptible. Increased renal endothelin expression is observed after a variety of injurious stimuli, including ischemia, and persists for days after resolution of the initial injury. Autoinduction of its own production is likely to be a central mechanism underlying endothelin's prolonged effects. Furthermore, antagonizing endothelin reveals its role in maintaining the postischemic glomerular dysfunction that typifies ischemic acute renal failure.