Experimental Chronic Renal Insufficiency Research Articles

Vasopeptidase Inhibition Corrects the Structure and Function of the Small Arteries in Experimental Renal Insufficiency

Background: We studied whether vasopeptidase inhibition corrects the structure and function of the small arteries in experimental chronic renal insufficiency (CRI). Methods: After 5/6 nephrectomy (NX) surgery was performed on rats, there was a 14-week follow-up, allowing CRI to become established. Omapatrilat (40 mg/kg/day in chow) was then given for 8 weeks, and the small mesenteric arterial rings were investigated in vitro using wire and pressure myographs. Results: Plasma and ventricular B-type natriuretic peptide (BNP) concentrations were increased 2- to 2.7-fold, while systolic blood pressure (BP) increased by 32 mm Hg after NX. Omapatrilat treatment normalized the BNP and reduced the BP by 45 mm Hg in the NX rats. Endothelium-dependent vasorelaxation was impaired but the response to acetylcholine was normalized after omapatrilat treatment. Vasorelaxations induced by nitroprusside, isoprenaline and levcromakalim were enhanced after omapatrilat, and the responses were even more pronounced than in untreated sham-operated rats. Arterial wall thickness and wall-to-lumen ratio were increased after NX, whereas omapatrilat normalized these structural features and improved the strain-stress relationship in the small arteries; this suggests improved arterial elastic properties. Conclusion: Omapatrilat treatment reduced BP, normalized volume overload, improved vasorelaxation and corrected the dimensions and passive elastic properties of the small arteries in the NX rats. Therefore, we consider vasopeptidase inhibition to be an effective treatment for CRI-induced changes in the small arteries.

Phosphate Binding Reduces Aortic Angiotensin-Converting Enzyme and Enhances Nitric Oxide Bioactivity in Experimental Renal Insufficiency

Background: Disturbed calcium-phosphorus metabolism is associated with increased kidney angiotensin-converting enzyme (ACE) in experimental chronic renal insufficiency (CRI). However, information about the effects of phosphate binding and loading on vascular ACE is lacking. Methods: Fifteen weeks after 5/6 nephrectomy (NX), rats were placed on a phosphate-binding (NX+Ca, 3.0% Ca), phosphate-loading (NX+Pi, 1.5% Pi), or control diet for 12 weeks (NX and sham). Results: Aortic ACE, blood pressure, plasma phosphate, and parathyroid hormone were increased in the NX and NX+Pi groups, but were reduced with phosphate binding. Endothelium-mediated relaxations of isolated mesenteric conduit artery rings to acetylcholine were impaired in the NX and NX+Pi groups, but did not differ from sham in NX+Ca rats. Experiments with nitric oxide (NO) synthase inhibition in vitro suggested that the NO-mediated component of acetylcholine response was lower in the NX and NX+Pi groups, but did not differ from sham in NX+Ca rats. In all NX groups, aortic endothelial NO synthase (eNOS) was reduced, while plasma and urine concentrations of NO metabolites were increased. Aortic nitrated proteins and calcification were increased in the NX and NX+Pi groups when compared with the NX+Ca and sham groups. Conclusion: Hypertension in the NX model of CRI was associated with reduced vasorelaxation, decreased eNOS, and increased ACE and nitrated proteins in the aorta. Phosphate binding with calcium carbonate enhanced vasorelaxation via endogenous NO and suppressed elevation of ACE and nitrated proteins, suggesting reduced vascular oxidative stress. Our findings support the view that correction of the calcium-phosphorus balance prevents CRI-induced vascular pathophysiology.

Mechanistic analysis for time-dependent effects of cinacalcet on serum calcium, phosphorus, and parathyroid hormone levels in 5/6 nephrectomized rats

This study investigates the time-dependent effects of cinacalcet on serum calcium, phosphorus, and parathyroid hormone (PTH) levels in 5/6 nephrectomized (NX) rats with experimental chronic renal insufficiency. In this study, 5/6 NX male, Sprague–Dawley rats were treated with vehicle or cinacalcet (10 mg/kg, oral, 1× daily). On Day 0 (before treatment), Day 12 and 13 after treatment (to approximate the clinical practice), and also at 0, 1, 4, 8, 16, and 24 hours after the last dosing, blood was collected for analysis. After 12 or 13 days of cinacalcet treatment, modest changes were observed in serum Ca and phosphorus (Pi), while PTH decreased by >45% to Sham levels (152 ± 15 pg/mL). Detailed mapping found that cinacalcet caused a significant time-dependent decrease in serum Ca following dosing, reaching a lowest point at 8 hours (decrease by 20% to 8.43 ± 0.37 mg/dL), and then returning to normal at 24 hours. Cinacalcet also caused a significant increase in serum Pi levels (by 18%). To investigate the potential mechanism of action, a broad approach was taken by testing cinacalcet in a panel of 77 protein-binding assays. Cinacalcet interacted with several channels, transporters, and neurotransmitter receptors, some of which are involved in brain and heart, and may impact Ca homeostasis. Cinacalcet dose-dependently increased brain natriuretic peptide (BNP) mRNA expression by 48% in cardiomyocytes, but had no significant effects on left ventricular hypertrophy and cardiac function. The results suggest that cinacalcet's hypocalcemic effect may be due to its nonspecific interaction with other receptors in brain and heart.

Vitamin D Receptor Activation Mitigates the Impact of Uremia on Endothelial Function in the 5/6 Nephrectomized Rats

Endothelial dysfunction increases cardiovascular disease risk in chronic kidney disease (CKD). This study investigates whether VDR activation affects endothelial function in CKD. The 5/6 nephrectomized (NX) rats with experimental chronic renal insufficiency were treated with or without paricalcitol, a VDR activator. Thoracic aortic rings were precontracted with phenylephrine and then treated with acetylcholine or sodium nitroprusside. Uremia significantly affected aortic relaxation (−50.0 ± 7.4% in NX rats versus −96.2 ± 5.3% in SHAM at 30 μM acetylcholine). The endothelial-dependent relaxation was improved to –58.2 ± 6.0%, –77.5 ± 7.3%, and –90.5 ± 4.0% in NX rats treated with paricalcitol at 0.021, 0.042, and 0.083 μg/kg for two weeks, respectively, while paricalcitol at 0.042 μg/kg did not affect blood pressure and heart rate. Parathyroid hormone (PTH) suppression alone did not improve endothelial function since cinacalcet suppressed PTH without affecting endothelial-dependent vasorelaxation. N-omega-nitro-L-arginine methyl ester completely abolished the effect of paricalcitol on improving endothelial function. These results demonstrate that VDR activation improves endothelial function in CKD.

Reduced expression of the renal calcium-sensing receptor in rats with experimental chronic renal insufficiency.

Chronic renal insufficiency is associated with elevated serum parathyroid hormone (PTH) levels (2 degrees HPT), deficiency of 1,25-dihydroxyvitamin D (1,25(OH)2D), and hypocalciuria. In chronic renal insufficiency, the 2 degrees HPT may result from reduced expression of the parathyroid gland extracellular Ca(2+)-sensing receptor (CaSR). Since the CaSR was cloned from rat and human kidney, this study examined in rats whether expression of the renal CaSR is altered in experimental chronic renal insufficiency. Four weeks after chronic renal insufficiency was induced by 5/6 nephrectomy (Nx) in Sprague Dawley rats, the serum creatinine concentration was 0.96+/-0.06 mg/dl compared with 0.35+/-0.02 mg/dl in sham-operated animals (P < 0.05). The serum total Ca2+ and phosphorus concentrations were not different. In the Nx group, the serum concentration of amino-PTH was higher (65+/-8 pg/ml), and the concentration of 1,25(OH)2D was significantly lower (47+/-5 pg/ml) compared with 45+/-5 pg/ml and 61+/-4 pg/ml (P = 0.05) in the sham group, respectively. In a subset of rats studied, the Nx group was hypocalciuric (1.4+/-0.5 mg/kg per d) compared with the sham group (3.7+/-0.5 mg/kg per d) (P < 0.05). In the Nx rats, CaSR mRNA expression and CaSR protein levels were found to be reduced by 35 and 38%, respectively, than those observed in controls. These results suggest that reduced renal CaSR expression in chronic renal insufficiency may play a role in disordered mineral ion homeostasis, including hypocalciuria.

Changes in cyclosporine A kinetics after experimental reduction of renal parenchyma.

Experimental chronic renal insufficiency (produced by 5/6 ablation of renal parenchyma) is associated with changes in the kinetics of oral (intragastric) cyclosporine A (CyA). Compared with animals with intact renal parenchyma, significantly lower levels of CyA are reached under these conditions. The factors responsible for reduced CyA availability under these conditions have not yet been identified.

Benefit of moderate dietary protein restriction on growth in the young animal with experimental chronic renal insufficiency: importance of early growth.

Using a model of unilateral nephrectomy and contralateral heminephrectomy in weanling rats, we studied the effect of severe (6%) and moderate (14%) protein restriction on growth and renal function up to 24 wk of age. One-half the normal (N) and chronic renal insufficiency (CRI) rats underwent a cross-over protocol 6%----14% and 14%----6% at 10wk of age to analyze the contribution of early growth on ultimate wt and length. N or CRI fed a 6% protein diet were the smallest. Animals switched from 6%----14% protein diet did not catch up to their counterparts started on a 14% protein diet (N or CRI). However, N or CRI animals started on a 14% protein diet and switched to a 6% protein diet grew along a curve similar to animals fed a 14% protein diet. Creatinine clearances for CRI rats were not different among the groups. However, protein excretion was highest in those animals completing the study on a 14% protein diet. Growth failure caused by severe protein restriction occurred primarily in the early accelerated growth phase (3-10 wk). We conclude that moderate protein restriction during the early rapid growth phase maintains growth, whereas severe protein restriction is detrimental to growth. Switching to a more restrictive protein intake after the rapid growth phase did appear to offer more renal protection (lower urinary protein excretion) at the end of 24 wk. Severe protein restriction to preserve renal function is a poor strategy for the young rapidly growing individual with chronic renal disease.

Tryptophan distribution and metabolism in experimental chronic renal insufficiency

Several aspects of tryptophan distribution and metabolism in chronic renal insufficiency (CRI) were investigated in a rat model prepared by partial nephrectomy. Partially nephrectomized (pNx) rats with a moderate degree of CRI demonstrated a 40% decrease in plasma total tryptophan concentration between 5 and 11 weeks after the acute reduction of renal functional mass. This decrease was accompanied by hypoalbuminemia, polyuria, albuminuria, and tryptophanuria. After 5 weeks of sustained plasma total tryptophan deficiency (from Week 6 to Week 11), the plasma free tryptophan concentration, the plasma concentrations of large neutral amino acids, and the tryptophan levels in red cells, liver, and kidney of the pNx rats were similar to those of the controls. However, evidence for abnormal brain tryptophan metabolism in pNx rats after 11 weeks of CRI included 16% reductions of tryptophan levels in the midbrain and pons and 65% increases in the serotonin contents of the hypothalamus and medulla. Monoamine oxidase activities in hypothalamus and cerebellum of pNx rats were the same as those of the controls. These studies indicate that tryptophanuria is an important factor in the development of the plasma tryptophan deficiency in the pNx model. In addition, the results support the hypothesis that regional abnormalities in tryptophan metabolism contribute to the neurological and neuroendocrine dysfunction of CRI.

Thymic functions in uremic rats: evidence for thymosin alpha 1 deficiency.

Changes in the circulating lymphocyte populations and thymus glands were studied in rats with experimental chronic renal insufficiency (CRI). Compared to normal or sham-operated animals, rats with CRI had significant reduction in the percentages of circulating T and B lymphocytes. CRI was also associated with marked thymic atrophy and reduction in the numbers of small cortical and medullary thymic lymphocytes. Quantitative microfluorometry revealed a significant reduction in the intrathymic concentration of a potent immunomodulator, thymosin alpha 1 in all uremic animals. There was a significant positive correlation between the percentages of circulating T lymphocytes and the intrathymic concentrations of thymosin alpha 1.

Increased histidine and histamine content in the brain of chronic uremic rats. Cause of enhanced cerebral cyclic adenosine monophosphate in uremia?

In rats with experimental chronic renal insufficiency (90% nephrectomy) the histidine content in brain was increased (+ 35%) in spite of normal plasma values and decreased concentrations in the striated muscle (− 23%). The finding of a raised histidine level in the brain seems to be a uremia specific disorder, probably caused by a local disturbance in histidine metabolism. In addition an increase of the histidine decarboxylation product histamine could be observed in the brain of rats with chronic renal insufficiency, as compared to pair-fed controls. This increase was directly related to the severity of azotemia. In the pathogenesis of the histamine alteration the increased histidine content in the brain of uremic rats must be considered, since the specific histidine decarboxylase is not saturated by the normal endogenous level of the amino acid precursor. Probably the increased histamine contributes to the raised cerebral cyclic AMP in the brain of uremic rats.

Hyperlipoproteinemia in experimental chronic renal insufficiency in the rat

Lipid metabolism was studied in experimental uremia. Uremic (U) rats were compared with sham-operated, pair-fed (PF) controls and with ad-lib-fed (AL) controls. In U animals, fasting glucose concentrations were normal, immunoreactive serum insulin (IRI) levels were decreased, and immunoreactive glucagon levels were increased. A significant increase in the serum concentration of all lipid classes was observed: triglycerides were elevated 10-fold above the values in PF and AL controls; phospholipids, twofold; total cholesterol, threefold; and free cholesterol, sixfold. Cholesterol concentration was increased in beta- and pre-beta-lipoproteins and even more so in alpha- and pre-alpha-lipoproteins. There was an increase in the ratio of free cholesterol/total cholesterol. The fatty acid composition of serum lipoproteins was unchanged. Concomitantly, in liver tissue, there was no change in lipid content (triglyceride, cholesterol) and fatty acid composition. These findings argue against glucose- or insulin-mediated changes in hepatic de novo fatty acid synthesis, chain elongation, or poly-desaturation. In U animals, the HMG-CoA-reductase activity of liver microsomes was slightly, but not significantly, reduced as was tritiated water incorporation into cholesterol in isolated perfused liver preparations. In adipose tissue, there was a decrease in triglyceride content. The results provide evidence against insulin-mediated hepatic overproduction as a major cause of hyperlipoproteinemia in this model of experimental renal insufficiency and point to peripheral under-utilization of lipoproteins.

Activities of intestinal enzymes in experimental chronic renal insufficiency.

Rats with chronic uremia following five-sixths nephrectomy showed a significant fall in the sucrase and maltase activities in the small intestinal mucosa, the lactase and cellobiase activities in contrast remained uninfluenced. The activity of the L-leucyl-L-proline and L-methionyl-L-proline dipeptidases in the small intestinal mucosa was significantly increased, while the activities of seven other dipeptidases studied were unaffected. The mucosal protein and DNA content likewise remained unchanged. Occasional slight alterations of the mucosa were the only finding at histology.

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.