MoErv14 mediates the intracellular transport of cell membrane receptors to govern the appressorial formation and pathogenicity of Magnaporthe oryzae.

Magnaporthe oryzae causes rice blasts posing serious threats to food security worldwide. During infection, M. oryzae utilizes several transmembrane receptor proteins that sense cell surface cues to induce highly specialized infectious structures called appressoria. However, little is known about the mechanisms of intracellular receptor tracking and their function. Here, we described that disrupting the coat protein complex II (COPII) cargo protein MoErv14 severely affects appressorium formation and pathogenicity as the ΔMoerv14 mutant is defective not only in cAMP production but also in the phosphorylation of the mitogen-activated protein kinase (MAPK) MoPmk1. Studies also showed that either externally supplementing cAMP or maintaining MoPmk1 phosphorylation suppresses the observed defects in the ΔMoerv14 strain. Importantly, MoErv14 is found to regulate the transport of MoPth11, a membrane receptor functioning upstream of G-protein/cAMP signaling, and MoWish and MoSho1 function upstream of the Pmk1-MAPK pathway. In summary, our studies elucidate the mechanism by which the COPII protein MoErv14 plays an important function in regulating the transport of receptors involved in the appressorium formation and virulence of the blast fungus.

Enriched Single-Nucleus RNA-Sequencing Reveals Unique Attributes of Distal Convoluted Tubule Cells.

SIGNIFICANCE STATEMENT: High-resolution single-nucleus RNA-sequencing data indicate a clear separation between primary sites of calcium and magnesium handling within distal convoluted tubule (DCT). Both DCT1 and DCT2 express Slc12a3, but these subsegments serve distinctive functions, with more abundant magnesium-handling genes along DCT1 and more calcium-handling genes along DCT2. The data also provide insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand. BACKGROUND: The distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. METHODS: To understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining "Isolation of Nuclei Tagged in specific Cell Types" and sodium chloride cotransporter-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting and performed single-nucleus transcriptomics. RESULTS: Among 25,183 DCT cells, 75% were from DCT1 and 25% were from DCT2. In addition, there was a small population (<1%) enriched in proliferation-related genes, such as Top2a , Cenpp , and Mki67 . Although both DCT1 and DCT2 expressed sodium chloride cotransporter, magnesium transport genes were predominantly expressed along DCT1, whereas calcium, electrogenic sodium, and potassium transport genes were more abundant along DCT2. The transition between these two segments was gradual, with a transitional zone in which DCT1 and DCT2 cells were interspersed. The expression of the homeobox genes by DCT cells suggests that they develop along different trajectories. CONCLUSIONS: Transcriptomic analysis of an enriched rare cell population using a genetically targeted approach clarifies the function and classification of distal cells. The DCT segment is short, can be separated into two subsegments that serve distinct functions, and is speculated to derive from different origins during development.

Dysregulation of the WNK4-SPAK/OSR1 pathway has a minor effect on baseline NKCC2 phosphorylation.

The with-no-lysine kinase 4 (WNK4)-sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway mediates activating phosphorylation of the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and the thiazide-sensitive NaCl cotransporter (NCC). The commonly used pT96/pT101-pNKCC2 antibody cross-reacts with pT53-NCC in mice on the C57BL/6 background due to a five amino acid deletion. We generated a new C57BL/6-specific pNKCC2 antibody (anti-pT96-NKCC2) and tested the hypothesis that the WNK4-SPAK/OSR1 pathway strongly regulates the phosphorylation of NCC but not NKCC2. In C57BL/6 mice, anti-pT96-NKCC2 detected pNKCC2 and did not cross-react with NCC. Abundances of pT96-NKCC2 and pT53-NCC were evaluated in Wnk4-/-, Osr1-/-, Spak-/-, and Osr1-/-/Spak-/- mice and in several models of the disease familial hyperkalemic hypertension (FHHt) in which the CUL3-KLHL3 ubiquitin ligase complex that promotes WNK4 degradation is dysregulated (Cul3+/-/Δ9, Klhl3-/-, and Klhl3R528H/R528H). All mice were on the C57BL/6 background. In Wnk4-/- mice, pT53-NCC was almost absent but pT96-NKCC2 was only slightly lower. pT53-NCC was almost absent in Spak-/- and Osr1-/-/Spak-/- mice, but pT96-NKCC2 abundance did not differ from controls. pT96-NKCC2/total NKCC2 was slightly lower in Osr1-/- and Osr1-/-/Spak-/- mice. WNK4 expression colocalized not only with NCC but also with NKCC2 in Klhl3-/- mice, but pT96-NKCC2 abundance was unchanged. Consistent with this, furosemide-induced urinary Na+ excretion following thiazide treatment was similar between Klhl3-/- and controls. pT96-NKCC2 abundance was also unchanged in the other FHHt mouse models. Our data show that disruption of the WNK4-SPAK/OSR1 pathway only mildly affects NKCC2 phosphorylation, suggesting a role for other kinases in NKCC2 activation. In FHHt models NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide sensitivity of FHHt.NEW & NOTEWORTHY The renal cation cotransporters NCC and NKCC2 are activated following phosphorylation mediated by the WNK4-SPAK/OSR1 pathway. While disruption of this pathway strongly affects NCC activity, effects on NKCC2 activity are unclear since the commonly used phospho-NKCC2 antibody was recently reported to cross-react with phospho-NCC in mice on the C57BL/6 background. Using a new phospho-NKCC2 antibody specific for C57BL/6, we show that inhibition or activation of the WNK4-SPAK/OSR1 pathway in mice only mildly affects NKCC2 phosphorylation.

Prognostic factors and outcomes in anti-neutrophil cytoplasmic antibody-associated glomerulonephritis: A retrospective single-center study from southern China.

Objective: To investigate the prognostic factors and outcomes in patients with anti-neutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN) in Southern China. Methods: A retrospective analysis of medical records of patients with ANCA-GN admitted to Shenzhen Hospital of Southern Medical University and Nanfang Hospital of Southern Medical University between September 2011 and September 2021 was performed. The clinical presentation, biological, and renal pathology were collected. In addition, the risk factors for end-stage renal disease (ESRD) and short-term overall survival in patients with ANCA-GN were analyzed. Results: A total of 93 patients with ANCA-GN were included in the study. Of them, 91.4%, were perinuclear anti-neutrophil cytoplasmic antibodies (MPO-pANCA)-positive. Approximately one-quarter (24.7%) of patients had progressed to ESRD, and 7.5% died within six months. Most patients presented with hematuria (94.6%), proteinuria (78.5%), elevated serum creatinine (86.0%), anemia (90.3%), and increased erythrocyte sedimentation rate (ESR) (44.1%). The majority (94.6%) of patients presented with crescent formations at histopathological examination. Serum creatinine, hemoglobin, and Birmingham vasculitis activity score (BVAS) were all independent factors for ESRD (P<0.05). Moreover, while ANCA renal risk score (ARRS) has an impact on prognosis of nephropathy, it did not influence ESRD independently (P>0.05). The effect of Berden's histopathologic classification on ESRD has not been confirmed. Age at onset, ESR and cardiovascular involvement were all independent factors affecting short-term overall survival of patients with ANCA-GN (P<0.05). Conclusions: Serum creatinine, hemoglobin, and BVAS were all independent risk factors of ESRD, while ARRS and Berden's histopathologic classification were not. Age at onset, ESR, and cardiovascular involvement were independent risk factors for the overall six-month survival rate in patients with ANCA-GN.

Combined Kelch-like 3 and Cullin 3 Degradation is a Central Mechanism in Familial Hyperkalemic Hypertension in Mice.

BACKGROUND: Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) gene cause the disease familial hyperkalemic hypertension (FHHt). In the kidney, mutant CUL3 (CUL3-Δ9) increases abundance of With-No-Lysine (K) Kinase 4 (WNK4), inappropriately activating sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK), which then phosphorylates and hyperactivates the Na+Cl- cotransporter (NCC). The precise mechanism by which CUL3-Δ9 causes FHHt is unclear. We tested the hypothesis that reduced abundance of CUL3 and of Kelch-like 3 (KLHL3), the CUL3 substrate adaptor for WNK4, is mechanistically important. Because JAB1, an enzyme that inhibits CUL3 activity by removing the ubiquitin-like protein NEDD8, cannot interact with CUL3-Δ9, we also determined whether Jab1 disruption mimicked the effects of CUL3-Δ9 expression. METHODS: We used an inducible renal tubule-specific system to generate several mouse models expressing CUL3-Δ9, mice heterozygous for both CUL3 and KLHL3 (Cul3+/-/Klhl3+/- ), and mice with short-term Jab1 disruption (to avoid renal injury associated with long-term disruption). RESULTS: Renal KLHL3 was higher in Cul3-/- mice, but lower in Cul3-/-/Δ9 mice and in the Cul3+/-/Δ9 FHHt model, suggesting KLHL3 is a target for both WT and mutant CUL3. Cul3+/-/Klhl3+/- mice displayed increased WNK4-SPAK activation and phospho-NCC abundance and an FHHt-like phenotype with increased plasma [K+] and salt-sensitive blood pressure. Short-term Jab1 disruption in mice lowered the abundance of CUL3 and KLHL3 and increased the abundance of WNK4 and phospho-NCC. CONCLUSIONS: Jab1-/- mice and Cul3+/-/Klhl3+/- mice recapitulated the effects of CUL3-Δ9 expression on WNK4-SPAK-NCC. Our data suggest degradation of both KLHL3 and CUL3 plays a central mechanistic role in CUL3-Δ9-mediated FHHt.

DMC (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone) enhances exercise tolerance via the AMPK-SIRT1-PGC-1α pathway in mice fed a high-fat diet.

Obesity is caused by an imbalance between energy intake and energy expenditure. This study aimed to determine the effects and mechanisms of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) on exercise tolerance in high-fat diet (HFD)-fed mice. Male C57BL/6J mice were randomly divided into two categories (7 groups [n = 8]): sedentary (control [CON], HFD, 200 mg/kg DMC, and 500 mg/kg DMC) and swimming (HFD, 200 mg/kg DMC, and 500 mg/kg DMC). Except the CON group, all other groups were fed HFD with or without DMC intervention for 33 days. The swimming groups were subjected to exhaustive swimming (three sessions/week). Changes in swimming time, glucolipid metabolism, body composition, biochemical indicators, histopathology, inflammation, metabolic mediators, and protein expression were assessed. DMC combined with regular exercise improved endurance performance, body composition, glucose and insulin tolerance, lipid profile, and the inflammatory state in a dose-dependent manner. Further, DMC alone or combined with exercise could restore normal tissue morphology, reduce fatigue-associated markers, and boost whole-body metabolism and the protein expression of phospho-AMP-activated protein kinase alpha/total-AMP-activated protein kinase alpha (AMPK), sirtuin-1 (SIRT1), peroxisome-proliferator-activated receptor gamma coactivator 1alpha (PGC-1α), and peroxisome proliferator-activated receptor alpha in the muscle and adipose tissues of HFD-fed mice. DMC exhibits antifatigue effects by regulating glucolipid catabolism, inflammation, and energy homeostasis. Furthermore, DMC exerts a synergistic exercise-related metabolic effect via the AMPK-SIRT1-PGC-1α signaling pathway, suggesting that DMC is a potential natural sports supplement with mimicked or augmented exercise effects for obesity prevention.

COP9 signalosome deletion promotes renal injury and distal convoluted tubule remodeling.

Cullin-RING ligases are a family of E3 ubiquitin ligases that control cellular processes through regulated degradation. Cullin 3 targets with-no-lysine kinase 4 (WNK4), a kinase that activates the Na+-Cl- cotransporter (NCC), the main pathway for Na+ reabsorption in the distal convoluted tubule (DCT). Mutations in the cullin 3 gene lead to familial hyperkalemic hypertension by increasing WNK4 abundance. The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) regulates the activity of cullin-RING ligases by removing the ubiquitin-like protein neural precursor cell expressed developmentally downregulated protein 8. Genetic deletion of the catalytically active CSN subunit, Jab1, along the nephron in mice (KS-Jab1-/-) led to increased WNK4 abundance; however, NCC abundance was substantially reduced. We hypothesized that the reduction in NCC resulted from a cortical injury that led to hypoplasia of the segment, which counteracted WNK4 activation of NCC. To test this, we studied KS-Jab1-/- mice at weekly intervals over a period of 3 wk. The results showed that NCC abundance was unchanged until 3 wk after Jab1 deletion, at which time other DCT-specific proteins were also reduced. The kidney injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin demonstrated kidney injury immediately after Jab1 deletion; however, the damage was initially limited to the medulla. The injury progressed and expanded into the cortex 3 wk after Jab1 deletion coinciding with loss of the DCT. The data indicate that nephron-specific disruption of the cullin-RING ligase system results in a complex progression of tubule injury that leads to hypoplasia of the DCT.NEW & NOTEWORTHY Cullin 3 (CUL3) targets with-no-lysine-kinase 4 (WNK4), which activates Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Renal-specific genetic deletion of the constitutive photomorphogenesis 9 signalosome, an upstream regulator of CUL3, resulted in a reduction of NCC due to DCT hypoplasia, which coincided with cortical kidney injury. The data indicate that nephron-specific disruption of the cullin-RING ligase system results in a complex progression of tubule injury leading to hypoplasia of the DCT.

MoErv29 promotes apoplastic effector secretion contributing to virulence of the rice blast fungus Magnaporthe oryzae.

During plant-pathogenic fungi and host plants interactions, numerous pathogen-derived proteins are secreted resulting in the activation of the unfolded protein response (UPR) pathway. For efficient trafficking of secretory proteins, including those important in disease progression, the cytoplasmic coat protein complex II (COPII) exhibits a multifunctional role whose elucidation remains limited. Here, we discovered that the COPII cargo receptor MoErv29 functions as a target of MoHac1, a previously identified transcription factor of the UPR pathway. In Magnaporthe oryzae, deletion of MoERV29 severely affected the vegetative growth, conidiation and biotrophic invasion of the fungus in susceptible rice hosts. We demonstrated that MoErv29 is required for the delivery of secreted proteins through recognition and binding of the amino-terminal tripeptide motifs following the signal peptide. By using bioinformatics analysis, we predicted a cargo spectrum of MoErv29 and found that MoErv29 is required for the secretion of many proteins, including extracellular laccases and apoplastic effectors. This secretion is mediated through the conventional endoplasmic reticulum-Golgi secretion pathway and is important for conferring host recognition and disease resistance. Taken together, our results revealed how MoErv29 operates on effector secretion, and our findings provided a critical link between COPII vesicle trafficking and the UPR pathway.

Mulberry Leaf Extract Alleviates Staphylococcus aureus-Induced Conjunctivitis in Rabbits via Downregulation of NLRP3 Inflammasome and Upregulation of the Nrf2 System and Suppression of Pro-Inflammatory Cytokines.

INTRODUCTION: Mulberry (Morus alba L.) leaves are widely used in traditional Chinese medicine for their antioxidant, anti-inflammatory, antibacterial, anti-obesity, antidiabetic, antiatherosclerotic, and anticancer properties. The current study aimed to investigate the effect of mulberry leaf extract (MLE) on Staphylococcus aureus (S. aureus)-induced conjunctivitis (5 × 109 colony-forming units, 0.5 mL/eye) in a rabbit model. METHODS: Rabbits were treated with MLE (5 mL/kg·d-1 and 10 mL/kg·d-1), 0.9% saline, pearl bright eye (PBE) drops, or erythromycin eye ointment (EEO) group for 5 days. The ocular infection symptoms, bacterial negative conversion rate, and conjunctival histopathological changes of rabbits in each group were observed. The expression of caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain, NOD-like receptor leucine-rich pyrin domain-containing protein 3 (NLRP3), interleukin (IL)-18, IL-6, IL-1ß, TNFα, Keap1, and nuclear factor erythroid 2-related factor 2 (Nrf2) in conjunctival tissue of rabbits were detected by quantitative real-time reverse transcription PCR and/or Western blot analysis. RESULTS: The results showed that MLE treatment significantly reduced the clinical sign scores of conjunctivitis, alleviated clinical signs, and decreased bacterial load, and histological damage in a time- and dose-dependent manner was compared to that in the control group. The antibacterial and anti-inflammatory activities of MLE (10 mL/kg·d-1) were similar to those of the positive control drug PBE and EEO. In addition, MLE significantly decreased the levels of pro-inflammatory cytokines, downregulated the NLRP3 inflammasome, and upregulated the Nrf2 system. CONCLUSIONS: MLE is effective in alleviating S. aureus-induced conjunctivitis in rabbits, and this mechanism is associated with the inhibition of the NLRP3 inflammasome and activation of the Nrf2 system to regulate pro-inflammatory signaling.

Large-Scale Proteomic Assessment of Urinary Extracellular Vesicles Highlights Their Reliability in Reflecting Protein Changes in the Kidney.

BACKGROUND: Urinary extracellular vesicles (uEVs) are secreted into urine by cells from the kidneys and urinary tract. Although changes in uEV proteins are used for quantitative assessment of protein levels in the kidney or biomarker discovery, whether they faithfully reflect (patho)physiologic changes in the kidney is a matter of debate. METHODS: Mass spectrometry was used to compare in an unbiased manner the correlations between protein levels in uEVs and kidney tissue from the same animal. Studies were performed on rats fed a normal or high K+ diet. RESULTS: Absolute quantification determined a positive correlation (Pearson R=0.46 or 0.45, control or high K+ respectively, P<0.0001) between the approximately 1000 proteins identified in uEVs and corresponding kidney tissue. Transmembrane proteins had greater positive correlations relative to cytoplasmic proteins. Proteins with high correlations (R>0.9), included exosome markers Tsg101 and Alix. Relative quantification highlighted a monotonic relationship between altered transporter/channel abundances in uEVs and the kidney after dietary K+ manipulation. Analysis of genetic mouse models also revealed correlations between uEVs and kidney. CONCLUSION: This large-scale unbiased analysis identifies uEV proteins that track the abundance of the parent proteins in the kidney. The data form a novel resource for the kidney community and support the reliability of using uEV protein changes to monitor specific physiologic responses and disease mechanisms.

Aggregation of Fe2O3 nanoparticle in the presence of organophosphate esters (OPEs).

Aggregation is a key process for determining the environmental behavior and impact of a nanoparticle (NP). Since organophosphate esters (OPEs), which are recognized as emerging contaminants, are distributed widely in the natural aquatic environment, they may contribute to interacting with NPs and ultimately influence their transport and fate. Here, we investigated two typical organophosphate esters OPEs on aggregation the Fe2O3 NP in aquatic environments. The results showed that both tri-ethylhexyl phosphate (TEHP) and tris (chloroisopropyl) phosphate (TCPP) improved the colloidal stability of Fe2O3 NP in artificial water and environmental matrices. TEHP exhibited an obvious effect than TCPP on the Zeta potential and aggregation rates of Fe2O3 NP in artificial water. In the presence of electrolyte, 10 mg/L TCPP and TEHP increased the critical coagulation concentration (CCC) by 3.6 times and 17.4 times, respectively. Compared with pore-water, the aggregation rates of Fe2O3 NP in river water were slightly higher than those in pore-water, which can be attributed to the higher DOC in pore-water. We suggested that the high hydrophobicity and molecular weight of OPEs were considered important factors against the aggregation of Fe2O3 NP in water. Greater surface charge and steric hindrance originating from TCPP and TEHP dominated the colloidal stability of Fe2O3 NP.

Renal Tubule Nedd4-2 Deficiency Stimulates Kir4.1/Kir5.1 and Thiazide-Sensitive NaCl Cotransporter in Distal Convoluted Tubule.

BACKGROUND: The potassium channel Kir4.1 forms the Kir4.1/Kir5.1 heterotetramer in the basolateral membrane of the distal convoluted tubule (DCT) and plays an important role in the regulation of the thiazide-sensitive NaCl cotransporter (NCC). Kidney-specific deletion of the ubiquitin ligase Nedd4-2 increases expression of NCC, and coexpression of Nedd4-2 inhibits Kir4.1/Kir5.1 in vitro. Whether Nedd4-2 regulates NCC expression in part by regulating Kir4.1/Kir5.1 channel activity in the DCT is unknown. METHODS: We used electrophysiology studies, immunoblotting, immunostaining, and renal clearance to examine Kir4.1/Kir5.1 activity in the DCT and NCC expression/activity in wild-type mice and mice with kidney-specific knockout of Nedd4-2, Kir4.1, or both. RESULTS: Deletion of Nedd4-2 increased the activity/expression of Kir4.1 in the DCT and also, hyperpolarized the DCT membrane. Expression of phosphorylated NCC/total NCC and thiazide-induced natriuresis were significantly increased in the Nedd4-2 knockout mice, but these mice were normokalemic. Double-knockout mice lacking both Kir4.1/Kir5.1 and Nedd4-2 in the kidney exhibited increased expression of the epithelial sodium channel α-subunit, largely abolished basolateral potassium ion conductance (to a degree similar to that of kidney-specific Kir4.1 knockout mice), and depolarization of the DCT membrane. Compared with wild-type mice, the double-knockout mice displayed inhibited expression of phosphorylated NCC and total NCC and had significantly blunted thiazide-induced natriuresis as well as renal potassium wasting and hypokalemia. However, NCC expression/activity was higher in the double-knockout mice than in Kir4.1 knockout mice. CONCLUSIONS: Nedd4-2 regulates Kir4.1/Kir5.1 expression/activity in the DCT and modulates NCC expression by Kir4.1-dependent and Kir4.1-independent mechanisms. Basolateral Kir4.1/Kir5.1 activity in the DCT partially accounts for the stimulation of NCC activity/expression induced by deletion of Nedd4-2.

Distal convoluted tubule Cl- concentration is modulated via K+ channels and transporters.

Cl--sensitive with-no-lysine kinase (WNK) plays a key role in regulating the thiazide-sensitive Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT). Cl- enters DCT cells through NCC and leaves the cell across the basolateral membrane via the Cl- channel ClC-K2 or K+-Cl- cotransporter (KCC). While KCC is electroneutral, Cl- exit via ClC-K2 is electrogenic. Therefore, an alteration in DCT basolateral K+ channel activity is expected to influence Cl- movement across the basolateral membrane. Although a role for intracellular Cl- in the regulation of WNK and NCC has been established, intracellular Cl- concentrations ([Cl-]i) have not been directly measured in the mammalian DCT. Therefore, to measure [Cl-]i in DCT cells, we generated a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl- fluorescent imaging in the isolated DCT. Basal measurements indicated that the mean [Cl-]i was ~7 mM. Stimulation of Cl- exit with low-Cl- hypotonic solutions decreased [Cl-]i, whereas inhibition of KCC by DIOA or inhibition of ClC-K2 by NPPB increased [Cl-]i, suggesting roles for both KCC and ClC-K2 in the modulation of [Cl-]i . Blockade of basolateral K+ channels (Kir4.1/5.1) with barium significantly increased [Cl-]i. Finally, a decrease in extracellular K+ concentration transiently decreased [Cl-]i, whereas raising extracellular K+ transiently increased [Cl-]i, further suggesting a role for Kir4.1/5.1 in the regulation of [Cl-]i. We conclude that the alteration in ClC-K2, KCC, and Kir4.1/5.1 activity influences [Cl-]i in the DCT.

A novel distal convoluted tubule-specific Cre-recombinase driven by the NaCl cotransporter gene.

Cre-lox technology has revolutionized research in renal physiology by allowing site-specific genetic recombination in individual nephron segments. The distal convoluted tubule (DCT), consisting of distinct early (DCT1) and late (DCT2) segments, plays a central role in Na+ and K+ homeostasis. The only established Cre line targeting the DCT is Pvalb-Cre, which is limited by noninducibility, activity along DCT1 only, and activity in neurons. Here, we report the characterization of the first Cre line specific to the entire DCT. CRISPR/Cas9 targeting was used to introduce a tamoxifen-inducible IRES-Cre-ERT2 cassette downstream of the coding region of the Slc12a3 gene encoding the NaCl cotransporter (NCC). The resulting Slc12a3-Cre-ERT2 mice were crossed with R26R-YFP reporter mice, which revealed minimal leakiness with 6.3% of NCC-positive cells expressing yellow fluorescent protein (YFP) in the absence of tamoxifen. After tamoxifen injection, YFP expression was observed in 91.2% of NCC-positive cells and only in NCC-positive cells, revealing high recombination efficiency and DCT specificity. Crossing to R26R-TdTomato mice revealed higher leakiness (64.5%), suggesting differential sensitivity of the floxed site. Western blot analysis revealed no differences in abundances of total NCC or the active phosphorylated form of NCC in Slc12a3-Cre-ERT2 mice of either sex compared with controls. Plasma K+ and Mg2+ concentrations and thiazide-sensitive Na+ and K+ excretion did not differ in Slc12a3-Cre-ERT2 mice compared with controls when sex matched. These data suggest genetic modification had no obvious effect on NCC function. Slc12a3-Cre-ERT2 mice are the first line generated demonstrating inducible Cre recombinase activity along the entire DCT and will be a useful tool to study DCT function.

WNK bodies cluster WNK4 and SPAK/OSR1 to promote NCC activation in hypokalemia.

K+ deficiency stimulates renal salt reuptake via the Na+-Cl- cotransporter (NCC) of the distal convoluted tubule (DCT), thereby reducing K+ losses in downstream nephron segments while increasing NaCl retention and blood pressure. NCC activation is mediated by a kinase cascade involving with no lysine (WNK) kinases upstream of Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1). In K+ deficiency, WNKs and SPAK/OSR1 concentrate in spherical cytoplasmic domains in the DCT termed "WNK bodies," the significance of which is undetermined. By feeding diets of varying salt and K+ content to mice and using genetically engineered mouse lines, we aimed to clarify whether WNK bodies contribute to WNK-SPAK/OSR1-NCC signaling. Phosphorylated SPAK/OSR1 was present both at the apical membrane and in WNK bodies within 12 h of dietary K+ deprivation, and it was promptly suppressed by K+ loading. In WNK4-deficient mice, however, larger WNK bodies formed, containing unphosphorylated WNK1, SPAK, and OSR1. This suggests that WNK4 is the primary active WNK isoform in WNK bodies and catalyzes SPAK/OSR1 phosphorylation therein. We further examined mice carrying a kidney-specific deletion of the basolateral K+ channel-forming protein Kir4.1, which is required for the DCT to sense plasma K+ concentration. These mice displayed remnant mosaic expression of Kir4.1 in the DCT, and upon K+ deprivation, WNK bodies developed only in Kir4.1-expressing cells. We postulate a model of DCT function in which NCC activity is modulated by plasma K+ concentration via WNK4-SPAK/OSR1 interactions within WNK bodies.

Kidney Is Essential for Blood Pressure Modulation by Dietary Potassium.

Eating more potassium may reduce blood pressure and the occurrence of other cardiovascular diseases by actions on various systems, including the vasculature, the sympathetic nervous system, systemic metabolism, and body fluid volume. Among these, the kidney plays a major role in the potassium-rich diet-mediated blood pressure reduction. PURPOSE OF REVIEW: To provide an overview of recent discoveries about the mechanisms by which a potassium-rich diet leads to natriuresis. RECENT FINDINGS: Although the distal convoluted tubule (DCT) is a short part of the nephron that reabsorbs salt, via the sodium-chloride cotransporter (NCC), it is highly sensitive to changes in plasma potassium concentration. Activation or inhibition of NCC raises or lowers blood pressure. Recent work suggests that extracellular potassium concentration is sensed by the DCT via intracellular chloride concentration which regulates WNK kinases in the DCT. High-potassium diet targets NCC in the DCT, resulting in natriuresis and fluid volume reduction, which are protective from hypertension and other cardiovascular problems.

Kir4.1/Kir5.1 Activity Is Essential for Dietary Sodium Intake-Induced Modulation of Na-Cl Cotransporter.

BACKGROUND: Dietary sodium intake regulates the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT). Whether the basolateral, inwardly rectifying potassium channel Kir4.1/Kir5.1 (a heterotetramer of Kir4.1/Kir5.1) in the DCT is essential for mediating the effect of dietary sodium intake on NCC activity is unknown. METHODS: We used electrophysiology, renal clearance techniques, and immunoblotting to examine effects of Kir4.1/Kir5.1 in the DCT and NCC in wild-type and kidney-specific Kir4.1 knockout mice. RESULTS: Low sodium intake stimulated basolateral Kir4.1/Kir5.1 activity, increased basolateral K+ conductance, and hyperpolarized the membrane. Conversely, high sodium intake inhibited the potassium channel, decreased basolateral K+ currents, and depolarized the membrane. Low sodium intake increased total and phosphorylated NCC expression and augmented hydrochlorothiazide-induced natriuresis; high sodium intake had opposite effects. Thus, elevated NCC activity induced by low sodium intake was associated with upregulation of Kir4.1/Kir5.1 activity in the DCT, whereas inhibition of NCC activity by high sodium intake was associated with diminished Kir4.1/Kir5.1 activity. In contrast, dietary sodium intake did not affect NCC activity in knockout mice. Further, Kir4.1 deletion not only abolished basolateral K+ conductance and depolarized the DCT membrane, but also abrogated the stimulating effects induced by low sodium intake on basolateral K+ conductance and hyperpolarization. Finally, dietary sodium intake did not alter urinary potassium excretion rate in hypokalemic knockout and wild-type mice. CONCLUSIONS: Stimulation of Kir4.1/Kir5.1 by low intake of dietary sodium is essential for NCC upregulation, and inhibition of Kir4.1/Kir5.1 induced by high sodium intake is a key step for downregulation of NCC.

Deletion of Kir5.1 Impairs Renal Ability to Excrete Potassium during Increased Dietary Potassium Intake.

BACKGROUND: The basolateral potassium channel in the distal convoluted tubule (DCT), comprising the inwardly rectifying potassium channel Kir4.1/Kir5.1 heterotetramer, plays a key role in mediating the effect of dietary potassium intake on the thiazide-sensitive NaCl cotransporter (NCC). The role of Kir5.1 (encoded by Kcnj16) in mediating effects of dietary potassium intake on the NCC and renal potassium excretion is unknown. METHODS: We used electrophysiology, renal clearance, and immunoblotting to study Kir4.1 in the DCT and NCC in Kir5.1 knockout (Kcnj16-/- ) and wild-type (Kcnj16+/+ ) mice fed with normal, high, or low potassium diets. RESULTS: We detected a 40-pS and 20-pS potassium channel in the basolateral membrane of the DCT in wild-type and knockout mice, respectively. Compared with wild-type, Kcnj16-/- mice fed a normal potassium diet had higher basolateral potassium conductance, a more negative DCT membrane potential, higher expression of phosphorylated NCC (pNCC) and total NCC (tNCC), and augmented thiazide-induced natriuresis. Neither high- nor low-potassium diets affected the basolateral DCT's potassium conductance and membrane potential in Kcnj16-/- mice. Although high potassium reduced and low potassium increased the expression of pNCC and tNCC in wild-type mice, these effects were absent in Kcnj16-/- mice. High potassium intake inhibited and low intake augmented thiazide-induced natriuresis in wild-type but not in Kcnj16-/- mice. Compared with wild-type, Kcnj16-/- mice with normal potassium intake had slightly lower plasma potassium but were more hyperkalemic with prolonged high potassium intake and more hypokalemic during potassium restriction. CONCLUSIONS: Kir5.1 is essential for dietary potassium's effect on NCC and for maintaining potassium homeostasis.

Kir4.1/Kir5.1 in the DCT plays a role in the regulation of renal K+ excretion.

The aim of this mini review is to provide an overview regarding the role of inwardly rectifying potassium channel 4.1 (Kir4.1)/Kir5.1 in regulating renal K+ excretion. Deletion of Kir4.1 in the kidney inhibited thiazide-sensitive NaCl cotransporter (NCC) activity in the distal convoluted tubule (DCT) and slightly suppressed Na-K-2Cl cotransporter (NKCC2) function in the thick ascending limb (TAL). Moreover, increased dietary K+ intake inhibited, whereas decreased dietary K+ intake stimulated, the basolateral potassium channel (a Kir4.1/Kir5.1 heterotetramer) in the DCT. The alteration of basolateral potassium conductance is essential for the effect of dietary K+ intake on NCC because deletion of Kir4.1 in the DCT abolished the effect of dietary K+ intake on NCC. Since potassium intake-mediated regulation of NCC plays a key role in regulating renal K+ excretion and potassium homeostasis, the deletion of Kir4.1 caused severe hypokalemia and metabolic alkalosis under control conditions and even during increased dietary K+ intake. Finally, recent studies have suggested that the angiotensin II type 2 receptor (AT2R) and bradykinin-B2 receptor (BK2R) are involved in mediating the effect of high dietary K+ intake on Kir4.1/Kir5.1 in the DCT.

GR and Foxa1 promote the transcription of ANGPTL4 in bovine adipocytes.

ANGPTL4 (angiopoietin-like 4) is a secreted protein involved in triacylglycerol homeostasis. It is a key enzyme in lipolysis, which stimulates the oxidation of fatty acids and inhibits fat accumulation by inhibiting the activity of lipoprotein lipase (LPL). Using quantitative Real-Time PCR (qRT-PCR) to investigate the mRNA expression pattern of the bovine ANGPTL4 gene in different tissues and organs, we found that bovine ANGPTL4 had the highest expression level in the liver followed by subcutaneous adipose tissue. To clarify the molecular mechanism involved in the regulation of bovine ANGPTL4, we identified the transcriptional start site (TSS) of the ANGPTL4 gene and obtained 2011 bp of the 5' regulatory region. A series of 5' deletion promoter luciferase reporter assays revealed that the minimum functional promoter region of bovine ANGPTL4 was located at -568 bp to -261 bp relative to TSS. Two transcription factors, GR and Foxa1, were identified and considered as important transcriptional activators of ANGPTL4 by mutational analysis and RNA interference assays in combination with electrophoretic mobility shift assays (EMSA) in bovines. In conclusion, GR and Foxa1 were determined to be responsible for the regulation of ANGPTL4 transcription. Our results may provide a basis for further investigation of ANGPTL4 regulation and a reference for improvement of beef quality in cattle.