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| Effect of chronic loop diuretic administration on DCT activity and morphology. (Upper panel) Sodium reabsorption normally mediated by the thick ascending limb Na-K-2Cl cotransporter (NKCC2) is blocked by loop diuretics, such as furosemide and bumetanide. This results in enhanced Na 1 delivery to the DCT, which likely acts as a stimulus for DCT hypertrophy. (Lower panel) DCT hypertrophy manifests as an increase in mitochondrial size and basolateral membrane infoldings. The increase in NCC-mediated apical sodium transport coupled with enhanced activity of the sodium/potassium pump at the basolateral membrane results in a vectorial increase in sodium reabsorption and diuretic resistance.
Source publication
The distal convoluted tubule is the nephron segment that lies immediately downstream of the macula densa. Although short in length, the distal convoluted tubule plays a critical role in sodium, potassium, and divalent cation homeostasis. Recent genetic and physiologic studies have greatly expanded our understanding of how the distal convoluted tubu...
Contexts in source publication
Context 1
... most common clinical scenario in which this phenom- enon is encountered is loop diuretic resistance (Figure 4) (37). Chronic treatment with diuretics that inhibit the Na-K-2Cl cotransporter in the loop of Henle (bumetanide-sensitive sodium-potassium-chloride cotransporter [NKCC2, SLC12A1]), such as furosemide, results in a sustained increase in luminal Na 1 delivery to the DCT. ...
Context 2
... WNK4 mutants can phosphorylate SPAK and OSR1 (50), but many of them cannot block NCC plasma membrane trafficking (51,52). Thus, increased mutant WNK4 protein expression stimulates NCC phosphorylation and traffick- ing to the cell surface, resulting in NCC overactivation ( Figure 6B) (44,46). Like WNK4, the kinase active form of WNK1 phosphor- ylates and activates SPAK and OSR1 (53). ...
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Citations
... ATC was proven to be compatible with fluorescent reporters in zebrafish, murine, and 3D stem cell models. The advantageous properties of ATC readily facilitated 3D imaging of the cardiac conduction system of the murine heart 11 , as well as the gross intact structure of adjacent renal connecting and distal convoluted tubule nephron segments that had previously remained undocumented 12,13 . Moreover, ATC enabled multiplex 3D imaging of distinct glomerular tissue layers in human kidney biopsies, with cellular resolution 14 . ...
... Thus, we used ATC to image parvalbumin and ENAC that mark adjacent distal convoluted and connecting tubule nephron segments, respectively 12,13 . As shown in Fig. 3 and Supplemental Movie 2, these tissue regions were distinctly visualized. ...
3D reconstructive imaging is a powerful strategy to interrogate the global architecture of tissues. We developed Atacama Clear (ATC), a novel method that increases 3D imaging signal-to-noise ratios (SNRs) while simultaneously increasing the capacity of tissue to be cleared. ATC potentiated the clearing capacity of all tested chemical reagents currently used for optical clearing by an average of 68%, and more than doubled SNRs. This increased imaging efficacy enabled multiplex interrogation of tough fibrous tissue and specimens that naturally exhibit high levels of background noise, including the heart, kidney, and human biopsies. Indeed, ATC facilitated visualization of previously undocumented adjacent nephron segments that exhibit notoriously high autofluorescence, elements of the cardiac conduction system, and the distinct human glomerular tissue layers, at single cell resolution. Moreover, ATC was validated to be compatible with fluorescent reporter proteins in murine, zebrafish, and 3D stem cell model systems. These data establish ATC for 3D imaging studies of challenging tissue types.
... Upon sensing of the extracellular potassium concentration, the channel changes the membrane potential and adjusts the intracellular chloride concentration [12,13]. Furthermore, Kir4.1/Kir5.1 channels showed to be involved in sodium reabsorption and potassium secretion in the CD, where sodium reabsorption is facilitated by the epithelial Na + channel (ENaC) [14] and potassium secretion is facilitated by the outer medullary potassium channel (ROMK) [15], which is considered one of the main routes of potassium secretion in the kidney. ...
Background
the KCNJ16 gene has been associated with a novel kidney tubulopathy phenotype, viz. disturbed acid-base homeostasis, hypokalemia and altered renal salt transport. KCNJ16 encodes for Kir5.1, which together with Kir4.1 constitutes a potassium channel located at kidney tubular cell basolateral membranes. Preclinical studies provided mechanistical links between Kir5.1 and a disease phenotype, however, the disease pathology remains poorly understood. Here, we aimed at generating and characterizing a novel advanced in vitro human kidney model that recapitulates the disease phenotype to investigate further the pathophysiological mechanisms underlying the disease and potential therapeutic interventions.
Methods
we used CRISPR/Cas9 to generate KCNJ16 mutant ( KCNJ16 +/- and KCNJ16 -/- ) cell lines from healthy human induced pluripotent stem cells (iPSC) KCNJ16 control ( KCNJ16 WT ) . The iPSCs were differentiated following an optimized protocol into kidney organoids in an air-liquid interface.
Results
KCNJ16 -depleted kidney organoids showed transcriptomic and potential functional impairment of key voltage-dependent electrolyte and water-balance transporters. We observed cysts formation, lipid droplet accumulation and fibrosis upon Kir5.1 function loss. Furthermore, a large scale, glutamine tracer flux metabolomics analysis demonstrated that KCNJ16 -/- organoids display TCA cycle and lipid metabolism impairments. Drug screening revealed that treatment with statins, particularly the combination of simvastatin and C75, prevented lipid droplet accumulation and collagen-I deposition in KCNJ16 -/- kidney organoids.
Conclusions
mature kidney organoids represent a relevant in vitro model for investigating the function of Kir5.1. We discovered novel molecular targets for this genetic tubulopathy and identified statins as a potential therapeutic strategy for KCNJ16 defects in the kidney.
Significance Statement
In this study, the use of CRISPR/Cas9 technology resulted in the establishment of a KCNJ16- depleted kidney organoid model, instrumental in elucidating the pathophysiology of the recently reported KCNJ16 -associated kidney tubulopathy. Our study substantiates the role of Kir5.1 (KCNJ16) in kidney disease, confirming already described phenotypes, as well as aiding to gain insight in the causal role of Kir5.1 loss in the disease phenotype. Our approach increases the knowledge on KCNJ16 -related kidney phenotype, and it states the importance of combining CRISPR/Cas9 technology and advanced in vitro models for complex disease modeling and therapy testing. Furthermore, we encourage the application of our approach to the in vitro modeling of rare and/or underrepresented genetic kidney diseases, for which the availability of patient material is limited.
... At its distal end, a group of tightly packed cells (the macula densa) forms the connection of the TAL with the DCT [20], the shortest segment of the nephron. The DCT, which can be subdivided into a proximal DCT1 and a distal DCT2, plays an important role in the homeostasis of potassium, sodium, magnesium, and calcium [30,34]. A key marker of the entire DCT is the thiazide-sensitive Na-Cl cotransporter NCC (encoded by SLC12A3). ...
The kidney is a highly complex organ equipped with a multitude of miniscule filter-tubule units called nephrons. Each nephron can be subdivided into multiple segments, each with its own morphology and physiological function. To date, conventional manual approaches to isolate specific nephron segments are very laborious, time-consuming, often limited to only a specific segment, and typically have low yield. Here, we describe a novel, unconventional method that is superior in many aspects to previous protocols by combining low-cost fluorophore-conjugated lectins or agglutinins (Flaggs) with flow sorting. This allows the simultaneous separation of different nephron segments with preserved 3D morphology from mouse or human samples in under 3 h. Using a 200-µm nozzle and 5 psi, glomeruli, proximal, or distal convoluted tubules are sorted with Cy3-labeled Sambucus Nigra agglutinin (SNA-Cy3), Fluorescein-labeled Lotus Tetragonolobus lectin (LTL-FITC), or Pacific Blue-labeled soybean agglutinin (SBA-PB), respectively. Connecting tubules and collecting ducts are sorted by double-positive SBA-PB and SNA-Cy3 signals, while thick ascending limb segments are characterized by the absence of any Flaggs labeling. From two mouse kidneys, this yields 37–521 ng protein/s or 0.71–16.71 ng RNA/s, depending on the specific nephron segment. The purity of sorted segments, as assessed by mRNA expression level profiling of 15 genes, is very high with a 96.1-fold median enrichment across all genes and sorted segments. In summary, our method represents a simple, straightforward, cost-effective, and widely applicable tool yielding high amounts of pure and morphologically largely intact renal tubule materials with the potential to propel nephron segment-specific research.
... MDCK cells, derived from the kidney, specifically from the distal convoluted tubule [91], which play a role in potassium reabsorption [92]. TRPV4 channels, along with TRPP2 channels, have been reported to be involved in this process [93,94]. ...
Ouabain, a substance originally obtained from plants, is now classified as a hormone because it is produced endogenously in certain animals, including humans. However, its precise effects on the body remain largely unknown. Previous studies have shown that ouabain can influence the phenotype of epithelial cells by affecting the expression of cell–cell molecular components and voltage-gated potassium channels. In this study, we conducted whole-cell clamp assays to determine whether ouabain affects the activity and/or expression of TRPV4 channels. Our findings indicate that ouabain has a statistically significant effect on the density of TRPV4 currents (dITRPV4), with an EC50 of 1.89 nM. Regarding treatment duration, dITRPV4 reaches its peak at around 1 h, followed by a subsequent decline and then a resurgence after 6 h, suggesting a short-term modulatory effect related to on TRPV4 channel activity and a long-term effect related to the promotion of synthesis of new TRPV4 channel units. The enhancement of dITRPV4 induced by ouabain was significantly lower in cells seeded at low density than in cells in a confluent monolayer, indicating that the action of ouabain depends on intercellular contacts. Furthermore, the fact that U73122 and neomycin suppress the effect caused by ouabain in the short term suggests that the short-term induced enhancement of dITRPV4 is due to the depletion of PIP2 stores. In contrast, the fact that the long-term effect is inhibited by PP2, wortmannin, PD, FR18, and IKK16 suggests that cSrc, PI3K, Erk1/2, and NF-kB are among the components included in the signaling pathways.
... (Changes may still occur before final publication.) October 17, 2023 13:44 In mammals, one example epithelium that likely leverages crowding-dependent WNK signaling and phase behavior to activate transcellular ion transport is the kidney's distal convoluted tubule (DCT) (104). WNK kinases are highly active in this nephron segment, as they drive the phosphorylation-dependent activation of NCC (SLC12A3), a sodium chloride cotransporter closely related to NKCC1. ...
The cytoplasm is densely packed with molecules that contribute to its nonideal behavior. Cytosolic crowding influences chemical reaction rates, intracellular water mobility, and macromolecular complex formation. Overcrowding is potentially catastrophic; to counteract this problem, cells have evolved acute and chronic homeostatic mechanisms that optimize cellular crowdedness. Here, we provide a physiology-focused overview of molecular crowding, highlighting contemporary advances in our understanding of its sensing and control. Long hypothesized as a form of crowding-induced microcompartmentation, phase separation allows cells to detect and respond to intracellular crowding through the action of biomolecular condensates, as indicated by recent studies. Growing evidence indicates that crowding is closely tied to cell size and fluid volume, homeostatic responses to physical compression and desiccation, tissue architecture, circadian rhythm, aging, transepithelial transport, and total body electrolyte and water balance. Thus, molecular crowding is a fundamental physiologic parameter that impacts diverse functions extending from molecule to organism.
Expected final online publication date for the Annual Review of Physiology, Volume 86 is February 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Several studies have reported that chemotherapeutic agents can affect various renal components, including the glomerulus, tubules, interstitium, or the renal microvasculature. Clinical manifestations can range from asymptomatic elevation of serum creatinine to acute renal failure necessitating dialysis [53]. As adverse renal effects often occur due to drug toxicity, probiotics and their secondary metabolites present a promising alternative to chemotherapy. ...
... WNK1 is a serine-threonine kinase, it can regulate various ion channels, which participate in the sodium and chloride transport in the kidney (Náray-F et al., 2004;Cope et al., 2006). NCC is a critical sodium reabsorption channel located in the DCT, and it is a key regulator resulting in volume expansion and hypertension when it is hyperactive (Simon et al., 1996;Subramanya et al., 2014). The reabsorption of NCC must be switched on with direct phosphorylation by Ste20-like prolinealanine rich kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1), phosphorylated and activated by a family of proteins Frontiers in Genetics frontiersin.org ...
Background: Essential hypertension (EH) is a complex disorder resulting from interaction of genetic and environmental factors. Lysine deficient protein kinase 1 (WNK1) plays a very important role in maintaining renal potassium, sodium and chlorine ions balance as well as the regulation of blood pressure, so the WNK1 gene is considered a key gene for EH. This study thus sought to evaluate possible genetic associations between the WNK1 genetic variants and EH risk in the Northern Han Chinese population in Beijing.
Methods: This study included 476 hypertensive subjects and 491 normotensive subjects. A total of 12 tag SNVs of WNK1 gene were genotyped successfully by TaqMan assay. Comparisons of the genotypic and allelic frequency between cases and controls were made by using the chi-square test. Logistic regression analyses were performed under different genetic models, and haplotype analysis was also conducted.
Results: A total of 12 SNVs were identified as the tag SNVs for WNK1 gene. Significant associations were observed between WNK1 gene rs7305099 variant and EH risk, and T allele influenced hypertension risk in a protective manner. After correcting for multiple testing using Bonferroni, the significance remained for the SNV of rs7305099 in three genetic models [allele comparison, p < 0.0002, OR = 0.627, 95%CI (0.491–0.801); homozygote comparison, p < 0.0003, OR = 0.278, 95%CI (0.140–0.552); additive model, p < 0.0003, OR = 0.279, 95%CI (0.140–0.553)]. In the haplotype analyses, we found that the haplotype A-A-A-C-G-G-G was significantly associated with increased risk for EH (p = 0.043, OR = 1.23).
Conclusion: Our data suggested that the rs7305099 genetic variant and the haplotype A-A-A-C-G-G-G on WNK1 gene might be associated with the susceptibility of EH in the Northern Han Chinese population. These could provide evidences to the risk assessment, early prevention and individualized therapy of EH to some extent.
... As previously mentioned, the kidney is a mitochondria-rich organ (Bhargava and Schnellmann, 2017), but the distribution of these organelles varies across different segments of the nephron. Studies have documented that the proximal and distal tubules have a high mitochondrial density (Subramanya and Ellison, 2014;McCormick and Ellison, 2015;Molitoris et al., 2022). In contrast, podocytes exhibit notably lower mitochondrial density (Brinkkoetter et al., 2019), a trend also observed in the collecting ducts (Pfaller and Rittinger, 1980;Ahmad et al., 2021). ...
Acute Kidney Injury (AKI), a critical clinical syndrome, has been strongly linked to mitochondrial malfunction. Mitochondria, vital cellular organelles, play a key role in regulating cellular energy metabolism and ensuring cell survival. Impaired mitochondrial function in AKI leads to decreased energy generation, elevated oxidative stress, and the initiation of inflammatory cascades, resulting in renal tissue damage and functional impairment. Therefore, mitochondria have gained significant research attention as a potential therapeutic target for AKI. Mitochondrial dynamics, which encompass the adaptive shifts of mitochondria within cellular environments, exert significant influence on mitochondrial function. Modulating these dynamics, such as promoting mitochondrial fusion and inhibiting mitochondrial division, offers opportunities to mitigate renal injury in AKI. Consequently, elucidating the mechanisms underlying mitochondrial dynamics has gained considerable importance, providing valuable insights into mitochondrial regulation and facilitating the development of innovative therapeutic approaches for AKI. This comprehensive review aims to highlight the latest advancements in mitochondrial dynamics research, provide an exhaustive analysis of existing studies investigating the relationship between mitochondrial dynamics and acute injury, and shed light on their implications for AKI. The ultimate goal is to advance the development of more effective therapeutic interventions for managing AKI.
... In the renal collecting ducts, the CaSR prevents the development of hypercalciuria-mediated nephrocalcinosis by increasing urinary acidification and water excretion [60]. The CaSR detects increases in tubular fluid Ca 2þ in the DCT, which might increase Ca 2þ reabsorption via the apical TRPV5 channel [61]. ...
Purpose of review:
Regulation of the sodium chloride cotransporter (NCC) in the distal convoluted tubule (DCT) plays a crucial role in renal salt handling. The calcium-sensing receptor (CaSR) has been shown to activate NCC through the WNK4-SPAK pathway, which is independent of the Renin-Angiotensin-Aldosterone system. In this review, we examine new information about the mechanism of how the CaSR regulates NCC through the WNK4-SPAK pathway and its physiological and therapeutic implications.
Recent findings:
The activation of CaSR in TALH cells during hypercalcemia inhibits NKCC2 and ROMK activity, reducing paracellular Ca2+ reabsorption but decreasing salt reabsorption. This pathway enables NaCl reabsorption in the DCT while promoting Ca2+ excretion. CaSR activation in the apical DCT stimulates a signaling pathway involving PKC, WNK4, and SPAK, which increases NCC activation to recover the NaCl not reabsorbed in TAHL. Glucose or fructose acting as calcimimetics enhance apical CaSR sensitivity, increasing NCC activity, which contribute to the mechanism of hypertension prevalence in diabetic patients or in those with high fructose consumption.
Summary:
These findings reveal the importance of the CaSR-mediated activation of the WNK4-SPAK pathway in regulating salt and calcium homeostasis and its potential as a therapeutic target for hypertension and related diseases.
... These kinases are activated by upstream enzymes including SGK1 (glucocorticoid regulated kinase 1) and WNKs (with no lysine kinases) in response to distinct mediators such as aldosterone and angiotensin II. 2 Upon activation by SGK1 and WNKs, SPAK and OSR1 can phosphorylate serine and threonine residues in the cytoplasmic domain of the NCC, which allow NaCl absorption from the lumen of the distal convoluted tubule. Fan et al. 1 showed that critical residues for phosphorylation are located at the interaction region of the regulatory cytosolic NTD and CTD. ...
