Sodium butyrate ameliorates insulin resistance and renal failure in CKD rats by modulating intestinal permeability and mucin expression.

BACKGROUND: The associated increase in the lipopolysaccharide (LPS) levels and uremic toxins in chronic kidney disease (CKD) has shifted the way we focus on intestinal microbiota. This study shows that a disruption of the intestinal barrier in CKD promotes leakage of LPS from the gut, subsequently decreasing insulin sensitivity. Butyrate treatment improved the intestinal barrier function by increasing colonic mucin and tight junction (TJ) proteins. This modulation further ameliorated metabolic functions such as insulin intolerance and improved renal function. METHODS: Renal failure was induced by 5/6th nephrectomy (Nx) in rats. A group of Nx and control rats received sodium butyrate in drinking water. The Nx groups were compared with sham-operated controls. RESULTS: The Nx rats had significant increases in serum creatinine, urea and proteinuria. These animals had impaired glucose and insulin tolerance and increased gluconeogenesis, which corresponded with decreased glucagon-like peptide-1 (GLP-1) secretion. The Nx animals suffered significant loss of intestinal TJ proteins, colonic mucin and mucin 2 protein. This was associated with a significant increase in circulating LPS, suggesting a leaky gut phenomenon. 5'adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, known to modulate epithelial TJs and glucose metabolism, was significantly reduced in the intestine of the Nx group. Anti-inflammatory cytokine, interleukin 10, anti-bacterial peptide and cathelicidin-related antimicrobial peptide were also lowered in the Nx cohort. Butyrate treatment increased AMPK phosphorylation, improved renal function and controlled hyperglycemia. CONCLUSIONS: Butyrate improves AMPK phosphorylation, increases GLP-1 secretion and promotes colonic mucin and TJ proteins, which strengthen the gut wall. This decreases LPS leakage and inflammation. Taken together, butyrate improves metabolic parameters such as insulin resistance and markers of renal failure in CKD animals.

High Fat High Cholesterol Diet (Western Diet) Aggravates Atherosclerosis, Hyperglycemia and Renal Failure in Nephrectomized LDL Receptor Knockout Mice: Role of Intestine Derived Lipopolysaccharide.

A high fat meal, frequently known as western diet (WD), exacerbates atherosclerosis and diabetes. Both these diseases are frequently associated with renal failure. Recent studies have shown that lipopolysaccharide (LPS) leaks into the circulation from the intestine in the setting of renal failure and after WD. However, it is not clear how renal function and associated disorders are affected by LPS. This study demonstrates that circulatory LPS exacerbates renal insufficiency, atherosclerosis and glucose intolerance. Renal insufficiency was induced by 2/3 nephrectomy in LDL receptor knockout mice. Nx animals were given normal diet (Nx) or WD (Nx+WD). The controls were sham operated animals on normal diet (control) and WD (WD). To verify if LPS plays a role in exaggerating renal insufficiency, polymyxin (PM), a known LPS antagonist, and curcumin (CU), a compound known to ameliorate chronic kidney disease (CKD), was given to Nx animals on western diet (Nx+WD+PM and Nx+WD+CU, respectively). Compared to control, all other groups displayed increased circulatory LPS. The Nx+WD cohort had the highest levels of LPS. Nx group had significant renal insufficiency and glucose intolerance but not atherosclerosis. WD had intense atherosclerosis and glucose intolerance but it did not show signs of renal insufficiency. Compared to other groups, Nx+WD had significantly higher cytokine expression, macrophage infiltration in the kidney, renal insufficiency, glucose intolerance and atherosclerosis. PM treatment blunted the expression of cytokines, deterioration of renal function and associated disorders, albeit not to the levels of Nx, and was significantly inferior to CU. PM is a non-absorbable antibiotic with LPS binding properties, hence its beneficial effect can only be due to its effect within the GI tract. We conclude that LPS may not cause renal insufficiency but can exaggerate kidney failure and associated disorders following renal insufficiency.

Reciprocal changes of renal neuronal nitric oxide synthase-α and -ß associated with renal progression in a neonatal 5/6 nephrectomized rat model.

BACKGROUND: Nitric oxide (NO) deficiency contributes to chronic kidney disease progression. NO deficiency could occur for many reasons, one of which is decreased NO synthase (NOS) abundance and/or activity. METHODS: In these experiments, we studied two groups of male Sprague Dawley rats given sham or surgical excision of both poles of the left kidney (at 2 days of age) followed by sham or surgical removal of the right kidney at 10 days. Rats were sacrificed at 9 weeks of age and the kidneys examined for abundance of neuronal NOS (nNOS)-α and -ß, endothelial NOS, arginase II, argininosuccinate synthase and lysate, protein arginine methyltransferase 1, dimethylarginine dimethylamino-hydrolase 1 and 2, as well as renal pathology. RESULTS: The 5/6 nephrectomy (NX) group showed renal dysfunction, severe rapidly progressing glomerulosclerosis, and hypertension. Renal cortical nNOSα abundance was significantly reduced, whereas nNOSß abundance was increased in the 5/6 NX group versus sham. Renal endothelial NOS was unchanged. Next, renal protein arginine methyltransferase 1 abundance was higher, whereas dimethylarginine dimethylamino-hydrolase 2 expression was lower in the 5/6 NX group versus sham. Renal arginase II, argininosuccinate synthase, and argininosuccinate lysate abundances were significantly decreased in 5/6 NX rats than those in sham. CONCLUSION: The neonatal kidney is very susceptible to 5/6 NX-induced injury, and, as in adults, reciprocal changes in the nNOSα and nNOSß in renal cortex occur during progression of chronic kidney disease and may contribute to the injury.

Optimal timing of surgery after chemoradiation for advanced rectal cancer: preliminary results of a multicenter, nonrandomized phase II prospective trial.

OBJECTIVE: To determine whether extending the interval between chemoradiation (CRT) and surgery, and administering additional chemotherapy during the waiting period has an impact on tumor response, CRT-related toxicity and surgical complications in patients with advanced rectal cancer. BACKGROUND: Locally advanced rectal cancer is usually treated with preoperative CRT followed by surgery approximately 6 weeks later. The Timing of Rectal Cancer Response to Chemoradiation Consortium designed a prospective, multicenter, Phase II clinical trial to investigate extending the interval between CRT and surgery, and administering additional chemotherapy during the waiting period. Here, we present preliminary results of this trial, reporting the tumor response, CRT-related toxicity and surgical complications. METHODS: Stage II and III rectal cancer patients were treated concurrently with 5-Fluorouracil (FU) and radiation for 5 to 6 weeks. Patients in study group (SG) 1 underwent total mesorectal excision (TME) 6 weeks later. Patients in SG2 with evidence of a clinical response 4 weeks after CRT received 2 cycles of modified FOLFOX-6 (mFOLFOX-6) followed by TME 3 to 5 weeks later. Tumor response, CRT-related toxicity and surgical complications were recorded. RESULTS: One hundred and forty-four patients were accrued. One hundred and thirty-six (66, SG1; 70, SG2) were evaluated for CRT-related toxicity. One hundred and twenty-seven (60, SG1; 67, SG2) were assessed for tumor response and surgical complications. A similar proportion of patients completed CRT per protocol in both SGs, but the cumulative dose of sensitizing 5-FU and radiation was higher in SG2. CRT-related toxicity was comparable between SGs. Average time from CRT-to-surgery was 6 (SG1) and 11 weeks (SG2). Pathologic complete response (pCR) was 18% (SG1) and 25% (SG2). Postoperative complications were similar between SGs. CONCLUSIONS: Intense neoadjuvant therapy consisting of CRT followed by additional chemotherapy (mFOLFOX-6), and delaying surgery may result in a modest increase in pCR rate without increasing complications in patients undergoing TME for locally advanced rectal cancer.

Curcumin prevents cardiac remodeling secondary to chronic renal failure through deactivation of hypertrophic signaling in rats.

The prevalence of left ventricular hypertrophy (LVH) is frequent in patients with end-stage renal disease following chronic renal failure (CRF). We investigated the therapeutic efficacy of curcumin, the principal curcuminoid of the Indian curry spice turmeric, in attenuation of LVH and sought to delineate the associated signaling pathways in blunting the hypertrophic response in nephrectomized rats. Adult Sprague-Dawley rats underwent nephrectomy (Nx) by removal of 5/6 of the kidneys. Four groups were studied for 7 wk: 1) control (sham), 2) Nx, 3) Nx + curcumin (150 mg/kg bid), and 4) Nx + enalapril (15 mg/kg bid) as positive control. Subtotal nephrectomy caused renal dysfunction, as evidenced by a gradual increase in proteinuria and elevation in blood urea nitrogen and plasma creatinine. Nx rats showed a significant hypertrophic response and increased diameter of inferior vena cava at inspiration, which was inhibited by treatment with curcumin or enalapril. Moreover, the Nx rats demonstrated changes in the signaling molecules critically involved in the hypertrophic response. These include increased glycogen synthase kinase-3ß phosphorylation, ß-catenin expression, calcineurin, phosphorylated (p) nuclear factor of activated T cells, pERK, and p-cAMP-dependent kinase. Both curcumin and enalapril variably but effectively deactivated these pathways. Curcumin attenuates cardiac hypertrophy and remodeling in nephrectomized rats through deactivation of multiple hypertrophic signaling pathways. Considering the safety of curcumin, these studies should facilitate future clinical trials in suppressing hypertrophy in patients with CRF.

Cardiac hypertrophy in neonatal nephrectomized rats: the role of the sympathetic nervous system.

Cardiac hypertrophy is frequently encountered in patients with renal failure and represents an independent risk factor for cardiovascular morbidity and mortality. The pathogenesis of cardiac hypertrophy is related to multiple factors, including excess adrenergic activity. This study investigated how renal injury in the early stages of life affects the adrenergic system and thereby potentially influences cardiac growth. Biomarkers of cardiac hypertrophy were used to assess adrenergic function. Newborn male Sprague-Dawley rats were allocated to three groups of five rats each: 5/6 nephrectomy (Nx), pair-fed controls (PF), and sham-operated (SH). Nx animals had significantly higher plasma urea nitrogen, serum creatinine, and mean arterial blood pressure. The heart-weight/body-weight ratio of the Nx cohort was higher than SH and PF (p < 0.001) groups. Plasma norepinephrine (NE) of Nx animals was almost twofold higher than SH and PF (p < 0.01) animals. Compared with SH and PF, Nx animals had higher alpha1A-receptor protein expression, lower cardiac beta1- and beta2-receptor protein expression (p < 0.05), but higher G-protein-coupled receptor kinase-2 (GRK2) expression (p < 0.05). Norepinephrine transporter protein (NET) and renalase protein expression in cardiac tissue from Nx pups were significantly lower than SH and PF. Our data suggest that early age Nx animals have increased circulating catecholamines due to decreased NE metabolism. Enhancement of cardiac GRK2 and NE can contribute to cardiac hypertrophy seen in Nx animals. Furthermore, AKT (activated via alpha1A receptors), as well as increased alpha1A receptors and their agonist NE, might contribute to the observed hypertrophy.

Growth hormone (GH) secretion, GH-dependent gene expression, and sexually dimorphic body growth in young rats with chronic renal failure.

Chronic renal disease results in growth failure in children. This study sought to determine the influences of early renal failure on body growth, growth hormone (GH) secretion, and GH-dependent hepatic gene expression. Neonatal animals were subjected to five-sixth nephrectomy (Nephr) and monitored during growth. Sham-operated male (Sham) and female (Fem) rats served as controls. Whereas Nephr of adult animals causes renal insufficiency, neonatal nephrectomy leads to frank renal failure. In male Nephr compared with Sham animals, GH half-life and GH pulse frequency increased by 1.55- and 1.33-fold, respectively, and GH secretory-burst size decreased by 80%. Approximate entropy analysis quantified more disorderly patterns of GH secretion in Nephr animals, which differed from Sham males, but not from Fem rats. Expression of liver P450 CYP2C11 mRNA, which is dependent upon the male GH pattern, became undetectable, whereas expression of liver P450 CYP2C12 mRNA, which is dependent upon the female GH pattern, increased multifold. Renal failure in young rats abrogates the male pattern of GH pulsatility, abolishes the sexual dimorphism of body weight gain, and induces a female pattern of hepatic gene expression. These data raise the possibility that disruption of pulsatile GH secretion contributes to the growth failure of renal disease.

Evaluation of planned treatment breaks during radiation therapy for anal cancer: update of RTOG 92-08.

PURPOSE: Radiation Therapy Oncology Group (RTOG) 92-08 began as a single arm, Phase II trial for patients with anal cancer consisting of radiation (RT) + 5-flourouracil + mitomycin-C with a mandatory 2-week break and was amended after completion to evaluate the same treatment regimen without a treatment break. Long-term efficacy and late toxicity reporting are the specific aims of this study. METHODS AND MATERIALS: Survivals were estimated with the Kaplan-Meier method. Overall survival (OS) was compared with RTOG 87-04 with the log-rank test. Time to local failure, regional failure, locoregional failure (LRF), distant metastases, second primary, and colostomy failure were estimated by the cumulative incidence method. LRF was compared with RTOG 87-04 using the Gray's test. RESULTS: Forty-seven patients entered in the mandatory treatment break cohort. The study was reopened in 1995 to the no mandatory treatment break cohort completing accrual with 20 patients in 1996. Of 67 total patients, 1 patient in the mandatory treatment break portion of the study did not receive any protocol treatment and is excluded from analyses. After adjusting for tumor size, neither cohort showed a statistically significant difference in OS or LRF compared with the RTOG 87-04 mitomycin-C arm. No patient in either cohort experienced a Grade 3 or higher late toxicity. CONCLUSIONS: No statistically significant differences were seen in OS or LRF when compared to the mitomycin-C arm of RTOG 87-04, but the sample sizes for the mandatory break cohort and the no mandatory break cohort are small. Late toxicity was low and similar for the treatment cohorts.

Higher radiation dose with a shorter treatment duration improves outcome for locally advanced carcinoma of anal canal.

AIM: To assess whether radiation dose and duration of treatment influence local control and survival of patients with locally advanced anal cancer treated with definitive chemoradiation. METHODS: Twenty-eight consecutive patients who were treated with definitive radiation therapy for bulky anal cancers (> 5 cm in size) were reviewed. Nineteen patients had T3 lesions, 8 patients had T4 lesions, and 15 patients had lymph node involvement. The median tumor size was 7.5 cm. All but one patient received concurrent chemoradiation. The median radiation dose was 54 Gy. The median duration of treatment was 58 d. RESULTS: With a median follow-up of 2.5 years in all patients and 7.8 years in living patients, the 2-year local recurrence-free probability was 57% and overall survival rate was 67%. Neither radiation dose nor duration of treatment alone was predictive of either time to local failure or overall survival. However, longer treatment breaks can potentially mask an advantage over higher radiation doses. Therefore, we examined those patients who received >or= 54 Gy within 60 d, comparing them to the rest of the patients. Of patients who received >or= 54 Gy within 60 d, local progression-free probability was 89% versus 42% for the rest of the group (P=0.01). CONCLUSION: Local failure is a significant problem in locally advanced carcinomas of the anal canal. Higher radiation doses with limited treatment breaks may offer an increase in local control and survival.

Effect of chronic renal insufficiency on hepatic and renal udp-glucuronyltransferases in rats.

Significant evidence exists regarding altered CYP450 enzymes in chronic renal insufficiency (CRI), although none exists for the phase II enzymes. The objective of this study was to investigate the effect of CRI on hepatic and renal UDP-glucuronyltransferase (UGT) enzymes. Three groups of rats were included: CRI induced by the 5/6th nephrectomy model, control, and control pair-fed (CPF) rats. UGT activities were determined in liver and kidney microsomes by the 3- and 17-glucuronidation of beta-estradiol (E2-3G and E2-17G), glucuronidation of 4-methylumbelliferone (4-MUG), and 3-glucuronidation of morphine (M3G). UGT isoforms responsible for these catalytic activities were screened using recombinant rat UGT1A1, UGT1A2, UGT1A3, UGT1A7, UGT2B2, UGT2B3, and UGT2B8. UGT protein levels were examined by Western blot analysis using polyclonal antibodies. There was no significant difference between CRI and CPF rats in hepatic and/or renal E2-3G (UGT1A1), E2-17G (UGT2B3), 4-MUG (UGT1A6), and M3G (UGT2B1) formation. Formation of E2-17G and 4-MUG in the liver and E2-3G and 4-MUG in the kidney was significantly reduced (p < 0.05) in CPF and CRI rats compared with control rats. The down-regulated glucuronidation activities were accompanied by corresponding reductions in protein content of specific UGT isoforms. These results suggest that CRI does not seem to influence the protein levels or catalytic activity of most of the major hepatic or renal UGT enzymes. The observed down-regulation of hepatic and renal UGTs in CRI and CPF rats could be caused by restricted food intake in these groups of rats.

Down-regulation of hepatic CYP3A in chronic renal insufficiency.

PURPOSE: The objective of this study was to investigate the mechanisms underlying the decrease in hepatic clearance of some drugs metabolized by CYP450 enzymes in chronic renal insufficiency (CRI). METHODS: CRI was induced in male Sprague-Dawley rats (n = 7) by the remnant kidney model (RKM); control animals (C) (n = 12) underwent sham surgery, of which n = 6 rats were pair-fed (CPF) with CRI rats and others (n = 6) had free access to food. Serum creatinine (Scr) and urea nitrogen (SUN) were monitored every 2 weeks. On day 36, livers were isolated, and microsomes were prepared. Catalytic activities were measured through O-demethylation (CYP2D) and N-demethylation of dextromethorphan (CYP3A) and O-deethylation of 7-ethoxyresorufin (CYP1A2). CYP450 protein and mRNA levels were also measured. RESULTS: Compared with CPF, Scr and SUN levels in CRI rats were increased twofold (p < 0.01) and 2.5-fold (p < 0.01), respectively. No effect on CYP1A2 and CYP2D activities, mRNA, or protein levels was observed between the groups. There was a reduction (41.8 +/- 20%, p < 0.01) in CYP3A activity, mRNA (p < 0.05), and protein levels (p < 0.05) in CRI rats compared to CPF. CONCLUSIONS: CRI induced by RKM does not have an effect on hepatic CYP1A2 and CYP2D enzymes but does reduce CYP3A activity, probably through down-regulation of CYP3A2.

Effects of deflazacort and cortisone on cellular proliferation in the rat thymus.

Deflazacort is a relatively new glucocorticoid with significant immunosuppressant activity and presumably fewer side effects. The present study was designed to compare the effects of deflazacort on the proliferative activity of thymus cells and thymolysis with the growth inhibition. We treated Long-Evans rats for nine days with cortisone (CORT, 5.0 mg/day), deflazacort (DFZ, 0.15 mg/day), and control vehicle (CTRL). Animals were sacrificed 1 hour after injection of bromodeoxyuridine (BrdUrd) on day 10. BrdUrd-labeled thymic cells were quantified without knowledge of treatment. A Labeling Index (LI), expressed as the number of BrdUrd BrdUrd-labeled cells per 100 total cells and the Numerical Density (ND), expressed as the total number of cells per 100 microm(2) were calculated. Treatment with either glucocorticoid resulted in a significant and equal decrease of thymus weight, indicating a marked reduction in total immunogenic tissue. A general alteration of thymic histological structure occurred in the CORT group. The LI was not different between CTRL and DFZ groups, 6.9 and 7.9% of cells were labeled respectively. In the CORT group, the LI was 2.5%. With respect to Numerical Density, the CTRL group had the greatest value (14.6 +/- 0.4 cells/100 microm(2)), with the DFZ (12.3 +/- 0.06 cells/100 microm(2)) and CORT groups being significantly lower (10.4 +/- 0.5 cells/100 microm(2)). Although regression analysis of thymus weight pointed to bioequivalence of the glucocorticoid dosages used, BrdUrd-labeling raised the possibility that the cells still present in the thymus of DFZ-treated animals retained, at least partially, their normal capacities for proliferation.

Growth hormone and growth hormone-related mRNA in uremic rats: effect of a growth hormone secretagogue.

In experimental animals, the decreased growth during mild uremia is not accompanied by a loss in the capacity of the pituitary gland to secrete growth hormone (GH). With the development of orally administered GH "secretagogues" (GHS), it might be possible to stimulate growth during uremia without injections. This study was designed to determine the effects of the GHS, L-163,255. Uremia was induced by 5/6 nephrectomy (NX). GHS was given orally, 3 mg/kg, twice a week. Four groups of animals included: (1) sham-operated, (2) sham-operated, pair-fed, (3) uremic (NX), and (4) uremic, GHS-treated (NX+GHS). Blood sampling was conducted via intra-atrial catheters, and GH was quantitated. Pituitary GH mRNA was measured by Northern blot, and liver GH receptor and insulin-like growth factor-I mRNAs by RNAase protection. Untreated NX animals had a specific decrease in the "mass" of the GH pulses. A burst of GH was induced by GHS, but the pulsatile pattern of GH secretion over 6 h was not affected. An increase or a return to non-uremic levels of GH-related mRNAs occurred after GHS. Thus, GHS stimulated an acute burst of GH secretion and increased specific mRNAs encoding GH-related proteins in uremic animals.

Effect of the GH secretagogue L-163,255 and restricted feeding time on GH pulsatility in the rat.

OBJECTIVE: To determine the effect of repeated treatments with the growth hormone secretagogue (GHS) L-163,255 on the pulsatile release of GH when administered in meal-fed rats before and after feeding. DESIGN: The first group of rats (AL, n=6) had food available ad libitum. The second (restricted, R, n=6), third (GHSB, n=6), and fourth (GHSA, n=6) groups were fed from 1100 to 1400 h. Groups GHSB and GHSA were given GHS by gavage, 3.0 mg/kg L-163,255, at 1000 h (before feeding, B) and at 1500 h (after feeding, A) respectively. Three weeks after the initiation of the treatment, blood samples were collected at 10-min intervals over 6 h, and GH levels were determined. RESULTS: In group R, the concentrations of GH were higher before feeding (17.6+/-2.4 ng/ml) than during feeding (11.2+/-1.2 ng/ml), P<0.05. The average concentrations of the peak in response to GHS were higher when GHS was administered before (121.70+/-33.68 ng/ml) than after (49.67+/-17.87 ng/ml) feeding. The mass of GH, as calculated by deconvolution analysis was also higher in the GHSB group than in the GHSA group (251.6+/-64.1 ng/ml per min vs 85.3+/-22.9 ng/ml per min respectively, P<0.05). CONCLUSION: L-163,255 is effective in inducing GH release after repeated oral administration in rats. The effectiveness is greater when GHS is administered before rather than after feeding in meal-fed animals.