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Cadmium (Cd) is a toxic metal with a long half-life in biological systems. This half-life is partly as a result of metallothioneins (MTs), metal-binding proteins with a high affinity for Cd. The high retention properties of the kidneys reside in proximal tubular cells that possess transport mechanisms for Cd-MT uptake, ultimately leading to more Cd accumulation. Researchers have studied MT-metal interactions using various techniques including quantitative real-time PCR (qPCR), an efficient tool for quantifying gene expression. Often a poor choice of reference genes, which is represented by their instability and condition dependency, leads to inefficient normalization of gene expression data and misinterpretations. This study demonstrates the importance of an efficient normalization strategy in toxicological research. A selection of stable reference genes was proposed in order to acquire reliable and reproducible gene quantification under metal stress using MT expression as an example. Moreover, in vitro and in vivo setups were compared to identify the influence of toxicological compounds in function of the experimental design. This study shows that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine monooxygenase/tryptophan5-monooxygenase activation-protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) are the most stable reference genes in a kidney proximal tubular cell line exposed to moderate and high Cd concentrations, applied as CdCl2. A slightly different sequence in reference gene stability was found in renal cells isolated from rats in vivo exposed to Cd. It was further shown that three reference genes are required for efficient normalization in this experimental setup. This study demonstrates the importance of an efficient normalization strategy in toxicological research. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Mutation of the K-ras gene is thought to be an early and important event in pancreatic carcinogenesis. In order to study the role of this molecular alteration in the transition from the normal to the neoplastic pancreatic cell, bovine pancreatic duct cells were first immortalized by SV40 large T antigen (Ag) complementary (c)DNA transfection and then transfected with a mutated K-ras gene. As did primary duct cells, the immortalized duct cells (more than 100 passages) expressed cytokeratins, carbonic anhydrase type-II, cystic fibrosis transmembrane conductance regulator (CFTR), and multidrug resistance (mdr). They grew as a single layer after transplantation under plastic domes and formed three-dimensional structures resembling ducts when grown on Matrigel. Cell growth was stimulated by insulin, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, but cells did not respond to gastrin and CCK-8. They did not form colonies in soft agar nor did they form tumors in nude mice. Immortalized cells transfected with mutated K-ras acquired the ability to form tumors after orthotopic injection into the nude mouse pancreas. It is concluded that SV 40 immortalized bovine pancreatic [ABSTRACT FROM AUTHOR]

The properties of transporters (or channels) for monovalent cations in the membrane of isolated pancreatic zymogen granules were characterized with an assay measuring bulk cation influx driven by a proton diffusion potential. The proton diffusion potential was generated by suspending granules in an isotonic monovalent cation/acetate solution and increasing the proton conductance of the membrane with a protonophore. Monovalent cation conductance had the sequence Rb+ > K+ > NA+ > Cs+ > LI+ > N-methyl glucamine+. The conductance could be inhibited by Ca2+, Mg2+, Ba2+, and pharmacological agents such as quinine, quinidine, glyburide and tolbutamide, but not by 5 mM tetra-ethyl ammonium or 5 mM 4-aminopyridine, when applied to the cytosolic surface of the granule membrane. Over 50% of K+ conductance could be inhibited by millimolar concentrations of ATP or MgATP. The inhibition by MgATP, but not by ATP itself, was reversed by the K+ channel opener diazoxide. The inhibitory effect is probably by a noncovalent interaction since it could be mimicked by nonhydrolyzable analogs of ATP and by ADP. The reversal of MgATP inhibition by diazoxide may be mediated by phosphorylation since it was not affected by dilution, and was blocked by the protein kinase inhibitor H7. The properties of the K+ conductance of pancreatic zymogen granule membranes are similar to those of ATP-sensitive K+ channels found in the plasma membrane of insulin-secreting islet cells, neurons, muscle, and renal cells. [ABSTRACT FROM AUTHOR]

We have measured Ca2+ uptake and Ca2+ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca2+ uptake into cells was monitored with a Ca2+ electrode, whereas Ca2+ uptake into membrane vesicles was measured with 45Ca2+. Using inhibitors of known action, such as the H+ ATPase inhibitors NBD-Cl and NEM, the Ca2+ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP3) and its analog inositol 1,4,5-trisphosphorothioate (IPS3), we could functionally differentiate two nonmitochondrial Ca2+ pools. Ca2+ uptake into the IP3-sensitive Ca2+ pool (IsCaP) occurs by a MgATP-dependent Ca2+ uptake mechanism that exchanges Ca2+ for H+ ions. In the absence of ATP Ca2+ uptake can occur to some extent at the expense of an H+ gradient that is established by a vacuolar-type MgATP-dependent H+ pump present in the same organelle. The other Ca2+ pool takes up Ca2+ by a vanadate-sensitive Ca2+ ATPase and is insensitive to IP3 (IisCaP). The IsCaP is filled at "higher" Ca2+ concentrations (approximately 10(-6) mol/liter) which may occur during stimulation. The low steady-state [Ca2+] of approximately 10(-7) mol/liter is adjusted by the IisCaP. It is speculated that both Ca2+ pools can communicate with each other, the possible mechanism of which, however, is at present unknown. [ABSTRACT FROM AUTHOR]

In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H+ transport was evaluated by measuring H+-dependent accumulation of acridine orange (AO). Active H+ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (Km for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H+ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H+ accumulation was abolished by protonophores, such as nigericin (10(-6) mol/liter) or CCCP (10(-5) mol/liter), and by inhibitors of nonmitochondrial H+ ATPases, such as NEM or NBD-Cl, at a concentration of 10(-5) mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H+ pumps, such as DCCD (10(-5) mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H+ transport by about 90%. Vanadate (2 x 10(-3) mol/liter), a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H+ transport. The stilbene derivative DIDS (10(-4) mol/liter), which inhibits anion transport systems, abolished H+ transport completely. MgATP-dependent H+ transport was found to be anion dependent in the sequence Cl- greater than Br- greater than gluconate-; in the presence of SO2-4, CH3COO- or No-3, no H+ transport was observed. MgATP-dependent H+ accumulation was also cation dependent in the sequence K+ greater than Li+ greater than Na+ = choline+. As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl- and a K+ conductance, as well as a small H+ conductance, were found in the microsomal membranes. When membranes containing the H+ pump were further purified by Percoll gradient centrifugation (ninefold enrichment compared to homogenate), no correlation with markers for endoplasmic reticulum, mitochondria, plasma membranes, zymogen granules or Golgi membranes was found. The present data indicate that the H+ pump located in microsomes from rat exocrine pancreas is a vacuolar- or "V" -type H+ ATPase and has most similarities to that described in endoplasmic reticulum, Golgi apparatus or endosomes. [ABSTRACT FROM AUTHOR]

Abstract Background The class 1 carcinogen cadmium (Cd2+) disrupts the E-cadherin/β-catenin complex of epithelial adherens junctions (AJs) and causes renal cancer. Deregulation of E-cadherin adhesion and changes in Wnt/β-catenin signaling are known to contribute to carcinogenesis. Results We investigated Wnt signaling after Cd2+-induced E-cadherin disruption in sub-confluent cultured kidney proximal tubule cells (PTC). Cd2+ (25 μM, 3-9 h) caused nuclear translocation of β-catenin and triggered a Wnt response measured by TOPflash reporter assays. Cd2+ reduced the interaction of β-catenin with AJ components (E-cadherin, α-catenin) and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c-Myc, cyclin D1 and ABCB1) were up-regulated by Cd2+, electromobility shift assays showed increased TCF4 binding to cyclin D1 and ABCB1 promoter sequences with Cd2+. Overexpression of wild-type and mutant TCF4 confirmed Cd2+-induced Wnt signaling. Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. Overexpression of E-cadherin reduced Wnt signaling, PTC proliferation and Cd2+ toxicity. Cd2+ also induced reactive oxygen species dependent expression of the pro-apoptotic ER stress marker and Wnt suppressor CHOP/GADD153 which, however, did not abolish Wnt response and cell viability. Conclusions Cd2+ induces Wnt signaling in PTC. Hence, Cd2+ may facilitate carcinogenesis of PTC by promoting Wnt pathway-mediated proliferation and survival of pre-neoplastic cells.

The kidney is the main organ that senses changes in systemic O 2 pressure by hypoxia-PHD-HIFa (HPH) signaling, resulting in adaptive target gene activation, including erythropoietin (EPO). The non-essential transition metal cadmium (Cd) is nephrotoxic and disrupts the renal HPH pathway, which may promote Cd-associated chronic renal disease (CKD). A deeper molecular understanding of Cd interference with renal HPH signaling is missing, and no data with renal cell lines are available. In rat kidney NRK-52E cells, which model the proximal tubule, and murine fibroblastoid atypical interstitial kidney (FAIK3-5) cells, which mimic renal EPO-producing cells, the chemical hypoxia mimetic dimethyloxalylglycine (DMOG; 1 mmol/l) or hypoxia (1% O 2 ) activated HPH signaling. Cd 2+ (2.5-20 µmol/l for ≤ 24 h) preferentially induced necrosis (trypan blue uptake) of FAIK3-5 cells at high Cd whereas NRK-52E cells specially developed apoptosis (PARP-1 cleavage) at all Cd concentrations. Cd (12.5 µmol/l) abolished HIFa stabilization and prevented upregulation of target genes (quantitative real-time polymerase chain reaction and immunoblotting) induced by DMOG or hypoxia in both cell lines, which was caused by the formation of insoluble HIFa aggregates. Strikingly, hypoxic preconditioning (1% O 2 for 18 h) reduced apoptosis of FAIK3-5 and NRK-52E cells at low Cd concentrations and decreased insoluble HIFa proteins. Hence, drugs mimicking hypoxic preconditioning could reduce CKD induced by chronic low Cd exposure., (© 2024. The Author(s).)

Dent disease (DD) is a hereditary renal disorder characterized by low molecular weight (LMW) proteinuria and progressive renal failure. Inactivating mutations of the CLCN5 gene encoding the 2Cl - /H + exchanger ClC-5 have been identified in patients with DD type 1. ClC-5 is essentially expressed in proximal tubules (PT) where it is thought to play a role in maintaining an efficient endocytosis of LMW proteins. However, the exact pathological roles of ClC-5 in progressive dysfunctions observed in DD type 1 are still unclear. To address this issue, we designed a mouse model carrying the most representative type of ClC-5 missense mutations found in DD patients. These mice showed a characteristic DD type 1 phenotype accompanied by altered endo-lysosomal system and autophagy functions. With ageing, KI mice showed increased renal fibrosis, apoptosis and major changes in cell metabolic functions as already suggested in previous DD models. Furthermore, we made the interesting new discovery that the Lipocalin-2-24p3R pathway might be involved in the progression of the disease. These results suggest a crosstalk between the proximal and distal nephron in the pathogenesis mechanisms involved in DD with an initial PT impairment followed by the Lipocalin-2 internalisation and 24p3R overexpression in more distal segments of the nephron. This first animal model of DD carrying a pathogenic mutation of Clcn5 and our findings pave the way aimed at exploring therapeutic strategies to limit the consequences of ClC-5 disruption in patients with DD type 1 developing chronic kidney disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cellular responses to toxic metals depend on metal accessibility to intracellular targets, reaching interaction sites, and the intracellular metal concentration, which is mainly determined by uptake pathways, binding/sequestration and efflux pathways. ATP-binding cassette (ABC) transporters are ubiquitous in the human body-usually in epithelia-and are responsible for the transfer of indispensable physiological substrates (e.g. lipids and heme), protection against potentially toxic substances, maintenance of fluid composition, and excretion of metabolic waste products. Derailed regulation and gene variants of ABC transporters culminate in a wide array of pathophysiological disease states, such as oncogenic multidrug resistance or cystic fibrosis. Cadmium (Cd) has no known physiological role in mammalians and poses a health risk due to its release into the environment as a result of industrial activities, and eventually passes into the food chain. Epithelial cells, especially within the liver, lungs, gastrointestinal tract and kidneys, are particularly susceptible to the multifaceted effects of Cd because of the plethora of uptake pathways available. Pertinent to their broad substrate spectra, ABC transporters represent a major cellular efflux pathway for Cd and Cd complexes. In this review, we summarize current knowledge concerning transport of Cd and its complexes (mainly Cd bound to glutathione) by the ABC transporters ABCB1 (P-glycoprotein, MDR1), ABCB6, ABCC1 (multidrug resistance related protein 1, MRP1), ABCC7 (cystic fibrosis transmembrane regulator, CFTR), and ABCG2 (breast cancer related protein, BCRP). Potential detoxification strategies underlying ABC transporter-mediated efflux of Cd and Cd complexes are discussed., (© 2024. The Author(s).)

Levels and chemical species of reactive oxygen/nitrogen species (ROS/RNS) determine oxidative eustress and distress. Abundance of uptake pathways and high oxygen consumption for ATP-dependent transport makes the renal proximal tubule particularly susceptible to cadmium (Cd 2+ )-induced oxidative stress by targeting ROS/RNS generation or antioxidant defence mechanisms, such as superoxide dismutase (SOD) or H 2 O 2 -metabolizing catalase (CAT). Though ROS/RNS are well-evidenced, the role of distinct ROS profiles in Cd 2+ concentration-dependent toxicity is not clear. In renal cells, Cd 2+ (10-50 µM) oxidized dihydrorhodamine 123, reaching a maximum at 2-3 h. Increases (up to fourfold) in lipid peroxidation by TBARS assay and H 2 O 2 by Amplex Red were evident within 30 min. ROS and loss in cell viability by MTT assay with 50 µM Cd 2+ could not be fully reversed by SOD mimetics Tempol and MnTBAP nor by SOD1 overexpression, whereas CAT expression and α-tocopherol were effective. SOD and CAT activities were attenuated below controls only with >6 h 50 µM Cd 2+ , yet augmented by up to 1.5- and 1.2-fold, respectively, by 10 µM Cd 2+ . Moreover, 10 µM, but not 25-50 µM Cd 2+ , caused 1.7-fold increase in superoxide anion (O 2 •- ), detected by dihydroethidium, paralled by loss in cell viability, that was abolished by Tempol, MnTBAP, α-tocopherol and SOD1 or CAT overexpression. H 2 O 2 -generating NADPH oxidase 4 (NOX4) was attenuated by ~50% with 10 µM Cd 2+ at 3 h compared to upregulation by 50 µM Cd 2+ (~1.4-fold, 30 min), which was sustained for 24 h. In summary, O 2 •- predominates with low-moderate Cd 2+ , driving an adaptive response, whereas oxidative stress by elevated H 2 O 2 at high Cd 2+ triggers cell death signaling pathways.Highlights Different levels of reactive oxygen species are generated, depending on cadmium concentration. Superoxide anion predominates and H 2 O 2 is suppressed with low cadmium representing oxidative eustress. High cadmium fosters H 2 O 2 by inhibiting catalase and increasing NOX4 leading to oxidative distress. Superoxide dismutase mimetics and overexpression were less effective with high versus low cadmium. Oxidative stress profile could dictate downstream signalling pathways., (© 2024. The Author(s).)

The transmembrane protein SLC22A17 [or the neutrophil gelatinase-associated lipocalin/lipocalin-2 (LCN2)/24p3 receptor] is an atypical member of the SLC22 family of organic anion and cation transporters: it does not carry typical substrates of SLC22 transporters but mediates receptor-mediated endocytosis (RME) of LCN2. One important task of the kidney is the prevention of urinary loss of proteins filtered by the glomerulus by bulk reabsorption of multiple ligands via megalin:cubilin:amnionless-mediated endocytosis in the proximal tubule (PT). Accordingly, overflow, glomerular, or PT damage, as in Fanconi syndrome, results in proteinuria. Strikingly, up to 20% of filtered proteins escape the PT under physiological conditions and are reabsorbed by the distal nephron. The renal distal tubule and collecting duct express SLC22A17, which mediates RME of filtered proteins that evade the PT but with limited capacity to prevent proteinuria under pathological conditions. The kidney also prevents excretion of filtered essential and nonessential transition metals, such as iron or cadmium, respectively, that are largely bound to proteins with high affinity, e.g., LCN2, transferrin, or metallothionein, or low affinity, e.g., microglobulins or albumin. Hence, increased uptake of transition metals may cause nephrotoxicity. Here, we assess the literature on SLC22A17 structure, topology, tissue distribution, regulation, and assumed functions, emphasizing renal SLC22A17, which has relevance for physiology, pathology, and nephrotoxicity due to the accumulation of proteins complexed with transition metals, e.g., cadmium or iron. Other putative renal functions of SLC22A17, such as its contribution to osmotic stress adaptation, protection against urinary tract infection, or renal carcinogenesis, are discussed.

Oncolytic viruses are a promising technology to attack cancer cells and to recruit immune cells to the tumor site. Since the Lipocalin-2 receptor (LCN2R) is expressed on most cancer cells, we used its ligand LCN2 to target oncolytic adenoviruses (Ads) to cancer cells. Therefore, we fused a Designed Ankyrin Repeat Protein (DARPin) adapter binding the knob of Ad type 5 (knob5) to LCN2 to retarget the virus toward LCN2R with the aim of analyzing the basic characteristics of this novel targeting approach. The adapter was tested in vitro with Chinese Hamster Ovary (CHO) cells stably expressing the LCN2R and on 20 cancer cell lines (CCLs) using an Ad5 vector encoding luciferase and green fluorescent protein. Luciferase assays with the LCN2 adapter (LA) showed 10-fold higher infection compared with blocking adapter (BA) in CHO cells expressing LCN2R and in cells not expressing the LCN2R. Most CCLs showed an increased viral uptake of LA-bound virus compared with BA-bound virus and for five CCLs viral uptake was comparable to unmodified Ad5. Flow cytometry and hexon immunostainings also revealed increased uptake of LA-bound Ads compared with BA-bound Ads in most tested CCLs. Virus spread was studied in 3D cell culture models and nine CCLs showed increased and earlier fluorescence signals for LA-bound virus compared with BA-bound virus. Mechanistically, we show that the LA increases viral uptake only in the absence of its ligand Enterobactin (Ent) and independently of iron. Altogether, we characterized a novel DARPin-based system resulting in enhanced uptake demonstrating potential for future oncolytic virotherapy.

The kidney is the main organ that senses changes in systemic oxygen tension, but it is also the key detoxification, transit and excretion site of transition metals (TMs). Pivotal to oxygen sensing are prolyl-hydroxylases (PHDs), which hydroxylate specific residues in hypoxia-inducible factors (HIFs), key transcription factors that orchestrate responses to hypoxia, such as induction of erythropoietin (EPO). The essential TM ion Fe is a key component and regulator of the hypoxia-PHD-HIF-EPO (HPHE) signaling axis, which governs erythropoiesis, angiogenesis, anaerobic metabolism, adaptation, survival and proliferation, and hence cell and body homeostasis. However, inadequate concentrations of essential TMs or entry of non-essential TMs in organisms cause toxicity and disrupt health. Non-essential TMs are toxic because they enter cells and displace essential TMs by ionic and molecular mimicry, e. g. in metalloproteins. Here, we review the molecular mechanisms of HPHE interactions with TMs (Fe, Co, Ni, Cd, Cr, and Pt) as well as their implications in renal physiology, pathophysiology and toxicology. Some TMs, such as Fe and Co, may activate renal HPHE signaling, which may be beneficial under some circumstances, for example, by mitigating renal injuries from other causes, but may also promote pathologies, such as renal cancer development and metastasis. Yet some other TMs appear to disrupt renal HPHE signaling, contributing to the complex picture of TM (nephro-)toxicity. Strikingly, despite a wealth of literature on the topic, current knowledge lacks a deeper molecular understanding of TM interaction with HPHE signaling, in particular in the kidney. This precludes rationale preventive and therapeutic approaches to TM nephrotoxicity, although recently activators of HPHE signaling have become available for therapy., (© 2022. The Author(s).)

Oncogenic multidrug resistance is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1 + clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (approximately twofold) in chemoresistant ABCB1 high (A498, Caki-1) compared with chemosensitive ABCB1 low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1 high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B 1 ) in cell viability assays. Downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123 + efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and SM composition to ultra long-chain species (C22-C26). Inhibitors of endoplasmic reticulum-associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Hemolysis is known to cause acute kidney injury (AKI). The iron regulatory hormone hepcidin, produced by renal distal tubules, is suggested to exert a renoprotective role during this pathology. We aimed to elucidate the molecular mechanisms of renal hepcidin synthesis and its protection against hemoglobin-induced AKI. In contrast to known hepatic hepcidin induction, incubation of mouse cortical collecting duct (mCCD cl1 ) cells with IL-6 or LPS did not induce Hamp1 mRNA expression, whereas iron (FeS) and hemin significantly induced hepcidin synthesis ( p < 0.05). Moreover, iron/heme-mediated hepcidin induction in mCCD cl1 cells was caused by the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, as indicated by increased nuclear Nrf2 translocation and induced expression of Nrf2 downstream targets GCLM ( p < 0.001), NQO1 ( p < 0.001), and TXNRD1 ( p < 0.005), which could be prevented by the known Nrf2 inhibitor trigonelline. Newly created inducible kidney-specific hepcidin KO mice demonstrated a significant reduction in renal Hamp1 mRNA expression. Phenylhydrazine (PHZ)-induced hemolysis caused renal iron loading and oxidative stress in both wildtype (Wt) and KO mice. PHZ treatment in Wt induced inflammatory markers ( IL-6 , TNFα ) but not Hamp1 . However, since PHZ treatment also significantly reduced systemic hepcidin levels in both Wt and KO mice (both p < 0.001), a dissection between the roles of systemic and renal hepcidin could not be made. Combined, the results of our study indicate that there are kidney-specific mechanisms in hepcidin regulation, as indicated by the dominant role of iron and not inflammation as an inducer of renal hepcidin, but also emphasize the complex interplay of various iron regulatory mechanisms during AKI on a local and systemic level.

Kidney iron deposition may play a role in the progression of tubulointerstitial injury during chronic kidney disease. Here, we studied the molecular mechanisms of kidney iron loading in experimental focal segmental glomerulosclerosis (FSGS) and investigated the effect of iron-reducing interventions on disease progression. Thy-1.1 mice were injected with anti-Thy-1.1 monoclonal antibody (mAb) to induce proteinuria. Urine, blood and tissue were collected at day (D)1, D5, D8, D15 and D22 after mAb injection. Thy-1.1 mice were subjected to captopril (CA), iron-deficient (ID) diet or iron chelation (deferoxamine; DFO). MAb injection resulted in significant albuminuria at all time points (p < 0.01). Kidney iron loading, predominantly in distal tubules, increased in time, along with urinary kidney injury molecule-1 and 24p3 concentration, as well as kidney mRNA expression of Interleukin-6 (Il-6) and Heme oxygenase-1 (Ho-1). Treatment with CA, ID diet or DFO significantly reduced kidney iron deposition at D8 and D22 (p < 0.001) and fibrosis at D22 (p < 0.05), but not kidney Il-6. ID treatment increased kidney Ho-1 (p < 0.001). In conclusion, kidney iron accumulation coincides with progression of tubulointerstitial injury in this model of FSGS. Reduction of iron loading halts disease progression. However, targeted approaches to prevent excessive kidney iron loading are warranted to maintain the delicate systemic and cellular iron balance., (© 2022. The Author(s).)

The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe 2+ overload. The nephrotoxic non-essential metal ion Cd 2+ can displace Fe 2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe 2+ and Cd 2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD 3 ] collecting duct cell lines. Cells were exposed to equipotent Cd 2+ (0.5-5 μmol/l) and/or Fe 2+ (50-100 μmol/l) for 4-24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe 2+ -induced mIMCD 3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd 2+ -induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe 2+ prevented Cd 2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe 2+ with desferrioxamine augmented Cd 2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe 2+ , but not Cd 2+ . Because Fe 2+ and Cd 2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate., (© 2021. The Author(s).)

Background: The proximal tubule (PT) is the major target of cadmium (Cd 2+ ) nephrotoxicity. Current dogma postulates that Cd 2+ complexed to metallothionein (MT) (CdMT) is taken up through receptor-mediated endocytosis (RME) via the PT receptor megalin:cubilin, which is the predominant pathway for reuptake of filtered proteins in the kidney. Nevertheless, there is evidence that the distal parts of the nephron are also sensitive to damage induced by Cd 2+ . In rodent kidneys, another receptor for protein endocytosis, the 24p3 receptor (24p3R), is exclusively expressed in the apical membranes of distal tubules (DT) and collecting ducts (CD). Cell culture studies have demonstrated that RME and toxicity of CdMT and other (metal ion)-protein complexes in DT and CD cells is mediated by 24p3R. In this study, we evaluated the uptake of labeled CdMT complex through 24p3R after acute kidney injury (AKI) induced by gentamicin (GM) administration that disrupts PT function. Subcutaneous administration of GM at 10 mg/kg/day for seven days did not alter the structural and functional integrity of the kidney's filtration barrier. However, because of PT injury, the concentration of the renal biomarker Kim-1 increased. When CdMT complex coupled to FITC was administered intravenously, both uptake of the CdMT complex and 24p3R expression in DT increased and also colocalized after PT injury induced by GM. Although megalin decreased in PT after GM administration, urinary protein excretion was not changed, which suggests that the increased levels of 24p3R in the distal nephron could be acting as a compensatory mechanism for protein uptake. Altogether, these results suggest that PT damage increases the uptake of the CdMT complex through 24p3R in DT (and possibly CD) and compensate for protein losses associated with AKI.

Regulation of body fluid homeostasis is a major renal function, occurring largely through epithelial solute transport in various nephron segments driven by Na + /K + -ATPase activity. Energy demands are greatest in the proximal tubule and thick ascending limb where mitochondrial ATP production occurs through oxidative phosphorylation. Mitochondria contain 20-80% of the cell's iron, copper, and manganese that are imported for their redox properties, primarily for electron transport. Redox reactions, however, also lead to reactive, toxic compounds, hence careful control of redox-active metal import into mitochondria is necessary. Current dogma claims the outer mitochondrial membrane (OMM) is freely permeable to metal ions, while the inner mitochondrial membrane (IMM) is selectively permeable. Yet we recently showed iron and manganese import at the OMM involves divalent metal transporter 1 (DMT1), an H + -coupled metal ion transporter. Thus, iron import is not only regulated by IMM mitoferrins, but also depends on the OMM to intermembrane space H + gradient. We discuss how these mitochondrial transport processes contribute to renal injury in systemic (e.g., hemochromatosis) and local (e.g., hemoglobinuria) iron overload. Furthermore, the environmental toxicant cadmium selectively damages kidney mitochondria by "ionic mimicry" utilizing iron and calcium transporters, such as OMM DMT1 or IMM calcium uniporter, and by disrupting the electron transport chain. Consequently, unraveling mitochondrial metal ion transport may help develop new strategies to prevent kidney injury induced by metals., (Copyright © 2020 Thévenod, Lee and Garrick.)

Ever increasing environmental presence of cadmium as a consequence of industrial activities is considered a health hazard and is closely linked to deteriorating global health status. General animal and human cadmium exposure ranges from ingestion of foodstuffs sourced from heavily polluted hotspots and cigarette smoke to widespread contamination of air and water, including cadmium-containing microplastics found in household water. Cadmium is promiscuous in its effects and exerts numerous cellular perturbations based on direct interactions with macromolecules and its capacity to mimic or displace essential physiological ions, such as iron and zinc. Cell organelles use lipid membranes to form complex tightly-regulated, compartmentalized networks with specialized functions, which are fundamental to life. Interorganellar communication is crucial for orchestrating correct cell behavior, such as adaptive stress responses, and can be mediated by the release of signaling molecules, exchange of organelle contents, mechanical force generated through organelle shape changes or direct membrane contact sites. In this review, cadmium effects on organellar structure and function will be critically discussed with particular consideration to disruption of organelle physiology in vertebrates.