Renal Proximal Cells Research Articles

Functional and metabolomic analysis of urinary extracellular vesicles from juvenile mice with renal compensatory hypertrophy

Unilateral nephrectomy, a procedure reducing kidney mass, triggers a compensatory response in the remaining kidney, increasing its size and function to maintain a normal glomerular filtration rate (GFR). Recent research has highlighted the role of extracellular vesicles (EVs) in renal physiology and disease, although their involvement in unilateral nephrectomy has been underexplored. In this study, unilateral nephrectomy was performed on young mice, and urinary extracellular vesicles (uEVs) characterization and cargo were analyzed. Kidney volume increased significantly post-nephrectomy, demonstrating compensatory hypertrophy. Serum creatinine, cystatin C, and urinary electrolytes concentrations were similar in both nephrectomized and control groups. Western blot analysis revealed upregulation of sodium-glucose cotransporter 2 (SGLT2) and sodium chloride cotransporter (NCC), and downregulation of sodium‑potassium-chloride co-transporter (NKCC2) and epithelial sodium channel (ENaC) in the nephrectomized group. Metabolomic analysis of uEVs showed an enrichment of certain metabolites, including citrate and stachydrine. Interestingly, uEVs from the nephrectomized group demonstrated a protective effect, downregulating signal transducer and activator of transcription 3 (STAT3) and reducing reactive oxygen species (ROS) in renal proximal cells, compared to uEVs from the control group. This study suggests that uEVs contain bioactive components capable of inducing protective, anti-inflammatory, anti-fibrinolytic, and antioxidative effects in renal cells. These findings contribute to our understanding of uEVs' role in renal compensatory mechanisms after unilateral nephrectomy and may hold promise for future therapeutic interventions in renal diseases.

Differentiation and Subculturing of Renal Proximal Tubular-like Cells Derived from Human iPSC.

Recently, we have developed a protocol to differentiate human induced pluripotent stem cells (iPSC) into proximal tubular-like cells (PTL) (Chandrasekaran etal., 2021). These cells express proximal tubular-specific markers, including megalin, and form a polarized monolayer expressing tight junction proteins, including ZO-3 and occludin. Furthermore, PTL display functional properties, including megalin-facilitated endocytosis, P-glycoprotein (ABCB1) efflux, and respond to parathyroid hormone. Here, we report step-by-step protocols to culture iPSC prior to differentiation (Basic Protocol 1), to differentiate PTL from iPSC (Basic Protocol 2), and to passage and freeze-thaw PTL (Basic Protocol 3). Additionally, we provide a protocol (Basic Protocol 4) to culture PTL on microporous growth supports (transwells). Immunofluorescence stainings for characteristic markers, including megalin, are shown for unpassaged (Basic Protocol 2) and passaged (Basic Protocol 3) PTL. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: iPSC culture Basic Protocol 2: iPSC-derived PTL differentiation Basic Protocol 3: PTL passaging, culturing, and freezing Basic Protocol 4: PTL culturing on transwells Support Protocol 1: Preparation of Geltrex-coated cell culture plates Support Protocol 2: Preparation of RPTEC/TERT1 or fHDF/TERT166-ECM-coated cell culture plates Support Protocol 3: Preparation of human collagen IV-coated cell culture plates Support Protocol 4: Immunofluorescence staining.

#5403 PTEN-INDUCED KINASE 1 HAS ASSOCIATION WITH RENAL AGING PROCESS THROUGH THE CGAS -STING PATHWAY

Abstract Background and Aims The global increase of an aging population has encouraged the research on renal aging, as the kidney is one of the organs that shows the greatest change as aging progresses. Although the mechanism of renal aging has not been clearly identified, dysfunctional mitochondria has been suggested as one of the factors that induce inflammation of the kidney, which is the major mediator of the pro-aging process of chronic kidney disease. PTEN-induced kinase 1(PINK1) is a protein involved in the quality control of mitochondria and regulates mitochondrial dysfunction. Although it is known that the mitochondrial DNA release promoted by PINK1 deficiency stimulates cyclic GMP–AMP synthase (cGAS) - stimulator of interferon genes (STING) pathway eventually resulting in the inflammatory response, the role of PINK1 and cGAS-STING pathway in renal aging has not yet been clarified. This study aimed to investigate the relationship between PINK1 and renal aging, especially through the cGAS-STING pathway. Method To determine the role of PINK1 on the renal aging process, renal fibrosis, and tubular injury were compared in 4- and 24-month-old wild-type (Pink1+/+) and PINK1 knockout (Pink1-/-) mice. To establish in vitro senescence model, hydrogen peroxide (H2O2) treatment on human renal proximal cells (HKC-8) was used. The changes in gene expression levels related to PINK1 were analyzed by RNA sequencing, applying transcriptomic and metabolomic analyses. To validate the results of RNA sequencing, we measured mitochondrial oxygen consumption rate (OCR) by Seahorse Mito Stress Test. To investigate the relationship between PINK1 and renal aging through the cGAS-STING pathway, we explored the change of cGAS-STING expression on senescence-induced HKC-8 cells and additionally used H-151 treatment, a specific STING inhibitor. Results The renal fibrosis and tubular injury were significantly aggravated in 24-month-old Pink1-/- mice compared to 24-month-old Pink1+/+ mice. Western blot and RT-qPCR confirmed remarkably increased senescence markers and senescence-associated secretory phenotype (SASPs) in 24-month Pink1-/- mice and senescence-induced HKC-8 cells. The RNA sequencing of mice kidneys showed that inflammation-related pathways significantly increased in 24 months Pink1-/-mice, and transcriptomic and metabolomic analyses showed that PINK1 has an association with mitochondrial metabolism dysregulation. On OCR measurement, the basal respiration, maximal respiration, ATP production, and respiratory capacity significantly declined in H2O2-treated siPINK1 cells, suggesting that PINK1 deficiency might have effects on mitochondrial dysfunction. Finally, the STING pathway was significantly activated in 24-month Pink1-/- mice and senescence-induced HKC-8 cells, which was inhibited by a specific inhibitor of STING, H-151. Conclusion In conclusion, PINK1 is associated with renal aging, and the dysregulation of mitochondria caused by PINK1 deficiency might lead to aging-related inflammatory responses through the cGAS-STING pathway.

Acidosis Activates the Nrf2 Pathway in Renal Proximal Tubule-Derived Cells through a Crosstalk with Renal Fibroblasts

Crosstalk of renal epithelial cells with interstitial fibroblasts plays an important role in kidney pathophysiology. A previous study showed that crosstalk between renal epithelial cells and renal fibroblasts protects against acidosis-induced damage. In order to gain further mechanistic insight into this crosstalk, we investigated the effect of acidosis on the transcriptome of renal epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) in co-culture by RNASeq, bioinformatics analysis and experimental validation. Cells were exposed to acidic media or control media for 48 h. RNA and protein from whole cell lysate were isolated. In addition, cells were fractionated into cytosol, nucleus and chromatin. RNASeq data were analyzed for differential expression and pathway enrichment (ingenuity pathway analysis, IPA, QIAGEN). Total and phosphorylated protein expression was assessed by Western blot (WB). Transcription factor activity was assessed by luciferase reporter assay. Bioinformatic analysis using differentially expressed genes according to RNASeq (7834 for NRK-52E and 3197 for NRK-49F) predicted the antioxidant and cell-protective Nrf2 pathway as acidosis-induced in NRK-52E and NRK-49F cells. Activation of Nrf2 comprises enhanced Nrf2 phosphorylation, nuclear translocation, DNA binding and initiation of a cell protective transcriptional program. Our data show that acidosis enhances chromatin-associated Nrf2 expression and the abundance of phosphorylated Nrf2 in the chromatin fraction of NRK-52E cells in co-culture but not in monoculture. Furthermore, acidosis enhances the activity of a reporter for Nrf2 (ARE-luciferase). Despite the bioinformatics prediction, NRK-49F cells did not respond with Nrf2 activation. Transketolase (TKT) is an important regulator of antioxidant and homeostatic responses in the kidney and a canonical Nrf2 target gene. We show that protein and mRNA expression of TKT is increased in NRK-52E cells under co-culture but not under monoculture conditions. In conclusion, our data show that extracellular acidosis activates the cytoprotective transcription factor Nrf2 in renal epithelial cells co-cultivated with renal fibroblasts, thereby enhancing the expression of cytoprotective TKT. This protective response is not observed in monoculture. Activation of the Nrf2 pathway represents a co-operative cellular strategy of protection against acidosis.

Capturing time-dependent activation of genes and stress-response pathways using transcriptomics in iPSC-derived renal proximal tubule cells

Transcriptomic analysis is a powerful method in the utilization of New Approach Methods (NAMs) for identifying mechanisms of toxicity and application to hazard characterization. With this regard, mapping toxicological events to time of exposure would be helpful to characterize early events. Here, we investigated time-dependent changes in gene expression levels in iPSC-derived renal proximal tubular-like cells (PTL) treated with five diverse compounds using TempO-Seq transcriptomics with the aims to evaluate the application of PTL for toxicity prediction and to report on temporal effects for the activation of cellular stress response pathways. PTL were treated with either 50 μM amiodarone, 10 μM sodium arsenate, 5 nM rotenone, or 300 nM tunicamycin over a temporal time course between 1 and 24 h. The TGFβ-type I receptor kinase inhibitor GW788388 (1 μM) was used as a negative control. Pathway analysis revealed the induction of key stress-response pathways, including Nrf2 oxidative stress response, unfolding protein response, and metal stress response. Early response genes per pathway were identified much earlier than 24 h and included HMOX1, ATF3, DDIT3, and several MT1 isotypes. GW788388 did not induce any genes within the stress response pathways above, but showed deregulation of genes involved in TGFβ inhibition, including downregulation of CYP24A1 and SERPINE1 and upregulation of WT1. This study highlights the application of iPSC-derived renal cells for prediction of cellular toxicity and sheds new light on the temporal and early effects of key genes that are involved in cellular stress response pathways.

Mechanical stretch‐driven ROS generation stimulates endothelin production in neonatal pig renal epithelial and vascular endothelial cells

The circulating level of endothelin (ET), a potent vasoactive peptide, decreases with postnatal age. ET is also of pathophysiological significance in the kidney. ET is generated via endothelin converting enzyme (ECE)‐induced proteolytic processing of pro‐molecule big ET to biologically active peptides. Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), transactivates the promoter of ECE1. Oxyradicals contribute to kidney pathophysiology, but whether ROS regulates ET production in the kidneys remains unresolved. The present study shows that ECE1 is the predominant isoform in neonatal pig kidneys. We also demonstrated that H2O2 induces ECE1‐dependent ET1‐3 production in the neonatal pig renal proximal tubules (PT) and vascular endothelial cells (EC). A uniaxial stretch of neonatal pig PT and EC increased NADPH oxidase (NOX) 2 and 4 levels and stimulated H2O2production. Mechanical stretch increased cellular ECE1 production in the PC and EC cells, which catalase mimetic EUK 134 and NOX inhibitor apocynin reversed. Mechanical stretch also triggered cellular ET1‐3 generation in PT and EC. Mechanical stretch‐induced ET1‐3 production was attenuated by EUK 134, apocynin, and a selective ECE1 inhibitor CGS 35066. Short‐term unilateral urinary tract obstruction (UUO), an inducer of the mechanical stretch of kidney cells and oxyradical production, increased ECE1‐dependent ET1‐3 production in neonatal pig kidneys. Despite removing the obstruction, acute UUO generated ROS, reduced kidney perfusion, and increased renal vascular resistance (RVR). UUO also elevated early biomarkers of acute kidney injury (AKI). Inhibition of ECE1 and ET receptors protected against UUO‐induced increase in RVR and early AKI. These data suggest that mechanical stretch‐driven ROS generation stimulates ET production in neonatal pig renal epithelial and vascular endothelial cells. ET then promotes AKI by causing renal hypoperfusion. This signaling pathway may underlie UUO‐induced renal insufficiency in infants.

Temporal transcriptomic alterations of cadmium exposed human iPSC-derived renal proximal tubule-like cells

Cadmium is a well-studied environmental pollutant where the kidney and particularly the proximal tubule cells are especially sensitive as they are exposed to higher concentrations of cadmium than other tissues. Here we investigated the temporal transcriptomic alterations (TempO-Seq) of human induced pluripotent stem cell (iPSC)-derived renal proximal tubule-like (PTL) cells exposed to 5 μM cadmium chloride for 1, 2, 4, 8, 12, 16, 20, 24, 72 and 168 h. There was an early activation (within 4 h) of the metal and oxidative stress responses (metal-responsive transcription factor-1 (MTF1) and nuclear factor erythroid-2-related factor 2 (Nrf2) genes). The Nrf2 response returned to baseline within 24 h. The Activator Protein 1 (AP-1) regulated genes HSPA6 and FOSL-1 followed the Nrf2 time course. While the MTF1 genes also spiked at 4 h, they remained strongly elevated over the entire exposure period. The data and cell culture model utilised will be useful in further research aimed at the refinement of safe human exposure limits for cadmium, other metals and their mixtures.

Generation and characterization of iPSC-derived renal proximal tubule-like cells with extended stability

The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.

Chemosensitizing activity of peptide from Lentinus squarrosulus (Mont.) on cisplatin-induced apoptosis in human lung cancer cells

The limitations of cisplatin, a standard chemotherapy for lung cancer, have been documented with serious adverse effects and drug resistance. To address the need for novel therapy, this study firstly reveals the potential of peptide from Lentinus squarrosulus (Mont.) as a chemotherapeutic adjuvant for cisplatin treatment. The purified peptide from L. squarrosulus aqueous extracts was obtained after eluting with 0.4 M NaCl through FPLC equipped with anion exchange column. Preincubation for 24 h with 5 µg/mL of the peptide at prior to treatment with 5 µM cisplatin significantly diminished %cell viability in various human lung cancer cells but not in human dermal papilla and proximal renal cells. Flow cytometry indicated the augmentation of cisplatin-induced apoptosis in lung cancer cells pretreated with peptide from L. squarrosulus. Preculture with the peptide dramatically inhibited colony formation in lung cancer cells derived after cisplatin treatment. Strong suppression on integrin-mediated survival was evidenced with the diminution of integrins (β1, β3, β5, α5, αV) and down-stream signals (p-FAK/FAK, p-Src/Src, p-Akt/Akt) consequence with alteration of p53, Bax, Blc-2 and Mcl-1 in cisplatin-treated lung cancer cells preincubated with peptide from L. squarrosulus. These results support the development of L. squarrosulus peptide as a novel combined chemotherapy with cisplatin for lung cancer treatment.

Nephrotoxicity of cypermethrin in rats. Histopathological aspects.

Cypermethrin (CYP) is an important type II pyrethroid pesticide widely used to protect crops against pests and insect infestations. However, its toxicity is a risk to both human health and the surrounding environment. The present study was conducted to investigate the nephrotoxic effect and histopathological changes caused by Cypermethrin in the kidney tissues of adult Wistar rats. In this study, 30 Wistar rats were equally divided into three groups. G1, control animals; G2 and G3 treated with various sub lethal doses of CYP for 30 days as follows: G2, administered low dose (1/100 of LD50) of CYP; G3, administered high dose (1/50 of LD50) of CYP. The damage to different organelles of renal proximal and distal cells was observed using transmission electron microscopy. Histopathological damage in kidney samples was confirmed using morphological and histological measures. The results showed that CYP caused significant histopathological damage to the renal proximal and distal tubules of treated rats. Compared to control samples, CYP caused marked alterations in the dimensions of nucleus, ovoid and filamentous mitochondria of the treated cells. In conclusion, Cypermethrin is found to be toxic to mammals. It caused marked ultrastructural damage to the renal proximal and distal tubules of Wistar rats and the intensity of nephrotoxicity correlated with the dose of oral administration.

Differential interactions of the β-lactam cloxacillin with human renal organic anion transporters (OATs).

The β-lactam penicillin antibiotic cloxacillin (CLX) presents wide inter-individual pharmacokinetics variability. To better understand its molecular basis, the precise identification of the detoxifying actors involved in CLX disposition and elimination would be useful, notably with respect to renal secretion known to play a notable role in CLX elimination. The present study was consequently designed to analyze the interactions of CLX with the solute carrier transporters organic anion transporter (OAT) 1 and OAT3, implicated in tubular secretion through mediating drug entry at the basolateral pole of renal proximal cells. CLX was first shown to block OAT1 and OAT3 activity in cultured OAT-overexpressing HEK293 cells. Half maximal inhibitory concentration (IC50 ) value for OAT3 (13µm) was however much lower than that for OAT1 (560µm); clinical inhibition of OAT activity and drug-drug interactions may consequently be predicted for OAT3, but not OAT1. OAT3, unlike OAT1, was next shown to mediate CLX uptake in OAT-overexpressing HEK293 cells. Kinetic parameters for this OAT3-mediated transport of CLX (Km =10.7µm) were consistent with a possible in vivo saturation of this process for high CLX plasma concentrations. OAT3 is consequently likely to play a pivotal role in renal CLX secretion and consequently in total renal CLX elimination, owing to the low plasma unbound fraction of the antibiotic. OAT3 genetic polymorphisms as well as co-administered drugs inhibiting in vivo OAT3 activity may therefore be considered as potential sources of CLX pharmacokinetics variability.

Renogenic characterization and in vitro differentiation of rat amniotic fluid stem cells into renal proximal tubular- and juxtaglomerular-like cells.

The aim of the present study was to investigate the renogenic characteristics of amniotic fluid stem cells (AFSCs) and to evaluate their in vitro differentiation potential into renal proximal tubular-like cells and juxtaglomerular-like cells. We culture expanded AFSCs derived from rat amniotic fluid. The AFSCs grew as adherent spindle-shaped cells and expressed mesenchymal markers CD73, CD90, and CD105 as well as renal progenitor markers WT1, PAX2, SIX2, SALL1, and CITED1. AFSCs exhibited an in vitro differentiation potential into renal proximal tubular epithelial-like cells, as shown by the upregulation of expression of proximal tubular cell-specific genes like AQP1, CD13, PEPT1, GLUT5, OAT1, and OCT1. AFSCs could also be differentiated into juxtaglomerular-like cells as demonstrated by the expression of renin and α-SMA. The AFSCs also expressed pluripotency markers OCT4, NANOG, and SOX2 and could be induced into embryoid bodies with differentiation into all the three germ layers, highlighting the pluripotent nature of these cells. Our results show that amniotic fluid contains a population of primitive stem cells that express renal-progenitor markers and also possess the propensity to differentiate into two renal lineage cell types and, thus, may have a therapeutic potential in renal regenerative medicine.

Effect of Fuyuan capsule on the expression of transforming growth factor-β1, insulin-like growth factor-1, osteocalcin and type I collagen in rats with fractures

Objective To investigate the effect of Fuyuan capsule on expression of transforming growth factor-β1 (TGF-β1), insulin-like growth factor-1 (IGF-1), osteocalcin and type Ⅰ collagen in serum of rats with diabetic fractures. Methods Forty-eight male SD rats were divided into Fuyuan capsule group (group A, n=24, 8 rats in high, medium and low dose subgroups, respectively), insulin group (group B, n=8), model group (group C, n=8) and blank control group (group D, n=8). Except group D, the other groups were treated with intraperitoneal injection of streptozotocin to induce diabetes. All rats were treated with reduction and external fixation with splint intervention after establishing left tibia upper oblique fracture models. Group A were treated with intragastric administration of Fuyuan capsule according to high, medium and low dose (1.23, 0.82, 0.41 g/kg). Group B were treated with 6-8 U insulin, while group C and group D were treated with local injection of normal saline.1 week, 3 weeks and 5 weeks after modeling, the expressions of TGF-β1, IGF-1, osteocalcin and type Ⅰ collagen in serum was detected.5 weeks after modeling, the rats were killed and the main organs were examined. Results The expression levels of TGF-β1 [(116.52±10.55), (117.25±10.37), (117.82±10.24) ng/L], IGF-1 [(3.16±0.42), (3.18±0.44), (3.21±0.41) ng/ml], osteocalcin [(3.03±0.91), (4.06±0.92), (12.05±2.91) U/ml] and type Ⅰ collagen [(5.32±1.15), (5.36±1.18), (14.34±2.15) pg/ml] in serum of group C at different time points were significantly lower than those in the other groups (F=10.369, 16.836, 20.583, 9.364, 9.025, 10.352, 9.684, 18.364, 22.536, 8.365, 14.834, 16.286, P=0.014, 0.000, 0.000, 0.019, 0.020, 0.014, 0.018, 0.000, 0.000, 0.022, 0.000, 0.000). 1 week after modeling, the levels of serum indexes in group B and three subgroups of group A were slightly higher than those in group D (P=0.752, 0.694, 0.633, 0.645, 0.160, 0.153, 0.120, 0.134, 0.853, 0.805, 0.820, 0.822, 0.909, 0.884, 0.857, 0.870). 3 and 5 weeks after modeling, the levels of serum indexes in group B and group A were significantly higher than those in group D (P=0.005, 0.003, 0.000, 0.000, 0.021, 0.024, 0.017, 0.018, 0.000, 0.000, 0.007, 0.007, 0.000, 0.000, 0.013, 0.014) but significantly lower than those in the medium and low dose groups of group A (P=0.032, 0.030, 0.045, 0.042, 0.033, 0.030, 0.036, 0.034, 0.016, 0.016, 0.015, 0.016, 0.020, 0.023, 0.017, 0.020, 0.002, 0.001, 0.001, 0.001, 0.021, 0.022, 0.020, 0.021, 0.003, 0.004, 0.003, 0.003, 0.010, 0.011, 0.010, 0.010). There were no significant differences in levels of serum indexes between the medium and low dose groups at different time points (P=0.988, 0.979, 0.978, 0.964, 0.966, 0.932, 1.000, 0.995, 0.985, 0.987, 0.946, 0.985). After 5 weeks of continuous administration, except there was 1 rat with local mild edema of renal proximal convoluted tubule epithelial cells in high dose group of group A, there were no abnormal pathological changes in the other groups. Conclusion Fuyuan Capsule can effectively regulate expression of TGF-β1, IGF-1, osteocalcin and type Ⅰ collagen in serum of rats with diabetic fractures, which is good for fracture healing. Key words: Fuyuan capsule; Rat; Fracture; Bone healing

Megalin-targeted enhanced transfection efficiency in cultured human HK-2 renal tubular proximal cells using aminoglycoside-carboxyalkyl- polyethylenimine -containing nanoplexes

Non-viral vectors are of interest as therapeutic gene delivery agents in gene therapy, because they are simple to prepare, easy to modify and have definable safety profiles compared to viral vectors. The potential of gene therapy in the treatment of renal diseases is limited by a lack of effective kidney-targeted gene delivery systems. Aminoglycoside antibiotics gentamicin and neomycin were connected by amide linkages to carboxyl groups on carboxyalkylated-PEI25 (25kDa PEI) or carboxyalkylated-PEI10 (10kDa PEI). Aminoglycoside-carboxyalkylated-PEI conjugates were characterized with respect to size, surface charge density, DNA condensation ability, and buffering capacity. Polyplexes prepared by electrostatic interaction between aminoglycoside-carboxyalkylated-PEIs and enhanced green fluorescent protein-expressing (EGFP) plasmid DNA had appropriate nano-scale size (143–173nm). Their targeting potential was investigated in cultured HK-2 immortalized human cortex/proximal tubule kidney epithelial cells, which expresses megalin, a scavenger receptor that mediates endocytosis of a diverse group of ligands, including aminoglycoside antibiotics. Aminoglycoside-carboxyalkylated-PEIs significantly increased EGFP gene transfection efficiency in HK-2 cells by ∼13-fold for aminoglycoside-carboxyalkylated-PEI25 and ∼7-fold increase for aminoglycoside-carboxyalkylated-PEI10 relative to the corresponding PEIs without aminoglycosides. The transfection efficiency of polyplexes was dependent on the weight ratio of aminoglycoside-containing ligand in the carrier. In the presence of a range of concentrations of human serum albumin, which competes for megalin binding, aminoglycoside-carboxyalkylated-PEI-mediated transfection was reduced to background levels. These results suggest that aminoglycoside-carboxyalkylated-PEI polyplexes can target megalin-expressing kidney-derived cells in vitro resulting in improved transfection efficiency with low cytotoxicity.

Protective effect of ginsenoside Rh3 against anticancer drug-induced apoptosis in LLC-PK1 kidney cells

BackgroundGinsenosides are active components of Panax ginseng that exert various health benefits including kidney protection effect. The medicinal activity of ginsenosides can be enhanced by modulating their stereospecificity by heat processing. Ginsenosides Rk2 and Rh3 represent positional isomers of the double bond at C-20(21) or C-20(22). MethodsThe present study investigated the kidney-protective effects of ginsenosides Rk2 and Rh3 against cisplatin, a platinum based anticancer drug, induced apoptotic damage in renal proximal LLC-PK1 cells. ResultsAs a result, ginsenoside Rh3 shows a stronger protective effect than that shown by Rk2. Cisplatin-induced elevated protein levels of phosphorylated c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38, and cleaved caspase-3 decreased after cotreatment with ginsenoside Rh3. The increase in the percentage of apoptotic LLC-PK1 cells induced by cisplatin treatment also significantly reduced after cotreatment with ginsenoside Rh3. ConclusionThese results demonstrate that inhibition of the JNK and ERK mitogen-activated protein kinase signaling cascade plays a critical role in mediating the renoprotective effect of ginsenoside Rh3.

Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome.

The eukaryotic initiation factor 5A (eIF5A), which is highly conserved throughout evolution, has the unique characteristic of post-translational activation through hypusination. This modification is catalyzed by two enzymatic steps involving deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Notably, eIF5A may be involved in regulating the lifespan of Drosophila during long-term hypoxia. Therefore, we investigated the possibility of a link between eIF5A hypusination and cellular resistance to hypoxia/anoxia. Pharmacologic targeting of DHPS by N1-guanyl-1,7-diaminoheptane (GC7) or RNA interference-mediated inhibition of DHPS or DOHH induced tolerance to anoxia in immortalized mouse renal proximal cells. Furthermore, GC7 treatment of cells reversibly induced a metabolic shift toward glycolysis as well as mitochondrial remodeling and led to downregulated expression and activity of respiratory chain complexes, features characteristic of mitochondrial silencing. GC7 treatment also attenuated anoxia-induced generation of reactive oxygen species in these cells and in normoxic conditions, decreased the mitochondrial oxygen consumption rate of cultured cells and mice. In rats, intraperitoneal injection of GC7 substantially reduced renal levels of hypusinated eIF5A and protected against ischemia-reperfusion-induced renal injury. Finally, in the preclinical pig kidney transplant model, intravenous injection of GC7 before kidney removal significantly improved graft function recovery and late graft function and reduced interstitial fibrosis after transplant. This unconventional signaling pathway offers an innovative therapeutic target for treating hypoxic-ischemic human diseases and organ transplantation.

Triiodothyronine regulates cell growth and survival in renal cell cancer.

Triiodothyronine plays an important role in the regulation of kidney cell growth, differentiation and metabolism. Patients with renal cell cancer who develop hypothyreosis during tyrosine kinase inhibitor (TKI) treatment have statistically longer survival. In this study, we developed cell based model of triiodothyronine (T3) analysis in RCC and we show the different effects of T3 on renal cell cancer (RCC) cell growth response and expression of the thyroid hormone receptor in human renal cell cancer cell lines from primary and metastatic tumors along with human kidney cancer stem cells. Wild-type thyroid hormone receptor is ubiquitously expressed in human renal cancer cell lines, but normalized against healthy renal proximal tube cell expression its level is upregulated in Caki-2, RCC6, SKRC-42, SKRC-45 cell lines. On the contrary the mRNA level in the 769-P, ACHN, HKCSC, and HEK293 cells is significantly decreased. The TRβ protein was abundant in the cytoplasm of the 786-O, Caki-2, RCC6, and SKRC-45 cells and in the nucleus of SKRC-42, ACHN, 769-P and cancer stem cells. T3 has promoting effect on the cell proliferation of HKCSC, Caki-2, ASE, ACHN, SK-RC-42, SMKT-R2, Caki-1, 786-0, and SK-RC-45 cells. Tyrosine kinase inhibitor, sunitinib, directly inhibits proliferation of RCC cells, while thyroid hormone receptor antagonist 1-850 (CAS 251310‑57-3) has less significant inhibitory impact. T3 stimulation does not abrogate inhibitory effect of sunitinib. Renal cancer tumor cells hypostimulated with T3 may be more responsive to tyrosine kinase inhibition. Moreover, some tumors may be considered as T3-independent and present aggressive phenotype with thyroid hormone receptor activated independently from the ligand. On the contrary proliferation induced by deregulated VHL and or c-Met pathways may transgress normal T3 mediated regulation of the cell cycle.

Tuberin regulates reactive oxygen species in renal proximal cells, kidney from rodents, and kidney from patients with tuberous sclerosis complex

Reactive oxygen species (ROS) are an important endogenous source of DNA damage and oxidative stress in all cell types. Deficiency in tuberin resulted in increased oxidative DNA damage in renal cells. In this study, the role of tuberin in the regulating of ROS and NADPH oxidases was investigated. Formation of ROS and activity of NADPH oxidases were significantly higher in mouse embryonic fibroblasts and in primary culture of rat renal proximal tubular epithelial tuberin‐deficient cells compared to wild‐type cells. In addition, expression of NADPH oxidase (Nox)1, Nox2, and Nox4 (Nox isoforms) was higher in mouse embryonic fibroblasts and renal proximal tubular epithelial tuberin‐deficient cells compared to wild‐type cells. Furthermore, activity levels of NADPH oxidases and protein expression of all Nox isoforms were higher in the renal cortex of rat deficient in tuberin. However, treatment of tuberin‐deficient cells with rapamycin showed significant decrease in protein expression of all Nox. Significant increase in protein kinase C βII expression was detected in tuberin‐deficient cells, whereas inhibition of protein kinase C βII by bisindolylmaleimide I resulted in decreased protein expression of all Nox isoforms. In addition, treatment of mice deficient in tuberin with rapamycin resulted in significant decrease in all Nox protein expression. Moreover, protein and mRNA expression of all Nox were highly expressed in tumor kidney tissue of patients with tuberous sclerosis complex compared to control kidney tissue of normal subjects. These data provide the first evidence that tuberin plays a novel role in regulating ROS generation, NADPH oxidase activity, and Nox expression that may potentially be involved in development of kidney tumor in patients with tuberous sclerosis complex.

Our panel of experts highlight the most important research articles across the spectrum of topics relevant to the field of regenerative medicine.

Regenerative MedicineVol. 11, No. 2 Research HighlightsOur panel of experts highlight the most important research articles across the spectrum of topics relevant to the field of regenerative medicineThomas Bollenbach, Jizeng Qiao & Amber Kerstetter-FogleThomas Bollenbach Research & Development, Harvard Apparatus Regenerative Technology, 84 October Hill Road, Holliston, MA 01746, USASearch for more papers by this author, Jizeng Qiao Organogenesis, Inc., 150 Dan Rd, Canton, MA 02021, USASearch for more papers by this author & Amber Kerstetter-Fogle Case Western Reserve University, Cleveland, OH, USASearch for more papers by this authorPublished Online:29 Feb 2016https://doi.org/10.2217/rme.16.2AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleReferences1 Hiki N, Nunobe S, Kubota T et al. Function-preserving gastrectomy for early gastric cancer. Ann. Surg. Oncol. 20, 2683–2692 (2013).Crossref, Medline, Google Scholar2 Watanabe M, Shin'oka T, Tohyama S et al. Tissue-engineered vascular autograft: inferior vena cava replacement in a dog model. Tissue Eng. 7, 429–439 (2001).Crossref, Medline, CAS, Google Scholar3 Nigam SK, Wu W, Bush KT. In vitro kidney development, tissue engineering and systems biology. Organogenesis 4(3), 137–143 (2008).Crossref, Medline, Google Scholar4 Grobstein C. Trans-filter induction of tubules in mouse metanephrogenic mesenchyme. Exp. Cell Res. 10, 424–440 (1956).Crossref, Medline, CAS, Google Scholar5 Herzlinger D, Qiao J, Cohen D, Ramakrishna N, Brown AMC. Induction of kidney epithelial morphogenesis by cells expression Wnt-1. Dev. Biol. 166, 815–818 (1994).Crossref, Medline, CAS, Google Scholar6 Qiao J, Cohen D, Herzlinger D. The metanephric blastema differentiates into collecting system and nephron epithelial in vitro. Development 121, 3207–3214 (1995).Crossref, Medline, CAS, Google Scholar7 Qiao J, Sakurai H, Nigam SK. Branching morphogenesis independent of mesenchymal–epithelial contact in the developing kidney. Proc. Natl Acad. Sci. USA 96, 7330–7335 (1999).Crossref, Medline, CAS, Google Scholar8 Rosines E, Sampogna RV, Johkura K et al. Staged in vitro reconstitution and implantation of engineered rat kidney tissue. Proc. Natl Acad. Sci. USA 104, 20938–20943 (2007).Crossref, Medline, CAS, Google Scholar9 Song B, Smink AM, Hones CV et al. The directed differentiation of human iPS cells into kidney podocytes. PLoS ONE 7(9), e4645 (2012).Crossref, Google Scholar10 Xia Y, Nivet E, Sanchi-Martinez I et al. Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells. Nat. Cell Biol. 15(12), 1507–1515 (2013).Crossref, Medline, CAS, Google Scholar11 Narayanan K, Schumacher KM, Tasnim F et al. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells. Kidney Int. 83, 593–603 (2013).Crossref, Medline, CAS, Google Scholar12 Takasato M, Er PX, Becroft M et al. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat. Cell Biol. 16, 118–126 (2014).Crossref, Medline, CAS, Google Scholar13 Araoka T, Mae S, Kurose Y et al. Efficient and rapid induction of human iPSCs/ESCs into nephrogenic intermediate mesoderm using small molecule-based differentiation methods. PLoS ONE 9(1), e84881 (2014).Crossref, Medline, Google Scholar14 Xinaris C, Benedetti V, Rizzo P et al. In vivo maturation of functional renal organoids formed from embryonic cell suspensions. J. Am. Soc. Nephrol. 23, 1857–1868 (2012).Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Vol. 11, No. 2 Follow us on social media for the latest updates Metrics Downloaded 190 times History Published online 29 February 2016 Published in print March 2016 Information© Future Medicine LtdFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

Discovery of Novel Pyrazole Derivatives as Potent Neuraminidase Inhibitors against Influenza H1N1 Virus.

Ten pyrazole derivatives were synthesized and evaluated for their ability to inhibit the replication of influenza virions. All the compounds were synthesized in good-to-excellent yield, and the structures were ascertained with the help of (1) H NMR, (13) C NMR, mass, and elemental analysis. Among the tested series, compound 4i was identified as the most potent analog against the H1N1 virus, with IC50 = 5.4 µM, while the rest of the compounds showed mild-to-moderate inhibition of infection. Moreover, these compounds showed excellent inhibitory activity against influenza A neuraminidase (NA), with IC50 values ranging from 2.15 to 7.54 µM, among which compound 4i showed the most prominent inhibition with IC50 = 1.32 µM. To further exemplify the molecular contacts with NA, a molecular docking study of 4i was conducted with the 3D crystal structure of enzyme H5N1-NA in complex. Results showed that target molecules interact in a similar fashion with oseltamivir and zanamivir by creating interatomic contacts with Trp178, Glu227, and Arg371. Moreover, in the toxicity assay with the porcine renal proximal cell line, LLC-PK1, the confocal images showed no appreciable change in morphological character at the highest tested dose.