Induction Of Uremia Research Articles

Uremia does not affect neointima formation in mice

Atherosclerotic cardiovascular disease is a major complication of chronic kidney disease (CKD). CKD leads to uremia, which modulates the phenotype of aortic smooth muscle cells (SMCs). Phenotypic modulation of SMCs plays a key role in accelerating atherosclerosis. We investigated the hypothesis that uremia potentiates neointima formation in response to vascular injury in mice. Carotid wire injury was performed on C57BL/6 wt and apolipoprotein E knockout (Apoe−/−) mice two weeks after induction of uremia by 5/6 nephrectomy. Wire injury led to neointima formation and downregulation of genes encoding classical SMC markers (i.e., myocardin, α-smooth muscle actin, SM22-alpha, and smooth muscle myosin heavy chain) in both wt and Apoe−/− mice. Contrary to our expectations, uremia did not potentiate neointima formation, nor did it affect intimal lesion composition as judged from magnetic resonance imaging and histological analyses. Also, there was no effect of uremia on SMC marker gene expression in the injured carotid arteries, suggesting that there may be different effects of uremia on SMCs in different vascular beds. In conclusion, uremia does not accelerate neointima formation in response to wire injury of the carotid artery in mice.

Signaling of Serum Amyloid A Through Receptor for Advanced Glycation End Products as a Possible Mechanism for Uremia-Related Atherosclerosis.

Cardiovascular disease is the leading cause of death in patients with end-stage renal disease. Serum amyloid A (SAA) is an acute phase protein and a binding partner for the multiligand receptor for advanced glycation end products (RAGE). We investigated the role of the interaction between SAA and RAGE in uremia-related atherogenesis. We used a mouse model of uremic vasculopathy, induced by 5 of 6 nephrectomy in the Apoe(-/-) background. Sham-operated mice were used as controls. Primary cultures of Ager(+/+) and Ager(-/-) vascular smooth muscle cells (VSMCs) were stimulated with recombinant SAA, S100B, or vehicle alone. Relevance to human disease was assessed with human VSMCs. The surface area of atherosclerotic lesions at the aortic roots was larger in uremic Apoe(-/-) than in sham-operated Apoe(-/-) mice (P<0.001). Furthermore, atherosclerotic lesions displayed intense immunostaining for RAGE and SAA, with a pattern similar to that of α-SMA. Ager transcript levels in the aorta were 6× higher in uremic animals than in controls (P<0.0001). Serum SAA concentrations were higher in uremic mice, not only after 4 weeks of uremia but also at 8 and 12 weeks of uremia, than in sham-operated animals. We investigated the functional role of RAGE in uremia-induced atherosclerosis further, in animals lacking RAGE. We found that the induction of uremia in Apoe(-/-) Ager(-/-) mice did not accelerate atherosclerosis. In vitro, the stimulation of Ager(+/+) but not of Ager(-/-) VSMCs with SAA or S100B significantly induced the production of reactive oxygen species, the phosphorylation of AKT and mitogen-activated protein kinase-extracellular signal-regulated kinases and cell migration. Reactive oxygen species inhibition with N-acetyl cysteine significantly inhibited both the phosphorylation of AKT and the migration of VSMCs. Similar results were obtained for human VSMCs, except that the phosphorylation of mitogen-activated protein kinase-extracellular signal-regulated kinases, rather than of AKT, was subject to specific redox-regulation by SAA and S100B. Furthermore, human aortic atherosclerotic sections were positively stained for RAGE and SAA. Uremia upregulates SAA and RAGE expression in the aortic wall and in atherosclerotic lesions in mice. Ager(-/-) animals are protected against the uremia-induced acceleration of atherosclerosis. SAA modulates the functions of murine and human VSMCs in vitro in a RAGE-dependent manner. This study, therefore, identifies SAA as a potential new uremic toxin involved in uremia-related atherosclerosis through interaction with RAGE.

Role of Local Versus Systemic Vitamin D Receptors in Vascular Calcification

Calcitriol and various analogs are commonly used to suppress secondary hyperparathyroidism in chronic kidney disease but may also exacerbate vascular calcification. Although this could be because of increased intestinal calcium and phosphate absorption, direct effects through vitamin D receptors (VDRs) on vascular smooth muscle have also been proposed. The role of these receptors was investigated by examining gene regulation in rat aortas treated with calcitriol ex vivo and in vivo and by transplanting aortas from VDR-null (VDR(-/-)) mice into wild-type mice before induction of uremia and treatment with calcitriol. In cultured rat aortas, calcitriol increased the expression of mRNA for CYP24A1 but not mRNA for any bone-related or calcification-related genes. Gene expression in aortas in vivo was not altered by doses of calcitriol that promote calcification. Calcitriol markedly increased aortic calcification in uremic mice and this did not differ between VDR(-/-) aortic allografts and VDR(+/+) recipient aortas. Calcitriol promotes vascular calcification through a systemic action rather than through a direct vascular action.

Antiatherogenic effect of pioglitazone on uremic apolipoprotein E knockout mice by modulation of the balance of regulatory and effector T cells

ObjectiveUremia markedly accelerates atherogenesis, but the pathogenesis remains to be elucidated and effective anti-atherogenic treatments are needed. The aim of this study was to investigate the relationship between accelerated atherosclerosis (AS) and the balance of regulatory/effector T cells (Treg/Teff) in uremic apolipoprotein E knockout (apoE−/−) mice, and the effect of pioglitazone on uremic AS and possible mechanisms. Methods and resultsUremia was induced surgically in 8-week-old male apoE−/− mice. Two weeks after induction of uremia, the mice were randomized to receive pioglitazone (daily oral gavage with 20mg/kg) or vehicle. Control apoE−/− mice were sham-operated and received vehicle. After 8 weeks’ treatment, all mice were sacrificed. The cross-sectional area of atherosclerotic lesions at the aortic root was significantly larger and plaques were unstable in uremic mice, which was associated with a Treg/Teff imbalance (Treg down-regulated/Teff up-regulated) compared with controls. Renal function and the percentage of Treg cells in splenocytes were negatively correlated in control and uremic mice that received vehicle. Treatment with pioglitazone dramatically inhibited AS progression, stabilized plaque and modulated the Treg/Teff imbalance (up-regulated Treg/down-regulated Teff) in uremic mice, without influencing serum lipid profiles and blood glucose. In vitro, oxidized low density lipoprotein induced a Treg/Teff imbalance in splenocytes from uremic mice. Pioglitazone modulated the imbalance by upregulating Treg cells and downregulating Teff cells. The former was not abolished by the peroxisome proliferator-activated receptor (PPAR)γ antagonist GW9662, whereas the latter was completely abolished by GW9662. ConclusionPioglitazone ameliorates accelerated AS in uremic apoE−/− mice, probably through PPARγ-independent and -dependent mechanisms to modulate the Treg/Teff imbalance.

Progressive accumulation of lanthanum in the liver of normal and uremic rats

The loss of renal function in chronic kidney disease (CKD) patients leads to a variety of metabolic disturbances that promote the development of secondary hyperparathyroidism (SH) and bone disease. One of these alterations, hyperphosphatemia, is a persistent problem in patients with CKD and is an important factor in the development and severity of SH as well as coronary artery and cardiac valve calcification. To help control serum phosphorus (P), phosphate binders are used to minimize intestinal absorption of P in these patients. Since the use of aluminum- and calcium-based phosphate binders can result in severe side effects, other phosphate binders have been proposed. One of these is lanthanum (La) carbonate. Since there is some controversy as to the degree La is absorbed by the intestine, whether it is deposited in tissues, and what effect its prolonged use would have in patients with chronic renal failure, we studied normal and uremic rats fed a control diet or a diet with La carbonate (1.5% elemental La) for 45, 90, or 110 days. As expected, plasma creatinine increased after the induction of uremia and continued to increase with time. At 45 and 90 days, dietary La produced a reduction in plasma P in normal rats (N + La) [45 days: 3.15 +/- 0.35 vs. normal control (NC) 4.41 +/- 0.15 mg/dL, P < 0.01; 90 days: 3.20 +/- 0.30 vs. 4.09 +/- 0.15 mg/dL, P < 0.05], but not at 110 days. Plasma P was initially lower (45 days) in both groups of La-treated rats (U + La: 3.47 +/- 0.28 vs. UC: 4.91 +/- 0.27 mg/dL, P < 0.01), but was not at 90 and 110 days. Whole blood La content was not increased in rats fed the La diet and is, therefore, not a good indicator of the La content of tissue. La was increased in kidney, femur, and especially liver in normal and uremic rats fed the La diet. Uremia markedly enhanced tissue accumulation of La (liver, 110 days: N + La: 849 +/- 152 vs. U + La: 2676 +/- 479 ng/g wet wt, P < 0.01), which increased in a time-dependent manner. The increase in La in the liver of normal rats was 44-fold compared to control and 98-fold in the liver of uremic rats compared to uremic control. La accumulates in tissues, most strikingly in the liver, and renal failure accelerates the accumulation. While this was a relatively short-term study, La accumulation increased in a time-dependent manner. The effect of long-term La treatment on patients with chronic renal insufficiency needs to be evaluated.

A new analog of 1,25-(OH) 2D 3, 19-NOR-1,25-(OH) 2D 2, suppresses serum PTH and parathyroid gland growth in uremic rats without elevation of intestinal vitamin D receptor content

We have previously reported that 19-nor-1,25-(OH) 2D 2, a new analog of 1,25-(OH) 2D 3, suppresses parathyroid hormone (PTH) secretion in uremic rats in the absence of hypercalcemia or hyperphosphatemia. In the current study, we examined the effect of 19-nor-1,25-(OH) 2D 2on parathyroid gland growth and intestinal vitamin D receptor (VDR) content. After induction of uremia by 5/6 nephrectomy, rats were divided into five experimental groups and received intraperitoneal injections of vehicle, 1,25-(OH) 2D 3 (2 or 6 ng/rat), or 19-nor-1,25-(OH) 2D 2 (25 or 100 ng/ rat) three times a week for 8 weeks. Twelve normal rats received vehicle and served as the normal control group. During the course of the study, rats were maintained on a 1.0% calcium and 0.8% phosphorus diet. The higher dose of 1,25-(OH) 2D 3, 6 ng, significantly decreased PTH from 52.7 ± 10.2 pg/mL in the uremic control group to 25.7 ± 6.7 pg/mL ( P < 0.01). This dose of 1,25-(OH) 2D 3, however, increased serum levels of both ionized calcium (4.71 ± 0.05 to 4.85 ± 0.06 mg/dL; P < 0.05) and phosphorus (4.34 ± 0.30 to 6.67 ± 0.63 mg/dL; P < 0.01). Both doses of 19-nor-1,25-(OH) 2D 2 decreased serum PTH as effectively as 1,25-(OH) 2D 3 without changes in serum calcium or phosphorus. The 100-ng dose of 19-nor-1,25-(OH) 2D 2 decreased PTH to 20.7 ± 3.1 pg/mL ( P < 0.01) and suppressed parathyroid gland growth by more than 50%. Both doses of 19-nor-1,25-(OH) 2D 2 also decreased endogenous 1,25-(OH) 2D 3 levels compared with uremic control rats (25 ng: 30.4 ± 2.0, P < 0.05, and 100 ng: 27.9 ± 3.2, P < 0.01, v 48.4 ± 6.6 pg/mL). The 6-ng dose of 1,25-(OH) 2D 3 elevated intestinal VDR content (138.5 ± 20.0 fmol/mg protein) compared with animals receiving both doses of 19-nor-1,25-(OH) 2D 2 (25 ng: 84.0 ± 11.9, P < 0.05, and 100 ng: 78.4 ± 10.9, P < 0.01). This was probably attributable to the marked decrease in endogenous 1,25-(OH) 2D 3 levels caused by both doses of 19-nor-1,25-(OH) 2D 2 because intestinal VDR correlated directly with serum 1,25-(OH) 2D 3 ( r = 0.963; P = 0.008). Thus, 19-nor-1,25-(OH) 2D 2 appears to exert a selective action on the parathyroid glands compared with the intestine. Its low calcemic and phosphatemic properties may result from the decreased endogenous 1,25-(OH) 2D 3 levels that lead to a reduction in intestinal VDR. This selectivity makes this analog ideal for the treatment of secondary hyperparathyroidism.

An Animal Model for the Study of Amino Acid Metabolism in Uremia and during Peritoneal Dialysis

We tried to determine the suitability of the rabbit as an animal model to study amino acid (AA) metabolism in continuous ambulatory peritoneal dialysis. We also measured the effect of intraperitoneal (ip) infusion of AA on blood AA changes and food consumption. Plasma AA levels were measured in 10 normal rabbits after an overnight fasting and 30, 60, and 120 minutes after a meal. Following these baseline observations, rabbits were randomly divided into two groups. One group of five rabbits was made uremic after surgical partial nephrectomy, whereas the remaining (controls) underwent sham operations. Two weeks after the induction of uremia we measured the effect of chronic renal failure on fasting and postprandial (30, 60, 120 minutes) plasma AA levels. Upon the completion of the second experiment (4 weeks after the induction of uremia) we studied the effect of an ip AA on plasma AA profile 1, 2, 4, and 6 hours after the infusion in both uremic and control rabbits. We also measured the food intake in all experiments. The results of our experiments showed the following: 1. plasma AA in the rabbits decreased after induction of chronic renal failure and increased after food ingestion and ip infusion of AA solution; 2. neither induction of uremia nor ip AA infusion have an effect on food consumption; 3. the majority of the alterations in plasma AA levels we observed in the uremic rabbits were similar to those observed in humans, indicating that the rabbit may be a suitable model for the study of AA metabolism in chronic renal failure and during peritoneal dialysis.

Expression of hepatic microsomal cytochrome P450s as altered by uremia

The proportions of different hepatic microsomal cytochrome P450s expressed in uremic rats were studied with specific antibodies and with a steroid hydroxylase assay. In male uremic rats, the hepatic levels of P450 2C11, a male-specific form, and 3A2, a male-dominant form, were decreased to about 30% at 5 weeks after the induction of uremia. These changes were paralleled by decreases in the activities of testosterone 2α-, 16α-, and 6β-hydroxylation. The level of P450 2A1, abundant in immature rats, was increased 2-fold by uremia and accompanied by an increase in testosterone 7α-hydroxylation activity. The levels of P450 2C6 and 2E1 were not changed by uremia. The levels of male-specific and male-dominant forms such as P450 2C11 and 3A2 are affected by the serum level of testosterone, which was decreased in the male rats with uremia. Therefore, castrated rats were prepared to compare the effects of testosterone on hepatic cytochrome P450s in uremic rats with those in castrated rats. When testosterone was administered to the castrated rats, the decreased levels of both P450 2C11 and 3A2 returned to normal. However, the administration of testosterone to the uremic rats did not prevent the decrease in the levels of these P450s. In female rats, changes in the levels of cytochrome P450s were not as great during uremia as those in male uremic rats. The level of P450 2C12, a female-specific form, was not changed; the level of P450 2A1 was increased by 50%, that of 3A2 which is barely detected in female rats was increased by 60%, and that of 2E1 was increased by 25%. These results suggested that the changes in the hepatic levels of cytochrome P450s were affected by factors other than testosterone in uremic rats.

Cerebral vascular changes associated with hemorrhagic stroke in hypertension

There are a number of alterations that protect the cerebrovasculature from hemorrhagic stroke development during hypertension. The upper limit of cerebral blood flow autoregulation is shifted to higher blood pressure levels; this allows a constant blood flow to be maintained during hypertension. Studies we have performed have indicated that the middle cerebral arteries (MCA) of Wistar-Kyoto stroke-prone spontaneously hypertensive rats (spSHR) lose their ability to constrict in response to elevations in transmural pressure. The decline in such function precedes stroke development and totally disappears at an age where there is a 100% mortality from stroke. Prior to stroke development, spSHR also develop uremic conditions and signs of renal failure. The induction of uremia in stroke-resistant SHR (srSHR) via nephrectomy induces these animals to develop stroke. Like prestroke spSHR, prestroke uremic srSHR also have MCA with attenuated pressure-dependent myogenic function. It is hypothesized that the inability to increase vascular resistance in response to elevations in pressure might promote overperfusion of the more distal vasculature leading to cerebral hemorrhage formation. Since uremia promotes bleeding tendencies, such alterations along with the loss of cerebrovascular myogenic function could initiate or aggravate hemorrhage formation.

Phosphorus restriction reverses hyperparathyroidism in uremia independent of changes in calcium and calcitriol

Phosphorus is a well-known modulator of renal 1 alpha-hydroxylase activity. In early and moderate renal failure it is proposed that dietary Pi reduction ameliorates secondary hyperparathyroidism through increased circulating levels of calcitriol (i.e, 1 alpha, 25-dihydroxycholecalciferol). To gain further insight into the mechanisms by which a low-Pi diet ameliorates secondary hyperparathyroidism in advanced renal insufficiency, studies were performed in five dogs before and 6 mo after the induction of uremia by 5/6 nephrectomy. Glomerular filtration rate decreased from 69.0 +/- 2.3 to 10.5 +/- 0.5 ml/min, immunoreactive parathyroid hormone (irPTH) increased from 66.0 +/- 8.8 to 321.0 +/- 46 pg/ml, and calcitriol decreased from 39.0 +/- 10.4 to 27.0 +/- 6.2 pg/ml. Thereafter, dietary Pi was decreased gradually every 2 wk from 0.95% to 0.6, 0.45, and 0.3%, respectively. Dietary Ca was reduced from 1.6 to 0.6% to prevent development of hypercalcemia. Ionized Ca (ICa) decreased from 5.4 +/- 0.04 to 5.2 +/- 0.05 mg/dl (P less than 0.02), and plasma Pi decreased from 6.3 +/- 0.7 to 4.7 +/- 0.2 mg/dl (P less than 0.05). Calcitriol remained low (23.3 +/- 4.7 pg/ml). However, irPTH gradually decreased from 321.0 +/- 46.0 to 94.7 +/- 22.9 pg/ml (P less than 0.005). These studies indicate that a decrease in dietary Pi from 0.95 to 0.3% suppressed irPTH by approximately 70%. Reduction of irPTH was observed in the absence of a concomitant increase in levels of ICa or calcitriol. These studies suggest that reduction in dietary Pi in advanced renal insufficiency improves secondary hyperparathyroidism by a mechanism that is independent of the levels of calcitriol or plasma ICa.

Kinetics of infused acetate and effect on plasma pyruvate and lipid concentrations in uremic and non-uremic dogs.

During acetate infusion at a rate of 10 millimoles/kg/hr arterial blood acetate levels rose progressively to maximums of 7.9 +/- 1.7 mM in uremic dogs and 10.8 +/- 1.4 mM in non-uremic dogs. Following cessation of infusion, removal of acetate followed first order kinetics. Acute uremia had no significant effect on mean clearance rates of acetate (1.28 +/- 0.28 L/kg/hr uremics vs 0.92 +/- 0.22 in non-uremics) or upon blood half-life of acetate following infusion (9.7 min. vs 10.9 min.). Plasma pyruvate levels rose during infusion from 1.5 to 4.4 mg/dl in the uremic dogs and following infusion rose further to 6.5 mg/dl. In the non-uremic dogs pyruvate was not significantly elevated until 30 min. post-infusion. Plasma free fatty acids increased from 79 to 131 mumoles/dl during acetate infusion in the uremic dogs, but did not change significantly in the non-uremic group. Plasma cholesterol and triglycerides increased after induction of uremia, but showed no significant changes as a result of acetate infusion in either group. These results suggest that the electrolyte and lipid abnormalities that occur in hemodialyzed uremic patients may be related to the acetate load these patients receive during dialysis.

Skeletal resistance to the calcemic action of parathyroid hormone in uremia: Role of 1,25(OH)2D3

Studies were carried out to investigate the role of 1,25(OH)2D3 in the skeletal resistance to the calcemic action of parathyroid hormone. The change in serum calcium after the intravenous infusion of 2 U of parathyroid extract (PTE)/kg body wt/hr for eight hours was evaluated in thyroparathyroidectomized (T-PTX) dogs before, and one, two and three days after, induction of uremia by bilateral ureteral ligation (11 dogs) or by bilateral nephrectomy (8 dogs). In another six nephrectomized and T-PTX dogs, 0.68 ug of 1, 25 (OH)2D3/day was given on the day of nephrectomy and for two days thereafter. Serum creatinine in each day of the study was not different among the three groups. The study also included the evaluation of the effect of sham operation (five dogs) and the administration of 1,25 (OH)2D3 to dogs with normal renal function (four dogs) on the calcemic response to PTE, as well as the reproducibility of such a response in the same animal. The results showed that 1) the calcemic response to PTE was markedly impaired after one day of bilateral ureteral ligation or nephrectomy, but the impairment was more severe after nephrectomy; 2) the calcemic response to PTE after two or three days of bilateral ureteral ligation was similar to that seen at one day after nephrectomy; 3) 1, 25 (OH)2D3 partially restored the calcemic response to PTE in the nephrectomized animals to levels similar to those seen after one day of bilateral ureteral ligation; 4) sham operation did not affect the response to PTE, and repeated infusion of PTE produced similar changes in the concentrations of serum calcium. The data indicate that (a) a deficiency of 1,25 (OH)2D3 is at least partly responsible for the skeletal resistance to the calcemic action of PTH in uremia; (b) uremia, per se, may also contribute to this phenomenon; and (c) the kidney after one day of complete bilateral ureteral ligation may still produce 1,25 (OH)2D3, but this ability is compromised after two days of ureteral obstruction.

Changes in the electroencephalogram in acute uremia. Effects of parathyroid hormone and brain electrolytes.

Studies were carried out in order to evaluate the effects of changes in brain calcium and the influence of parathyroidectomy and administration of parathyroid extract on the electroencephalogram (EEG) of normal and uremic dogs. Manual analysis of frequency and power distribution of the EEG in uremic dogs revealed a significant increase in both the percentage distribution and the area or power occupied by frequencies below 5 Hz. In addition, high amplitude bursts of delta activity were apparent in the uremic dog. These changes were largely prevented by parathyroidectomy before the induction of uremia, but the administration of parathyroid extract to either normal dogs, or to previously parathyroidectomized uremic dogs, induced EEG changes similar to those noted in uremic animals with intact parathyroid glands. In all groups of animals which showed EEG changes, brain content of calcium was significantly higher than in either normal dogs or previously parathyroidectomized uremic dogs. Changes in arterial pH and bicarbonate, or in the concentrations of Na+, K+, urea, or creatinine in plasma or cerebrospinal fluid were similar in uremic animals with intact parthyroid glands and in previously parathyroidectomized uremia dogs. The results indicate that the EEG changes found in dogs with acute renal failure require the presence of excess parathyroid hormone in blood, and they may be related to the observed changes in brain content of calcium.

Effect of Experimental Chronic Renal Insufficiency on Bone Mineral and Collagen Maturation

The effect of chronic renal disease on bone matrix and mineral maturation was evaluated in rats with experimental renal insufficiency of 2-11 wk duration utilizing bromoform-toluene gradient fractionation of bone powder, pulse labeling experiments with (45)Ca and proline-(3)H differential extraction, and X-ray diffraction techniques.Maturation defects in both collagen and mineral ((45)Ca) metabolism were documented as early as 2 wk after the induction of uremia, when total bone calcium, inorganic phosphate, and hydroxyproline content were unchanged. The maturational defect progressed with advancing uremia despite insignificant changes in plasma pH and calcium, and normal bone carbonate levels. Although circulating levels of 25-hydroxycholecalciferol were significantly lower than normal in the uremic animals, pretreatment with either this vitamin D metabolite or vitamin D(3) itself failed to alter the observed changes in skeletal maturation.