A Focus on the Proximal Tubule Dysfunction in Dent Disease Type 1.

Dent disease type 1 is a rare X-linked recessive inherited renal disorder affecting mainly young males, generally leading to end-stage renal failure and for which there is no cure. It is caused by inactivating mutations in the gene encoding ClC-5, a 2Cl-/H+ exchanger found on endosomes in the renal proximal tubule. This transporter participates in reabsorbing all filtered plasma proteins, which justifies why proteinuria is commonly observed when ClC-5 is defective. In the context of Dent disease type 1, a proximal tubule dedifferentiation was shown to be accompanied by a dysfunctional cell metabolism. However, the exact mechanisms linking such alterations to chronic kidney disease are still unclear. In this review, we gather knowledge from several Dent disease type 1 models to summarize the current hypotheses generated to understand the progression of this disorder. We also highlight some urinary biomarkers for Dent disease type 1 suggested in different studies.

Effect of metal-modified sewage sludge biochar tubule on immobilization of chromium in unsaturated soil: Groundwater table fluctuations induced by rainfall.

Many studies have studied biochar immobilizing chromium (Cr) in soil. However, few studies were conducted to reduce the environmental risks due to biochar aging in soil. In this study, we adopt FeCl3, MgCl2, and AlCl3 to activate sewage sludge to form modified biochar and produce biochar tubules. Then, the column experiments were carried out to study the effect of fluctuating groundwater table on Cr leaching behavior, total Cr, and fractions distribution with the insertion of biochar tubule. Results showed that the Cr immobilization performance was improved by metal-modification biochar, the biochar tubules can significantly decrease the Cr leaching amounts, retard the Cr downward migration in the soil, and there was a better effect with slightly Cr-contaminated soil. In addition, the immobilization effect is also impacted by the biochar's application mode and the hydrodynamic conditions. Detailedly, the Cr leaching amounts maximally decreased by 33.39%, the residual amounts maximally increased by 10.05% in the soil column, and the exchangeable (EX) and carbonates-bound (CB) fractions were maximally increased by 85.18%, 151.78% at the equal depth of soil column, respectively. BET, SEM-EDS, XRD, and FTIR analyses revealed that biochars' immobilization mechanisms on Cr involved reduction(predominately), physisorption, precipitation, and complexation. Risk assessment demonstrated that the sewage sludge biochar has very low environmental risk. This study indicates that the biochar tubule applied to immobilize Cr in soil has potential and provides new insights into reducing environmental risks due to biochar aging.

Factors influencing the bioactivity of natural matrices: The case of osmolarity-dependent modulation of cell viability by different dilutions of camel urines.

The widespread practice of dromedary urinotherapy as a remedy for various illnesses, including cancer, is well-established in traditional dromedary countries. Researchers attempted to demonstrate anticancer properties of camel urine through in vitro experiments with debated outcomes. Notably, two critical aspects remained unexplored in those assays: (i) the osmolarity of tested urines, which can significantly influence in vitro results; (ii) the potential morphological changes of cells, following exposure to camel urines. In this study, we addressed these gaps by evaluating the osmolarity-dependent modulation of cell viability in human renal cell lines. In this regard, we assessed the impact of hyperosmolar mannitol-based solutions and dromedary urine on the viability and morphology of human non-tumor (HK2) and tumor renal cells (Caki-1). The results indicate that cell viability or morphology in both HK2 and Caki-1 cells are not significantly affected only if mannitol-induced hyperosmolarity is lower than 500 mOsm/L. Notably, when exposed to urine solution, diluted to <500 mOsm/L, statistically significant antiproliferative effects were observed primarily in Caki-1 cells (in presence of two out of ten tested urine samples). Conversely, alterations in cell morphology were observed exclusively in HK2 cells when exposed to the same diluted camel urines. In order to investigate, at molecular level, the observed antiproliferative effects, a preliminary metabolomics analysis of the tested urine samples was performed to identify potential bioactive compounds. The Nuclear Magnetic Resonance (NMR) metabolic profiling revealed the presence of three antioxidant compounds, namely trigonelline, pyruvic acid and N-acetylglucosamine. In conclusion, our results highlight the importance of considering the critical role of osmolarity when evaluating the bioactive properties of camel urine in vitro, which should not be used to treat any illness as it is. Conversely, it can be considered the possibility to use camel urines as a source of bioactive compounds.

Evaluating nephrotoxicity reduction in a novel polymyxin B formulation: insights from a 3D kidney-on-a-chip model.

This study aimed to assess the nephrotoxicity associated with VRP-034 (novel formulation of polymyxin B [PMB]) compared to marketed PMB in a three-dimensional (3D) kidney-on-a-chip model. To model the human kidney proximal tubule for analysis, tubular structures were established using 23 triple-channel chips seeded with RPTEC/hTERT1 cells. These cells were exposed to VRP-034 or PMB at seven concentrations (1-200 µM) over 12, 24, and 48 h. A suite of novel kidney injury biomarkers, cell health, and inflammatory markers were quantitatively assessed in the effluent. Additionally, caspase and cytochrome C levels were measured, and cell viability was evaluated using calcein AM and ethidium homodimer-1 (EthD-1). Exposure to marketed PMB resulted in significantly elevated levels (P < 0.05) of four key biomarkers (KIM-1, cystatin C, clusterin, and OPN) compared to VRP-034, particularly at clinically relevant concentrations of ≥10 µM. At 25 µM, all biomarkers demonstrated a significant increase (P < 0.05) with marketed PMB exposure compared to VRP-034. Inflammatory markers (interleukin-6 and interleukin-8) increased significantly (P < 0.05) with marketed PMB at concentrations of ≥5 µM, relative to VRP-034. VRP-034 displayed superior cell health outcomes, exhibiting lower lactate dehydrogenase release, while ATP levels remained comparable. Morphological analysis revealed that marketed PMB induced more severe damage, disrupting tubular integrity. Both treatments activated cytochrome C, caspase-3, caspase-8, caspase-9, and caspase-12 in a concentration-dependent manner; however, caspase activation was significantly reduced (P < 0.05) with VRP-034. This study demonstrates that VRP-034 significantly reduces nephrotoxicity compared to marketed PMB within a 3D microphysiological system, suggesting its potential to enable the use of full therapeutic doses of PMB with an improved safety profile, addressing the need for less nephrotoxic polymyxin antibiotics.

Pik3c3 Expression Profiling in the Mouse Kidney and Its Role in Proximal Tubule Cell Physiology.

The class 3 phosphatidylinositol 3-kinase (Pik3c3) plays critical roles in regulating autophagy, endocytosis, and nutrient sensing, but its expression profile in the kidney remains undefined. Recently, we validated a Pik3c3 antibody through immunofluorescence staining of kidney tissues from cell type-specific Pik3c3 knockout mice. Immunohistochemistry unveiled significant disparities in Pik3c3 expression levels across various kidney cell types. Notably, renal interstitial cells exhibit minimal Pik3c3 expression. Further, co-immunofluorescence staining, utilizing nephron segment- or cell type-specific markers, revealed nearly undetectable levels of Pik3c3 expression in glomerular mesangial cells and endothelial cells. Intriguingly, although podocytes exhibit the highest Pik3c3 expression levels among all kidney cell types, the renal proximal tubule cells (RPTCs) express the highest level of Pik3c3 among all renal tubules. RPTCs are known to express the highest level of the epidermal growth factor receptor (EGFR) in adult kidneys; however, the role of Pik3c3 in EGFR signaling within RPTCs remains unexplored. Therefore, we conducted additional cell culture studies. The results demonstrated that Pik3c3 inhibition significantly delayed EGF-stimulated EGFR degradation and the termination of EGFR signaling in RPTCs. Mechanistically, Pik3c3 inhibition surprisingly did not affect the initial endocytosis process but instead impeded the lysosomal degradation of EGFR. In summary, this study defines, for the first time, the expression profile of Pik3c3 in the mouse kidney and also highlights a pivotal role of Pik3c3 in the proximal tubule cells. These findings shed light on the intricate mechanisms underlying Pik3c3-mediated regulation of EGFR signaling, providing valuable insights into the role of Pik3c3 in renal cell physiology.

Organic anion transporters in remote sensing and organ crosstalk.

The organic anion transporters, OAT1 and OAT3, regulate the movement of drugs, toxins, and endogenous metabolites. In 2007, we proposed that OATs and other SLC22 transporters are involved in "remote sensing" and organ crosstalk. This is now known as the Remote Sensing and Signaling Theory (RSST). In the proximal tubule of the kidney, OATs regulate signaling molecules such as fatty acids, bile acids, indoxyl sulfate, kynurenine, alpha-ketoglutarate, urate, flavonoids, and antioxidants. OAT1 and OAT3 function as key hubs in a large homeostatic network involving multi-, oligo- and monospecific transporters, enzymes, and nuclear receptors. The Remote Sensing and Signaling Theory emphasizes the functioning of OATs and other "drug" transporters in the network at multiple biological scales (inter-organismal, organism, organ, cell, organelle). This network plays an essential role in the homeostasis of urate, bile acids, prostaglandins, sex steroids, odorants, thyroxine, gut microbiome metabolites, and uremic toxins. The transported metabolites have targets in the kidney and other organs, including nuclear receptors (e.g., HNF4a, AHR), G protein-coupled receptors (GPCRs), and protein kinases. Feed-forward and feedback loops allow OAT1 and OAT3 to mediate organ crosstalk as well as modulate energy metabolism, redox state, and remote sensing. Furthermore, there is intimate inter-organismal communication between renal OATs and the gut microbiome. Extracellular vesicles containing microRNAs and proteins (exosomes) play a key role in the Remote Sensing and Signaling System as does the interplay with the neuroendocrine, hormonal, and immune systems. Perturbation of function with OAT-interacting drugs (e.g., probenecid, diuretics, antivirals, antibiotics, NSAIDs) can lead to drug-metabolite interactions. The RSST has general applicability to other multi-specific SLC and ABC "drug" transporters (e.g., OCT1, OCT2, SLCO1B1, SLCO1B3, ABCG2, P-gp, ABCC2, ABCC3, ABCC4). Recent high-resolution structures of SLC22 and other transporters, together with chemoinformatic and artificial intelligence methods, will aid drug development and also lead to a deeper mechanistic understanding of polymorphisms.

The ability of different diffusing enhancers to deliver chlorhexidine into dentinal tubules: An in vitro evaluation.

Background/purpose: Root canal irrigants are difficult to diffuse deep into the dentinal tubules for root canal disinfection. The purpose of this study was to assess the ability of different diffusing enhancers to deliver chlorhexidine (CHX) into dentinal tubules. Materials and methods: The diffusing property of five diffusing enhancers (acetone, DMSO, Triton X-100, JFC-E and azone) into dentinal tubules was firstly assessed using the Rhodamine B. The ability of the diffusing enhancers to deliver CHX into dentinal tubules was then evaluated both qualitatively and quantitatively. A deep dentinal tubule Enterococcus faecalis infection model was further established to test the bactericidal effect of CHX delivered by different diffusing enhancers. Finally, the in vitro cytotoxicity of these diffusing enhancers was tested using the CCK-8 method. Results: Azone, Triton X-100 and JFC-E exhibited the largest maximum diffusing depths at all root canal parts (P < 0.05). Azone group also showed the highest percentage of diffusing depths for all root canal parts, followed by Triton X-100 (P < 0.05). The percentage of dead bacteria in dentinal tubules close to cementum layer was significantly higher in the CHX + azone group than in the other groups (P < 0.05), followed by CHX + Triton X-100 and CHX + JFC-E groups. The concentration of CHX diffusing onto the exterior surface of root was significantly higher in CHX + azone group than in the other groups (P < 0.05). All the diffusing enhancers showed relatively low cytotoxicity. Conclusion: Azone showed the highest diffusing ability with low cytotoxicity and might be employed as a carrier agent for either intracanal irrigants or medications to achieve more thorough root canal disinfection.

Biophysical Analysis of a Minimalistic Kidney Model Expressing SGLT1 Reveals Crosstalk between Luminal and Lateral Membranes and a Plausible Mechanism of Isosmotic Transport.

We extended our model of the S1 tubular segment to address the mechanisms by which SGLT1 interacts with lateral Na/K pumps and tight junctional complexes to generate isosmotic fluid reabsorption via tubular segment S3. The strategy applied allowed for simulation of laboratory experiments. Reproducing known experimental results constrained the range of acceptable model outputs and contributed to minimizing the free parameter space. (1) In experimental conditions, published Na and K concentrations of proximal kidney cells were found to deviate substantially from their normal physiological levels. Analysis of the mechanisms involved suggested insufficient oxygen supply as the cause and, indirectly, that a main function of the Na/H exchanger (NHE3) is to extrude protons stemming from mitochondrial energy metabolism. (2) The water path from the lumen to the peritubular space passed through aquaporins on the cell membrane and claudin-2 at paracellular tight junctions, with an additional contribution to water transport by the coupling of 1 glucose:2 Na:400 H2O in SGLT1. (3) A Na-uptake component passed through paracellular junctions via solvent drag in Na- and water-permeable claudin-2, thus bypassing the Na/K pump, in agreement with the findings of early studies. (4) Electrical crosstalk between apical rheogenic SGLT1 and lateral rheogenic Na/K pumps resulted in tight coupling of luminal glucose uptake and transepithelial water flow. (5) Isosmotic transport was achieved by Na-mediated ion recirculation at the peritubular membrane.

Regulation of ClC-K/barttin by endocytosis influences distal convoluted tubule hyperplasia.

ClC-K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC-Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline-tyrosine motif in the C-terminus of barttin stimulates ClC-K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co-expressing ClC-K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC-K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock-in mouse. Comparing wild-type (WT) and knock-in mice under a standard diet, we could not observe any difference in ClC-K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high-sodium low-potassium diet, known to induce hyperplasia of distal convoluted tubules, knock-in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. KEY POINTS: It is revealed by mutagenesis and functional experiments that a previously identified proline-tyrosine motif regulating ClC-K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC-K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock-in mice with a mutation in this motif, under conditions of a high-sodium low-potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild-type animals, indicating a gain of function of the channel in vivo.

Recurrent Fanconi Syndrome Induced by Lenalidomide: A Case Report.

Fanconi syndrome (FS) is a complex disorder characterized by a reabsorption defect in the proximal renal tubule (PT), leading to urinary loss of molecules such as glucose, phosphate, calcium, amino acids, bicarbonate, potassium, and low-molecular-weight proteins. Its etiology can be genetic or acquired, with drug toxicity being a significant cause of the acquired forms. The heterogeneous manifestations of FS, whether in its partial or complete form, can pose challenges in the emergency department; nevertheless, it should be considered in certain patients, as understanding its cause is crucial for initiating effective treatment. We present the case of a 59-year-old female patient with FS who was treated with lenalidomide in the context of stage III IgG kappa multiple myeloma according to the Salmon Durie classification. We highlight the recurrent nature of this syndrome in this patient.

Apical tubular complement activation and the loss of kidney function in proteinuric kidney diseases.

Many kidney diseases are associated with proteinuria. Since proteinuria is independently associated with kidney function loss, anti-proteinuric medication, often in combination with dietary salt restriction, comprises a major cornerstone in the prevention of progressive kidney failure. Nevertheless, complete remission of proteinuria is very difficult to achieve, and most patients with persistent proteinuria slowly progress toward kidney failure. It is well-recognized that proteinuria leads to kidney inflammation and fibrosis via various mechanisms. Among others, complement activation at the apical side of the proximal tubular epithelial cells is suggested to play a crucial role as a cause of progressive loss of kidney function. However, hitherto limited attention is given to the pathophysiological role of tubular complement activation relative to glomerular complement activation. This review aims to summarize the evidence for tubular epithelial complement activation in proteinuric kidney diseases in relation to loss of kidney function.

Hypokalemic Quadriparesis Associated With Renal Glycosuria in Dengue Fever: A Rare Presentation.

Dengue fever is a viral hemorrhagic fever mainly transmitted by Aedes mosquitoes and is especially prevalent in equatorial regions. The presentation of dengue fever can range from mild symptoms, such as fever and body aches, to severe symptoms, such as hemorrhagic bleeding and shock. Although it is a non-neurotropic virus, it rarely manifests as a neurological abnormality. Previous data suggests that the incidence of electrolyte disturbance is increasing in patients with dengue. Here, we have described a case of dengue fever with hypokalemia and renal glycosuria. Studies show that the probable mechanism of developing hypokalemia is increased insulin and catecholamine, but it is still not well-established. We propose a mechanism that can explain both hypokalemia and renal glycosuria in our case.

BK channels promote action potential repolarization in skeletal muscle but contribute little to myotonia.

Patients with myotonia congenita suffer from slowed relaxation of muscle (myotonia), due to hyperexcitability caused by loss-of-function mutations in the ClC-1 chloride channel. A recent study suggested that block of large-conductance voltage- and Ca2+- activated K+ channels (BK) may be effective as therapy. The mechanism underlying efficacy was suggested to be lessening of the depolarizing effect of build-up of K+ in t-tubules of muscle during repetitive firing. BK channels are widely expressed in the nervous system and have been shown to play a central role in regulation of excitability, but their contribution to muscle excitability has not been determined. We performed intracellular recordings as well as force measurements in both wild type and BK-/- mouse extensor digitorum longus muscles. Action potential width was increased in BK-/- muscle due to slowing of repolarization, consistent with the possibility K+ build-up in t-tubules is lessened by block of BK channels in myotonic muscle. However, there was no difference in the severity of myotonia triggered by block of muscle Cl- channels with 9-anthracenecarboxylic acid (9AC) in wild type and BK-/- muscle fibers. Further study revealed no difference in the interspike membrane potential during repetitive firing suggesting there was no reduction in K+ build-up in t-tubules of BK-/- muscle. Force recordings following block of muscle Cl- channels demonstrated little reduction in myotonia in BK-/- muscle. In contrast, the current standard of care, mexiletine, significantly reduced myotonia. Our data suggest BK channels regulate muscle excitability, but are not an attractive target for therapy of myotonia.

Distal convoluted tubule-specific disruption of the COP9 signalosome but not its regulatory target Cullin 3 causes tubular injury.

The disease Familial Hyperkalemic Hypertension (FHHt; also known as Gordon Syndrome) is caused by aberrant accumulation of WNK4 activating the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Mutations in cullin 3 (CUL3) cause FHHt by disrupting interaction with the deneddylase COP9 signalosome (CSN). Deletion of Cul3 or Jab1 (the catalytically active CSN subunit) along the entire nephron causes a partial FHHt phenotype with activation of the WNK4-SPAK-NCC pathway. However, progressive kidney injury likely prevents hypertension, hyperkalemia, and hyperchloremic metabolic acidosis associated with FHHt. We hypothesized that DCT-specific deletion would more closely model the disease. We utilized Slc12a3-Cre-ERT2 mice to delete Cul3 (DCT-Cul3-/-) or Jab1 (DCT-Jab1-/-) only in DCT and examined the mice after short- and long-term deletion. Short-term, DCT-specific knockout of both Cul3 and Jab1 mice caused elevated WNK4, SPAK, and pNCC abundance. However, neither model demonstrated changes in plasma K+, Cl-, or TCO2, even though no injury was present. Long-term DCT-Jab1-/- mice showed significantly lower NCC and parvalbumin abundance, and higher abundance of kidney injury molecule 1 (KIM-1), a marker of proximal tubule injury. No injury, or reduction in NCC or parvalbumin were observed in long-term DCT-Cul3-/- mice. In summary, the prevention of injury outside the DCT did not lead to a complete FHHt phenotype despite activation of the WNK4-SPAK-NCC pathway, possibly due to insufficient NCC activation. Chronically, only DCT-Jab1-/- mice developed tubule injury and atrophy of the DCT, suggesting a direct JAB1 effect or dysregulation of other cullins as mechanisms for injury.

Genetic deletion of the kidney sodium/proton exchanger-3 (NHE3) does not alter calcium and phosphate balance due to compensatory responses.

The sodium/proton exchanger-3 (NHE3) plays a major role in acid-base and extracellular volume regulation and is also implicated in calcium homeostasis. As calcium and phosphate balances are closely linked, we hypothesized that there was a functional link between kidney NHE3 activity, calcium, and phosphate balance. Therefore, we examined calcium and phosphate homeostasis in kidney tubule-specific NHE3 knockout mice (NHE3loxloxPax8 mice). Compared to controls, these knockout mice were normocalcemic with no significant difference in urinary calcium excretion or parathyroid hormone levels. Thiazide-induced hypocalciuria was less pronounced in the knockout mice, in line with impaired proximal tubule calcium transport. Knockout mice had greater furosemide-induced calciuresis and distal tubule calcium transport pathways were enhanced. Despite lower levels of the sodium/phosphate cotransporters (NaPi)-2a and -2c, knockout mice had normal plasma phosphate, sodium-dependent 32Phosphate uptake in proximal tubule membrane vesicles and urinary phosphate excretion. Intestinal phosphate uptake was unchanged. Low dietary phosphate reduced parathyroid hormone levels and increased NaPi-2a and -2c abundances in both genotypes, but NaPi-2c levels remained lower in the knockout mice. Gene expression profiling suggested proximal tubule remodeling in the knockout mice. Acutely, indirect NHE3 inhibition using the SGLT2 inhibitor empagliflozin did not affect urinary calcium and phosphate excretion. No differences in femoral bone density or architecture were detectable in the knockout mice. Thus, a role for kidney NHE3 in calcium homeostasis can be unraveled by diuretics, but NHE3 deletion in the kidneys has no major effects on overall calcium and phosphate homeostasis due, at least in part, to compensating mechanisms.

HO-1 activation contributes to cadmium-induced ferroptosis in renal tubular epithelial cells via increasing the labile iron pool and promoting mitochondrial ROS generation.

Cadmium (Cd), a prevalent environmental contaminant, has attracted widespread attention due to its serious health hazards. Ferroptosis is a form of iron-dependent oxidative cell death that contributes to the development of various kidney diseases. However, the mechanisms underlying the occurrence of ferroptosis in Cd-induced renal tubular epithelial cells (TECs) have not been fully elucidated. Hereby, both in-vitro and in-vivo experiments were established to elucidate this issue. In this study, we found that Cd elicited accumulation of lipid peroxides due to intracellular ferrous ion (Fe2+) overload and glutathione depletion, contributing to ferroptosis. Inhibition of ferroptosis via chelation of Fe2+ or reduction of lipid peroxidation can significantly mitigate Cd-induced cytotoxicity. Renal transcriptome analysis revealed that the activation of heme oxygenase 1 (HO-1) was closely related to ferroptosis in Cd-induced TECs injury. Cd-induced ferroptosis and resultant TECs injury are significantly alleviated due to HO-1 inhibition, demonstrating the crucial role of HO-1 in Cd-triggered ferroptosis. Further studies showed that accumulation of lipid peroxides due to iron overload and mitochondrial ROS (mtROS) generation was responsible for HO-1-triggered ferroptosis in Cd-induced cytotoxicity. In conclusion, the current study demonstrates that excessively upregulating HO-1 promotes iron overload and mtROS overproduction to trigger ferroptosis in Cd-induced TECs injury, highlighting that targeting HO-1-mediated ferroptosis may provide new ideas for preventing Cd-induced nephrotoxicity.

Estimated Proximal Tubule Fluid Phosphate Concentration and Renal Tubular Damage Biomarkers in Early Stages of Chronic Kidney Disease.

OBJECTIVE: An increase in proximal tubule fluid phosphate concentration is caused by increased serum fibroblast growth factor-23 (FGF23) levels, which resulted in renal tubular damage in a mouse model of chronic kidney disease (CKD). However, few human studies have supported this concept. This study aimed to explore the association among estimated proximal tubule fluid phosphate concentration (ePTFp), serum FGF23 levels, and renal tubular damage biomarkers in middle-aged and older populations with mild decline in renal function. METHODS: This cross-sectional study included 218 participants aged ≥45 with CKD stages G2-G4. Anthropometric measurements, blood tests, spot urine biomarkers, renal ultrasonography, cardiovascular assessment, smoking status, and medication usage were obtained in the morning in fasted states. The ePTFp was calculated using serum creatinine, urine phosphate, and creatinine concentrations. Urinary ß2-microglobulin (ß2-MG) and liver-type fatty acid-binding protein (L-FABP) levels were evaluated to assess renal tubular damage. RESULTS: PTFp, serum FGF23, urinary ß2-MG, and urinary L-FABP levels increased with CKD stage progression (stages G2, G3, and G4). However, serum and urine phosphate concentrations were comparable across the CKD stages. Univariate analysis revealed a stronger correlation of ePTFp with serum FGF23, urinary ß2-MG, and urinary L-FABP levels than with the corresponding serum and urine phosphate concentrations. Multivariate analyses demonstrated that increased ePTFp was independently associated with elevated serum FGF23 and urinary ß2-MG levels, even after adjusting for potential covariates, including the estimated glomerular filtration rate and urinary albumin-to-creatinine ratio. CONCLUSIONS: Our results are consistent with the concept in mouse model and suggest that increased ePTFp are associated with increased serum FGF23 levels and renal tubular damage during the early stages of CKD.

Drug Screening Using Normal Cell and Cancer Cell Mixture in an Automated 3D Cell Culture System.

Three-dimensional (3D) cell culture creates a more physiologically relevant environment for enhanced drug screening capabilities using microcarriers. An automated 3D system that integrates robotic manipulators, liquid handling systems, sensors, and environment control systems has the capacity to handle multiple samples in parallel, perform repetitive tasks, and provide real-time monitoring and analysis. This chapter describes a potential 3D cell culture drug screening model by combining renal proximal tubule cells as a representative normal cell line with cancer cell lines. This combination is subjected to drug screening to evaluate the drug's efficacy in suppressing cancer cells while minimizing impact on normal cells with the added benefit of having the ability to separate the two cell types by magnetic isolation for high content screens including mass spectrometry-based proteomics. This study presents advancements in 3D cell culture techniques, emphasizing the importance of automation and the potential of microcarriers in drug screening and disease modeling.

Biology of the proximal tubule in body homeostasis and kidney disease.

The proximal tubule (PT) is known as the workhorse of the kidney, both for the range and magnitude of the functions that it performs. It is not only responsible for reabsorbing most solutes and proteins filtered by glomeruli, but also for secreting non-filtered substances including drugs and uremic toxins. The PT therefore plays a pivotal role in kidney physiology and body homeostasis. Moreover, it is the major site of damage in acute kidney injury and nephrotoxicity. In this review, we will provide an introduction to the cell biology of the PT and explore how it is adapted to the execution of a myriad of different functions and how these can differ between males and females. We will then discuss how the PT regulates phosphate, glucose and acid-base balance, and the consequences of alterations in PT function for bone and cardiovascular health. Finally, we explore why the PT is vulnerable to ischemic and toxic insults, and how acute injury in the PT can lead to maladaptive repair, chronic damage, and kidney fibrosis. In summary, we will demonstrate that knowledge of the basic cell biology of the PT is critical for understanding kidney disease phenotypes and their associated systemic complications, and for developing new therapeutic strategies to prevent these.

Megalin Knockout Reduces SGLT2 Expression and Sensitizes to Western Diet-induced Kidney Injury.

Megalin (Lrp2) is a multiligand receptor that drives endocytic flux in the kidney proximal tubule (PT) and is necessary for the recovery of albumin and other filtered proteins that escape the glomerular filtration barrier. Studies in our lab have shown that knockout (KO) of Lrp2 in opossum PT cells leads to a dramatic reduction in sodium-glucose co-transporter 2 (SGLT2) transcript and protein levels, as well as differential expression of genes involved in mitochondrial and metabolic function. SGLT2 transcript levels are reduced more modestly in Lrp2 KO mice. Here, we investigated the effects of Lrp2 KO on kidney function and health in mice fed regular chow (RC) or a Western-style diet (WD) high in fat and refined sugar. Despite a modest reduction in SGLT2 expression, Lrp2 KO mice on either diet showed increased glucose tolerance compared to control mice. Moreover, Lrp2 KO mice were protected against WD-induced fat gain. Surprisingly, renal function in male Lrp2 KO mice on WD was compromised, and the mice exhibited significant kidney injury compared with control mice on WD. Female Lrp2 KO mice were less susceptible to WD-induced kidney injury than male Lrp2 KO. Together, our findings reveal both positive and negative contributions of megalin expression to metabolic health, and highlight a megalin-mediated sex-dependent response to injury following WD.