Tubule Segments Research Articles

Roxadustat (FG-4592) protects against ischaemia/reperfusion-induced acute kidney injury through inhibiting the mitochondrial damage pathway in mice.

Ischaemia-reperfusion (I/R) is one of the main factors of acute kidney injury (AKI). mitochondrial damage pathway are important features of I/R induced-acute kidney injury (IRI-AKI). Hypoxia-inducible factor (HIF) expression in renal tubule segments is up-regulated during AKI. Herein, we investigated the role of FG-4592 in a mouse model of IRI-AKI to confirm whether FG-4592 is beneficial in AKI. We found that pretreatment with FG-4592significantly ameliorated renal function and renal histological damage in mice after IRI. Furthermore, these results suggest that pretreatment with FG-4592significantly reduced the tubular cells apoptosis (decreased TUNEL-positive cells, Bax, caspase12levels), attenuated mitochondrial damage (increased ATPβ, PPARγ, mitochondrial DNA copy number, and decreased cytoplasmic cytochrome C), and alleviated DNA damage after IRI. In conclusion, pretreatment with FG-4592may effectively prevent kidney from IRI possibly by via diminishing tubular cells injuries and protection of mitochondrial damage pathway. These results further validate that FG-4592may be an effective drug in the clinical treatment of IRI-AKI.

Drug Repurposing Patent Applications April–June 2021

Drug Repurposing Patent Applications April–June 2021

Mapping the genetic architecture of human traits to cell types in the kidney identifies mechanisms of disease and potential treatments.

The functional interpretation of genome-wide association studies (GWAS) is challenging due to the cell-type-dependent influences of genetic variants. Here, we generated comprehensive maps of expression quantitative trait loci (eQTLs) for 659 microdissected human kidney samples and identified cell-type-eQTLs by mapping interactions between cell type abundances and genotypes. By partitioning heritability using stratified linkage disequilibrium score regression to integrate GWAS with single-cell RNA sequencing and single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing data, we prioritized proximal tubules for kidney function and endothelial cells and distal tubule segments for blood pressure pathogenesis. Bayesian colocalization analysis nominated more than 200 genes for kidney function and hypertension. Our study clarifies the mechanism of commonly used antihypertensive and renal-protective drugs and identifies drug repurposing opportunities for kidney disease.

Molecular determinants of protein reabsorption in the amphibian kidneys

Participation of molecular determinants of endocytosis in the processes of glomerular filtration and tubular reabsorption of albumin and lysozyme in the mesonephros of grass frogs (Rana temporaria L.), lake frogs (Rana ridibunda P.), and newts (Triturus vulgaris L.) is investigated. In all studied species, the constitutive expression of endocytic receptors in proximal tubule (PT) cells is established using immunofluorescence microscopy and immunoblotting. The certain stages of lysozyme and albumin endocytosis involving megalin/LRP2, cubilin, clathrin and protein Rab11 are detailed, and the central role of ligand-induced megalin/LRP2 activity in this process is shown. Increased ligand-induced expression for clathrin and Rab11was also found. In grass frogs, the different patterns of endocytic receptors and both absorbed proteins in the initial parts of proximal tubules suggest the proximo-distal specialization of absorptive processes along these tubule segments, similar to this in more complex mammalian nephrons. This data, as well as the revealed peculiarities of ligand-receptor interactions during intracellular trafficking of proteins prove that megalin is mainly involved in the absorption of lysozyme. At the same time, albumin absorption is mediated by both receptors, or cubilin contributes the most. The detection of endocytic receptor in glomerular structural elements in frogs and newts suggests the participation of filtration barrier components in endocytosis of filterable proteins. The results represent a new contribution to the study of the fundamental mechanisms of renal protein uptake in the amphibian mesonephros as a more primitive kidney compared to mammalian metanephros.

"SLC-omics" of the kidney: solute transporters along the nephron.

The fluid in the 14 distinct segments of the renal tubule undergoes sequential transport processes that gradually convert the glomerular filtrate into the final urine. The solute carrier (SLC) family of proteins is responsible for much of the transport of ions and organic molecules along the renal tubule. In addition, some SLC family proteins mediate housekeeping functions by transporting substrates for metabolism. Here, we have developed a curated list of SLC family proteins. We used the list to produce resource webpages that map these proteins and their transcripts to specific segments along the renal tubule. The data were used to highlight some interesting features of expression along the renal tubule including sex-specific expression in the proximal tubule and the role of accessory proteins (β-subunit proteins) that are thought to be important for polarized targeting in renal tubule epithelia. Also, as an example of application of the data resource, we describe the patterns of acid-base transporter expression along the renal tubule.

FC 014INFLUENCE OF GENETIC VARIATION IN SLC7A13/AGT1 IN HUMAN CYSTINURIA

Abstract Background and Aims Cystinuria (CU) is an inherited renal disorder based on urinary wasting of dibasic amino acids, urinary precipitation, and consecutive cystine stone formation. It is caused by pathogenic variants in two distinct disease genes, SLC3A1 and SLC7A9, both of which encode subunits of a heterodimeric tubular amino acid transporter, rBAT/SLC3A1 and BAT1/SLC7A9, located at the apical membrane of proximal renal tubules. CU is marked by incomplete penetrance and substantial disease variability. Recently, a novel cystine transporter, consisting of the light chain AGT1/SLC7A13 and its heterodimeric partner rBAT/SLC3A1 has been identified in the S3 segment of murine proximal tubules. In this study, we aim at evaluating the role of AGT1 in cystinuric patients with or without mutations in either SLC3A1 or SLC7A9, analyzing the role of AGT1/SLC7A13 as novel disease gene or genetic modifier in CU. Method A multicenter European CU-cohort comprising 132 individuals was screened for pathogenic variants in SLC3A1, SLC7A9, and SLC7A13 using high-throughput multiplex PCR-based amplification and next-generation sequencing (MiSeq Illumina) followed by multiplex ligation-dependent probe amplification (MLPA) of SLC3A1 and SLC7A9. For functional in vitro studies, epitope-tagged human and murine rBAT and AGT1 proteins were transiently expressed in different cell systems. Heterodimer complex formation was analyzed by co-immunoprecipitation and western blot studies and membrane trafficking was evaluated by immunofluorescence microscopy. Results Genectic analysis of our CU-cohort did not reveal indiviuals with SLC7A13 variation only, however we found three patients harbouring heterozygous missense variants in addition to pathogenic or VUS variants in SLC3A1 or SLC7A9. To evaluate their influence on the generation of functional cystine transporters in vitro, different cell models were transiently transfected with plasmids expressing wildtype or mutant proteins. In line with previous reports, co-expression of AGT1 and rBAT wildtype allowed efficient complex formation as AGT1-induced maturation of rBAT was detected by increased mature N-glycosylation, co-immunoprecipitation and membrane insertion. Whereas AGT1 patient variants p.Met452Thr (SLC7A13 c.1355T>C) and p.Ile174Phe (SLC7A13 c.520A>T) behaved comparable to wildtype AGT1, variants p.Asn45Lys (SLC7A13 c.135C>G) and p.Leu270Phe (SLC7A13 c.808C>T) led to clearly reduced glycosylation patterns and trafficking deficits of rBAT wildtype protein. Next, the mutual influence of pathogenic variation in both, AGT1 and rBAT, will unravel the consequences of patient-specific molecular interactions on the functional expression of cystine transporter complexes. Conclusion Here, we report three CU-patients with variants in SLC7A13 combined with either SLC3A1 or SLC7A9. For two of these variants, in vitro functional analysis revealed pathogenic molecular mechanisms disturbing complex formation, maturation and trafficking of rBAT. We hypothesize that specific pathogenic variants in SLC7A13 interfere with efficient membrane localization of heterodimeric cystine transporters, which results in modulation of cystine transport in the S3 segment of proximal tubules in CU-patients.

FC 016SPARSENTAN IMPROVES GLOMERULAR BLOOD FLOW AND AUGMENTS PROTECTIVE TISSUE REMODELING IN MOUSE MODELS OF FOCAL SEGMENTAL GLOMERULOSCLEROSIS (FSGS)

Abstract Background and Aims Preliminary preclinical and emerging clinical evidence indicates strong antiproteinuric actions of dual endothelin type A (ETA) and angiotensin II type 1 (AT1) receptor antagonism with sparsentan. These nephroprotective effects have been more pronounced in different experimental and clinical settings compared to current standard of care using an AT1 receptor blocker (ARB). Considering the broad spectrum of renal actions of endothelin (ET) and angiotensin II (Ang II), inhibition of both pathways using sparsentan is postulated to target multiple renal cell types via a variety of renoprotective mechanisms. The aim of this study was to determine glomerular action of sparsentan as compared to the ARB losartan (Los) by direct visualization of effects on renal hemodynamics and tissue remodeling in the intact living kidney. Method Intravital multiphoton microscopy (MPM) of the glomerular vasculature and filtration barrier structure and function was performed in genetically engineered mice combined with traditional urinalysis and histology-based phenotyping. Glomerular hemodynamic parameters (afferent and efferent arteriole (AA and EA) diameters and single nephron glomerular filtration rate (SNGFR)) and podocyte calcium entry, as a measure of cell injury, were quantitatively visualized in the FSGS model Pod-GCaMP5/Tomato TRPC6 transgenic mice (1.5 years of age), in which TRPC6 is overexpressed together with the calcium reporter GCaMP5 in podocytes. Single cell identification and fate tracking of cells of the renin lineage (CoRL) was performed over time using a second physiologic control mouse model, Ren1d-Confetti mice that feature a multicolor CFP/GFP/YFP/FP reporter. Three groups of mice in each model received treatment with either vehicle (CTRL), the ARB losartan (Los; 10 mg/kg/day), or sparsentan (120 mg/kg/day) for 6 weeks (FSGS model) or 2 weeks (control physiology model). Results Both Los and sparsentan treatment attenuated the acute ET + Ang II-induced elevation of podocyte calcium by ∼80%, and the development of albuminuria, and glomerulosclerosis and tissue fibrosis in the FSGS model. Notably, sparsentan prevented the ET + Ang II increases in podocyte calcium more than Los and was significantly more effective in dilating both AA and EA (Fig. 1A, B), increasing SNGFR (Fig. 1C), increasing capillary blood flow (2-fold; p<0.0001 vs. CTRL), and decreasing albuminuria (20%; p<0.05 vs. CTRL). Sparsentan also preserved p57+ podocyte number by 50% compared to Los (p<0.0001 vs. Los). Similarly, pretreatment with sparsentan was more effective in preventing glomerular arteriolar vasoconstriction induced by acute ET + Ang II iv injection compared to Los (p<0.05 vs. Los). Following a 2-week treatment in control healthy Ren1d-Confetti mice, sparsentan resulted in a more robust increase compared to Los in the number of Confetti+ cells, clones, and individual cells per clone in the glomeruli and AA (Fig. 1D-F). Renal tubule segments also showed active cellular remodeling in response to sparsentan. Conclusion Serial MPM imaging directly visualized several mechanisms underlying beneficial antiproteinuric and structural effects of sparsentan in both FSGS and in the normal mouse kidney and differences between dual ETA/AT1 receptor antagonism of sparsentan and a mono-selective ARB. The sparsentan-induced glomerular hemodynamic pattern was driven by both AA and EA dilation resulting in an increase in capillary blood flow. Compared to Los, sparsentan was more effective in attenuating ET/Ang II-induced podocyte injury and in activation of resident progenitor cells and tissue remodeling. These findings suggest multiple layers of renal protective actions by dual ETA and AT1 receptor antagonism.

Landscape of GPCR expression along the mouse nephron.

Kidney transport and other renal functions are regulated by multiple G protein-coupled receptors (GPCRs) expressed along the renal tubule. The rapid, recent appearance of comprehensive unbiased gene expression data in the various renal tubule segments, chiefly RNA sequencing and protein mass spectrometry data, has provided a means of identifying patterns of GPCR expression along the renal tubule. To allow for comprehensive mapping, we first curated a comprehensive list of GPCRs in the genomes of mice, rats, and humans (https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/) using multiple online data sources. We used this list to mine segment-specific and cell type-specific expression data from RNA-sequencing studies in microdissected mouse tubule segments to identify GPCRs that are selectively expressed in discrete tubule segments. Comparisons of these mapped mouse GPCRs with other omics datasets as well as functional data from isolated perfused tubule and micropuncture studies confirmed patterns of expression for well-known receptors and identified poorly studied GPCRs that are likely to play roles in the regulation of renal tubule function. Thus, we provide data resources for GPCR expression across the renal tubule, highlighting both well-known GPCRs and understudied receptors to provide guidance for future studies.

Roles of Sodium-Glucose Cotransporter 2 of Mesangial Cells in Diabetic Kidney Disease.

We have been studying the presence of sodium-glucose cotransporter 2 (SGLT2) in mesangial cells and pericytes since 1992. Recent large placebo-controlled studies of SGLT2 inhibitors in patients with type 2 diabetes mellitus have reported desirable effects of the inhibitors on the diabetic kidney and the diabetic heart. Most studies have indicated that these effects of SGLT2 inhibitors could be mediated by the tubuloglomerular feedback system. However, a recent study about urine sodium excretion in the presence of an SGLT2 inhibitor did not show any increases in urine sodium excretion. A very small dose of an SGLT2 inhibitor did not inhibit SGLT2 at the S1 segment of proximal tubules. Moreover, SGLT2 inhibition protects against progression in chronic kidney disease with and without type 2 diabetes. In these circumstances, the tubuloglomerular feedback hypothesis involves several theoretical concerns that must be clarified. The presence of SGLT2 in mesangial cells seems to be very important for diabetic nephropathy. We now propose a novel mechanism by which the desirable effects of SGLT2 inhibitors on diabetic nephropathy are derived from the direct effect on SGLT2 expressed in mesangial cells.

A Mathematical Model of the Rat Kidney: Ammonia Transport

Ammonia generated within the kidney is partitioned into a urinary fraction (the key buffer for net acid excretion), and an aliquot delivered to the systemic circulation. The physiology of this partitioning has yet to be examined in a kidney model, and that is undertaken in this work. This involves explicit representation of the cortical labyrinth, so that cortical interstitial solute concentrations are computed, rather than assigned. A detailed representation of cortical vasculature has been avoided by making the assumption that solute concentrations within interstitium and peritubular capillaries are likely to be identical, and that there is little to no modification of venous composition as blood flows to the renal vein. The model medullary ray has also been revised to include a segment of proximal straight tubule, which supplies ammonia to this region. The principal finding of this work is that cortical labyrinth interstitial ammonia concentration is likely to be several-fold higher than systemic arterial ammonia. This elevation of interstitial ammonia enhances ammonia secretion in both PCT and DCT, with uptake by Na,K-ATPases of both segments. Model prediction of urinary ammonia excretion is concordant with measured values, but at the expense of greater ammoniagenesis, with high rates of renal venous ammonia flux. This derives from a limited capability of the model medulla to replicate the high interstitial ammonia concentrations that are required to drive collecting duct ammonia secretion. Thus, renal medullary ammonia trapping appears key to diverting ammonia from renal vein to urine, but capturing the underlying physiology remains a challenge.

Roles of Zinc Transporters That Control the Essentiality and Toxicity of Manganese and Cadmium

Cellular transport systems for both essential and toxic trace elements remain elusive. In our studies on the transport systems for cadmium (Cd), we found that the cellular uptake of Cd is mediated by the transporter for manganese (Mn). We identified ZIP8 and ZIP14, members of the ZIP zinc (Zn) transporter family, as transporters having high affinities for both Cd and Mn. Notably, the uptake of Cd into rice root from soil is mediated by a transporter for Mn as well. We found that ZIP8 is highly expressed at the S3 segment of the kidney proximal tubule and can transport glomerulus-filtered Cd and Mn ions in the lumen into epithelial cells of the proximal tubule, suggesting that ZIP8 has an important role in the renal reabsorption of both toxic Cd and essential Mn. Mutations in ZIP8 and ZIP14 genes were found in humans having congenital disorders associated with the disturbed transport of Mn, although ZIP8 mutation causes whole-body Mn deficiency while ZIP14 mutation causes Mn accumulation in the brain. Mutations in ZnT10, a Zn transporter responsible for Mn excretion, also cause hyperaccumulation of Mn in the brain. Results of genome-wide association studies have indicated that ZIP8 SNPs are involved in a variety of common diseases. Thus, ZIP8, ZIP14, and ZnT10 play crucial roles in the transport of Mn and thereby control Mn- and Cd-related biological events in the body.

GPCR‐omics of the Nephron: Mapping Receptors Along the Renal Tubule

Kidney transport and other renal functions are regulated by multiple G-protein coupled receptors (GPCRs) expressed along the renal tubule. Development of unbiased ‘-omics’ approaches and next-generation sequencing (NGS)-based techniques, including transcriptomics in microdissected renal tubules, single-cell transcriptomics, and proteomics in microdissected tubules have provided a means of identifying patterns of GPCR expression. To allow for complete GPCR mapping, we first curated a comprehensive list of GPCRs in the genomes of rats, mice and humans (https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/). From this database we mapped the expression of 758 GPCRs across fourteen nephron segments using existing transcriptomic data, providing a complete picture of GPCR expression (https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/TubuleTPM.html). We used this list to mine segment-specific and cell-type specific expression data from RNA-seq studies to identify GPCRs that are selectively expressed in discrete tubule segments. Each GPCR found to be selectively expressed was also matched to known ligands, coupled G-proteins and known physiological roles in the segment in which it is selectively expressed. Comparisons of these mapped GPCRs with other transcriptomics and proteomics datasets, functional data from isolated perfused tubules, and micro-puncture studies in the literature confirms patterns of expression for well-known receptors and identifies lesser known GPCRs that are likely to play key roles in regulation of renal tubule transport and function. This study provides data resources for GPCR expression across the renal tubule, highlighting both well-known GPCRs and understudied receptors in order to provide guidance for future experiments.

Immunohistochemical localization and pharmacokinetics of the anti-MRSA drug teicoplanin in rat kidney using a newly developed specific antibody.

We prepared a polyclonal antibody against a teicoplanin (TEIC)-bovine serum albumin conjugate that was specific to both conjugated and free forms of TEIC. We demonstrated that this antibody could be used to detect the time-dependent localization of TEIC in rat kidneys. Immunohistochemistry revealed immunoreactivity specifically in the microvilli and apical cytoplasm of epithelial cells in proximal tubule segments S1 and S2, 1h after intravenous TEIC injection, with higher staining intensity in the S2 segments. The epithelial cells of S3 segments showed moderate immunostaining with a few cells exhibiting nuclear staining. Furthermore, we found that the distal tubules and collecting ducts contained both TEIC-positive and -negative cells. TEIC immunoreactivity decreased rapidly over time; only weak staining remained in the S3 segments, distal tubules, and collecting ducts 24h after administration. No staining was detected 7days after injection. These results were significantly different from those of our previous study obtained using vancomycin, which showed moderate staining in the proximal tubule segments S1 and S2, distal tubules, and the collecting ducts 8days after administration. The lower TEIC accumulation in tissues may account for a lower risk of adverse events compared to that using vancomycin.

Lower Urinary Tract Symptoms Should Be Queried When Initiating Sodium Glucose Co-Transporter 2 Inhibitors.

The sodium-glucose cotransporter 2 inhibitors (SGLT2i) reduce the risk of both macrovascular (myocardial infractions and stroke) (1) and microvascular events (progression of CKD) (2) in persons with type 2 diabetes. The beneficial effects of SGLT2i are derived from a cascade of effects subsequent to inhibition of glucose reabsorption in the S1 segment of the proximal tubule. Clinical trials have shown that SGLT2i reduce the absolute risk of kidney failure by 35% (95% CI, 54% to 81%) relative to placebo (2). Thus, SGLT2i may be one of the most important interventions provided for adults at high risk for kidney failure. With any intervention, side effects and adverse effects must be considered to identify patients appropriate for the intervention and to ensure that treated patients are educated regarding potential side effects and the appropriate coping mechanisms for bothersome side effects. For the SGLT2i drug class, adverse events includes hypoglycemia, ketoacidosis, volume depletion (especially when combined with loop diuretics), and genital urinary infections (3⇓⇓–6). Other important side effects that are not life threatening, but are nevertheless bothersome, are lower urinary tract symptoms (LUTS). Below, we describe reasons why LUTS should be queried in patients before initiating SGLT2i and methods for managing LUTS to maintain adherence with this drug class. LUTS include urinary frequency and urgency, with or without incontinence. Urinary urgency, or the sudden urge to urinate that may or may not be accompanied by urinary incontinence, is termed overactive bladder (OAB) syndrome (7). Over 10% of older adults cope with some degree of OAB, and prevalence increases with advancing age (7). Adults with diabetes, especially those with poor glucose control, are particularly at risk for OAB due to bladder dysfunction from neuropathy and osmotic diuresis, which can lead to detrusor muscle hypertrophy and reduction in bladder storage capacity …

Exploration of zebrafish larvae as an alternative whole-animal model for nephrotoxicity testing

Due to an increasing demand for testing of new and existing chemicals and legal restrictions for the use of animals, there is a strong need for alternative approaches to assess systemic toxicity. Embryonic and larval zebrafish (Danio rerio) are increasingly recognized as a promising alternative whole-animal model that may be able to overcome limitations of cell-based in vitro assays and bridge the gap between high-throughput in vitro screening and low-throughput in vivo tests in animals. Despite the relatively simple anatomical structure of the zebrafish larval kidney (pronephros) – composed of only two nephrons - the pronephros shares major functions and cell types with mammalian nephrons. Glomerular filtration begins at 48 h post fertilization. The aim of the present study was to investigate if early zebrafish larvae might be a suitable model for nephrotoxicity testing. On day 3 post fertilization, larval zebrafish were treated with selected nephrotoxins (aristolochic acid, cadmium chloride, potassium bromate, ochratoxin A, gentamicin) for 48 h. Histological evaluation of zebrafish larvae exposed to model nephrotoxins revealed tubule injury as evidenced by dilated tubules with loss of the brush border, tubule cell necrosis and disorganization of the tubular epithelium. These changes were most severe after treatment with gentamicin, which also impaired pronephros function as evidenced by reduced clearance of FITC-dextran. Whole-mount in situ hybridization showing loss of cdh17 expression revealed site-specific injury to the proximal tubule segment. Analysis of genes previously identified as novel biomarkers of kidney injury in mammals showed upregulation of the kidney injury marker genes heme oxygenase 1 (hmox1), clusterin (clu), secreted phosphoprotein/osteopontin (spp1), connective tissue growth factor (ctgf) and kim-1 (havcr-1) in response to nephrotoxin treatment, although the response of individual genes varied across compounds. Consistent with the severity of lesions and impaired kidney function, the most prominent gene expression changes occurred in larvae exposed to gentamicin. Overall, our results suggest that larval zebrafish may be a suitable alternative model organism for nephrotoxicity screening, yet further improvements and integration with quantitative in vitro to in vivo extrapolation will be needed to predict human toxicity.

A Comprehensive Map of mRNAs and Their Isoforms across All 14 Renal Tubule Segments of Mouse.

The repertoire of protein expression along the renal tubule depends both on regulation of transcription and regulation of alternative splicing that can generate multiple proteins from a single gene. A full-length, small-sample RNA-seq protocol profiled transcriptomes for all 14 renal tubule segments microdissected from mouse kidneys. This study identified >34,000 transcripts, including 3709 that were expressed in a segment-specific manner. All data are provided as an online resource (https://esbl.nhlbi.nih.gov/MRECA/Nephron/). Many of the genes expressed in unique patterns along the renal tubule were solute carriers, transcription factors, or G protein-coupled receptors that account for segment-specific function. Mapping the distribution of transcripts associated with Wnk-SPAK-PKA signaling, renin-angiotensin-aldosterone signaling, and cystic diseases of the kidney illustrated the applications of the online resource. The method allowed full-length mapping of RNA-seq reads, which facilitated comprehensive, unbiased characterization of alternative exon usage along the renal tubule, including known isoforms of Cldn10, Kcnj1 (ROMK), Slc12a1 (NKCC2), Wnk1, Stk39 (SPAK), and Slc14a2 (UT-A urea transporter). It also identified many novel isoforms with segment-specific distribution. These included variants associated with altered protein structure (Slc9a8, Khk, Tsc22d1, and Scoc), and variants that may affect untranslated, regulatory regions of transcripts (Pth1r, Pkar1a, and Dab2). Full-length, unbiased sequencing of transcripts identified gene-expression patterns along the mouse renal tubule. The data, provided as an online resource, include both quantitative and qualitative differences in transcripts. Identification of alternative splicing along the renal tubule may prove critical to understanding renal physiology and pathophysiology.

Single-Cell Imaging for Studies of Renal Uranium Transport and Intracellular Behavior

Nephrotoxicity is the primary health effect of uranium exposure. However, the renal transport and intracellular behavior of uranium remains to be clearly elucidated. In the present study, the intracellular uranium distribution was examined with the cell lines derived from the S3 segment of mouse renal proximal tubules, which is a toxic target site of uranium, using microbeam-based elemental analysis. Uranium exposure at 100 μM for 24 h (non-toxic phase) was performed in S3 cells. Two types of measurement specimens, including those that are adhesive cell specimens and cryosection specimens, were examined for the positional relationship of the intracellular localization of uranium. Based on the combined results of single-cell imaging from the two types of cell specimens, uranium was distributed inside the cell and localized in the cytoplasm near the cell nucleus. In some cells, uranium was colocalized with phosphorus and potassium. The amount of uranium accumulated in S3 cells was estimated using thin section-standards. The mean uranium content of three adhesive cells was hundreds of femtogram per cell. Thus, we believe that single-cell imaging would be useful for studies on renal uranium transportation and cellular behavior.

Modeling Distal Convoluted Tubule (Patho)Physiology: An Overview of Past Developments and an Outlook Toward the Future.

The kidneys are essential for maintaining electrolyte homeostasis. Blood electrolyte composition is controlled by active reabsorption and secretion processes in dedicated segments of the kidney tubule. Specifically, the distal convoluted tubule (DCT) and connecting tubule are important for regulating the final excretion of sodium, magnesium, and calcium. Studies unravelling the specific function of these segments have greatly improved our understanding of DCT (patho)physiology. Over the years, experimental models used to study the DCT have changed and the field has advanced from early dissection studies with rats and rabbits to the use of various transgenic mouse models. Developments in dissection techniques and cell culture methods have resulted in immortalized mouse DCT cell lines and made it possible to specifically obtain DCT fragments for ex vivo studies. However, we still do not fully understand the complex (patho)physiology of this segment and there is need for advanced human DCT models. Recently, kidney organoids and tubuloids have emerged as new complex cell models that provide excellent opportunities for physiological studies, disease modeling, drug discovery, and even personalized medicine in the future. This review presents an overview of cell models used to study the DCT and provides an outlook on kidney organoids and tubuloids as model for DCT (patho)physiology. Impact statement This study provides a detailed overview of past and future developments on cell models used to study kidney (patho)physiology and specifically the distal convoluted tubule (DCT) segment. Hereby, we highlight the need for an advanced human cell model of this segment and summarize recent advances in the field of kidney organoids and tubuloids with a focus on DCT properties. The findings reported in this review are significant for future developments toward an advanced human model of the DCT that will help to increase our understanding of DCT (patho)physiology.

Low retinoic acid levels mediate regionalization of the Sertoli valve in the terminal segment of mouse seminiferous tubules

In mammalian testes, undifferentiated spermatogonia (Aundiff) undergo differentiation in response to retinoic acid (RA), while their progenitor states are partially maintained by fibroblast growth factors (FGFs). Sertoli valve (SV) is a region located at the terminal end of seminiferous tubule (ST) adjacent to the rete testis (RT), where the high density of Aundiff is constitutively maintained with the absence of active spermatogenesis. However, the molecular and cellular characteristics of SV epithelia still remain unclear. In this study, we first identified the region-specific AKT phosphorylation in the SV Sertoli cells and demonstrated non-cell autonomous specialization of Sertoli cells in the SV region by performing a Sertoli cell ablation/replacement experiment. The expression of Fgf9 was detected in the RT epithelia, while the exogenous administration of FGF9 caused ectopic AKT phosphorylation in the Sertoli cells of convoluted ST. Furthermore, we revealed the SV region-specific expression of Cyp26a1, which encodes an RA-degrading enzyme, and demonstrated that the increased RA levels in the SV region disrupt its pool of Aundiff by inducing their differentiation. Taken together, RT-derived FGFs and low levels of RA signaling contribute to the non-cell-autonomous regionalization of the SV epithelia and its local maintenance of Aundiff in the SV region.

A Stereological Study of Key Histological Structures in the Kidneys of Rats from Young to Old Age

Quantitative (morphometric) study of the total amounts (per organ) and mean sizes of histological structures in individuals from young to old are essential for the understanding of age-related organ changes; stereological techniques are essential, reliable tools to obtain the quantitative data. Stereological study of all renal components, especially renal tubules, was lacking in age-related kidney studies and few studies used rats older than 24 months of age for a stereological analysis of the aging kidney. In the present study isotropic uniform random renal sections (methacrylate-embedded) were obtained from male Sprague-Dawley rats (89 per age-group) randomly sampled from a single cohort of normal animals at the ages of 3, 6, 12, 24 and 36 months, respectively. The sections were measured using various stereological methods to estimate the total amounts (per kidney) or mean sizes of key renal structures. The results demonstrated that the volume of kidney and the total volume or length of renal tubules increased continually from 3 to 24 months of age and then plateaued between 24 and 36 months of age. The total volume of renal corpuscles, glomeruli, Bowman’s space or interstitial tissue and the mean volume of renal corpuscles or glomeruli increased continually from 3 to 24 months and further until 36 months of age. The total number of glomeruli remained essentially constant and the relative volume of the cortex or medulla and the relative length of different segments of the renal tubules remained basically stable throughout the ages. Less than 5% of the renal corpuscular or tubular profiles were apparently atrophied at 24 or 36 months of age. The age-related results suggested that the rat renal tissues continued to develop adaptively or work actively, from young to old, to maintain normal physiological functions and the aging change in the kidney was primarily a compensatory or hypertrophic histological change.