Nephron Segments Research Articles

Uromodulin Regulates Murine Aquaporin-2 Activity via Thick Ascending Limb-Collecting Duct Cross-Talk during Water Deprivation.

Uromodulin, a urinary protein synthesized and secreted from the thick ascending limb (TAL) of the loop of Henle, is associated with hypertension through the activation of sodium reabsorption in the TAL. Uromodulin is a potential target for hypertension treatment via natriuresis. However, its biological function in epithelial cells of the distal nephron segment, particularly the collecting duct, remains unknown. Herein, we examined the regulation of uromodulin production during water deprivation in vivo as well as the effect of uromodulin on the activity of the water channel aquaporin−2 (AQP2) in vitro and in vivo using transgenic mice. Water deprivation upregulated uromodulin production; immunofluorescence experiments revealed uromodulin adhesion on the apical surface of the collecting duct. Furthermore, the activation of AQP2 was attenuated in mice lacking uromodulin. Uromodulin enhanced the phosphorylation and apical trafficking of AQP2 in mouse collecting duct cells treated with the vasopressin analog dDAVP. The uromodulin-induced apical trafficking of AQP2 was attenuated via endocytosis inhibitor treatment, suggesting that uromodulin activates AQP2 through the suppression of endocytosis. This study provides novel insights into the cross−talk between TAL and the collecting duct, and indicates that the modulation of uromodulin is a promising approach for diuresis and hypertension treatment.

Functional characterization of ion channels expressed in kidney organoids derived from human induced pluripotent stem cells.

Kidney organoids derived from human or rodent pluripotent stem cells have glomerular structures and differentiated/polarized nephron segments. Although there is an increasing understanding of the patterns of expression of transcripts and proteins within kidney organoids, there is a paucity of data regarding functional protein expression, in particular on transporters that mediate the vectorial transport of solutes. Using cells derived from kidney organoids, we examined the functional expression of key ion channels that are expressed in distal nephron segments: the large-conductance Ca2+-activated K+ (BKCa) channel, the renal outer medullary K+ (ROMK, Kir1.1) channel, and the epithelial Na+ channel (ENaC). RNA-sequencing analyses showed that genes encoding the pore-forming subunits of these transporters, and for BKCa channels, key accessory subunits, are expressed in kidney organoids. Expression and localization of selected ion channels was confirmed by immunofluorescence microscopy and immunoblot analysis. Electrophysiological analysis showed that BKCa and ROMK channels are expressed in different cell populations. These two cell populations also expressed other unidentified Ba2+-sensitive K+ channels. BKCa expression was confirmed at a single channel level, based on its high conductance and voltage dependence of activation. We also found a population of cells expressing amiloride-sensitive ENaC currents. In summary, our results show that human kidney organoids functionally produce key distal nephron K+ and Na+ channels.NEW & NOTEWORTHY Our results show that human kidney organoids express key K+ and Na+ channels that are expressed on the apical membranes of cells in the aldosterone-sensitive distal nephron, including the large-conductance Ca2+-activated K+ channel, renal outer medullary K+ channel, and epithelial Na+ channel.

Renal Deletion of LRRC8/VRAC Channels Induces Proximal Tubulopathy.

Volume-regulated anion channels (VRACs) are heterohexamers of LRRC8A with LRRC8B, -C, -D, or -E in various combinations. Depending on the subunit composition, these swelling-activated channels conduct chloride, amino acids, organic osmolytes, and drugs. Despite VRACs' role in cell volume regulation, and large osmolarity changes in the kidney, neither the localization nor the function of VRACs in the kidney is known. Mice expressing epitope-tagged LRRC8 subunits were used to determine the renal localization of all VRAC subunits. Mice carrying constitutive deletions of Lrrc8b-e, or with inducible or cell-specific ablation of Lrrc8a, were analyzed to assess renal functions of VRACs. Analysis included histology, urine and serum parameters in different diuresis states, and metabolomics. The kidney expresses all five VRAC subunits with strikingly distinct localization. Whereas LRRC8C is exclusively found in vascular endothelium, all other subunits are found in the nephron. LRRC8E is specific for intercalated cells, whereas LRRC8A, LRRC8B, and LRRC8D are prominent in basolateral membranes of proximal tubules. Conditional deletion of LRRC8A in proximal but not distal tubules and constitutive deletion of LRRC8D cause proximal tubular injury, increased diuresis, and mild Fanconi-like symptoms. VRAC/LRRC8 channels are crucial for the function and integrity of proximal tubules, but not for more distal nephron segments in spite of their larger need for volume regulation. LRRC8A/D channels may be required for the basolateral exit of many organic compounds, including cellular metabolites, in proximal tubules. Proximal tubular injury likely results from combined accumulation of several transported molecules in the absence of VRAC channels.

WNK1 in the kidney.

The aim of this manuscript was to review recent evidence uncovering the roles of the With No lysine (K) kinase 1 (WNK1) in the kidney. Analyses of microdissected mouse nephron segments have revealed the abundance of long-WNK1 and kidney-specific-WNK1 transcripts in different segments. The low levels of L-WNK1 transcripts in the distal convoluted tubule (DCT) stand out and support functional evidence on the lack of L-WNK1 activity in this segment. The recent description of familial hyperkalaemic hypertension (FHHt)-causative mutations affecting the acidic domain of WNK1 supports the notion that KS-WNK1 activates the Na+:Cl- cotransporter NCC. The high sensitivity of KS-WNK1 to KLHL3-targeted degradation and the low levels of L-WNK1 in the DCT, led to propose that this type of FHHt is mainly due to increased KS-WNK1 protein in the DCT. The observation that KS-WNK1 renal protein expression is induced by low K+ diet and recent reassessment of the phenotype of KS-WNK1-/- mice suggested that KS-WNK1 may be necessary to achieve maximal NCC activation under this condition. Evidences on the regulation of other renal transport proteins by WNK1 are also summarized. The diversity of WNK1 transcripts in the kidney has complicated the interpretation of experimental data. Integration of experimental data with the knowledge of isoform abundance in renal cell types is necessary in future studies about WNK1 function in the kidney.

Urinary extracellular vesicles as a source of protein-based biomarkers in feline chronic kidney disease and hypertension.

To validate a methodology for isolating feline urinary extracellular vesicles and characterise the urinary extracellular vesicle population and proteome in cats with normal renal function and cats with normotensive or hypertensive chronic kidney disease. Feline urinary extracellular vesicles were isolated using three different methods (precipitation alone, precipitation followed by size exclusion chromatography and ultrafiltration followed by size exclusion chromatography, which were compared via transmission electron microscopy and nanoparticle tracking analysis. Cats with normal renal function (n=9), normotensive chronic kidney disease (n=10) and hypertensive chronic kidney disease (n=9) were identified and urinary extracellular vesicles isolated from patient urine samples via ultrafiltration followed by size exclusion chromatography. Extracellular vesicle size and concentration were determined using nanoparticle tracking analysis, and subsequently underwent proteomic analysis using liquid chromatography with tandem mass spectrometry to identify differences in protein expression between categories. Urinary extracellular vesicle preparations contained particles of the expected size and morphology, and those obtained by ultrafiltration + size exclusion chromatography had a significantly higher purity (highest particle: protein ratio). The urinary extracellular vesicle proteomes contained extracellular vesicle markers and proteins originating from all nephron segments. Urinary extracellular vesicle concentration and size were unaffected by renal disease or hypertension. There were no differentially expressed proteins detected when comparing urinary extracellular vesicles derived from cats in the healthy category with the combined chronic kidney disease category, but five differentially expressed proteins were identified between the normotensive chronic kidney disease and hypertensive chronic kidney disease categories. Feline urinary extracellular vesicles can be successfully isolated from stored urine samples. Differentially expressed urinary extracellular vesicle proteins were discovered in cats with hypertensive chronic kidney disease, and warrant further investigation into their utility as biomarkers or therapeutic targets.

Spatially Resolved Transcriptomes of Mammalian Kidneys Illustrate the Molecular Complexity and Interactions of Functional Nephron Segments.

Available transcriptomes of the mammalian kidney provide limited information on the spatial interplay between different functional nephron structures due to the required dissociation of tissue with traditional transcriptome-based methodologies. A deeper understanding of the complexity of functional nephron structures requires a non-dissociative transcriptomics approach, such as spatial transcriptomics sequencing (ST-seq). We hypothesize that the application of ST-seq in normal mammalian kidneys will give transcriptomic insights within and across species of physiology at the functional structure level and cellular communication at the cell level. Here, we applied ST-seq in six mice and four human kidneys that were histologically absent of any overt pathology. We defined the location of specific nephron structures in the captured ST-seq datasets using three lines of evidence: pathologist's annotation, marker gene expression, and integration with public single-cell and/or single-nucleus RNA-sequencing datasets. We compared the mouse and human cortical kidney regions. In the human ST-seq datasets, we further investigated the cellular communication within glomeruli and regions of proximal tubules–peritubular capillaries by screening for co-expression of ligand–receptor gene pairs. Gene expression signatures of distinct nephron structures and microvascular regions were spatially resolved within the mouse and human ST-seq datasets. We identified 7,370 differentially expressed genes (padj < 0.05) distinguishing species, suggesting changes in energy production and metabolism in mouse cortical regions relative to human kidneys. Hundreds of potential ligand–receptor interactions were identified within glomeruli and regions of proximal tubules–peritubular capillaries, including known and novel interactions relevant to kidney physiology. Our application of ST-seq to normal human and murine kidneys confirms current knowledge and localization of transcripts within the kidney. Furthermore, the generated ST-seq datasets provide a valuable resource for the kidney community that can be used to inform future research into this complex organ.

Co-axial printing of convoluted proximal tubule for kidney disease modeling

Despite the increasing incidence of kidney-related diseases, we are still far from understanding the underlying mechanisms of these diseases and their progression. This lack of understanding is partly because of a poor replication of the diseases in vitro, limited to planar culture. Advancing towards three-dimensional models, hereby we propose coaxial printing to obtain microfibers containing a helical hollow microchannel. These recapitulate the architecture of the proximal tubule (PT), an important nephron segment often affected in kidney disorders. A stable gelatin/alginate-based ink was formulated to allow printability while maintaining structural properties. Fine-tuning of the composition, printing temperature and extrusion rate allowed for optimal ink viscosity that led to coiling of the microfiber’s inner channel. The printed microfibers exhibited prolonged structural stability (42 days) and cytocompatibility in culture. Healthy conditionally immortalized PT epithelial cells and a knockout cell model for cystinosis (CTNS -/-) were seeded to mimic two genotypes of PT. Upon culturing for 14 days, engineered PT showed homogenous cytoskeleton organization as indicated by staining for filamentous actin, barrier-formation and polarization with apical marker α-tubulin and basolateral marker Na+/K+-ATPase. Cell viability was slightly decreased upon prolonged culturing for 14 days, which was more pronounced in CTNS -/- microfibers. Finally, CTNS -/- cells showed reduced apical transport activity in the microfibers compared to healthy PT epithelial cells when looking at breast cancer resistance protein and multidrug resistance-associated protein 4. Engineered PT incorporated in a custom-designed microfluidic chip allowed to assess leak-tightness of the epithelium, which appeared less tight in CTNS -/- PT compared to healthy PT, in agreement with its in vivo phenotype. While we are still on the verge of patient-oriented medicine, this system holds great promise for further research in establishing advanced in vitro disease models.

Compact laboratory-based X-ray microscope enabling nondestructive 3D structure acquisition of mouse nephron with high speed and better user accessibility.

X-ray microscopes adopting computed tomography enable nondestructive 3D visualization of biological specimens at micron-level resolution without conventional 2D serial sectioning that is a destructive/laborious method and is routinely used for analyzing renal biopsy in clinical diagnosis of kidney diseases. Here we applied a compact commercial system of laboratory-based X-ray microscope to observe a resin-embedded osmium-stained 1-mm strip of a mouse kidney piece as a model of renal biopsy, toward a more efficient diagnosis of kidney diseases. A reconstructed computed tomography image from several hours of data collection using CCD detector allowed us to unambiguously segment a single nephron connected to a renal corpuscle, which was consistent with previous reports using serial sectioning. Histogram analysis on the segmented nephron confirmed that the proximal and distal tubules were distinguishable on the basis of their X-ray opacities. A 3D rendering model of the segmented nephron visualized a convoluted structure of renal tubules neighboring the renal corpuscle and a branched structure of efferent arterioles. Furthermore, another data collection using scientific complementary metal-oxide semiconductor detector with a much shorter data acquisition time of 15 min provided similar results from the same samples. These results suggest a potential application of the compact laboratory-based X-ray microscope to analyze mouse renal biopsy.

Inwardly rectifying K+ channels 4.1 and 5.1 (Kir4.1/Kir5.1) in the renal distal nephron.

The inwardly rectifying potassium channel (Kir) 4.1 (encoded by KCNJ10) interacts with Kir5.1 (encoded by KCNJ16) to form a major basolateral K+ channel in the renal distal convoluted tubule (DCT), connecting tubule (CNT), and the cortical collecting duct (CCD). Kir4.1/Kir5.1 heterotetramer plays an important role in regulating Na+ and K+ transport in the DCT, CNT, and CCD. A recent development in the field has firmly established the role of Kir4.1/Kir5.1 heterotetramer of the DCT in the regulation of thiazide-sensitive Na-Cl cotransporter (NCC). Changes in Kir4.1/Kir5.1 activity of the DCT are an essential step for the regulation of NCC expression/activity induced by dietary K+ and Na+ intakes and play a role in modulating NCC by type 2 angiotensin II receptor (AT2R), bradykinin type II receptor (BK2R), and β-adrenergic receptor. Since NCC activity determines the Na+ delivery rate to the aldosterone-sensitive distal nephron (ASDN), a distal nephron segment from late DCT to CCD, Kir4.1/Kir5.1 activity plays a critical role not only in the regulation of renal Na+ absorption but also in modulating renal K+ excretion and maintaining K+ homeostasis. Thus, Kir4.1/Kir5.1 activity serves as an important component of renal K+ sensing mechanism. The main focus of this review is to provide an overview regarding the role of Kir4.1 and Kir5.1 of the DCT and CCD in the regulation of renal K+ excretion and Na+ absorption.

Snapshots of nascent RNA can validate different nephron segment responses in acute kidney injury: a step toward clinically useful biomarkers?

Acute kidney injury (AKI) is a syndrome characterized by organ damage that occurs in response to a variety of types of injury. Across a spectrum of clinical severity and pathologic damage, AKI is associated with poor prognosis. Tubular epithelial cells respond to injury and start the repair process within minutes to hours; however, the specific molecular responses at each tubular segment in the nephron have not been well characterized. In a recent paper in JCI Insight, Shen et al. used novel methods to describe stimulus-specific, cell-specific, and time-dependent acute responses along the nephron.

Inverse Salt Sensitivity of Blood Pressure: Mechanisms and Potential Relevance for Prevention of Cardiovascular Disease.

To review the etiology of inverse salt sensitivity of blood pressure (BP). Both high and low sodium (Na+) intake can be associated with increased BP and cardiovascular morbidity and mortality. However, little is known regarding the mechanisms involved in the increase in BP in response to low Na+ intake, a condition termed inverse salt sensitivity of BP, which affects approximately 15% of the adult population. The renal proximal tubule is important in regulating up to 70% of renal Na+ transport. The renin-angiotensin and renal dopaminergic systems play both synergistic and opposing roles in the regulation of Na+ transport in this nephron segment. Clinical studies have demonstrated that individuals express a "personal salt index" (PSI) that marks whether they are salt-resistant, salt-sensitive, or inverse salt-sensitive. Inverse salt sensitivity results in part from genetic polymorphisms in various Na+ regulatory genes leading to a decrease in natriuretic activity and an increase in renal tubular Na+ reabsorption leading to an increase in BP. This article reviews the potential mechanisms of a new pathophysiologic entity, inverse salt sensitivity of BP, which affects approximately 15% of the general adult population.

Abstract 939: A novel lineage-tracing model of Vhldeletion reveals time-dependent cellular changes in the kidney

Abstract Clear cell renal cell carcinoma (ccRCC) is driven by biallelic inactivation of von Hippel Lindau (VHL) tumour suppressor gene. While ccRCC genesis has been associated with several signaling and metabolic pathways, the exact process by which VHL inactivation becomes tumorigenic remains unknown. We surmised that observing the behaviour and gene expression profiles of Vhl-inactivated cells in vivo with spatiotemporal resolution would help define cell types and conditions in which this genetic lesion is on balance tumorigenic. Here we report a novel lineage marking model of in vivo Vhl inactivation and describe the immediate consequences of Vhl loss in different nephron segments at cellular resolution. Studying Vhl inactivation with single-cell resolution requires a system in which Vhl-inactivated cells can be accurately identified. We developed a mouse model in which Vhl recombination is structurally linked to the expression of a tdTomato reporter cassette. This allows recombined cells to be visualised and isolated based on tdTomato expression. We coupled this model with the renal tubular epithelium (RTE) restricted Pax8-CreERT2 to induce biallelic (KO/KO) or monoallelic (wt/KO) inactivation of Vhl and observed the immediate fates of affected cells. Vhl is thought to be haplosufficient; thus, the wt/KO mice act as lineage-marked Vhl-competent controls to lineage-marked Vhl-null cells. We first observed the behavior of Vhl KO cells in the RTE over time. Vhl was inactivated by a controlled dose of tamoxifen that induced recombination in a subset of cells across the nephron. Three weeks after recombination, Vhl KO cells were morphologically normal but had stabilized HIF-2, confirming a reported ‘HIF-isoform’ switch. There were equal numbers of tdTomato-positive cells in the KO/KO and wt/KO kidneys, suggesting that initial recombination is equal between the genotypes and that there is no immediate distortion in cell fates following Vhl KO. However, four months after recombination, there were fewer tdTomato-positive cells in the KO/KO kidneys, suggesting a gradual removal or loss of Vhl KO from tissue. This was most prominent in the renal papilla and cortex. Additionally, some Vhl KO cells presented with morphological signs of cellular stress and altered differentiation. Taken together, we believe Vhl KO cells are subjected to profound selection in the nephron and that cell-type-specific, negative, and heterogenous consequences of Vhl KO impact on cell survival. One corollary is that many cells which ultimately survive in Vhl KO organs are in fact Vhl-competent. By intrinsically coupling Vhl-inactivation and lineage marking, our model allows these populations to be distinguished and followed over time. We are now studying the differential gene expression patterns of Vhl KO cells in cells of the RTE and aim to define genetic signatures for contexts in which Vhl KO cells survive and contribute to ccRCC genesis. Citation Format: Joanna D C Lima, Samvid Kurlekar, Chris W. Pugh, Julie Adam, Peter J. Ratcliffe. A novel lineage-tracing model of Vhldeletion reveals time-dependent cellular changes in the kidney [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 939.

A reference tissue atlas for the human kidney.

Kidney Precision Medicine Project (KPMP) is building a spatially specified human kidney tissue atlas in health and disease with single-cell resolution. Here, we describe the construction of an integrated reference map of cells, pathways, and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 56 adult subjects. We use single-cell/nucleus transcriptomics, subsegmental laser microdissection transcriptomics and proteomics, near-single-cell proteomics, 3D and CODEX imaging, and spatial metabolomics to hierarchically identify genes, pathways, and cells. Integrated data from these different technologies coherently identify cell types/subtypes within different nephron segments and the interstitium. These profiles describe cell-level functional organization of the kidney following its physiological functions and link cell subtypes to genes, proteins, metabolites, and pathways. They further show that messenger RNA levels along the nephron are congruent with the subsegmental physiological activity. This reference atlas provides a framework for the classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.

Ttc21b deficiency attenuates autosomal dominant polycystic kidney disease in a kidney tubular- and maturation-dependent manner

Primary cilia are sensory organelles built and maintained by intraflagellar transport (IFT) multiprotein complexes. Deletion of several IFT-B genes attenuates polycystic kidney disease (PKD) severity in juvenile and adult autosomal dominant polycystic kidney disease (ADPKD) mouse models. However, deletion of an IFT-A adaptor, Tulp3, attenuates PKD severity in adult mice only. These studies indicate that dysfunction of specific cilia components has potential therapeutic value. To broaden our understanding of cilia dysfunction and its therapeutic potential, we investigate the role of global deletion of an IFT-A gene, Ttc21b, in juvenile and adult mouse models of ADPKD. Both juvenile (postnatal day 21) and adult (six months of age) ADPKD mice exhibited kidney cysts, increased kidney weight/body weight ratios, lengthened kidney cilia, inflammation, and increased levels of the nutrient sensor, O-linked β-N-acetylglucosamine (O-GlcNAc). Deletion of Ttc21b in juvenile ADPKD mice reduced cortical collecting duct cystogenesis and kidney weight/body weight ratios, increased proximal tubular and glomerular dilations, but did not reduce cilia length, inflammation, nor O-GlcNAc levels. In contrast, Ttc21b deletion in adult ADPKD mice markedly attenuated kidney cystogenesis and reduced cilia length, inflammation, and O-GlcNAc levels. Thus, unlike IFT-B, the effect of Ttc21b deletion in mouse models of ADPKD is development-specific. Unlike an IFT-A adaptor, deleting Ttc21b in juvenile ADPKD mice is partially ameliorative. Thus, our studies suggest that different microenvironmental factors, found in distinct nephron segments and in developing versus mature stages, modify ciliary homeostasis and ADPKD pathobiology. Further, elevated levels of O-GlcNAc, which regulates cellular metabolism and ciliogenesis, may be a pathological feature of ADPKD.

EPO synthesis induced by HIF-PHD inhibition is dependent on myofibroblast transdifferentiation and colocalizes with non-injured nephron segments in murine kidney fibrosis.

Erythropoietin (EPO) is regulated by hypoxia-inducible factor (HIF)-2. In the kidney, it is produced by cortico-medullary perivascular interstitial cells, which transdifferentiate into collagen-producing myofibroblasts in response to injury. Inhibitors of prolyl hydroxylase domain (PHD) dioxygenases (HIF-PHIs) activate HIF-2 and stimulate kidney and liver EPO synthesis in patients with anemia of chronic kidney disease (CKD). We examined whether HIF-PHIs can reactivate EPO synthesis in interstitial cells that have undergone myofibroblast transdifferentiation in established kidney fibrosis. We investigated Epo transcription in myofibroblasts andcharacterized the histological distribution of kidney Epo transcripts by RNA in situ hybridization combined with immunofluorescence in mice with adenine nephropathy (AN) treated with HIF-PHI molidustat. Lectin absorption chromatography was used to assess liver-derived EPO. In addition, we examined kidney Epo transcription in Phd2 knockout mice with obstructive nephropathy. In AN, molidustat-induced Epo transcripts were not found in areas of fibrosis and did not colocalize with interstitial cells that expressed α-smooth muscle actin, a marker of myofibroblast transdifferentiation. Epo transcription was associated with megalin-expressing, kidney injury molecule 1-negative nephron segments and contingent on residual renal function. Liver-derived EPO did not contribute to serum EPO in molidustat-treated mice. Epo transcription was not associated with myofibroblasts in Phd2 knockout mice with obstructive nephropathy. Our studies suggest that HIF-PHIs do not reactivate Epo transcription in interstitial myofibroblasts and that their efficacy in inducing kidney EPO in CKD is dependent on the degree of myofibroblast formation, the preservation of renal parenchyma and the level of residual renal function.

Revisiting the role of Notch in nephron segmentation confirms a role for proximal fate selection during mouse and human nephrogenesis.

Notch signaling promotes maturation of nephron epithelia, but its proposed contribution to nephron segmentation into proximal and distal domains has been called into doubt. We leveraged single cell and bulk RNA-seq, quantitative immunofluorescent lineage/fate tracing, and genetically modified human induced pluripotent stem cells (iPSCs) to revisit this question in developing mouse kidneys and human kidney organoids. We confirmed that Notch signaling is needed for maturation of all nephron lineages, and thus mature lineage markers fail to detect a fate bias. By contrast, early markers identified a distal fate bias in cells lacking Notch2, and a concomitant increase in early proximal and podocyte fates in cells expressing hyperactive Notch1 was observed. Orthogonal support for a conserved role for Notch signaling in the distal/proximal axis segmentation is provided by the demonstration that nicastrin (NCSTN)-deficient human iPSC-derived organoids differentiate into TFA2B+ distal tubule and CDH1+ connecting segment progenitors, but not into HNF4A+ or LTL+ proximal progenitors.

The potential of RNA-based therapy for kidney diseases.

Inherited kidney diseases (IKDs) are a large group of disorders affecting different nephron segments, many of which progress towards kidney failure due to the absence of curative therapies. With the current advances in genetic testing, the understanding of the molecular basis and pathophysiology of these disorders is increasing and reveals new potential therapeutic targets. RNA has revolutionized the world of molecular therapy and RNA-based therapeutics have started to emerge in the kidney field. To apply these therapies for inherited kidney disorders, several aspects require attention. First, the mRNA must be combined with a delivery vehicle that protects the oligonucleotides from degradation in the blood stream. Several types of delivery vehicles have been investigated, including lipid-based, peptide-based, and polymer-based ones. Currently, lipid nanoparticles are the most frequently used formulation for systemic siRNA and mRNA delivery. Second, while the glomerulus and tubules can be reached by charge- and/or size-selectivity, delivery vehicles can also be equipped with antibodies, antibody fragments, targeting peptides, carbohydrates or small molecules to actively target receptors on the proximal tubule epithelial cells, podocytes, mesangial cells or the glomerular endothelium. Furthermore, local injection strategies can circumvent the sequestration of RNA formulations in the liver and physical triggers can also enhance kidney-specific uptake. In this review, we provide an overview of current and potential future RNA-based therapies and targeting strategies that are in development for kidney diseases, with particular interest in inherited kidney disorders.

MO123: The Correlation of Platinum-Based Chemotherapy with Decreased Blood Ionized Magnesium in Cancer Patients

Abstract BACKGROUND AND AIMS Platinum-based agents are prescribed as backbone chemotherapy for lung, colorectal, esophagogastric, breast, testicular and ovarian cancers, among others. Renal excretion of platinum-based agents is mediated by the cellular transporters localized in renal tubules. Accumulation of the agents in tubular cells leads to inflammation, injury and cell death. Meanwhile, one-tenth of total body magnesium (Mg) is filtered by kidney during a 24-h period, and 80–95% of the filtered Mg is reabsorbed by nephron segments, including the proximal tubule, thick ascending limb of the Loop of Henle and distal convoluted tubule. Thus, there is a potential that renal Mg loss can happen as a result of direct injury to those major Mg absorption sites in renal tubules by platinum-based agents. Mg serves as a cofactor in over 300 enzymes and is essential for many biological processes. Thus, monitoring for hypomagnesemia is important in these patients. We compared Mg loss in patients undergoing platinum-based therapy with those being treated with non-platinum-based agents to evaluate the potential for Mg loss in platinum-based chemotherapy. We evaluated Mg status using an assay for ionized Mg (iMg), which is the physiologically active form of Mg. METHOD A total of 78 patients suffering from various tumours and taking chemotherapy in the Fourth Hospital of Hebei Medical University (Hebei Tumor Hospital), China, between January and July 2019 were enrolled in the study. For each patient, blood iMg level was tested twice using Stat Profile Prime Plus® Critical Care Analyzer (Nova Biomedical, Waltham, MA, USA). The first iMg testing was conducted upon admission to the hospital before chemotherapy. The second iMg testing was conducted before discharge from the hospital after the chemotherapy course was completed. Generally, the time difference between the two iMg tests was several weeks up to 1 month. The difference of iMg levels in the two tests was evaluated, and the correlation of the degrees of iMg decrease after the chemotherapy course with the platinum-based agent group and non-platinum-based agent group was analysed using the Chi-square test. RESULTS Most of the patients suffered from lung cancer, followed by breast, esophagogastric, colon and rectal cancers. There were also sparse cases of other cancers. A total of 44 of them received a platinum-based agent, and 34 received non-platinum-based agents only. The platinum-based agents taken were cisplatin, oxaliplatin, carboplatin and nedaplatin. For patients aged over 60 years, the decrease in blood iMg levels in the platinum-based group was significantly greater than that in the non-platinum-based group (x2 = 4.353 and P &amp;lt; .05 for iMg decrease &amp;gt; 0.01 mmol/L, x2 = 4.403 and P &amp;lt; .05 for iMg decrease &amp;gt; 0.05 mmol/L, and x2 = 4.237 and P &amp;lt; .05 for iMg decrease &amp;gt;0.1 mmol/L). For male patients at all ages, the decrease in blood iMg levels in the platinum-based group was also significantly greater than that in the non-platinum-based group (x2 = 9.879 and P &amp;lt; .01 for iMg decrease &amp;gt;0.01 mmol/L, x2 = 11.481 and P &amp;lt; .001 for iMg decrease &amp;gt;0.01 mmol/L, and x2 = 10.000 and P &amp;lt; .01 for iMg decrease &amp;gt;0.1 mmol/L). There was no statistical significance in blood iMg levles between the platinum-based group and the non-platinum-based group for patients below 60 years old. There was also no statistical significance in blood iMg levels between the platinum-based group and the non-platinum-based group for all the female patients at all ages. CONCLUSION Our study showed that all patients aged over 60 years and male patients at all ages were liable to a decrease in blood iMg levels if treated with the platinum-based chemotherapy rather than non-platinum-based chemotherapy. It suggests that the iMg reabsorption process facilitated by the renal tubules is more likely to be affected by the platinum-based agents in those groups of patients. Monitoring renal function and blood iMg levels is important for patients receiving platinum-based chemotherapy.

Proximal Tubule‐Specific Transgenic Overexpression of Peroxisome Proliferator‐Activated Receptor Alpha Prevents the Development of Hypertension in Dahl Salt‐Sensitive Rats

The Dahl salt‐sensitive rat (Dahl SS) is a well investigated model of hypertension that mimics human salt sensitive hypertension. These rats develop a significant increase in blood pressure and renal disease when challenged with a high salt diet compared to salt‐resistant strains. Prolonged high salt feeding results in extensive renal pathology, including glomerular and proximal tubular pathology, tubular casting, and interstitial fibrosis. It has been shown that clofibrate, a lipid lowering peroxisome proliferator‐activated receptor alpha (PPARα) agonist, can prevent the hypertensive phenotype in Dahl SS rats. PPARα is a ligand activated transcription factor that regulates β‐oxidation of fatty acids (FAO). The proximal tubule is a very metabolically active tubular segment that relies primarily on FAO to meet its high energy requirements. Reduced FAO can lead to proximal tubular damage and renal dysfunction. To investigate the impact of PPARα‐mediated FAO during a high‐salt challenge, we developed a novel transgenic Dahl SS strain with a proximal tubule‐specific promoter from the sodium/glucose cotransporter 2 gene (Sglt2). This resulted in the overexpression of Ppara only in the proximal tubule (SSTgPTPpara). At 9 weeks of age, male wild‐type rats and heterozygous transgenic SSTgPTPpara rats were placed on a 4.0% NaCl, high salt diet (n=6/group). Following a 14‐day high salt challenge, rats were anesthetized for carotid blood pressure recording. Proximal tubule‐specific transgenic overexpression of PPARα in heterozygous SSTgPTPpararats had lower systolic blood pressure when compared to wild‐type littermates (161.2.6 ± 12.0 mmHg vs.122.8 ± 4.2 mmHg; mean ± SEM; p &lt;0.05, t‐test), as well as mean arterial pressure and diastolic blood pressure. Immunohistochemical analysis confirmed an increase in PPARA protein within proximal tubules of the renal cortex, relative to other nephron segments. In conclusion, proximal tubule specific‐transgenic overexpression of PPARα reduced salt‐sensitive hypertension, suggesting that proximal tubules may be one of the primary sites of anti‐hypertensive actions of clofibrate in Dahl SS rats. This also indicates that the SSTgPTPpara rat model could be useful for studying the contribution of dysregulated proximal tubule metabolism to salt‐sensitive hypertension and renal pathology in the Dahl SS rat.

Emx2is Required for Nephron Segment Development in the Zebrafish Pronephros

The empty spiracles homeobox gene 2 (emx2)encodes a transcription factor that is expressed during neuronal, auditory, olfactory, and renal development, but its role in the latter has not yet been fully elucidated. Here, our objective is to utilize the zebrafish animal model to investigate the functions of emx2 during the development of nephrons, which are the structural and functional units of the kidney. Using whole mount in situ hybridization (WISH), we detected emx2transcript beginning at the 6 somite‐stage in renal progenitors and this expression continued throughout the process of forming discrete proximal convoluted tubule (PCT) and proximal straight tubule (PST) segment regions at 24 hours post fertilization (hpf). Based on this spatiotemporal pattern of emx2 expression in renal progenitors, we hypothesized that emx2is essential for segmentation of the nephron into discrete proximal and distal regions. To investigate this, we generated emx2‐deficient embryos and compared them to wild‐type (WT) controls at various time points from 24‐96 hpf. Knockdown of emx2resulted in head necrosis and diminished yolk sac extension at 24 hpf, as well as development of smaller head, eye and body, pericardial edema, abnormal fin growth, yolk ball inflammation and vascular hemorrhage in the central nervous system from 2‐5 days post fertilization. We used WISH to stain for each nephron segment in fixed animals and then quantified segment length and cell number. WISH analysis revealed a significantly reduced podocyte area, diminished PCT and reduced multiciliated cell numbers in emx2‐deficient embryos compared to WT embryos. WISH results also revealed secondary phenotypes, such as reduced fin bud area and somite in emx2‐deficient embryos. Additionally, Alcian Blue staining at 96 hpf revealed jaw abnormalities and pharyngeal arch malformation in emx2‐deficient embryos. O‐Dianisidine staining showcased vascular hemorrhage in the eyes and head of emx2deficient embryos. We concluded that emx2is essential for kidney segmentation in zebrafish, especially in podocyte and PCT formation. Additionally, emx2deficiency causes secondary jaw, fin, cartilage blood accumulation and muscle phenotypes. Future studies will delineate the cellular defects in nephron development in emx2 deficient embryos and determine the relationship between Emx2 and pathways which are essential for proximal segment ontogeny, such as retinoic acid signaling.