Renal Slices Research Articles

The Potential Renoprotective Effect of Fedratinib in Renal Ischemia Reperfusion Injury in Male Rat Model

Renal Ischemia Reperfusion Injury (RIRI) is a serious condition that arises following kidney transplantation or other circumstances that result in decreased blood supply to the kidneys, these conditions cause a harm to the renal tissue. Gaining insight into the fundamental mechanisms of RIRI, including inflammation, cell death pathways, is essential for the development of successful therapeutic approaches. Objective: This study aimed to assess the effects of Fedratinib, a JAK2 inhibitor, in reducing RIRI in a rat model. The study involved rats divided into four groups: Sham group, control group, vehicle group, and Fedratinib group. Rats underwent anaesthesia and midline incision to expose renal pedicles, with blood flow halted using microvascular clamps. The wound was stitched, and the animals were left for 90 minutes to allow reperfusion. Blood samples were collected from the heart apex to assess serum urea and creatinine, and renal slices were used for PCR to detect the jak2/stat3 pathway. The Elisa was used to evaluate TNF, IL-6, NF-κB, Bax, Bcl2, and HMGB levels. Results: IL-6 and TNF-α, and HMGB1 were identified to have a role in the development of RIRI, inhibition of JAK2/STAT3 pathway by Fedratinib significantly decrease their concentration and minimizing RIRI. Suppression of JAK2/STAT3 pathway led to a significant inhibition in NF-κB and apoptosis. Conclusion: Fedratinib has a potential preventive effect against RIRI through their activity as JAK2 inhibitor and inhibition of the following downstream inflammatory pathways.

Research Methods and New Advances in Drug-Drug Interactions Mediated by Renal Transporters.

The kidney is critical in the human body's excretion of drugs and their metabolites. Renal transporters participate in actively secreting substances from the proximal tubular cells and reabsorbing them in the distal renal tubules. They can affect the clearance rates (CLr) of drugs and their metabolites, eventually influence the clinical efficiency and side effects of drugs, and may produce drug-drug interactions (DDIs) of clinical significance. Renal transporters and renal transporter-mediated DDIs have also been studied by many researchers. In this article, the main types of in vitro research models used for the study of renal transporter-mediated DDIs are membrane-based assays, cell-based assays, and the renal slice uptake model. In vivo research models include animal experiments, gene knockout animal models, positron emission tomography (PET) technology, and studies on human beings. In addition, in vitro-in vivo extrapolation (IVIVE), ex vivo kidney perfusion (EVKP) models, and, more recently, biomarker methods and in silico models are included. This article reviews the traditional research methods of renal transporter-mediated DDIs, updates the recent progress in the development of the methods, and then classifies and summarizes the advantages and disadvantages of each method. Through the sorting work conducted in this paper, it will be convenient for researchers at different learning stages to choose the best method for their own research based on their own subject's situation when they are going to study DDIs mediated by renal transporters.

V06-10 LEFT ROBOT-ASSISTED UPPER POLE HEMINEPHRECTOMY FOR UPPER URINARY TRACT UROTHELIAL CANCER IN LYNCH SYNDROME

You have accessJournal of UrologyCME1 Apr 2023V06-10 LEFT ROBOT-ASSISTED UPPER POLE HEMINEPHRECTOMY FOR UPPER URINARY TRACT UROTHELIAL CANCER IN LYNCH SYNDROME Abderrahmane Khaddad, Xavier Lacroix, Margue Gaëlle, Alezra Eric, Vincent Estrade, Grégoire Capon, Franck Bladou, Grégoire Robert, Jean-Jacques Patard, and Jean-Christophe Bernhard Abderrahmane KhaddadAbderrahmane Khaddad More articles by this author , Xavier LacroixXavier Lacroix More articles by this author , Margue GaëlleMargue Gaëlle More articles by this author , Alezra EricAlezra Eric More articles by this author , Vincent EstradeVincent Estrade More articles by this author , Grégoire CaponGrégoire Capon More articles by this author , Franck BladouFranck Bladou More articles by this author , Grégoire RobertGrégoire Robert More articles by this author , Jean-Jacques PatardJean-Jacques Patard More articles by this author , and Jean-Christophe BernhardJean-Christophe Bernhard More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003274.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Partial nephrectomy is not recommended in kidney urothelial carcinoma, but may be considered in exceptional cases, mainly in case of poor nephronic capital or a genetic disease with high risk of recurrence. We selected for this video one of these exceptional cases, performed by minimally invasive robotic approach. METHODS: The case is a 55-year-old patient in good general state followed for Lynch syndrome, with a history of subtotal colectomy. History of 2 localizations of low-grade urothelial carcinoma. The first one was in the upper calyx of the left kidney, treated conservatively with laser. The second one was in the bladder, treated by resection and mitomycin instillations. During follow-up, he presented with a recurrence in the superior calyx of the left kidney, which had become inaccessible endoscopically due to post-treatment stenosis. In this patient at high risk of recurrence, we performed a 3D image guided left robot-assisted upper pole heminephrectomy, with anatomical devascularisation and without opening of the excretory tract. The procedure was performed using the Da Vinci surgical robot (Intuitive Surgical) and the 3D model was created with Synapse 3D. RESULTS: The procedure begins with the complete release of the kidney and the precise dissection of the pedicle. The 3D model was used to perform the anatomical devascularization of the upper pole of the kidney. The tumor calicium stem was excluded before it section. The section of the parenchyma was performed without arterial clamping. Hemostasis of the renal slice was performed with V-lok wire, and Tachosyl sponge. The urinary tract was closed with absorbable monofilament, without drainage. Operative time was 5 h 45 and estimated blood loss was 250 ml. Patient discharge at post-operative day 1. Pathology report showed a high grade pT1 urothelial papillary carcinoma of complete resection. No upper tract recurence was observed at 7 months endoscopic control. CONCLUSIONS: Standard treatment of high-risk kidney urothelial carcinoma is radical nephroureterectomy. Partial nephrectomy and other conservative treatments may be considered in exceptional cases or poor nephronic capital, or high risk of recurrence (Lynch). In any case, the opening of the excretory tract must be avoided in order to avoid the risk of tumor dissemination. Moreover, all conservative treatment must require very regular and close endoscopic follow-up. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e511 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Abderrahmane Khaddad More articles by this author Xavier Lacroix More articles by this author Margue Gaëlle More articles by this author Alezra Eric More articles by this author Vincent Estrade More articles by this author Grégoire Capon More articles by this author Franck Bladou More articles by this author Grégoire Robert More articles by this author Jean-Jacques Patard More articles by this author Jean-Christophe Bernhard More articles by this author Expand All Advertisement PDF downloadLoading ...

Periglomerular afferent innervation of the mouse renal cortex.

Recent studies using a novel method for targeted ablation of afferent renal nerves have demonstrated their importance in the development and maintenance of some animal models of hypertension. However, relatively little is known about the anatomy of renal afferent nerves distal to the renal pelvis. Here, we investigated the anatomical relationship between renal glomeruli and afferent axons identified based on transient receptor potential vanilloid 1 channel (TRPV1) lineage or calcitonin gene related peptide (CGRP) immunolabeling. Analysis of over 6,000 (10,000 was accurate prior to the removal of the TH data during the review process) glomeruli from wildtype C57BL/6J mice and transgenic mice expressing tdTomato in TRPV1 lineage cells indicated that approximately half of all glomeruli sampled were closely apposed to tdTomato+ or CGRP+ afferent axons. Glomeruli were categorized as superficial, midcortical, or juxtamedullary based on their depth within the cortex. Juxtamedullary glomeruli were more likely to be closely apposed by afferent axon subtypes than more superficial glomeruli. High-resolution imaging of thick, cleared renal slices and subsequent distance transformations revealed that CGRP+ axons closely apposed to glomeruli were often found within 2 microns of nephrin+ labeling of glomerular podocytes. Furthermore, imaging of thick slices suggested that CGRP+ axon bundles can closely appose multiple glomeruli that share the same interlobular artery. Based on their expression of CGRP or tdTomato, prevalence near glomeruli, proximity to glomerular structures, and close apposition to multiple glomeruli within a module, we hypothesize that periglomerular afferent axons may function as mechanoreceptors monitoring glomerular pressure. These anatomical findings highlight the importance of further studies investigating the physiological role of periglomerular afferent axons in neural control of renal function in health and disease.

Mechanistic Study on the Effect of Renal Impairment on the Pharmacokinetics of Vildagliptin and its Carboxylic Acid Metabolite.

To clarify the mechanism of renal impairment leading to different degrees of increased plasma exposure to dipeptidyl peptidase 4 inhibitor vildagliptin and its major metabolite, M20.7. The 5/6 nephrectomized (5/6 Nx) rat model, to simulate chronic renal failure (CRF) patients, combined with kidney slices and transporter studies in vitro were used to assess this pharmacokinetic differences. After intragastric administration to 5/6 Nx rats, vildagliptin showed increased plasma levels by 45.8%, and M20.7 by 7.51 times, which was similar to patients with severe renal impairment. The recovery rate of M20.7 in urine and feces increased by less than 20%, showing limited effect of renal impairment on vildagliptin metabolism. In vitro studies found M20.7 to be the substrate for organic anion transporter 3 (OAT3). However, the active uptake of M20.7 in renal slices showed no difference between the 5/6 Nx and normal rats. In OAT3 overexpressed cells, the protein-bound uremic toxins, 3-carboxy-4-methyl-5propyl-2-furanpropionate (CMPF), hippuric acid (HA) and indoxyl sulfate (IS), which accumulate in CRF patients, inhibited M20.7 uptake with IC50 values of 5.75, 29.0 and 69.5μM respectively, far lower than plasma concentrations in CRF patients, and showed a mixed inhibition type. The large increase in plasma exposure of M20.7 could be attributed to the accumulation of uremic toxins in CRF patients, which inhibited OAT3 activity and blocked renal excretion of M20.7, while vildagliptin, with high permeability, showed a slight increase in plasma exposure due to reduced glomerular filtration.

Endogenous Taurine Downregulation Is Required for Renal Injury in Salt-Sensitive Hypertensive Rats via CBS/H2S Inhibition.

Although taurine is known to exert an antihypertensive effect, it is unclear whether it is involved in the mechanism for hypertension-related target organ injury. To reveal the role of endogenous taurine in renal injury formation during salt-sensitive hypertension and clarify its mechanisms, both salt-sensitive Dahl rats and salt-resistant SS-13BN rats were fed a high-salt diet (8% NaCl) and given 2% taurine for 6 weeks. Rat systolic blood pressure (SBP) was measured by the tail-cuff method and artery catheterization. Kidney ultrastructure was observed under an electron microscope. Taurine content and mRNA and protein levels of taurine synthases, cysteine dioxygenase type 1 (CDO1) and cysteine sulfinic acid decarboxylase (CSAD), were decreased in Dahl rats fed a high-salt diet. However, taurine supplementation and the resulting increase in renal taurine content reduced the increased SBP and improved renal function and structural damage in high-salt diet-fed Dahl rats. In contrast, taurine did not affect SS-13BN SBP and renal function and structure. Taurine intervention increased the renal H2S content and enhanced cystathionine-β-synthase (CBS) expression and activity in Dahl rats fed a high-salt diet. Taurine reduced the renin, angiotensin II, and aldosterone contents and the levels of oxidative stress indices in Dahl rat renal tissues but increased antioxidant capacity, antioxidant enzyme activity, and protein expression. However, taurine failed to achieve this effect in the renal tissue of SS-13BN rats fed a high-salt diet. Pretreatment with the CBS inhibitor HA or renal CBS knockdown inhibited H2S generation and subsequently blocked the effect of taurine on renin, superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2) levels in high-salt-stimulated Dahl renal slices. In conclusion, the downregulation of endogenous taurine production resulted in a decrease in the renal CBS/H2S pathway. This decrease subsequently promoted renin-angiotensin-aldosterone system (RAAS) activation and oxidative stress in the kidney, ultimately contributing to renal injury in salt-sensitive Dahl rats.

P0024TRIPARTITE MOTIF-CONTAINING 55 (TRIM55) PARTICIPATES IN THE IMMUNE RESPONSE IN THE EXPERIMENTAL ANTI-THY1 GLOMERULONEPHRITIS

Abstract Background and Aims Mesangial proliferative glomerulonephritis (MsPGN) is considered as an immune- related disease. In the previous study, tripartite motif family was reported to be closely related to immune regulation. Therefore, we performed a series of experiments in vivo and vitro to investigate the role of tripartite motif-containing 55 (TRIM55), a member of tripartite motif family, in the progression of MsPGN. Method 36 male SD rats were randomly divided into 6 groups to induce anti-Thy1 nephritis. Specimens of the negative control group were collected at 0d after the injection, while those of other groups were collected at 1d, 2d, 3d, 4d, 5d after the injection, respectively. Renal cortex slices were obtained for histological and immunohistochemistry assessmen of CD68. qPCR analysis of glomerulus was performed to examine the expression of TRIM55 . Primary RMCs with overexpression of TRIM55 were obtained through plasmid transfection. In addition, si-RNA tranfection was used to knock down the expression of TRIM55 in primary RMCs. qPCR analysis was conducted to detect the level of cytokines, such as TNF-α, CCL2, CXCL6, CXCL10, and IL-6. Results PAS staining results indicated mesangial dissolution occurred since 1d, followed by inflammatory cell infiltration. CD68 immunohistochemical staining results showed that macrophages infiltration peaked at 1d and then decreased gradually. The expression of TRIM55 mRNA also peaked at 1d and decreased gradually, which was consistent with the trend of macrophages infiltration. In primary RMCs, knockdown of TRIM55 led to down-regulation of the expression of cytokines (TNF-α, CCL2, CXCL6, CXCL10 and IL-6). On the other hand, the expression of those cytokines(TNF-α, CCL2, CXCL6, CXCL10 and IL-6) significantly increased in TRIM55-overexpressed primary RMCs. Conclusion The above results indicate that TRIM55 participates in the immune response in experimental anti-Thy1 glomerulonephritis by regulating the production of cytokines. We duce TRIM55 may be a promising therapeutic intervention to ameliorate leukocyte infiltration in MsPGN.

Concomitant lansoprazole ameliorates cisplatin-induced nephrotoxicity by inhibiting renal organic cation transporter 2 in rats.

Cisplatin is used widely for the treatment of multiple solid tumors. Cisplatin-induced nephrotoxicity is caused by renal accumulation of cisplatin via human organic cation transporter 2 (hOCT2). As lansoprazole, a proton pump inhibitor, is known to inhibit hOCT2 activity, lansoprazole might ameliorate cisplatin-induced nephrotoxicity. A previous study showed that concomitant lansoprazole administration ameliorated nephrotoxicity in patients receiving cisplatin. However, the detailed mechanism remains to be clarified. In the present study, the drug-drug interaction between lansoprazole and cisplatin was examined using hOCT2-expressing cultured cells and rat renal slices. Moreover, the effect of lansoprazole on cisplatin-induced nephrotoxicity and the pharmacokinetics of cisplatin in rats was investigated. In the uptake study, lansoprazole potently inhibited the uptake of cisplatin in hOCT2-expressing cultured cells and rat renal slices. The in vivo rat study showed that concomitant lansoprazole significantly ameliorated cisplatin-induced nephrotoxicity and reduced the renal accumulation of platinum up to approximately 60% of cisplatin alone at 72 h after cisplatin intraperitoneal administration. Furthermore, the renal uptake of platinum at 3 min after intravenous cisplatin administration in rats with cisplatin and lansoprazole decreased to 78% of rats with cisplatin alone. In addition, there was no significant difference in the plasma platinum concentration between rats treated with and without lansoprazole at 3 min after cisplatin intravenous administration. These findings suggested that concomitant lansoprazole ameliorated cisplatin-induced nephrotoxicity by inhibiting rOCT2-mediated cisplatin uptake in rats, thus decreasing cisplatin accumulation in the kidney. The present findings provided important information for the establishment of novel protective approaches to minimize cisplatin-induced nephrotoxicity.

Piperacillin enhances the inhibitory effect of tazobactam on β-lactamase through inhibition of organic anion transporter 1/3 in rats

To assess the mechanism of the pharmacokinetic interaction between piperacillin and tazobactam, renal excretion and pharmacokinetic studies of piperacillin/tazobactam were investigated in normal and bacteremia rats. A bacteremia model was established to investigate the pharmacokinetic properties of piperacillin and tazobactam under different conditions. Renal slices were taken to examine the uptake of piperacillin and tazobactam. Pharmacokinetic studies of β-lactamase in rats were performed to study the contribution of rOat1/3 to the inhibition of tazobactam on β-lactamase. The AUC (from 2.93 ± 0.58 to 6.52 ± 1.44 mg·min/ml) and the plasma clearance (CLP) (from 2.41 ± 1.20 to 0.961 ± 0.212 ml/min/kg) of tazobactam were both altered after the intravenous coadministration of piperacillin and tazobactam in the bacteremia rats. The renal clearance (CLR) of tazobactam decreased from 1.30 ± 0.50 to 0.361 ± 0.043 ml/min/kg. In summary, there was a beneficial interaction between piperacillin and tazobactam mediated by rOat1 and rOat3. Piperacillin enhances the inhibitory effect of tazobactam on β-lactamase through the inhibition of rOat1 and rOat3 in rats. The contribution rate of rOat1/3 for the synergistic effect was 20% when the two drugs were coadministered.

A5662 Effects of Neurogenic CGRP on Renal Afferent Peptidergic Neurons

Objectives: Although release of the proinflammatory vasodilator CGRP from peptidergic afferent nerves is unquestioned it is not known if released neurogenic CGRP will affect renal afferent innervation in return thus influencing sympathetic activity e.g. in hypertension. Hence we wanted to test the hypothesis that CGRP elicits action potential production related to TRPV1 receptor stimulation in cultured renal neurons. Methods: Cultured dorsal root ganglion neurons (Th11-L2) of rats with renal afferents were investigated in current clamp mode to asses action potential generation or in voltage clamp mode to investigate inward currents during stimulation of TRPV1 receptors with acid of pH 6 (−80 mV holding voltage) and/or administration of CGRP (0,5 μmol). Furthermore, renal slices were incubated to study CGRP release due to TRPV1 receptor stimulation. (CGRP concentrations assessed in organ bath). Results: More than 90 DRG neurons with renal afferents were tested. Addition of CGRP did not change action potential generation nor inward currents. Proton stimulation (ph 6) of TRPV1 markedly increased long-term inward currents (baseline −361,7 +/− 89,6 pA vs. −1393,3+/−337,3* pA, p < 0,05, mean +/- SEM). The co-stimulation of renal neurons with protons (pH 6) and CGRP impaired sustained inward current as compared to proton stimulation alone (baseline −478,2 +/−54,4 pA vs. −767,6+/−89,6* pA, p < 0,05). CGRP release of renal slices increased significantly after TRPV1 receptor stimulation (on average: 21+/−4 pg/ml CGRP). Conclusion: In contrast to our hypothesis CGRP could not elicit action potentials in afferent neurons related to the kidney but even impaired electric currents in general after TRPV1 receptor stimulation. Hence, renal CGRP secretion from peptidergic afferent nerves might decrease further neuronal CGRP release.

Effects of Neurogenic CGRP on Renal Afferent Peptidergic Neurons

Although release of the proinflammatory vasodilator CGRP from peptidergic afferent nerves is generally unquestioned the matter is far less investigated with respect to afferent renal innvervation that is said to be involved in sympathetic control and blood pressure regulation. Furthermore, it is not known in how far released neurogenic CGRP will affect renal afferent innervation in return. Hence we wanted to test the hypothesis that CGRP elicits action potential production related to TRPV1 receptor stimulation in cultured neurons with afferent projections from the kidney in vivo.Cultured dorsal root ganglion neurons (Th11‐L2) of rats with renal afferents in vivo were investigated in current clamp mode to asses action potential generation or in voltage clamp mode to investigate inward currents during stimulation of TRPV1 receptors with acid of pH 6 (−80 mV holding voltage) and/or administration of CGRP (0,5 μmol). Furthermore, renal slices were incubated during baseline, experimental stimulation period and recovery to study CGRP release due to TRPV1 receptor stimulation (capsaicin 10−6 M, increased proton concentrations). CGRP concentrations in the organ bath were assessed with a commercial kit. Capsazepine was used for TRPV1 receptor blockadeMore than 90 DRG neurons with renal afferents were tested. Addition of CGRP did not change action potential generation nor inward currents. Proton stimulation (ph 6) of TRPV1 markedly increased long‐term inward currents (baseline −361,7 +/− 89,6 pA vs. −1393,3+/−337,3 pA, p&lt;0,05, mean +/− SEM). The co‐stimulation of renal neurons with protons (pH 6) and CGRP led to an impairment of sustained inward current as compared to proton stimulation alone (baseline − 478,2 +/−54,4 pA vs. −767,6+/−89,6 pA, p&lt;0,05, mean +/− SEM). Baseline CGRP release of renal slices (2.5 to 6 pg/ml) increased significantly after TRPV1 receptor stimulation (on average: 21+4 pg/ml CGRP).In contrast to our hypothesis CGRP could not elicit action potentials in afferent neurons related to the kidney but even impaired electric currents in general after TRPV1 receptor stimulation. Hence, renal CGRP secretion from peptidergic afferent nerves might decrease further neuronal CGRP release.Support or Funding InformationDFG Deutsche Forschungsgemeinschaft VE 104/4‐1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NOD2 promotes endothelial-to-mesenchymal transition of glomerular endothelial cells via MEK/ERK signaling pathway in diabetic nephropathy

Endothelial-to-mesenchymal transition (EndMT) of glomerular vascular endothelial cells (GEnCs) is now considered to play a critical role in diabetic nephropathy (DN). NOD2 is newly discovered to be closely related to DN renal injury. However, the relationship between NOD2 and EndMT of GEnCs has never been reported. In the present study, we found that NOD2 over-expression was positively correlated with the severity of DN injury in human renal biopsy samples. Immunohistochemical staining of DN renal slices showed gradual absence of endothelial character and gain of mesenchymal character, both of which were associated with NOD2 over-expression. In high glucose stimulated GEnCs, NOD2 was increased. What's more, over-expression and activation of NOD2 could both promote EndMT of GEnCs. On the other hand, silencing of NOD2 markedly attenuated EndMT induced by high glucose. Mechanically, we further found that MEK/ERK signaling pathway was involved in NOD2-regulated EndMT. Collectively, our results indicate that NOD2 has a regulatory role in EndMT via activation of MEK/ERK in high glucose-treated GEnCs. Targeting this pathway is a promising strategy for intervention of DN endothelial dysfunction.

Interaction of green tea catechins with renal organic cation transporter 2

1. Green tea extract (GTE) and EGCG have previously shown to increase the uptake of MPP+ into Caco-2 cells. However, whether GTE and its derivatives interact with renal basolateral organic cation transporter 2 (Oct2) which plays a crucial role for cationic clearance remains unknown. Thus, this study assessed the potential of drug-green tea (GT) catechins and its derivatives interactions with rat Oct2 using renal cortical slices and S2 stably expressing rat Oct2 (S2rOct2).2. Both GTE and ECG inhibited MPP+ uptake in renal slices in a concentration-dependent manner (IC50 = 2.71 ± 0.360 mg/ml and 0.87 ± 0.151 mM), and this inhibitory effect was reversible. Inhibition of [3H]MPP+ transport in S2rOct2 by either GTE or ECG (IC50 = 1.90 ± 0.087 mg/ml and 1.67 ± 0.088 mM) was also observed.3. The weak and reversible interactions of GTE and ECG with rOct2 indicate that consumption of GT beverages could not interfere with cationic drugs secreted via renal OCT2 in humans. However, the rise of therapeutic use of GTE and ECG might have to take into account the significant possibility of adverse drug–green tea catechins interactions which could alter renal organic cation drug clearance.

Abstract P247: Tumor Necrosis Factor-alpha Receptors (Type 1 &amp; Type 2) are Differentially Expressed in Renal Tissues During Chronic Dietary Intake of High Salt and Angiotensin II Treatment

Tumor necrosis factor-alpha (TNF-α) exerts natriuresis that is mediated by its’ receptor type 1 (TNFR1) while its’ receptor type 2 (TNFR2) is involved in mediating inflammatory renal injury. To determine the differential roles of these receptors in angiotensin II (AngII) induced salt-sensitive hypertension and associated renal injury, protein expressions of TNFR1 and TNFR2 were examined in mice (n=6-7 in each group) chronically treated with or without AngII (25 ng/min; implanted minipump) for 4 weeks which were fed either normal (NS; 0.4% NaCl) or high salt (HS; 4% NaCl) diets. Systemic blood pressure (SBP) in these mice was measured by tail-cuff plethysmography and 24 hour urine collections were made using metabolic cages at the start and at the end of treatment period when the kidneys were harvested after sacrificing the mice with euthanasia. Immuno-histochemical analysis of TNFR1 and TNFR2 proteins in renal slices was performed by measuring the staining area as well as the intensity of receptors’ immunoreactivities using NIS Elements Software (Nikon), which allowed the semi-quantitation of positive staining and the intensity of these proteins. The results were expressed in percent area of positive staining and the relative intensity. HS intake alone did not alter mean SBP (HS; 77±1 vs NS; 76±3 vs mmHg) but it caused an exaggeration of AngII induced increases in mean SBP (AngII+HS; 104±2 vs AngII+NS; 95±2 mmHg). The area of TNFR1 staining was higher in HS (6.0± 0.9 vs 3.2 ±0.7%; P&lt;0.05) than NS group but it was not significantly different between AngII+HS (5.0± 0.7%) and AngII+NS groups (6.3±0.7%). Similar qualitative differences were also observed in relative intensity in protein expressions. TNFR2 immunoreactivity was minimal in NS and HS groups but it was high in AngII+NS group and even greater in AngII + HS group. These data suggest that the increases in TNFR1 activity due to HS alone facilitate salt excretion that results no change in SBP in response to HS intake. However, such HS induced increases in TNFR1 activity was compromised in elevated AngII condition causing more salt retention and thus, exaggerated hypertensive response. On the other hand, HS induced increases in TNFR2 activity in elevated AngII condition facilitates enhanced renal injury response.

Chronic treatment with atrial natriuretic peptide in spontaneously hypertensive rats: beneficial renal effects and sex differences.

ObjectiveThe aim of this study was to investigate the effects of chronic treatment with atrial natriuretic peptide (ANP) on renal function, nitric oxide (NO) system, oxidative stress, collagen content and apoptosis in kidneys of spontaneously hypertensive rats (SHR), as well as sex-related differences in the response to the treatment.Methods10 week-old male and female SHR were infused with ANP (100 ng/h/rat) or saline (NaCl 0.9%) for 14 days (subcutaneous osmotic pumps). Systolic blood pressure (SBP) was recorded and diuresis and natriuresis were determined. After treatment, renal NO synthase (NOS) activity and eNOS expression were evaluated. Thiobarbituric acid-reactive substances (TBARS), glutathione concentration and glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in the kidney. Collagen was identified in renal slices by Sirius red staining and apoptosis by Tunel assay.ResultsFemale SHR showed lower SBP, oxidative stress, collagen content and apoptosis in kidney, and higher renal NOS activity and eNOS protein content, than males. ANP lowered SBP, increased diuresis, natriuresis, renal NOS activity and eNOS expression in both sexes. Renal response to ANP was more marked in females than in males. In kidney, ANP reduced TBARS, renal collagen content and apoptosis, and increased glutathione concentration and activity of GPx and SOD enzymes in both sexes.ConclusionsFemale SHR exhibited less organ damage than males. Chronic ANP treatment would ameliorate hypertension and end-organ damage in the kidney by reducing oxidative stress, increasing NO-system activity, and diminishing collagen content and apoptosis, in both sexes.

Arsenic and Mercury Containing Traditional Chinese Medicine (Realgar and Cinnabar) Strongly Inhibit Organic Anion Transporters, Oat1 and Oat3,In Vivoin Mice

Toxic heavy metals, including mercury (Hg) and arsenic (As), accumulate preferentially in kidneys and always cause acute renal failure. The aim of this study was to investigate whether these samples affect organic anion transporters, Oat1 and Oat3, in vivo in mice kidney. Mice (n = 10) were orally treated with investigational samples. After last administration, all mice were i.v. p-aminohippuric acid (PAH), and the blood and kidneys samples were collected. The concentrations of PAH were quantified by spectrophotometry. mRNA expressions of Oat1 and Oat3 were assayed by real-time PCR. In comparison with corresponding control, major pharmacokinetic parameters of PAH in sera were significantly changed by investigational samples (p < 0.05), PAH accumulations in the kidney tissues were significantly higher (p < 0.05), PAH uptake by renal slices was greatly reduced, Oat1 and Oat3 mRNA expression were significantly inhibited in investigational sample groups. Arsenic and mercury containing traditional Chinese medicine (Realgar and Cinnabar) probably induce kidney damage through inhibiting several members of the organic anion transporters (such as OAT1 and OAT3).

Antidiabetic and Renoprotective Effects ofCladophora glomerataKützing Extract in Experimental Type 2 Diabetic Rats: A Potential Nutraceutical Product for Diabetic Nephropathy

Cladophora glomerata extract (CGE) has been shown to exhibit antigastric ulcer, anti-inflammatory, analgesic, hypotensive, and antioxidant activities. The present study investigated antidiabetic and renoprotective effects of CGE in type 2 diabetes mellitus (T2DM) rats. The rats were induced by high-fat diet and streptozotocin and supplemented daily with 1 g/kg BW of CGE for 12 weeks. The renal transport function was assessed by the uptake of para-aminohippurate mediated organic anion transporters 1 (Oat1) and 3 (Oat3), using renal cortical slices. These two transporters were known to be upregulated by insulin and PKCζ while they were downregulated by PKCα activation. Compared to T2DM, CGE supplemented rats had significantly improved hyperglycaemia, hypertriglyceridemia, insulin resistance, and renal morphology. The baseline uptake of para-aminohippurate was not different among experimental groups and was correlated with Oat1 and 3 mRNA expressions. Nevertheless, while insulin-stimulated Oat1 and 3 functions in renal slices were blunted in T2DM rats, they were improved by CGE supplementation. The mechanism of CGE-restored insulin-stimulated Oat1 and 3 functions was clearly shown to be associated with upregulated PKCζ and downregulated PKCα expressions and activations. These findings indicate that CGE has antidiabetic effect and suggest it may prevent diabetic nephropathy through PKCs in a T2DM rat model.

Resveratrol Reduces Cisplatin Mediated Oxidative Modifications of Proteins and Oxidative Stress Enzyme Activity in Renal Rat Tissue

Nephrotoxicity is an adverse effect associated with the cancer chemotherapeutic agent cisplatin. (RES) is a phytochemical found in grapes which possesses anticancer and antioxidant properties. This study investigated RES attenuation of cisplatin renal cytotoxicity and reduction in oxidative stress mediated by cisplatin. Kidneys were isolated from male Fischer 344 rats and renal slices were pre‐incubated with 30 ul ethanol (VEH) or 30 ug/ml resveratrol (RES, final concentration) for 30 min. Tissue were immediately incubated for a maximum of 120 min with 0–75 ug/mL cisplatin. Loss of membrane integrity was measured as lactate dehydrogenase (LDH) leakage. Cisplatin associated oxidative stress was evaluated by measuring total, Cu/Zn and Mn Superoxide dismutase (SOD), glutathione peroxidase and catalase enzyme activity in renal cortical slices. LDH leakage required a 120 min exposure to cisplatin. Cisplatin alter GSH peroxidase and SOD within 60 min when compared to VEH group. RES reduced oxidative stress by maintaining GSH peroxidase and SOD enzyme activity. An increase in protein carbonyls as detected by Oxyblot, was increased by cisplatin and totally prevented by RES. Our findings showed RES diminished cisplatin renal toxicity and early changes in oxidative stress. (Supported by NIH Grants INBRE 3P20RR016477‐09S4 and 5P20RR016477 to the West Virginia IDeA Network for Biomedical Research Excellence).

Modified quadruple inversion recovery prepulse for arterial spin labeling angiography without the need of subtraction

Arterial Spin Labeling (ASL) is a well-known technique that allows the non-invasive acquisition of angiograms without the need of a contrast agent [1][2]. ASL angiography is still clinically underused because of several challenges [3]: ASL requires two acquisitions (labeled and non labeled dataset) thereby doubling scan time. The need of subtraction increases the sensitivity to spatial misregistration and the need of the choice of an optimal inversion delay for best blood-to-background contrast introduces some “operator dependence”. An alternative technique not requiring image subtraction has been proposed (Figure ​(Figure1)1) [4]. This approach is based on a double inversion (DIR-ASL) prepulse and provides good background suppression if the surrounding tissues have similar T1 values (Figure ​(Figure1).1). However, DIR-ASL has only one optimal inversion delay and thus there is a trade-off between background suppression and visualized vessel extent. Our goal was to develop an ASL technique with improved background suppression and without the need of subtraction. Figure 1 Double inversion bright-blood MR angiography sequence Methods The proposed method is a modified Quadruple Inversion Recovery [5] sequence (mQIR-ASL), which provides excellent background signal suppression according to our simulations (Figure ​(Figure2).2). The first pair of RF pulses consists of a non-selective inversion RF pulse followed by a selective reinversion pulse and a navigator-restore pulse. The second pair of RF pulses consists of a non-selective inversion pulse followed by a navigator-restore pulse. With this configuration, the upstream labeled blood only “experiences” the second non-selective inversion pulse, while the static tissue “experiences” both non-selective inversion pulses. With the right choice of TI1 and TI2, signal from static tissue can be suppressed over a wide T1 range while maintaining the signal of target blood (Figure ​(Figure2).2). This pre-pulse was implemented on a 3T Achieva Gyroscan MR scanner (Philips Healthcare, Best, NL) and tested in 5 subjects. Figure 2 mQIR-ASL sequence, NS: Adiabatic Non-Selective Inversion Pulse. R: Selective Inversion Pulse, NR: Navigator Restore Pulse, N: Naviagtor, AQ: Acquisition Results Renal arteries including small branches were successfully visualized in all subjects with excellent suppression of background (Figure ​(Figure33,​,4).4). The plan scan and the maximum projection angiogram (MIP) are shown in Figure ​Figure3.3. Improved background suppression was observed with mQIR-ASL compared to DIR-ASL (Figure ​(Figure44). Figure 3 In vivo result with mQIR-ASL (TR/TI1/TI2 = 1000/480/162 ms). A- Planning procedure; B- and C- MIPs of aorta and renal arteries Figure 4 Transverse slice at the renal artery level demonstrating improved background suppression with mQIR-ASL (TW/TI1/TI2 = 480/162 ms) © compared with DIR-ASL (Inversion time (TI) of 300 ms (A) and 600 ms (B)). L=liver; M=muscle; K=kidneys. Conclusions We demonstrate a new ASL approach for non-contrast enhanced MR angiography with excellent background tissue suppression and without the need of subtraction. In comparison to DIR-ASL, mQIR-ASL yielded better background suppression and improved vessel delineation.

Sodium-Hydrogen Exchanger Regulatory Factor 1 (NHERF-1) Transduces Signals That Mediate Dopamine Inhibition of Sodium-Phosphate Co-transport in Mouse Kidney

Dopamine inhibited phosphate transport in isolated renal brush border membrane vesicles and in cultured renal proximal tubule cells from wild-type but not from NHERF-1 null mice. Co-immunoprecipitation experiments established that NHERF-1 associated with D1-like receptors. In wild-type mice, dopamine stimulated cAMP accumulation and protein kinase C (PKC) activity in renal proximal tubule cells, an effect that was abolished by SCH-23390, a D1-like receptor antagonist. In NHERF-1 null kidney tissue; however, dopamine failed to stimulate either cAMP accumulation or PKC activity. Infection of proximal tubule cells from NHERF-1 null mice with adenovirus-green fluorescent protein-NHERF-1 restored the ability of dopamine to stimulate cAMP and PKC. Finally, in (32)P-labeled wild-type proximal tubule cells and in opossum kidney cells, dopamine increased NHERF-1 phosphorylation at serine 77 of the PDZ I domain of NHERF-1, a site previously shown to attenuate binding of cellular targets including the Npt2a (sodium-dependent phosphate transporter 2a). Together, these studies establish that NHERF-1 plays a key role in dopamine signaling and is also a downstream target of D1-like receptors in the mouse kidney. These studies suggest a novel role for the PDZ adapter protein NHERF-1 in coordinating dopamine signals that inhibit renal phosphate transport.