Kidney Contrast Research Articles

Metabolomics analysis underlay mechanisms in the renal impairment of mice caused by combination of aflatoxin M1 and ochratoxin A

Aflatoxin M1 (AFM1) and ochratoxin A (OTA) are pernicious mycotoxins widely co-existing in the environment. However, nephrotoxicity and underlying mechanism induced by AFM1 coupled with OTA still remain to be explored. In this study, CD-1 mice were treated with 3.5 mg/kg b.w. AFM1, OTA, and AFM1 + OTA for 35 days, and UPLC-MS-based metabolomics method was effectuated to investigate metabolomic profiles of mice kidney. Subsequent experiments on human renal proximal tubular (HK-2) cells were performed to dig out the causal connections between distinguished differential metabolites and nephrotoxicity. Compared with DMSO vehicle group, all three toxin treatments (AFM1 and OTA alone, and in combination) significantly reduced final body weight, and remarkably elevated the concentration of serum creatinine (SCr) and caused abnormal histological phenotypes (shown by histopathological slices). OTA, AFM1 + OTA but not AFM1 reduced the relative weight index of kidney. These phenotypic results indicated that AFM1 and OTA were both toxic to the body, and it seemed that OTA exhibited a notable impairment to kidney while AFM1 had similar but limited effect compared with OTA. Further metabolomics analysis showed that when AFM1 and OTA were combined together, OTA exerted dominant effect on the alteration of metabolic processes. There were few differences in the number of changed metabolites between OTA and AFM1 + OTA group. Among the differentially expressed metabolites affected by OTA, and AFM1 + OTA, lysophosphatidylcholines (LysoPCs) were identified as the main type with significant upregulation, in which LysoPC (16:0) accounted for the most prime proportion. Western blotting results of HK-2 cells showed that single OTA and AFM1 + OTA increased the apoptotic protein expressions of Bax, caspase 3 and PARP, and decreased the expression of Bcl-2; while AFM1 only raised the expression of caspase 3. LysoPC (16:0) but not LysoPC (18:1) lifted the protein level of caspase 3 and PARP in HK-2 cells, and reduced the level of Bcl-2. Taken together, this study is the first effort trying to assess nephrotoxicity of AFM1 with OTA, and we guessed that OTA had a more pronounced toxicity to kidney in contrast to AFM1. No obvious synergism between AFM1 and OTA was found to contribute to the occurrence or development of nephropathy. LysoPC (16:0) might be the pivotal metabolite in response to single OTA and combined AFM1 + OTA engendering renal injury.

716. In vitro and In vivo Nonclinical Efficacy of AR-501 (Gallium Citrate)

BackgroundGallium nitrate citrate exhibits strong antibacterial activity and was recently shown to be safe and efficacious when intravenously administered to cystic fibrosis patients in a Phase 2 clinical study conducted by the University of Washington. We are developing an inhaled formulation of gallium citrate (AR-501), which is being tested in a Phase 1/2a clinical study. The in vitro antimicrobial activities, drug resistance profile, activities in combination with selected antibiotics, and in vivo animal efficacy if the inhaled vs. IV formulation is being presented.MethodsMIC tests were performed on strains using the CLSI susceptibility test standards. Resistance testing exposed bacteria to 20 cycles at ranges above and below the MIC level of the drug used.SPF mice (C57BL/6J, 7–9 weeks) were inoculated intranasally with P. aeruginosa under ketamine/xylazine anesthesia. Inhalation of AR-501 used an Aeroneb Solo nebulizer. Gallium levels were determined by elemental analysis using atomic absorption spectroscopy. CFU levels were measured by enumeration of bacterial colonies following serial dilution of tissue homogenates.Results In vitro efficacy: MIC testing demonstrates the efficacy of AR-501 against gram (−), gram (+) and several species of mycobacteria of clinical isolates and the comparative antibacterial response with antibiotics. Resistance testing showed that AR-501 exhibited lower propensity to develop resistance than the antibiotics tested. In vivo efficacy: AR-501 Inhalation also increased the median survival time compared with IV dosing in the murine model. Bacterial clearance was increased when Tobramycin and AR-501 are co-administered. Comparative analysis of AR-501 after IH route demonstrate increased gallium levels in BAL and reduced levels in the kidney in contrast to IV route.Conclusion In vitro studies demonstrate the susceptibility of gram (−), gram (+) and mycobacteria pathogens and the dose range of AR-501 compared with SOC antibiotics. In vivo studies confirm the therapeutic efficacy of AR-501 in bacterial pneumonia by IH delivery and demonstrate that bacterial clearance is enhanced when SOC antibiotics are used in combination with AR-501. Disclosures All authors: No reported disclosures.

Abstract 5559: Novel synthetic pyridyl analogues of CDDO-Im with improved stability and their potential use in cancer prevention

Abstract Synthetic oleanane triterpenoids are non-cytotoxic, multifunctional drugs with a broad spectrum of applications for prevention and treatment of cancer and for many other chronic diseases. CDDO-Im, 1[2-Cyano-3,12-dioxooleana-1,9(11-dien-28-oyl] imidazole, synthesized more than a decade ago, is one of the most potent triterpenoids known to date with marked anti-inflammatory, cytoprotective, antiproliferative, differentiative and proapoptotic activity on various human and murine tumor cell lines. However, pharmacokinetics of CDDO-Im are not optimal. Therefore, three new pyridyl analogues of CDDO-Im, namely CDDO-3P-Im, CDDO-2P-Im and CDDO-4P-Im, have been synthesized and screened for their possible use as chemopreventive or chemotherapeutic drugs. At low nanomolar concentrations, they were equivalent to CDDO-Im for induction of differentiation in U937 leukemia cells and at higher doses they induced apoptosis. As inflammation and oxidative stress contribute to carcinogenesis, we also assessed their cytoprotective potential. The new compounds suppressed inducible nitric oxide synthase expression in RAW264.7 macrophage-like cells and significantly induced heme oxygenase-1 and NADPH quinone reductase mRNA and protein levels in lung cancer cells as well as in various mouse tissues. Most importantly, pharmacokinetic studies performed in vitro in human plasma and in vivo revealed superior stability for each new analogue. While CDDO-Im was almost completely degraded after 30 min (< 12% of starting material remaining) in human plasma, the new compounds were more stable with > 50% still detectable. Six hours after gavage, much higher concentrations of the new derivatives were found in mouse liver, lung, pancreas and kidney in contrast to CDDO-Im. Thus, the new pyridyl analogues have better bioavailability, and because of their potent anti-inflammatory activity and improved stability, they will be tested in vivo in relevant carcinogenesis models. Citation Format: Martine Cao, Evans Onyango, Charlotte Williams, Darlene Royce, Gordon Gribble, Michael Sporn, Karen Liby. Novel synthetic pyridyl analogues of CDDO-Im with improved stability and their potential use in cancer prevention. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5559. doi:10.1158/1538-7445.AM2015-5559

Effects of aqueous leave extract of Ficus exasperata on pathophysiology and histopathogy of alloxan-induced diabetic albino rats

The effects of treatment with aqeous extract of Ficus exasperata on the pathophysiology and histopathology in alloxan-induced diabetic rats was studied. Thirty rats divided into five treatment and one control groups (n = 5 rats each) were used for the study. Rats in the treatment groups were induced by a single intraperitoneal injection of 150 mg/kg alloxan monohydrate. Diabetic groups were thereafter treated with 100, 200 and 300 mg/kg, respectively of acqeous extract of F. exasperata, the fourth group was treated with 10 mg/kg glibenclamide, the last group was not treated after induction while the control (non-diabetic) group received distilled water. Hyperglycemia was recorded in all induced ratsafter alloxan induction, while treatment with the different concentrations of the plant extract reversed hyperglycemia within four days. Values of Packed cell volume (PCV),Haemoglobin concentration (Hb) and Red blood cell count (RBC) were higher in rats treated with the extract than in rats treated with glibenclamide, while ionoregulatory distruptions observed in the diabetic groups reduced significantly (p < 0.05) in rats treated with the extract. Lipid profile parameters were higher in rats treated with glibenclamide compared to groups treated with the extract. Treatment with the plant extract ameliorated the various degenerations observed in the pancreas, liver and kidney in contrast to untreated group and group treated with glibenclamide. Results from this study demonstrated the ameliorative effects of acqeous leave extract of F. exasperata on the pathophysiological and histopathological complications of diabetes mellitus. Key words: Diabetes mellitus, Ficus exasperata, pathophysiology, hispathology.

Arterial Spin Labeling: A Noninvasive Method to Assess Renal Perfusion after Ischemia-Reperfusion Injury

Introduction: Acute kidney injury (AKI) is a common clinical problem caused by ischemia-reperfusion injury (IRI). Renal perfusion (RP) in AKI decreases as a consequence of vascular damages and interstitial edema, resulting in functional deterioration of the kidney. Assessment of RP in the early stage of AKI is therefore warranted. Although dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can be used to measure RP, contrast agents (CA) may also have adverse effects on the kidney and should preferably not be used in patients with impaired renal function (RF). To overcome this problem we used arterial spin labeling (ASL), a noninvasive measurement in MRI using the arterial blood as an endogenous tracer, as an innovative method for evaluating RP. Methods: We used a standardized warm ischemia model in four male Lewis rats (260g - 290g) with a 45min ischemia time. IRI was induced by clamping the left kidney vessels. To evaluate RP, ASL measurements were performed on a 3 Tesla MR scanner four days after ischemia. Because this method uses magnetically labeled protons in arterial blood, no other procedures during measurements were performed. To quantitate RP, ASL perfusion maps were calculated with an in-house MATLAB script (The MathWorks, Natick, MA, USA). After ASL measurements, we used DCE-MRI as a control to confirm our results. Thereafter, kidneys were processed for histology to evaluate cellular injury. The native right kidney served as non-ischemic damaged control. Results:Fig. 1 shows the comparison between DCE-MRI and ASL. Although both methods give crucial information about RP, ASL is a completely noninvasive measurement to use in case of injured kidneys. According to the perfusion map (Fig. 1), it is easily shown that RP of the diseased kidney (arrow) is worse than the native kidney. We evaluated a mean cortical perfusion of 277± 20ml/100g/min in the diseased kidney compared to 482± 54ml/100g/min in the native kidney. The medullary perfusion was also lower in the diseased kidney in contrast to the native kidney (58±2ml/100g/min vs. 150±30ml/100g/min).[Fig. 1: Images DCE-MRI and ASL]Moreover, on histological slides we could clearly determine that the kidneys with reduced RP are obviously damaged showing cellular swelling, tubular dilatation and necrosis that are main histological hallmarks of AKI. Conclusion: We demonstrate that ASL is able to assess RP showing changes between diseased and healthy kidneys. Histology was concordant to a reduced RP showing severe ischemic damages. Since in ASL there is no need using exogenous CA that may have adverse effects on RF, this method might be safely used particularly in patients with impaired RF or in renal transplant recipients in which immediate RF is not observed.

Gastrointestinal absorption of cadmium and metallothionein

Intestinal uptake and transport of cadmium (Cd) to different organs were studied in control and oral zinc pretreated rats using an in situ intestinal loop model. Intestinal loop was incubated with either CdCl 2 or Cd-metallothionein (Cd-MT) for 30 and 60 min in rats under anesthesia. Induction of MT by oral Zn pretreatment had little effect on intestinal uptake of Cd ion. However, when intestinal loop was incubated with exogenous Cd-MT, the uptake of Cd was significantly smaller than that from CdCl 2 incubation. About 50% of the Cd in the intestine of control rat after CdCl 2 incubation was recovered in the cytosol fraction and bound to high-molecular-weight (>60 kDa) proteins. In both Zn pretreated rats incubated with CdCl 2 and control rats incubated with Cd-MT, Cd was mostly recovered in the intestinal cytosol fraction (75–85%) and was mainly bound to MT. After 60 min incubation of control intestinal loop with CdCl 2, Cd was detected mainly in liver with small amounts in kidney and pancreas; with Cd-MT incubation, Cd was detected only in the kidney. The deposition of Cd in the liver was markedly decreased by Zn pretreatment. Both the uptake of Cd-MT by intestine and the induction of MT synthesis in the intestine by Zn pretreatment were demonstrated by immunohistochemistry using a specific antibody to rat liver MT. The results suggest a slow uptake of exogenous Cd-MT from the intestine and transport to kidney in contrast to deposition of Cd in the liver from CdCl 2. Although the intracellular presence of MT does not affect the uptake of Cd from lumen, it may decrease both the release of Cd from the intestine and its deposition in liver.

In vivo and in vitro fates of 8-hydroxyquinoline derivatives in rat.

The in vivo and in vitro fates of 8-hydroxyquinoline (8-OH), 5-chloro-8-hydroxyquinoline (MC), 5, 7-dichloro-8-hydroxyquinoline (DC), and 5-chloro-8-hydroxy-7-iodoquinoline (CF) in rat was studied. The results obtained were as follows. (1) These compounds were metabolized to glucuronides and sulfates after intravenous administration. Glucuronides were excreted in bile and urine, but sulfates were excreted exclusively in urine. Unmetabolized forms were almost not excreted. (2) Glucuronides were more excreted in bile as the molecular weights increased by halogen substituents. And glucuronides having the higher affinities to plasma protein and 15% liver 100000 g supernatant fraction were more excreted in bile. (3) Although sulfates were well hydrolyzed as glucuronides in the body, sulfates (especially, CF sulfate) resisted enzymatic hydrolysis in vitro by mitochondria-lysosomal and microsomal enzymes in both liver and kidney in contrast to susceptibilities of glucuronides to them. (4) The conjugates were hydrolyzed by Cu2+, Fe3+, Zn2+, and Mg2+. Especially in the presence of Cu2+ or Fe3+, DC sulfate and CF sulfate which were extremely stable in thh enzymatic hydrolysis were unstable. Accordingly, the effects of the metal ions as well as the hydrolytic enzymes were suggested for the hydrolysis of the conjugates in the body.

About degradation products of the inter-alpha-trypsin inhibitor in serum. II. Acid label derivatives of the inter-alpha-trypsin inhibitor (author's transl)

Simultaneous to the liberation of the acid stable trypsin-plasmin-inhibitor from the inter-alpha-trypsininhibitor in vivo, acid labile degradation products are set free. The main product can be estimated by immunological methods. This product is not excreted by the kidney in contrast to the acid stable inhibitor. The product accumulates in serum in different diseases. An increased concentration of this product indicates also an increased turn-over of the inter-alpha-trypsin inhibitor in the case when the concentraton of the intact inter-alpha-trypsin inhibitor or of the filtrable derivative are within normal range.

The 24-hydroxylation of 1,25-dihydroxyvitamin D3.

The production of 1,24,25-trihydroxyvitamin D3 in vivo in vitamin D-deficient rats has been demonstrated from either 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3. The stereochemical configuration of the hydroxyl on the 24 position of 1,24,25-trihydroxyvitamin D3 has also been unambiguously established to be R. Nephrectomy failed to eliminate the conversion of 1,25-dihydroxyvitamin D3 to the 1,24,25-trihydroxyvitamin D3, establishing that the 24-hydroxylase can be demonstrated in at least one organ in addition to kidney in contrast to the 1-hydroxylase. 1,24,25-Trihydroxyvitamin D3 can also be produced in vivo from 1alpha-hydroxyvitamin D3 or from 24-hydroxyvitamin D3, analogs of vitamin D3 which are not naturally occurring. Using chick kidney mitochondrial preparations it has been demonstrated that the 24-hydroxylase is able to utilize 1,25-dihydroxyvitamin D3 as a substrate whereas it is unable to utilize 1alpha-hydroxyvitamin D3. In addition, the chicke kidney 1-hydroxylase is known to convert 24,25-dihydroxyvitamin D3 to the 1,24,25-trihydroxyvitamin D3, but this hydroxylase is unable to act on the 24-hydroxyvitamin D3. These results demonstrate that the renal vitamin D hydroxylases require that a hydroxyl be on the 25 carbon of the vitamin D molecule before it can be 1- or 24-hydroxylated.

Bilateral Multicystic Dysplasia of the Kidneys

Five cases of bilateral multicystic dysplasia of the kidneys were found among 49,296 deliveries during a period of 14 years. Three of the patients were born within a period of 14 days. This suggests that this entity may occur more often than is evident from the literature. All patents were born to young, healthy mothers 3–12 weeks pre-term and died within 24 hours of birth. The possibility of the involvement of an exogenous, teratogenic factor must be left open, although we were unable to demonstrate one in the present study. In 1936 Schwartz proposed unilateral multicystic kidney as a name describing a cystic kidney disease affecting one kidney in contrast to polycystic kidney disease usually affecting both ( Schwartz , 1936). This was for years accepted as an entity ( Fine et al., 1959; Vellios et al., 1961; Uehling et al., 1966), but after reports of bilateral occurrence, the prefix unilateral was omitted ( Longino et al., 1958; Moe et al., 1963; Gleason et al., 1967; Lalli , 1967). When only a circumscribed portion of one kidney is affected, the term multilocular cysts may be used ( Dainko et al., 1963; Steiner et al., 1970). As the kidneys are also dysplastic, the term multicystic dysplasia has been suggested as more adequate ( Parkkulainen et al., 1959). The term polycystic kidneys, type 2 has also been used ( Osathanondh et al., 1964 b).

Renal contribution to overall metabolism of drugs I: Conversion of benzoic acid to hippuric acid

The kinetics of biotransformation of benzoic acid to hippuric acid during infusion in animals were studied at steady state. The contributions to the glycine conjugation of benzoic acid made by the kidney in contrast to the body as a whole were quantified by the utilization of apparent clearances, (Vel)app., and true renal clearances, (Vel)ex. Michaelis-Menten kinetics appear to describe the rate of biotransformation of both metabolic processes. The data result in estimation of the in vivo Km and Vmax. values.

Microinjections of L-leucine into tubules and peritubular capillaries of the rat. II. The maleic acid model.

Antiluminal and luminal transport of amino acids has been studied <i>in vivo </i>with a microinjection technique in maleate-treated rats. Upon microinjections of l -leucine-³H and inulin-¹⁴C in the lumen of proximal convoluted tubules and in peritubular capillaries of the cortical segments of the nephron, the amount of radioactivity measured in ureteral urine gave an indication of amino acid absorption. The results suggest that the entry of amino acids into the renal tubule cells is not impeded in maleate-treated rats and that maleic acid does not act as a competitor for amino acid transport. After microinjections into peritubular capillaries of the left kidney, <i>L</i>-leucine excretion was found to be greater in this experimental kidney than in the contralateral non-injected kidney. However, in maleate-treated rats, the excretion of leucine <i>preceded </i>that of inulin in the experimental kidney in contrast to the non-injected kidney where leucine and inulin excretion occurred simultaneously. It is proposed that this modification of amino acid excretion can be explained by an enhanced transtubular flux of amino acid, caused by a decreased ability of the cell to retain absorbed amino acids.