Genome-wide autozygosity is associated with lower general cognitive ability.

Inbreeding depression refers to lower fitness among offspring of genetic relatives. This reduced fitness is caused by the inheritance of two identical chromosomal segments (autozygosity) across the genome, which may expose the effects of (partially) recessive deleterious mutations. Even among outbred populations, autozygosity can occur to varying degrees due to cryptic relatedness between parents. Using dense genome-wide single-nucleotide polymorphism (SNP) data, we examined the degree to which autozygosity associated with measured cognitive ability in an unselected sample of 4854 participants of European ancestry. We used runs of homozygosity-multiple homozygous SNPs in a row-to estimate autozygous tracts across the genome. We found that increased levels of autozygosity predicted lower general cognitive ability, and estimate a drop of 0.6 s.d. among the offspring of first cousins (P=0.003-0.02 depending on the model). This effect came predominantly from long and rare autozygous tracts, which theory predicts as more likely to be deleterious than short and common tracts. Association mapping of autozygous tracts did not reveal any specific regions that were predictive beyond chance after correcting for multiple testing genome wide. The observed effect size is consistent with studies of cognitive decline among offspring of known consanguineous relationships. These findings suggest a role for multiple recessive or partially recessive alleles in general cognitive ability, and that alleles decreasing general cognitive ability have been selected against over evolutionary time.

Phenotypic transitions and fibrosis in diabetic nephropathy.

The cause of renal fibrosis in diabetic nephropathy is widely believed to be phenotypic switching of fibroblasts to an activated state. However, emerging evidence suggests that diabetes also alters the phenotype of normal, non-fibroblast kidney cells, such as mesangial cells, tubular epithelial cells, and bone marrow-derived progenitors. Experiments have shown that cytokines, high glucose, and advanced glycation end products induce profibrotic changes in kidney cell phenotype by the processes of myofibroblast transdifferentiation and epithelial-mesenchymal transition. As a result, differentiated kidney cells become reprogrammed to secrete and accumulate extracellular matrix. This revised view implies that inhibiting phenotypic transitions in nonfibroblasts might limit fibrosis in diabetic nephropathy.

Pneumoperitoneum upregulates preproendothelin-1 messenger RNA.

BACKGROUND: Laparoscopic pneumoperitoneum has been shown to decrease glomerular filtration rate (GFR) and urine volume (UV). Endothelin-1 (ET-1), a potent renal vasoconstrictor, has been implicated. The purpose of this study was to determine renal function, ET-1 gene expression, and peptide localization in kidneys subjected to CO2 pneumoperitoneum. METHODS: Experiments were performed in three groups of anesthetized Sprague-Dawley rats in which GFR and UV were measured before, during, and after insufflation. In the first group (n = 8), pneumoperitoneum (10 mmHg) was established for 30 min. The second group (n = 4) underwent a sham operation without pneumoperitoneum. In the final group (n = 4), kidneys were obtained from normal control animals without any prior surgical instrumentation. PreproET-1 (ppET-1) mRNA levels were measured by reverse transcription-polymerase chain reaction (RT-PCR). The ET-1 peptide was localized within kidneys by immunohistochemistry (IHC). RESULTS: Pneumoperitoneum caused a significant (p < 0.05) 87% decrease in GFR and a 79% decrease in UV from baseline, with a return to baseline values after desufflation. RT-PCR showed a significant (p < 0.05) increase in expression of ppET-1 mRNA in the laparoscopic group; it was 3.52 +/- 0.33 densitometric units (DU), as compared to 0.35 +/- 0.06 DU and 0.57 +/- 0.12 DU in the control and sham groups, respectively. IHC showed enhanced expression of the ET-1 peptide in the vascular endothelium and proximal tubular cells of the laparoscopic group compared to the control and sham groups. CONCLUSION: Pneumoperitoneum induces ET-1 gene and peptide upregulation in the kidney. Expression of ET-1 is increased in the renal vasculature and proximal tubular cells. The elevation of ET-1 and its localization may account for some of the renal dysfunction observed during pneumoperitoneum. This suggests that antagonism of ET-1 may be beneficial in patients with renal impairment undergoing prolonged laparoscopic procedures or in protecting allograft function during and after living donor nephrectomy.

Elevated endothelin-1 in tubular epithelium is associated with renal allograft rejection.

Vascular endothelin-1 (ET-1) levels are elevated in patients with renal allograft rejection, and the mitogenic and pressor actions of ET-1 might contribute to transplant vasculopathy, posttransplantation hypertension, and ischemia-reperfusion injury. In contrast, relatively little is known about tubular expression of ET-1 in acute or chronic rejection of renal allografts. We sought to determine whether tubular ET-1 levels were altered in patients with acute or chronic renal allograft rejection. Immunohistochemical analysis of tubular ET-1 was performed in renal biopsy specimens from 18 patients with acute rejection, 7 patients with chronic rejection, and 5 normal kidneys excised for localized neoplasm. The diagnosis of acute or chronic rejection in each patient was verified and graded using the Banff schema. Renal tubular epithelium from patients with allograft rejection had markedly elevated staining for ET-1 compared with normal kidneys. Tubular ET-1 levels were elevated in 18 of 18 patients with acute rejection and 5 of 7 patients with chronic rejection. Tubular ET-1 staining was graded from 0 to +3 as follows: normal kidneys, 1.2 +/- 0.2; acute rejection, 2.3 +/- 0.4 (P < 0.01); and chronic rejection, 2.2 +/- 0.5 (P < 0.01). ET-1 staining was prominent in both proximal and distal tubules, and we observed abundant ET-1 secretion from proximal tubular epithelium in culture. Moreover, ET-1 activated the c-fos immediate early gene promoter in proximal tubular cells transfected with a c-fos luciferase reporter. We conclude that elevated tubular ET-1 levels are associated with acute and chronic rejection of renal allografts. Our results also suggest distinct pathophysiological roles for the tubular and vascular ET-1 systems in renal allograft rejection.

Intracellular pathways linking hypoxia to activation of c-fos and AP-1.

Organisms respond to hypoxia through detection of blood oxygen levels by sensors at peripheral chemoreceptors and by receptors in certain key cells of the body. The pathways over which peripheral chemoreceptor signals are transmitted to respiratory muscles are well established. However, the intracellular pathways that transmit hypoxic stimulus to gene activation are just being identified. Using anti-sense c-fos strategy, we have shown that c-fos is essential for the activation of activator protein-1 transcription factor complex (AP-1) and subsequent stimulation of downstream genes such as tyrosine hydroxylase (TH; Mishra et al. 1998). The purpose of the present study was to identify intracellular pathways that link hypoxia to activation of c-fos. The results of the present study show that hypoxia causes Ca2+ influx through L-type voltage gated Ca2+ channels and that hypoxia-induced c-fos gene expression is Ca2+/calmodulin dependent. We also demonstrate that hypoxia activates the extracellular-regulated kinase (ERK) and p38, but not JNK. Further, phosphorylation of ERK is essential for c-fos activation via SRE cis-element. Further characterization of nuclear signalling pathways provides evidence for the involvement of Src, a non receptor protein tyrosine kinase, and Ras, a small G protein, in the hypoxia-induced c-fos gene expression. These results suggest a possible role for non-receptor protein tyrosine kinases in propagating signals from G-protein coupled receptors to the activation of immediate early genes such as c-fos during hypoxia.

L-type Ca(2+) channel activation regulates induction of c-fos transcription by hypoxia.

In the present study we examined the intracellular pathways that link hypoxia to activation of c-fos gene expression. Experiments were performed on rat pheocromocytoma-12 (PC-12) cells. c-fos mRNA and promoter activities were analyzed by RT-PCR and reporter gene assays, respectively. BAPTA, a Ca(2+) chelator, inhibited c-fos mRNA and promoter activation by hypoxia. Nitrendipine, an L-type Ca(2+)-channel blocker, abolished, whereas BAY K 8644, an L-type channel agonist, enhanced c-fos activation by hypoxia. Ca(2+) currents were augmented reversibly by hypoxia, suggesting that Ca(2+) influx mediated by L-type Ca(2+) channels is essential for c-fos activation by hypoxia. We next determined downstream pathways activated by intracellular Ca(2+) concentration. Immunoblot analysis revealed Ca(2+)/calmodulin-dependent kinase II (CaMKII) protein in PC-12 cells and revealed that hypoxia increased the enzyme activity. KN-93, a CaMK inhibitor, blocked CaMKII activation and c-fos promoter stimulation by hypoxia. Ectopic expression of an active mutant of CaMKII (pCaMKII290) stimulated c-fos promoter activity under normoxia. Hypoxia increased phosphorylation of CREB at the serine residue 133 (Ser-133), and KN-93 attenuated this effect. Point mutations at the Ca(2+)/cAMP-responsive cis-element (Ca/CRE) attenuated, whereas point mutations in the serum-responsive cis-element (SRE) abolished transcriptional activation of c-fos by hypoxia. These results demonstrate that c-fos activation by hypoxia involves CaMK activation and CREB phosphorylation at Ser-133 and requires Ca/CRE and SRE. These observations demonstrate that Ca(2+)-dependent signaling pathways play a crucial role in induction of c-fos gene expression, which may underlie long-term adaptive responses to hypoxia.

Inhibition of endothelin-converting enzyme attenuates transplant vasculopathy and rejection in rat cardiac allografts.

BACKGROUND: Transplant vasculopathy in kidney and heart allografts is associated with marked elevation of endothelin-1 (ET-1), but a role for ET-1 in the pathogenesis of transplant vasculopathy and chronic rejection has not been established. We, therefore, tested whether inhibition of ET-1-converting enzyme by phosphoramidon (PA) would attenuate rejection in a rat model of chronic cardiac allograft rejection (Lewis [LEW] to F344). METHODS: Donor LEW rats were pretreated 24 hr before transplantation with a bolus injection of vehicle (water) or PA. Twenty- four hour after transplantation, water or PA was continuously administered through an osmotic mini-pump. Plasma ET-1 levels in Fisher 344 (F344) recipients were 0.8+/-0.1 pg/ml in water-treated rats and 0.2+/-0.2 pg/ml (P<0.01) in PA-treated rats, demonstrating that the PA treatment protocol effectively lowered ET-1 biosynthesis. RESULTS: LEW cardiac allografts treated with water survived (i.e., palpable heart beat) for 16.0+/-0.5 days (n=6). Inhibition of ET-1 secretion by PA improved allograft survival to 28.8+/-3.3 days (P<0.01, n=8). An analysis of cardiac arteries demonstrated that PA treatment attenuated transplant vasculopathy. A morphometric scale of neointima formation (0-5) was 1.4+/-0.2 and 3.6+/-0.2 in PA- or water-treated rats, respectively (P<0.01). The percent of luminal occlusion, as measured by microscopic image analysis, was 19+/-6% in PA-treated animals and 38+/-6% (P<0.01) in animals treated with water. PA treatment also reduced infiltration of ED-1-positive monocytes/macrophages into the vascular neointima. CONCLUSIONS: We conclude that, even in the absence of concomitant immunosuppression, inhibition of ET-1 biosynthesis significantly attenuates transplant vasculopathy and improves survival of LEW to F344 cardiac allografts.

Cold ischemia induces endothelin gene upregulation in the preserved kidney.

BACKGROUND: Prolonged cold ischemia time (CIT) can lead to posttransplant renal dysfunction; however, the pathophysiology remains unclear. Endothelin (ET), a potent vasoconstrictive peptide, may play a role in this injury. The purpose of this study was to determine if cold ischemia could induce renal ET-1 gene upregulation and to localize ET-1 peptide expression in the hypothermic kidney. MATERIALS AND METHODS: Kidneys from Lewis rats were perfused with Viaspan, harvested, and stored at 4 degrees C for varying periods of CIT: 0, 6, 24, and 48 h. Preproendothelin-1 (ppET-1) gene upregulation was measured using a reverse-transcription polymerase-chain reaction. ET-1 peptide expression was localized using immunohistochemistry. RESULTS: Control kidneys (0 h CIT) had 0. 56 +/- 0.22 DU of ppET-1 mRNA. After 6 h of CIT, a 2.3-fold increase in this level was noted. Following 24 h of CIT, ppET-1 mRNA was significantly upregulated to 1.96 +/- 0.38 DU (P < 0.05). Immunohistochemistry revealed typical vascular ET-1 staining in control kidneys. At 6 h of CIT, a significant increase in the expression of ET-1 was noted in the peritubular capillaries and vasa recta. After 24 h, intense staining for ET-1 was seen in the medullary collecting ducts. After 48 h of CIT, early cellular necrosis was present along with global decreases in ET-1 expression and ppET-1 mRNA levels. CONCLUSIONS: This study demonstrates that 24 h of cold preservation can induce significant upregulation of the renal ET-1 gene and increase expression of the ET-1 peptide localized to both vascular endothelial and tubular epithelial surfaces of the kidney. Consequently, prolonged cold ischemia prior to transplantation may lead to delayed renal function following revascularization via endothelin-induced vasoconstriction and/or tubular impairment.

Endothelin up-regulation and localization following renal ischemia and reperfusion.

BACKGROUND: Endothelin (ET), a potent vasoconstrictor, is known to play a role in ischemic acute renal failure. Although preproET-1 (ppET-1) mRNA is known to be up-regulated following ischemia/reperfusion injury, it has not been determined which component of the injury (ischemia or reperfusion) leads to initial gene up-regulation. Likewise, although ET-1 peptide expression has been localized in the normal kidney, its expression pattern in the ischemic kidney has not been determined. Therefore, the purpose of this study was twofold: (a) to determine whether ischemia alone or ischemia plus reperfusion is required for the up-regulation of ppET-1 mRNA to occur, and (b) to localize ET-1 peptide expression following ischemia in the rat kidney to clarify better the role of ET in the pathophysiology of ischemia-induced acute renal failure. METHODS: Male Lewis rats underwent clamping of the right renal vascular pedicle for either 30 minutes of ischemia (group 1), 60 minutes of ischemia (group 2), 30 minutes of ischemia followed by 30 minutes of reperfusion (group 3), or 60 minutes of ischemia followed by three hours of reperfusion (group 4). The contralateral kidney acted as a control. ppET-1 mRNA up-regulation and ET-1 peptide expression were examined using the reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. RESULTS: Reverse transcription-polymerase chain reaction yielded a control (nonischemic) value of 0.6 +/- 0.2 densitometric units (DU) of ppET-1 mRNA in the kidney. Group 1 levels (30 min of ischemia alone) were 1.8 +/- 0.4 DU, a threefold increase (P < 0.05). Group 2 levels (60 min of ischemia alone) increased almost six times above baseline, 3.5 +/- 0.2 DU (P < 0.01), whereas both group 3 and group 4 (ischemia plus reperfusion) did not experience any further significant increases in mRNA levels (1.9 +/- 0.4 DU and 2.8 +/- 0.6 DU, respectively) beyond levels in group 1 or 2 animals subjected to similar ischemic periods. ET-1 peptide expression in the ischemic kidneys was significantly increased over controls and was clearly localized to the endothelium of the peritubular capillary network of the kidney. CONCLUSIONS: Initial ET-1 gene up-regulation in the kidney occurs secondary to ischemia, but reperfusion most likely contributes to sustaining this up-regulation. The marked increase of ET-1 in the peritubular capillary network suggests that ET-induced vasoconstriction may have a pathophysiological role in ischemic acute tubular necrosis.

Role of c-fos in hypoxia-induced AP-1 cis-element activity and tyrosine hydroxylase gene expression.

Previous studies have demonstrated that hypoxia stimulates expression of the c-fos gene in intact animals and isolated cells. The purpose of the present study was to assess the functional significance of c-fos activation during hypoxia. Using antisense c-fos strategy, we tested the hypothesis that c-fos is essential for activation of activator protein-1 transcription factor complex (AP-1) and subsequent stimulation of down stream genes such as tyrosine hydroxylase (TH) gene during hypoxia. Experiments were performed on rat pheochromocytoma 12 (PC12) cells. AP-1 activity was determined by a reporter gene assay using a luciferase expression vector driven by two copies of an AP-1 cis-element (AP-1-Luc). Cells transfected with AP-1-Luc construct were exposed to normoxia (21% O2) or to varying intensities and/or durations of hypoxia. AP-1 activity increased in response to hypoxia. The magnitude of the response depended on the intensity and duration of the hypoxic stimulus. Increases in AP-1 activity could not be elicited in neuroblastoma cells, indicating that hypoxia-induced increase in AP-1 activity is a cell selective phenomenon. Antisense c-fos abolished hypoxia-induced AP-1 activation in PC12 cells. Hypoxia increased tyrosine hydroxylase-chloramphenicol acetyl transferase activity (TH-CAT), and antisense c-fos and mutations at AP-1 binding sites in TH promoter abolished this effect. These results provide direct evidence that c-fos is essential for functional activation of AP-1 and subsequent activation of delayed response genes such as TH in PC12 cells.

Elevated neointimal endothelin-1 in transplantation-associated arteriosclerosis of renal allograft recipients.

BACKGROUND: Chronic renal allograft rejection is characterized histologically by transplantation-associated arteriosclerosis and glomerulosclerosis (Tx-AA and Tx-AGS). Recent studies in animal models implicate the mitogenic and pressor actions of endothelin-1 (ET-1) in Tx-AA. In humans, however, a link between elevated ET-1 secretion and Tx-AA or Tx-AGS remains unclear. In this study we analyzed expression of ET-1 in the vasculature of renal transplant patients with chronic or acute rejection and in normal controls. METHODS: Renal vascular and glomerular ET-1 was assessed by immunohistochemistry in 12 patients with clinically and histologically defined chronic rejection, in 11 patients with acute rejection, and in 5 normal kidneys. ET-1 staining was also correlated with various clinical parameters and with a morphometric index of neointima formation. ET-1 secretion was measured by ELISA in cultured human vascular cell types treated with T cell- and macrophage-associated cytokines. RESULTS: We found that renal allografts with chronic rejection and Tx-AA expressed 6.1-fold more ET-1 in the vasculature relative to allografts with acute rejection or to normal kidneys (P < 0.01). In Tx-AA, ET-1 was detected predominantly in the neointima, which contained mostly endothelial cells and smooth muscle cells. A strong positive correlation (r = 0.82, P < 0.01) was observed between vascular ET-1 peptide expression and hypertension in patients with chronic rejection. We also showed that macrophage-associated cytokines, but not T cell-associated cytokines, stimulated ET-1 secretion in human endothelial cells, vascular smooth muscle and mesangial cells. CONCLUSIONS: These results demonstrate that elevated ET-1 in the neointima is associated with Tx-AA and chronic rejection. In addition, these results point to an important role for endothelial dysfunction in chronic renal allograft rejection.

Vascular and glomerular expression of endothelin-1 in normal human kidney.

To understand better the function of endothelin-1 (ET-1) in renal physiology, we examined vascular and glomerular expression of ET-1 in normal human kidney and in lupus nephritis. Immunohistochemical analysis revealed that renal endothelium of glomeruli, arteries, veins, and capillaries expressed ET-1. Endothelial cells were the principal source of glomerular ET-1; positive immunostaining was detected only rarely in mesangial cells and vascular smooth muscle cells from normal kidney. However, mesangial staining for ET-1 was elevated in patients with lupus nephritis, suggesting that under certain conditions mesangial cells elaborate ET-1. Indeed cultured human mesangial cells from normal subjects secreted ET-1 peptide. ET-1 secretion was augmented by the protein kinase C activator phorbol ester and by transforming growth factor-beta1 (TGF-beta1), a cytokine implicated in the development of glomerulosclerosis. Transient transfection of cultured mesangial cells with a preproET-1 reporter construct showed that the preproET-1 promoter is transcriptionally active in mesangial cells and is stimulated by TGF-beta1, phorbol ester, or ectopic expression of protein kinase beta1. Cultured human mesangial cells have both ETA and ETB receptors that contribute to ET-1-stimulated mitogenesis. Taken together, these results demonstrate that ET-1 is expressed at sites where paracrine or autocrine signaling by ET-1 might control renal vasoconstriction, glomerular filtration rate, and remodeling of the glomerulus in renal disease.

GATA-6 induces p21(Cip1) expression and G1 cell cycle arrest.

GATA transcription factors represent a family of highly conserved zinc finger proteins with tissue-specific expression patterns. Previous studies have shown that GATA-6 is expressed in vascular smooth muscle cells (VSMCs) and rapidly down-regulated when VSMCs are induced to proliferate. Here we investigated whether the GATA-6 transcription factor can modulate cellular proliferation. Transient transfection with a GATA-6 expression vector inhibited S-phase entry in VSMCs and in mouse embryonic fibroblasts (MEFs) lacking both p53 alleles. The GATA-6-induced growth arrest correlated with a marked increase in the expression of the general cyclin-dependent kinase (Cdk) inhibitor p21. In contrast to p53-deficient MEFs and VSMCs, MEFs null for both p21 alleles were refractory to the GATA-6-induced growth inhibition. These data demonstrate that elevated GATA-6 expression can promote the quiescent phenotype in VSMCs.

Specific repression of the preproendothelin-1 gene in intracranial arteriovenous malformations.

Cerebrovascular arteriovenous malformations (AVMs) display abnormal vascular development and dysautoregulation of blood flow. Genetic mechanisms that contribute to the pathogenesis and phenotype of cerebral AVMs are unknown. As a first step in understanding the pathophysiology of AVMs, the authors investigated the hypothesis that endothelial dysfunction-specifically, deregulation of endothelin-1 (ET-1) secretion-contributes to the abnormal vascular phenotype and the lack of hemodynamic autoregulation elaborated by these lesions. Endothelin-1 peptide and preproendothelin-1 (ppET1) messenger RNA were not detected in the intranidal vasculature of all 17 patients with AVMs studied, but were prominently expressed in human control subjects with normal cerebrovasculature (p < 0.01). Although AVM vasculature lacked ET-1, its expression was prominent in vasculature distant from these lesions, suggesting local repression of the ppET-1 gene. Local repression of ET-1 was specific to AVMs; ET-1 in vascular malformations of patients with Sturge-Weber disease was actually elevated compared to normal controls (p < 0.01). Repression of the ppET-1 gene was an intrinsic phenotype of AVM endothelial cells and was not due to factors in the AVM microenvironment. The authors also showed that ETA receptor expression was low in AVM vasculature compared to normal controls. Together, these results demonstrate that the ppET-1 gene is locally repressed in AVM lesions and suggest a role for abnormal ppET-1 gene regulation in the pathogenesis and clinical sequelae of cerebral AVMs.