Kidney Slices Research Articles

Cyclooxygenase‐2 Regulates the Renal Expression of Indoleamine 2,3‐dioxygenase

Chronic kidney disease (CKD) affects approximately 10% of the population. In CKD patients, the majority of metabolic pathways are in disarray, which most likely contributes to the wide variety of comorbidities observed in these patients. However, few studies have focused on the unique regulatory aspects of enzyme systems during CKD development and progression. The cyclooxygenase‐2 (COX‐2)/prostaglandin (PG) system plays a dominant role in the progression of renal injury. Interestingly, it has been shown that the COX‐2/PG system modulates the activity of indoleamine 2,3‐dioxygenase (IDO) in tumors. Catabolism of tryptophan by IDO gives rise to numerous biologically active metabolites implicated in CKD progression. Therefore, the aim of this project is to explore the interplay between the COX‐2 and IDO enzyme systems during renal injury.We studied the gene expression of IDO, COX‐2 as well as several fibrosis and inflammatory markers in human and murine precision‐cut kidney slices (PCKS) and COX‐2 KO mice. Furthermore, we evaluated the gene expression profile after treatment with butaprost, an EP2 receptor agonist, in hPCKS and following treatment with SC‐236, a selective COX‐2 inhibitor, in mPCKS.Our results demonstrated that IDO gene expression was markedly increased in both human and mouse PCKS during the first 24h of culturing, after which levels dropped again. This expression profile was similar to the gene expression of various cytokines (e.g. IL‐6 and CXCL1), suggesting a potential pro‐inflammatory role of IDO. In addition, in COX‐2 KO animals, constitutive gene expression of IDO was increased as compared to wild‐type mice, and the knockout animals appeared to be more prone to renal fibrosis. Moreover, treatment with butaprost suppressed IDO gene expression, while exposure to SC‐236 induced IDO mRNA expression as well as the gene levels of IL‐6 and IL‐8.Taken together, it appears that COX‐2 suppresses IDO gene expression. In addition, our findings suggest a potential pro‐inflammatory role of IDO. However, the precise role of IDO in CDK development and progression requires further study.Support or Funding InformationThis work was kindly supported by Lundbeckfonden, grant number R231‐2016‐2344.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Administration of a selective prostaglandin E2 receptor agonist attenuates renal fibrosis in vitro and in vivo

Renal fibrosis plays a pivotal role in the development and progression of chronic kidney disease (CKD), which affects 10% of the adult population. Previously, it has been demonstrated that the cyclooxygenase‐2 (COX‐2)/prostaglandin (PG) system influences the progression of renal injury. PGE2 interacts with four different G‐protein‐coupled PGE2 EP receptors, known as EP1–EP4, and plays a role in several biological functions. Previous studies show that the EP2 receptor is involved in progression of lung fibrosis. In this study, we investigated the effect of an EP2 agonist (butaprost) on renal fibrogenesis on a cellular, renal tissue and organismal level using well‐established in vivo and in vitro models as well as a recently developed human model of renal fibrosis. This model is ideal to study multicellular pathological processes, e.g. fibrosis, directly in human tissue since cellular diversity and organ architecture is maintained in the slices. Butaprost (50 μM) reduced TGF‐β‐induced fibronectin (FN) expression, Smad2/3 phosphorylation and epithelial‐mesenchymal transition in MDCK cells. In addition, treatment with 4 mg/kg butaprost attenuated the development of fibrosis in mice that underwent unilateral ureteral obstruction (UUO) surgery, as illustrated by a significant reduction in the gene and protein expression of α‐smooth muscle actin, FN and collagen 1A1. More importantly, a similar anti‐fibrotic effect of butaprost was observed in human precision‐cut kidney slices exposed to TGF‐β. The mechanism of action of butaprost appeared to be a direct effect on TGF‐β/Smad signaling, which was independent of the cAMP/PKA pathway. In conclusion, this study demonstrates that stimulation of the EP2 receptor effectively mitigates renal fibrogenesis in various fibrosis models. These findings warrant further research into the clinical application of butaprost, or other EP2 agonists, for the inhibition of renal fibrosis.Support or Funding Information Sources of support: This work was kindly supported by Lundbeckfonden, grant number R231‐2016‐2344 as well as the Danish Council for Independent Research, grant number 6110‐00231B, Aarhus University Research Foundation, grant number AUFF‐E‐2015‐FLS‐8‐69 and Hildur and Dagny Jacobsens Foundation grant number 1295716‐1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Organic anion transporter 3 (OAT3)-mediated transport of dicaffeoylquinic acids and prediction of potential drug-drug interaction

Dicaffeoylquinic acids (DCQAs) are widely distributed in daily food and herb medicine (such as Dengzhanxiyin injection) with multiple health benefits and pharmacological activities. However, drug-drug Interactions (DDIs) between DCQAs and possible concomitant drugs were not fully understood in clinic. The purpose of present study was to investigate the role of organic anion transporters (OATs) in the transport of DCQAs and to explore the potential clinical DDIs using in vitro transporter assays. Uptake study using hOAT1/hOAT3-transfected HEK293 cells revealed that none of DCQAs was OAT1 substrate, while 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA were substrates of OAT3 with Km values of 119.7 ± 28.8, 269.3 ± 129.5 and 53.2 ± 32.1 μM, respectively. The docking analysis revealed that 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA were effectively embedded in the active site of OAT3 and fitted well with the cavity in three-dimensional space. Moreover, the classical substrates/inhibitors of OAT decreased the accumulation of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in kidney slices, suggesting potential DDI risks with co-administration of substrate drugs of OAT. In fact, antivirals, antibiotics, neuroprotective agents, and PPIs (proton pump inhibitors) all showed varying degrees of inhibition of OAT3-mediated uptake of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in vitro. For cefaclor, ceftizoxime, pantoprazole, and zidovudine, in particular, their IC50 values were <10 times the maximal free plasma concentration, indicating potential clinically relevant DDIs when used together with DCQAs. These findings provided useful information for the prediction of DDIs between DCQAs and OAT3 inhibitors, and rational application of herbal medicines containing DCQAs in clinic.

Protein Phosphatase 1 Inhibitor-1 Mediates the cAMP-Dependent Stimulation of the Renal NaCl Cotransporter.

A number of cAMP-elevating hormones stimulate phosphorylation (and hence activity) of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Evidence suggests that protein phosphatase 1 (PP1) and other protein phosphatases modulate NCC phosphorylation, but little is known about PP1's role and the mechanism regulating its function in the DCT. We used ex vivo mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor-1 (I1), mediates cAMP's effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions. Treating isolated DCTs with forskolin and IBMX increased NCC phosphorylation via a protein kinase A (PKA)-dependent pathway. Ex vivo incubation of mouse kidney slices with isoproterenol, norepinephrine, and parathyroid hormone similarly increased NCC phosphorylation. The cAMP-induced stimulation of NCC phosphorylation strongly correlated with the phosphorylation of I1 at its PKA consensus phosphorylation site (a threonine residue in position 35). We also found an interaction between NCC and the I1-target PP1. Moreover, PP1 dephosphorylated NCC in vitro, and the PP1 inhibitor calyculin A increased NCC phosphorylation. Studies in kidney slices and isolated perfused kidneys of control and I1-KO mice demonstrated that I1 participates in the cAMP-induced stimulation of NCC. Our data suggest a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKA-dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hormones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity.

Intake of Heated Leaf Extract of Coriandrum sativum Contributes to Resistance to Oxidative Stress via Decreases in Heavy Metal Concentrations in the Kidney.

Coriandrum sativum (coriander) is an annual herb of the Apiaceae family and has been used as a traditional remedy. Here we examined whether heated leaf extract of coriander decreases the concentrations of heavy metals in tissues. Male ddY mice were given a drinking water containing 0.25% of heated leaf extract of coriander for 8weeks. Eight weeks after the intake, the concentrations of zinc, iron, copper, arsenic, and cadmium were measured in the liver and kidney. The intake of coriander did not modify the concentrations of all heavy metals tested in the liver, but decreased the concentrations of iron, arsenic, and cadmium in the kidney. Because heavy metals can induce oxidative stress, the effect of coriander intake on hydrogen peroxide-induced oxidative stress was compared between slices from the kidney and liver. The slices were immersed in Ringer solution containing 100μM hydrogen peroxide and aminophenyl fluorescein (APF), a probe for detecting reactive oxygen species (ROS). APF fluorescence was markedly increased in the control kidney slices, while the increase was completely blocked in kidney slices from coriander intake group. In contrast, APF fluorescence was also markedly increased in the control liver slices, while the increase was not blocked by coriander intake. The present study indicates that intake of coriander leaf extract contributes to powerful resistance to oxidative stress in the kidney, probably via decreased concentrations in heavy metals. It is likely that decrease in arsenic concentration to the detection limit is a major factor for the resistance.

Cadmium disrupts melanocortin 2 receptor signaling in rainbow trout

Cadmium is an endocrine disruptor and inhibits corticosteroid production, but the mechanisms are far from clear. We tested the hypothesis that sublethal exposure to environmentally realistic levels of cadmium impairs cortisol production by disrupting the melanocortin 2 receptor (MC2R) signaling in rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of cadmium (0.75 or 2.0 μg/L) in a flow-through system for 7 d and subjected to an acute secondary stressor to evoke a cortisol response. Cadmium exposure for 7 d did not affect plasma cortisol concentrations, but head kidney mc2r mRNA levels were higher than in control fish. The cortisol stress performance to a secondary-stressor was attenuated in the cadmium groups, and this corresponded with transient reduction in transcript abundance of mc2r and the gene encoding its accessory protein MRAP1 but not MRAP2 in the head kidney. Furthermore, in vivo cadmium exposure attenuated the adrenocorticotropic hormone (ACTH)-, but not 8-br-cAMP-stimulated cortisol production in head kidney slices ex vivo. This corresponded with reduced transcript abundance of mc2r and mrap1, but not mrap2 in these tissue slices. Also, reporter assays with CHO cells transiently transfected with rainbow trout mc2r and zebrafish mrap1 revealed a dose-independent inhibition in ACTH-stimulated luciferase activity by cadmium. Collectively, waterborne exposure to environmentally realistic concentration of cadmium compromises the stressor-induced cortisol response, and a mode of action involves the disruption of MC2R signaling in rainbow trout.

Quantitative Proteomics Using Formalin-fixed, Paraffin-embedded Biopsy Tissues in Inflammatory Disease.

Background:Investigations in human disease pathogenesis have been hampered due to paucity of access to fresh-frozen tissues (FFT) for use in global, data-driven methodologies. As an alternative, formalin-fixed, paraffin-embedded (FFPE) tissues are readily available in pathology banks. However, the use of formalin for fixation can lead to the loss of proteins that appear during inflammation, thus introducing an inherent sample bias. To address this, we compared FF and FFPE tissue proteomics to determine whether FFPE-tissue can be used effectively in inflammatory diseases.Methods:Adjacent kidney slices from lupus nephritic mice were processed as FFPE or FFTs. Their tissue lysates were run together using proteomics workflow involving filter-aided sample preparation, in-solution dimethyl isotope labeling, StageTip fractionation, and nano-LC MS/MS through an Orbitrap XL MS.Results:We report a >97% concordance in protein identification between adjacent FFPE and FFTs in murine lupus nephritic kidneys. Specifically, proteins representing pathways, namely, ‘systemic lupus erythematosus’, ‘interferon-α’, ‘TGF-β’, and ‘extracellular matrix’, were reproducibly quantified between FFPE and FFTs. However, 12%−29% proteins were quantified differently in FFPE compared to FFTs, but the differences were consistent across experiments. In particular, certain proteins represented in pathways, including ‘inflammatory response’ and ‘innate immune system’ were quantified less in FFPE than in FFTs. In a pilot study of human FFPE tissues, we identified proteins relevant to pathogenesis in lupus nephritic kidney biopsies compared to control kidneys.Conclusion:This is the first report of lupus nephritis kidney proteomics using FFPE tissue. We concluded that archived FFPE tissues can be reliably used for proteomic analyses in inflammatory diseases, with a caveat that certain proteins related to immunity and inflammation may be quantified less in FFPE than in FFTs.

Organic anion transporters also mediate the drug–drug interaction between imipenem and cilastatin

This study aimed to clarify that organic anion transporters (OATs) mediate the drug–drug interaction (DDI) between imipenem and cilastatin. After co-administration with imipenem, the plasma concentrations and the plasma concentration-time curve (AUC) of cilastatin were significantly increased, while renal clearance and cumulative urinary excretion of cilastatin were decreased. At the same time, imipenem significantly inhibited the uptake of cilastatin in rat kidney slices and in human OAT1 (hOAT1)-HEK293 and human OAT3 (hOAT3)-HEK293 cells. Probenecid, p-aminohippurate, and benzylpenicillin inhibited the uptake of imipenem and cilastatin in rat kidney slices and in hOAT1- and hOAT3-HEK 293 cells, respectively. The uptakes of imipenem and cilastatin in hOAT1- and hOAT3-HEK 293 cells were significantly higher than that in mock-HEK-293 cells. Moreover, the Km values of cilastatin were increased in the presence of imipenem with unchanged Vmax, indicating that imipenem inhibited the uptake of cilastatin in a competitive manner. When imipenem and cilastatin were co-administered, the level of imipenem was higher compared with imipenem alone both in vivo and in vitro. But, cilastatin significantly inhibited the uptake of imipenem when dehydropeptidase-1 (DPEP1) was silenced by RNAi technology in hOAT1- and hOAT3-HEK 293 cells. In conclusion, imipenem and cilastatin are the substrates of OAT1 and OAT3. OAT1 and OAT3 mediate the DDI between imipenem and cilastatin. Meanwhile, cilastatin also reduces the hydrolysis of imipenem by inhibiting the uptake of imipenem mediated by OAT1 and OAT3 in the kidney as a complement.

Progression of Repair and Injury in Human Liver Slices.

Human liver slice function was stressed by daily dosing of acetaminophen (APAP) or diclofenac (DCF) to investigate injury and repair. Initially, untreated human liver and kidney slices were evaluated with the global human U133A array to assess the extended culture conditions. Then, drug induced injury and signals of repair in human liver slices exposed to APAP or DCF (1 mM) were evaluated via specific gene expression arrays. In culture, the untreated human liver and kidney slices remained differentiated and gene expression indicated that repair pathways were activated in both tissues. Morphologically the human liver slices exhibited evidence of repair and regeneration, while kidney slices did not. APAP and DCF exposure caused a direct multi-factorial response. APAP and DCF induced gene expression changes in transporters, oxidative stress and mitochondria energy. DCF caused a greater effect on heat shock and endoplasmic reticulum (ER) stress gene expression. Concerning wound repair, APAP caused a mild repression of gene expression; DCF suppressed the expression of matrix collagen genes, the remodeling metalloproteases, cell adhesion integrins, indicating a greater hinderance to wound repair than APAP. Thus, human liver slices are a relevant model to investigate the mechanisms of drug-induced injury and repair.

Involvement of organic cation transporter 2 in the metformin-associated increased lactate levels caused by contrast-induced nephropathy

The application of iodinated contrast medium has become a risk factor for metformin-associated lactic acidosis (MALA), which leads to the accumulation of metformin in vivo is one of the principal reasons for MALA. However, the molecular mechanism of the adverse event is not yet clear. In this study, iohexol injection was used as a model drug. The contrast agent acute kidney injury rat model, in vivo rat pharmacokinetic study, kidney slices and HK-2 cells were performed to elucidate the pharmacokinetic molecular mechanism of accumulation of metformin caused by contrast-induced nephropathy (CIN). Plasma exposure of metformin was increased significantly in the CIN group compared with that in the normal and control groups. The AUC of metformin was from 2791 ± 382 μg min mL−1 to 4784 ± 767 μg min mL−1. The cumulative urinary excretion of metformin was also reduced in the CIN group. The uptake of metformin decreased in kidney slices in the CIN group. Compared with the normal and control groups, the blood lactate concentration was increased after intravenous administration of metformin in the CIN group followed a similar trend to the plasma concentrations of metformin. After treatment with contrast medium, the expression of OCT2 was reduced in rat kidney and HK-2 cells. These findings highlight that OCT2 deficiency was associated with increased lactate levels during metformin treatment caused by CIN.

Effect of tyrosine kinase inhibitors on renal handling of creatinine by MATE1

Creatinine is actively secreted across tubular epithelial cells via organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1). We previously showed that the tyrosine kinase inhibitor (TKI) crizotinib inhibits OCT2-mediated transport of creatinine. In the present work, we examined the inhibitory potency of TKIs, including crizotinib, on MATE1-mediated transport of creatinine. Then, we used the kinetic parameters estimated in this and the previous work to predict the potential impact of TKIs on serum creatinine level (SCr) via reversible inhibition of creatinine transport. Crizotinib inhibited [14C]creatinine uptake by MATE1-overexpressing cells, and the inhibitory effect increased with incubation time, being greater in the case of pre-incubation or combined pre-incubation/co-incubation (pre/co-incubation) than in the case of co-incubation alone. The inhibition was non-competitive, with Ki values of 2.34 μM, 0.455 μM and 0.342 μM under co-, pre- or pre/co-incubation conditions, respectively. Similar values were obtained for inhibition of [3H]MPP+ uptake by MATE1-overexpressing cells. Gefitinib, imatinib, pazopanib, sorafenib, and sunitinib also inhibited MATE1-mediated creatinine uptake. Further, all these TKIs except pazopanib inhibited [14C]creatinine uptake by OCT2-overexpressing cells. In rat kidney slices, the ratio of unbound tissue accumulation of TKIs to extracellular concentration ranged from 2.05 to 3.93. Prediction of the influence of TKIs on SCr based on the renal creatinine clearance and plasma maximum unbound concentrations of TKIs suggested that crizotinib and imatinib might increase SCr by more than 10% in the clinical context. Accordingly, it is necessary to be cautious in diagnosing TKI-induced renal failure only on the basis of an increase of SCr.

Quantitative Bioimaging of Platinum via Online Isotope Dilution-Laser Ablation-Inductively Coupled Plasma Mass Spectrometry.

A new calibration strategy for elemental bioimaging based on online isotope dilution analysis (IDA) and laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) was developed and applied for the quantification of platinum in rat kidney tissues. A dry 194Pt spike aerosol was added in a post-cell setup, and the natural 194Pt/195Pt isotope ratio of the sample aerosol from laser ablation was changed accordingly. Spike mass flow determination was carried out based on reversed IDA using a reference standard. Quantitative data obtained by the new approach correlated well with those obtained by external calibration when analyzing parallel tissue slices of rat kidney from cisplatin perfusion studies. The novel quantification approach is traceable to SI units, as IDA is an definitive method. Signal drifts are compensated as the second isotope acts as an internal standard.

Puerarin improves methotrexate-induced renal damage by up-regulating renal expression of Oat1 and Oat3 in vivo and in vitro

The regulation of renal transporters such as organic anion transporter (OATs) is a new target for treatment of acute renal failure. The purpose of this study was to investigate whether the effect of puerarin (Pur) on renal damage induced by methotrexate (MTX) is related to the expression of renal Oat1/3 in vivo and in vitro, and to explore the related mechanisms. Effect of Pur on the renal damage caused by MTX was evaluated by assessment of the changes of endogenous metabolites, toxins and H&E staining. Furthermore, Real-time PCR and western blot methods were taken to evaluate the modulation of Oat1/3 in rats. Then, the regulation of Oat1/3 by B-cell CLL/lymphoma (BCL)6 was explored by siRNA assay using NRK-52E cells in vitro. Pur reduced levels of endogenous metabolites and toxins, like creatinine, urea nitrogen and indoxyl sulfate in plasma in MTX-treated rats. Moreover, plasma concentration of MTX was significantly decreased, while the cumulative urinary excretion of MTX and the uptake of MTX by kidney slices were strongly increased after administration of multiple-dose of Pur via up-regulation of renal Oat1/3 expression. Knockdown of BCL6 by siRNA abrogated the Pur-induced Oat1/3 expression in NRK-52E cells. Pur improved MTX-induced renal toxicity through promotion of renal excretion of toxins by up-regulating renal Oat1/3 via BCL6. Pur was beneficial for the improvement of MTX-induced renal toxicity.

Tiger nut and walnut extracts modulate extracellular metabolism of ATP and adenosine through the NOS/cGMP/PKG signalling pathway in kidney slices

BackgroundTiger nut (Cyperus esculentus L.) and walnut (Tetracarpidium conophorum Müll. Arg.) have been reportedly used in the treatment of inflammatory diseases such as atherosclerosis, prevent heart attack and improve blood circulation, reduce serum cholesterol level as well as inhibit oxidation reactions. PurposeThis study investigated the effect of tiger nut and walnut hydro-alcoholic extracts on extracellular metabolism of ATP through the NOS/cGMP/PKG signaling pathway induced by Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) in kidney slices. MethodsThe plants were extracted for 24 h in 10 ml of 70% ethanol and 30% distilled water per gram milled material on a mechanical shaker and filtered using Whatman filter paper. The effect of the extracts on ecto-nucleotidases (NTPDase and 5′ nucleotidase) and adenosine deaminase activities, nitrites and nitrates levels (NO, markers of NO production) as well as lipid and protein oxidation reactions in kidney slices were evaluated. Also, the phenolic components of the nut samples were determined using High Performance Liquid Chromatography (HPLC). ResultsThe results revealed a protective effect of tiger nut and walnut on co-incubation with L-NAME of the enzyme activities, increased NO significantly (P < 0.05) when compared to the vehicle. L-NAME also increased the thiobabituric reactive substances but co-incubation with the extracts caused a significant reduction while protein oxidation across groups showed no significant difference when compared to the vehicle group. HPLC finger printing revealed the presence of quercetin and kaempferol as the most abundant phenolic compounds in tiger nut and walnut respectively. ConclusionTiger nut and walnut extracts showed a protective effect on L-NAME induced kidney slices by reducing the activities of NTPDase (ATP as substrate) and adenosine deaminase, increased NO levels as well as prevent oxidative damage. The effect observed may be attributed to the phenolic compounds present in both nuts as depicted by HPLC finger printing.

An In Situ Kidney Slice Model for Studying Angiotensin II‐ and TRPC5‐Mediated Calcium Signaling

Current approaches to study calcium signaling in glomeruli use isolated glomeruli or cultured cells, however the structure and physiology of the tissue is likely to be perturbed by the isolation process in both models. We used kidney slices maintained in physiological solution to better preserve the macroscopic structure of glomeruli and measured the impact of angiotensin II receptor activation and blockade of Transient Potential Receptor Canonical 5 (TRPC5) to examine their possible interaction in a number of pre‐ and intraglomerular cell types using spinning‐disk confocal calcium imaging.Calcium transients and tissue distortion in intact kidney slices of mice expressing genetically modified calcium‐sensors (GCaMP5) in Cx40‐positive cells were recorded and analyzed using particle analysis.We observed spontaneous calcium transients in preglomerular cells located at the vascular pole that were blocked by the SERCA pump inhibitor, cyclopiazonic acid (CPA). Stimulation with angiotensin II increased calcium activity and contraction in these cells and induced calcium transients in distinct populations of intraglomerular cells that were also blocked with CPA. While removal of external calcium did not change angiotensin II‐induced calcium transients in intraglomerular cells, the frequency of calcium transients in preglomerular cells was significantly reduced. In angiotensin II‐preincubated kidney slices, spontaneous calcium transients in the preglomerular area were diminished by the specific TRPC4/5 inhibitor, ML204. If given alone, ML204 completely inhibited all preglomerular calcium transients.Thick kidney slices maintained in vitro appear to better preserve the structure and responses of different cell types in glomeruli as measured using calcium transients and contractions. In our experimental set‐up, angiotensin II induced calcium transients in intraglomerular cells, while its impact on preglomerular cells appeared more indirect. As the TRPC5 antagonist was more effective at inhibiting calcium transients in preglomerular cells when the tissue was not prestimulated with angiotensin II, we suggest that TRPC5‐mediated calcium signaling is at least party modulated by angiotensin II.Support or Funding InformationSupported by Deutsche Forschungsgemeinschaft (DFG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The β‐adrenergic stimulation of the renal NaCl cotransporter is mediated via a cAMP‐dependent activation of protein phosphatase 1 inhibitor 1

Sympathetic β‐adrenergic activation of the thiazide‐sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT) was suggested to contribute to the development of salt‐sensitive hypertension. Previous studies demonstrated that β‐adrenergic agonists (e.g. norepinephrine and isoproterenol) increase cytoplasmic cAMP and the phosphorylation and hence the activity of NCC in the mouse DCT. Recently, we identified the endogenous inhibitor 1 (I1) of protein phosphatase 1 (PP1) as a DCT enriched gene product that controls NCC phosphorylation and blood pressure. As I1 is activated by cAMP‐dependent protein kinase A (PKA), we now tested the hypothesis that β‐adrenergic agonists inhibit PP1 via a cAMP/PKA‐dependent activation of I1 ultimately stimulating NCC phosphorylation. To test this hypothesis we performed experiments on MDCK cells stably transfected with NCC and on ex vivo preparations of kidneys from wildtype (WT) and I1‐knockout (I1‐KO) mice. The exposure of isolated DCTs to the cAMP‐elevating drugs forskolin and IBMX rapidly increased the phosphorylation of NCC via a protein kinase A (PKA)‐dependent pathway. The forskolin/IBMX‐induced phosphorylation of NCC was paralleled by I1 phosphorylation at its PKA‐consensus phosphorylation site (T35). Moreover, the forskolin/IBMX‐induced phosphorylation of NCC was diminished in kidney slices of from I1‐KO mice. The transgenic overexpression of a phosphomimetic I1 mutant (T35D) in kidneys of I1‐KO mice restored NCC phosphorylation, but made NCC resistant to forskolin/IBMX stimulation. Using an unbiased yeast‐two‐hybrid screen and co‐immunoprecipitation assays, we demonstrate that NCC and PP1 physically interact in transfected MDCK cells. Finally, the isoproterenol stimulated increase of NCC‐phosphorylation was impaired in I1‐KO mice as assessed in kidney slices and isolated‐perfused mouse kidneys. Remarkably, this occurred independent from any change in the phosphorylation of the kinases SPAK/OSR1. Thus, our data establish a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKA‐dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway likely accounts for beta‐adrenergic NCC activation and hence may contribute to salt‐sensitive hypertension in patients with sympathetic hyperactivity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Protective Role of TRPC6 Knockout in the Progression of Diabetic Nephropathy

Diabetic nephropathy (DN) is a chronic kidney pathology that leads to end stage renal disease (ESRD). It was previously found that a gain‐of‐function mutation in the Transient Receptor Potential Canonical 6 (TRPC6) channel, a non‐selective cation channel that is highly permeable for calcium ions, causes Focal Segmental Glomerulosclerosis (FSG) that is characterized with podocyte loss. Previous studies from our laboratory indicate that there is an association between the development of DN and over‐activation of the TRPC6 channel. We have demonstrated that there is an increase in TRPC6 expression and activity in the streptozotocin (STZ)‐induced diabetic Dahl Salt‐sensitive (SS) rat, an established model of type 1 DN. We have created the TRPC6 knock‐out on a SS rat background (SSTRPC6−/−), and test the hypothesis that the deletion of TRPC6 will protect podocytes and glomeruli during the development of DN. Four groups of animals – control SS, SSTRPC6−/−, and STZ‐treated SS and SSTRPC6−/− – were utilized in this study. Diabetes development was monitored for 11 weeks after STZ injection with periodic weight, glucose, and urinary output measurements. Glomerular filtration rate (GFR) was measured and tissue collections were made at the end of the protocol. Freshly isolated glomeruli of perfused kidneys were used from all four groups for either electron microscopy or confocal fluorescence microscopy to assess the foot process effacement and basal level of intracellular calcium ([Ca2+]i), respectively. Histochemical analysis of whole kidney slices was done to assess renal injury. There was a decrease in urinary output (49±29 vs 73±39 mL/day/100g BW) and a trend for blunted increase in albuminuria (97±34 vs 112±19 mg/day x 100g BW) in the STZ‐treated SSTRPC6−/− rats compared to the STZ‐SS animals. There was no significant difference in GFR between either of the 4 experimental groups; however there does appear to be a trend for hyperfiltration in the STZ‐treated animals. Importantly, pathological increases in basal [Ca2+]i levels and foot process damage of podocytes during the development of DN was attenuated in the STZ‐treated SSTRPC6−/− animals. Overall, our data indicates that inhibiting the TRPC6 channel may have renoprotective effects and the potential to lead to the development of new pharmacological tools to treat or prevent the progression of DN and kidney damage.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Db/db Obese Mice Exhibit Enhanced Phosphorylation of p38, ERK1/2 and AKT in the Kidney

Altered metabolic states due to obesity and diabetes have a direct impact on renal structure and function. Obesity and diabetes leads to several key morphological, subcellular fibrotic remodeling, nephropathy and renal failure. However the clear signaling mechanism is not known. Leptin receptor deficiency is associated with obesity and hypercholesterolemia. We showed that acute cholesterol depletion increased cellular function and phosphorylation of p38, ERK and AKT kinases. Hence we hypothesized that obesity enhances phosphorylation of p38, ERK1/2 and AKT kinases in the kidney. To test our hypothesis we used kidneys from 25 weeks old four leptin receptor deficient db/db obese mice and their age matched four c57BL6 (wild type) mice. Transverse slices were made from similar position of all kidneys. Kidney slices were chopped into very small pieces and homogenized under the ice cold lysis buffer with protease and phospahtase inhibitors. The low spin pellets were discarded and the supernatants were collected. Kidney lysates were run through 7–12% poly acrylamide gel electrophoresis. Phosphorylations of p38, ERK1/2 and AKT were measured using western blot and cell signaling antibodies. The results were normalized to β‐actin as a control for protein loading using NIH image J software. Data of phospho proteins expressed in arbitrary units for p38 (14477 ±1703 vs. 22488±2773, p&lt;0.03), ERK1/2 (12408 ±1965 vs. 21807±1467, p&lt;0.01) and AKTThr308 (10173 ±462 vs. 18250±1198, p&lt;0.01) were more in obese db/db mice compared to wild type mice. Total p38, ERK1/2 and AKT were not different between db/db and wild type mice. We conclude that obesity is associated with enhanced phosphorylation of p38, ERK and AKTThr308 which could be associated with altered renal structure and function.Support or Funding InformationHamad Medical Research Center #16304This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

OAT3 Mediated Interaction between Steviol Acyl Glucuronide and Probenecid: In Vitro and In Vivo Investigations

Steviol acyl glucuronide (SVAG) is a circulating metabolite formed after the ingestion of rebaudioside A, a naturally occurring sweetner with functional activities. Our previous studies have demonstrated that transporters particularly OAT3 played an important role in renal clearance of SVAG. The present study aimed to investigate the inhibitory effect of probenecid on OAT3 mediated SVAG transport using in vitro and in vivo systems. In the hOAT3 stably transfected HEK293 cell line, probenecid exhibited a dose‐dependent inhibition of SVAG uptake, with an IC50 value of 4.95 μM. In rat kidney slices, the uptake of SVAG was markedly inhibited by probenecid (67.5%), estrone‐3‐sulfate (41.9%), and para‐aminohippurate (PAH, 27.0%), respectively. In rats given an oral dose of 15 mg/kg of rebaudioside A, the Tmax, Cmax and AUC6–8 of SVAG were 6.25 hr, 176 ng/mL and 213.8 ng/mL*hr, respectively. In contrast, an intravenous dose of probenecid (20 mg/kg) led to an increase of Cmax and AUC by 2.15 and 1.71 folds, respectively, while Tmax remained relatively unchanged. Taken together, the inhibition effect on SVAG uptake in the presence of probenecid was mediated via OAT3. As OAT3 is a major uptake transporter in the kidney, the inhibition of OAT3 activity may alter SVAG's renal clearance. It is thus prudent to investigate transporter‐mediated food‐drug interactions and ensure safe and effective use of those products.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Three‐dimensional analysis of potassium deprivation‐induced tubular remodeling using optical clearing

Tubular remodeling can be induced by various stimuli and plays a critical role in functional adaptation. For instance, hypokalemia is associated with increased kidney mass caused by cellular hypertrophy and hyperplasia. However, accurate quantitative analysis of tubular remodeling is still lacking since most kidney morphometry was performed with two‐dimensional techniques. Here, we combined optical clearing and advanced light microscopy as a new approach for three‐dimensional analysis of tubular remodeling in mouse kidneys, upon dietary potassium (K+) restriction.Adult C57BL/6J mice were fed for 3 days with K+‐deficient diet (approx. 0.002% K+) or normal K+ diet (1.2% K+) and were injected with Bromodeoxyuridine (BrdU) intraperitoneally before kidneys were perfusion‐fixed 4 h later. Kidneys were cut into 1.0–1.5 mm thick slices and stained for BrdU then optically cleared with Ethyl Cinnamate. Confocal microscopy was used to identify the total number of BrdU positive cells per unit volume. In addition, 4 μm sections were used for co‐staining of BrdU with segment‐specific antibodies to localize proliferating cells. Whole blood was collected via terminal cardiac puncture and analyzed using i‐STAT analyzer (Abbot Point of Care Inc.).As expected, serum K+ levels were significantly lower in mice on K+ ‐deficient diet than in mice on normal diet (3.7 ± 0.1 mmol/L vs. 4.2 ± 0.1 mmol/L, p&lt;0.005, n=6–7). After optical clearing, confocal microscopy allowed high‐resolution imaging of BrdU positive cells within the whole thick kidney slice. Quantification of BrdU positive cells revealed that the cell proliferation rate was low in control mice, but that K+ restriction strongly induced cell proliferation (3748 ± 392 cells/mm3 vs. 1089 ± 341 cells/mm3 p&lt;0.05, n=3). Co‐localization using thin sections and segment‐specific markers revealed that this increase in proliferation was restricted to the S3 segment of the proximal tubule and outer medullary collecting duct intercalated cells.In this study, we validated a combination of optical clearing and advanced light microscopy as a new toolbox for imaging and quantification of tubular remodeling, which will decrease our reliance on biased two‐dimensional morphometric techniques and time‐consuming stereological approaches. Our analysis revealed that dietary K+ restriction caused preferential hyperplasia in S3 segments and in intercalated cells. The former observation might reflect a functional adaptation after low‐K+ induced injury of proximal tubules, whereas the latter likely represent a compensatory response to increase renal potassium uptake.Support or Funding InformationDFG German Research Foundation Fellowship (332853055) and Else Kröner‐Fresenius Stiftung (2015_A197) to TS, Humboldt Foundation and National Health and Medical Research Council of Australia Research Fellowships to VGP, R01DK098141 to JAM, and R01DK054983 to DHE.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.