Thick Ascending Limb Research Articles

Validity of Serum Uromodulin as Early Diagnosis Marker of Diabetic Nephropathy in T2DM

Increased prevalence of DM is accompanied by increased in its various complications include diabetic nephropathy, which can lead to end stage renal disease (ESRD). Urinary albumin-creatinine ratio (uACR) is the gold standard for diabetic nephropathy; however, it has several limitations, including the inability to early diagnose due to the absence of increased level in uACR. Uromodulin is produced by thick ascending limb (TAL) at Henle’s loop and early distal tubule of nephron. Uromodulin will decrease when tubular atrophy occurs in early stage of renal impairment with normoalbuminuria. The aim of this study was to determine the validity of serum uromodulin in type 2 diabetes mellitus (T2DM) in identifying diabetic nephropathy by comparing it with uACR as gold standard. This study was a cross-sectional analytical observational study at Dr. Hasan Sadikin (RSHS) Bandung from June to August 2020. Subjects consisted of 62 patiens with T2DM. Results showed that the serum uromodulin level had a sensitivity of 93.3%, a specificity of 88.2%, a positive predictive value of 95.5%, a negative predictive value of 83.3%, and an accuracy of 91,9% and an AUC value of 0.975. The cut-off point of serum uromodulin in this study was 47.195 ng/mL. In conclusion, serum uromodulin, when compared to the uACR as the gold standard, has good sensitivity and specificity for identifying diabetic nephropathy.

Acute effect of alcohol on working memory updating.

To examine the acute effects of alcohol on working memory (WM) updating, including potential variation across the ascending limb (AL) and descending limb (DL) of the blood alcohol concentration (BAC) time-course. A two-session experiment in which participants were randomly assigned to one of three beverage conditions [alcohol (males: 0.80g/kg; females: 0.72g/kg), active placebo (0.04g/kg) or non-alcohol control (tonic)] and one of two BAC limb testing conditions (AL and DL or DL-only) for the second session, yielding a 3 (beverage)×2 (time-points tested)×3 (time-point) mixed factorial design with repeated measures on the latter factor. One of the repeated assessments is 'missing by design' in the DL-only condition. A psychology laboratory at the University of Missouri campus in Columbia, MO, USA. Two hundred thirty-one community-dwelling young adults (51% female; aged 21-34years) recruited from Columbia, MO, USA, tested between 2011 and 2013. Latent WM updating performance as indexed by shared variance in accuracy on three WM updating tasks (letter memory, keep track, spatial 2-back) at three time-points. Multi-group modeling of latent WM updating indicated that performance among participants who consumed placebo or control beverages improved during the second session at time-points corresponding to AL (∆ from baseline in latent mean±standard error (SE)+0.5±0.01, P<0.001) and DL (+0.08±0.01, P<0.001). Alcohol consumption did not impair WM updating (∆ from baseline in latent mean±SE, at AL: +0.01±0.01, P=0.56; at DL: +0.05±0.01, P<0.001), but attenuated performance improvements (equality of latent means across beverage groups at AL or DL: Δχ2(1) ≥7.53, P<0.01). Acute alcohol-induced impairment in working memory updating may be limited, but dampening of practice effects by alcohol could interfere with the completion of novel, unpracticed tasks.

The FSGS protein Actinin‐4 (ACTN4) is expressed in Thick Ascending Limbs and interacts with NKCC2 and ALMS1 to regulate NKCC2 trafficking

NKCC2 is a Cl-dependent co-transporter that mediates all the NaCl absorption in the thick ascending limb (TAL). In humans, loss-of-function mutations in NKCC2 result in Bartter syndrome type 1, characterized by inability in concentrating urine, polyuria, and low blood pressure. Importantly, enhanced NKCC2 activity is associated with hypertension in humans and animal models. NKCC2 is present on the apical membrane and sub-apical space in the TAL and macula densa cells. We and others have shown that trafficking of NKCC2 to the apical membrane determines NKCC2-mediated NaCl absorption. These trafficking steps are controlled by signaling and protein-protein interactions. Very little is known about how NKCC2 endocytosis is controlled and only a handful of proteins that mediate NKCC2 internalization and trafficking are known. To identify new NKCC2 interacting partners we immunoprecipitated NKCC2 from TAL lysates using a C-terminus antibody and identified interacting proteins by mass spectrometry. We identified Actinin-4 (ACTN4), a protein studied in podocytes, that when mutated causes Focal segmental glomerulo-sclerosis (FSGS) in humans. We hypothesized that in addition to podocytes, ACTN4 is expressed along the nephron and is part of protein complex that binds apical NKCC2 and other proteins such as Alstrom Syndrome 1 (ALMS1) and promotes its endocytosis. To begin studying these mechanisms we used a targeted proteomics screen to identify new binding partners for ACTN4 in the TAL. We confirmed that glutathione-S-transferase (GST) ACTN4 (full length) pulled down NKCC2 and ALMS1 from TAL lysates identified by mass spectrometry (LC-MS) or Western blot. GST-ALMS1 also pulled down ACTN4, identified by LC-MS. Because there are no studies on the role of ACTN4 in NaCl reabsorption along the nephron, we studied its localization in the kidney. ACTN4 was abundant in glomeruli but also localized in cells along the nephron in a punctate vesicular pattern. ACTN4 was expressed in TALs and partially co-localized with NKCC2 the subapical space. To study if ACTN4 mediates NKCC2 trafficking, we generated adenoviruses expressing ACTN4 shRNA. In vivo transduction of the rat renal medulla decreased ACTN4 expression in TALs by 66±9% (p<0.04). Silencing ACTN4 in rat TALs increased the surface to total NKCC2 ratio by 49±16% (p<0.01) but did not affect total NKCC2, suggesting that ACTN4 regulates NKCC2 trafficking. To study the relationship of ACTN4 with ALMS1 and NKCC2, we generated nephron-specific ALMS1 knockout mice and measured surface NKCC2 and ACTN4. Deletion of ALMS1 increased surface to total NKCC2 ratio by 55±19% (p<0.05), but also decreased ACTN4 expression by 45±9% (p<0.05). We conclude that ACTN4 binds NKCC2 and ALMS1 and it is required for proper NKCC2 trafficking. It is likely that a large protein complex containing ACTN4 and ALMS1 mediates NKCC2 endocytosis to control renal salt reabsorption.

Genetic Deletion of the Prostaglandin EP3 Receptor in the Kidney Tubule of Adult Mice Has No Impact on Kidney Water Handling

Prostaglandin E2 (PGE2) is an important lipid mediator modulating various aspects of kidney function. PGE2 exerts its effects via four PGE2 receptors, EP1-EP4. The EP3 receptor is expressed in the thick ascending limb (TAL) and the collecting duct, where it is proposed to inhibit cAMP generation and NaCl and water reabsorption. However, EP3 is also expressed in endothelial cells of arteries and arterioles, which also play a role in kidney function. Therefore, to assess the tubular role of EP3 in adult mice we generated a mouse model based on the Pax8Cre system with doxycycline-dependent deletion of EP3 along the renal tubule and assessed their renal phenotype in respect to water handling. RNAscope confirmed that EP3 was highly expressed in cortical and medullary TAL and collecting ducts, but it was non-detected in proximal tubule and thin limbs. Two weeks after treatment with doxycycline, EP3 mRNA expression was reduced by >80% in whole kidney (RT-q-PCR) and non-detectable (RNAscope) in tubules of knockout mice compared to control mice. There were no compensatory changes in other EP receptors. Under basal conditions, there were no significant differences in food and water intake, bodyweight, urinary output or plasma and urine biochemistries in both male or female control and knockout mice. There were no differences between genotypes in their renal handling of water during an acute water load, or in their response to the vasopressin V2 receptor agonist dDAVP. Rats drinking 1% NaCl for several days in combination with an EP3 antagonist have increased urine output (Hao et al., 2016). However, relative to controls, we could not detect significant differences in urine volume or osmolality in tubular EP3 knockout mice during 1% NaCl intake. In conclusion, EP3 in the renal tubule is not important for renal water handling or compensatory mechanisms exist. This new model provides a novel tool for examination of the role of EP3 in other aspects of renal function or kidney disease independently of potential developmental abnormalities or systemic effects.

Urinary miRNA Biomarkers of Drug-Induced Kidney Injury and Their Site Specificity Within the Nephron.

Drug-induced kidney injury (DIKI) is a major concern in both drug development and clinical practice. There is an unmet need for biomarkers of glomerular damage and more distal renal injury in the loop of Henle and the collecting duct (CD). A cross-laboratory program to identify and characterize urinary microRNA (miRNA) patterns reflecting tissue- or pathology-specific DIKI was conducted. The overall goal was to propose miRNA biomarker candidates for DIKI that could supplement information provided by protein kidney biomarkers in urine. Rats were treated with nephrotoxicants causing injury to distinct nephron segments: the glomerulus, proximal tubule, thick ascending limb (TAL) of the loop of Henle and CD. Meta-analysis identified miR-192-5p as a potential proximal tubule-specific urinary miRNA candidate. This result was supported by data obtained in laser capture microdissection nephron segments showing that miR-192-5p expression was enriched in the proximal tubule. Discriminative miRNAs including miR-221-3p and -222-3p were increased in urine from rats treated with TAL versus proximal tubule toxicants in accordance with their expression localization in the kidney. Urinary miR-210-3p increased up to 40-fold upon treatment with TAL toxicants and was also enriched in laser capture microdissection samples containing TAL and/or CD versus proximal tubule. miR-23a-3p was enriched in the glomerulus and was increased in urine from rats treated with doxorubicin, a glomerular toxicant, but not with toxicants affecting other nephron segments. Taken together these results suggest that urinary miRNA panels sourced from specific nephron regions may be useful to discriminate the pathology of toxicant-induced lesions in the kidney, thereby contributing to DIKI biomarker development needs for industry, clinical, and regulatory use.

Localization and regulation of claudin-14 in experimental models of hypercalcemia.

Variations in the claudin-14 (CLDN14) gene have been linked to increased risk of hypercalciuria and kidney stone formation. However, the exact cellular localization of CLDN14 and its regulation remain to be fully delineated. To this end, we generated a novel antibody that allowed the detection of CLDN14 in paraffin-embedded renal sections. This showed CLDN14 to be detectable in the kidney only after induction of hypercalcemia in rodent models. Protein expression in the kidney is localized exclusively to the thick ascending limbs (TALs), mainly restricted to the cortical and upper medullary portion of the kidney. However, not all cells in the TALs expressed the tight junction protein. In fact, CLDN14 was primarily expressed in cells also expressing CLDN16 but devoid of CLDN10. CLDN14 appeared in very superficial apical cell domains and near cell junctions in a belt-like formation along the apical cell periphery. In transgenic mice, Cldn14 promotor-driven LacZ activity did not show complete colocalization with CLDN14 protein nor was it increased by hypercalcemia, suggesting that LacZ activity cannot be used as a marker for CLDN14 localization and regulation in this model. In conclusion, CLDN14 showed a restricted localization pattern in the apical domain of select cells of the TAL.

Mechanism of Diuresis and Natriuresis by Cannabinoids: Evidence for Inhibition of Na+-K+-ATPase in Mouse Kidney Thick Ascending Limb Tubules.

The endocannabinoid, anandamide (AEA), stimulates cannabinoid receptors (CBRs) and is enriched in the kidney, especially the renal medulla. AEA infused into the renal outer medulla of mice stimulates urine flow rate and salt excretion. Here we show that these effects are blocked by the CBR type 1 (CB1) inverse agonist, rimonabant. Immunohistochemical analysis demonstrated the presence of CB1 in thick ascending limb (TAL) tubules. Western immunoblotting demonstrated the presence of CB1 (52 kDa) in the cortex and outer medulla of mouse kidney. The effect of direct [CP55940 (CP) or AEA] or indirect [fatty acyl amide hydrolase (FAAH) inhibitor, PF3845 (PF)] cannabinoidimetics on Na+ transport in isolated mouse TAL tubules was studied using the Na+-sensitive dye, SBFI-AM. Switching from 0 Na+ solution to control Ringer's solution (CR) rapidly increased TAL cell [Na+]i Addition of CP to CR produced a further elevation, similar in magnitude to that of ouabain, a Na+-K+-ATPase inhibitor. This [Na+]i-elevating effect of CP was time-dependent, required the presence of Na+ in the bathing solution, and was insensitive to Na+-K+-2Cl- cotransporter inhibition. Addition of PF to CR elevated [Na+]i in FAAH wild-type but not FAAH knockout (KO) TALs, whereas the additions of CP and AEA to PF-treated FAAH KO TALs increased [Na+]i An interaction between cannabinoidimetics and ouabain (Ou) was observed. Ou produced less increase in [Na+]i after cannabinoidimetic treatment, whereas cannabinoidimetics had less effect after Ou treatment. It is concluded that cannabinoidimetics, including CP and AEA, inhibit Na+ transport in TALs by inhibiting Na+ exit via Na+-K+-ATPase. SIGNIFICANCE STATEMENT: Cannabinoids including endocannabinoids induce renal urine and salt excretion and are proposed to play a physiological role in the regulation of blood pressure. Our data suggest that the mechanism of the cannabinoids involves inhibition of the sodium pump, Na+-K+-ATPase, in thick ascending limb cells and, likely, other proximal and distal tubular segments of the kidney nephron.

Single-Cell RNA Sequencing Reveals Transcriptomes of DCT1, DCT2, Macula Densa, and Two Subtypes of Cortical Thick Ascending Limb Cells

Single-Cell RNA Sequencing Reveals Transcriptomes of DCT1, DCT2, Macula Densa, and Two Subtypes of Cortical Thick Ascending Limb Cells

Transcriptomic Profiling of Thick Ascending Limb Cells In Vivo Reveals the Complex Network of Genes Regulated by Uromodulin

Transcriptomic Profiling of Thick Ascending Limb Cells In Vivo Reveals the Complex Network of Genes Regulated by Uromodulin

Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka

The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl- permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl- currents time-dependently within several minutes. Of note, the same effect on Cl- currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca2+ chelator BAPTA-AM (30 μM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl- current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic.

SO040ROLE OF INTRACELLULAR CA2+ IN THE REGULATION OF THE RENAL CHLORIDE CHANNEL CLC-KA

Abstract Background and Aims It has been recognized that the physiological role of ClC-Ka and its mouse homolog ClC-K1 in the nephron is to confer high Cl- permeability in the thin Ascending Limb of loop of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla in the countercurrent system. Accordingly ClC-K1 KO mice display a severe polyuria resembling a Nephrogenic Diabetes Insipidus (NDI) phenotype. Thus, ClC-Ka in human nephron could be an interesting target for diuretic and anti-hypertensive drugs. Unfortunately the regulation of ClC-Ka in and its electrophysiological properties are still not completely known. Method We analyzed, by whole-cell voltage-clamp, Cl- currents in HEK293 cells co-expressing GFP-ClC-Ka and the accessory m-Cherry-tagged barttin subunit, upon intracellular Ca2+ mobilization. The patch clamp protocol consists of a holding potential from 0 mV, which is stepped to various test potentials from -195 and +125 mV for 150 ms. Pulses ended with a tail pulse to -125 mV for 20 ms. Concomitantly we monitored by live confocal microscopy, the membrane expression of either GFP-ClC-Ka or barttin-mCherry. Results In control conditions we found a linear current/voltage relationship over a broad range of membrane voltages except of hyperpolarization below -100 mV, where the channel open probability was reduced, providing the well-known hook-shaped appearance of the I-V curve for ClC-Ka channel. Interestingly, both 100 µM ATP and 400 µg/ml of dandelion root extract (DRE), both increasing intracellular Ca2+ in HEK293, significantly inhibited Cl- currents in a time dependent way. Pre-treatment of cells with either the Ca2+ chelator BAPTA-AM or the PCK inhibitor Calphostin C, reverted the inhibitory effects of both ATP and DRE. Interestingly, 1µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of ATP and DRE on Cl- currents. We finally demonstrated that 30 µM Heclin, an inhibitor of E3 ubiquitin ligase, did not revert the DRE- and ATP- induced Cl- currents inhibition, thus suggesting that the ClC-Ka inhibition is independent of channel ubiquitination. Accordingly, live confocal analysis showed that all the Ca2+ mobilizing compounds tested did not induced neither ClC-Ka nor Barttin internalization. We hypothesized that phosphorylation in the putative PKC consensus sites present in ClC-Ka amino acidic sequence may be involved in the inhibition of channel activity. Conclusion In conclusion, we demonstrated for the first time that the activity of ClC-Ka in renal cells could by inhibited by either receptor agonists or natural molecules able to activate an intracellular Ca2+/PKC pathway, thus opening venues for the development of new diuretic drugs.

Generation and Characterization of Thick Ascending Limb‐Specific NHE3 Knockout Mice

The Na+/H+ exchanger 3 (NHE3) mediates Na+ absorption in the intestine and reabsorption in the kidney, thereby playing a key role in fluid homeostasis and blood pressure regulation. NHE3 is expressed in the luminal membrane of the renal proximal tubule (PT), where the majority of renal Na+ reabsorption (~67%) occurs. In addition, NHE3 is expressed substantially in the thick ascending limb (TAL). Whole body NHE3 knockout mice exhibit hypovolemia, hypotension, diarrhea, metabolic acidosis and renal Na+ wasting. Studies in kidney‐specific (PT and TAL) NHE3 knockout mice showed a rather mild phenotype in terms of Na+ homeostasis and no acid‐base phenotype (Kidney International; 2017,92:397–414). To determine the physiological role of NHE3 in the TAL we generated a novel tamoxifen (Tam)‐inducible TAL‐specific NHE3 knockout (NHE3TAL‐KO) mouse model by crossing NHE3loxlox (Control, Con) with uromodulin Cre (UmodCreERT2) mice. After Tam administration (3 days, 67 mg/kg via oral gavage), TAL‐specific NHE3 knockout was confirmed by quantitative RT‐qPCR for NHE3 in microdissected inner medulla/inner stripe outer medulla (ISOM) and renal cortex and by immunofluorescent labeling with NHE3 (PT and TAL), NKCC2 (TAL marker) and lotus tetragonolobus lectin (LTL, PT marker). NHE3 mRNA levels in NHE3TAL‐KO mice (n=8) were reduced by 30% in the renal cortex and by 94% in the ISOM. Control mice showed colocalization of NHE3 with LTL and NKCC2; in contrast, no immunofluorescence was observed for NHE3 in the TAL of NHE3TAL‐KO mice. NHE3TAL‐KO mice were born at predicted Mendelian frequencies, appeared grossly indistinguishable from Con mice, and developed normally. Two weeks after Tam administration, NHE3TAL‐KO mice (n=11) showed a ~25% lower urine osmolality compared to Con mice (n=10) (2224±90 versus 1826±93 mOsm/kg, P&lt;0.05) associated with ~20% higher water intake (5.3±0.2 versus 6.3±0.3 mL/day, P&lt;0.05). Blood pH was slightly but significantly increased in NHE3TAL‐KO mice (n=20) versus Con mice (n=19) (7.39±0.01 versus 7.37±0.01, P&lt;0.05), which was associated with a tendency of higher blood HCO3− in NHE3TAL‐KO mice (26.1±0.4 versus 25.5±0.3 mmol, P=0.07). However, no differences were observed for urinary pH (6.1±0.1 vs 6.2±0.1, NS), urinary Na+/creatinine (37±5 vs 41±5 mmol/mmol, NS), K+/creatinine (81±6 vs 94±7 mmol/mmol, NS), plasma Na+ (147±1 versus 147±1 mmol, NS) or plasma K+ (4.2±0.1 vs 4.4±0.1 mmol, NS). In conclusion, we successfully generated TAL‐specific NHE3 knockout mouse and found that NHE3 in the TAL contributes to urinary concentrating ability and acid‐base regulation. This mouse model will further be useful to delineate the TAL‐specific role of NHE3 for renal function.Support or Funding InformationTR is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK110621) and American Heart Association Transformational Research Award (19TPA34850116), LT was supported by an American Heart Association Postdoctoral Fellowship (19POST34400026) and JX was supported by an American Heart Association Predoctoral Fellowship (18PRE33990236).

The FSGS protein Actinin‐4 (ACTN4) is expressed in Thick Ascending Limbs and interacts with NKCC2 and ALMS1 to regulate NKCC2 trafficking.

Thick ascending limb (TAL) of the loop of Henle reabsorbs 30% NaCl via the apical Na/K/2Cl cotransporter (NKCC2). Maintenance of steady‐state surface NKCC2 levels is results from a balance between endocytosis, exocytosis and recycling. These trafficking steps are controlled by signaling and protein‐protein interactions. Very little is known about how NKCC2 endocytosis is controlled and only a handful of proteins that mediate NKCC2 internalization and trafficking are known. To identify new NKCC2 interacting partners we immunoprecipitated NKCC2 from TAL lysates using a C‐terminus antibody and identified interacting proteins by mass spectrometry. We identified Actinin‐4 (ACTN4), a protein studied in podocytes, that when mutated causes Focal segmental glomerulo‐sclerosis (FSGS) in humans. We hypothesized that in addition to podocytes, ACTN4 is expressed along the nephron and is part of protein complex that binds apical NKCC2 and other proteins such as Alstrom Syndrome 1 (ALMS1) and promotes its endocytosis. To begin studying these mechanisms we used a targeted proteomics screen to identify new binding partners for ACTN4 in the TAL. We confirmed that glutathione‐S‐transferase (GST) ACTN4 (full length) pulled down NKCC2 and ALMS1 from TAL lysates identified by mass spectrometry (LC‐MS) or Western blot. GST‐ALMS1 also pulled down ACTN4, identified by LC‐MS. Because there are no studies on the role of ACTN4 in NaCl reabsorption along the nephron, we studied its localization in the kidney. ACTN4 was abundant in glomeruli but also localized in cells along the nephron in a punctate vesicular pattern. ACTN4 was expressed in TALs and partially co‐localized with NKCC2 the subapical space. To study if ACTN4 mediates NKCC2 trafficking, we generated adenoviruses expressing ACTN4 shRNA. In vivo transduction of the rat renal medulla decreased ACTN4 expression in TALs by 66±9% (p&lt;0.04). Silencing ACTN4 in rat TALs increased the surface to total NKCC2 ratio by 49±16% (p&lt;0.01) but did not affect total NKCC2, suggesting that ACTN4 regulates NKCC2 trafficking. To study the relationship of ACTN4 with ALMS1 and NKCC2, we generated nephron‐specific ALMS1 knockout mice and measured surface NKCC2 and ACTN4. Deletion of ALMS1 increased surface to total NKCC2 ratio by 55±19% (p&lt;0.05), but also decreased ACTN4 expression by 45±9% (p&lt;0.05). We conclude that ACTN4 binds NKCC2 and ALMS1 and it is required for proper NKCC2 trafficking. It is likely that a large protein complex containing ACTN4 and ALMS1 mediates NKCC2 endocytosis to control renal salt reabsorption.Support or Funding InformationNIH

Kctd15 Regulates Nephron Segment Differentiation by Repressing Tfap2a Activity

A functional vertebrate kidney relies on structural units called nephrons, which are epithelial tubules that contain a sequence of segments each expressing a distinct repertoire of solute transporters. To date, the transcriptional codes driving regional specification, solute transporter program activation, and terminal differentiation of segment populations remains poorly understood. We demonstrate for the first time that the KCTD15 paralogs, kctd15a and kctd15b, function in concert to restrict distal early (DE)/thick ascending limb (TAL) segment lineage assignment in the developing zebrafish pronephros by repressing Tfap2a activity. During renal ontogeny, expression of these factors co‐localized with tfap2a in distal tubule precursors. kctd15 loss primed nephron cells to adopt distal fates by driving expansions in slc12a1, kcnj1a.1, and stc1 marker expression. These phenotypes were resultant of Tfap2a hyperactivity, where kctd15a/b‐deficient embryos exhibited increased abundance of this transcription factor. Interestingly, tfap2a can reciprocally promote kctd15 transcription, unveiling a circuit of autoregulation operating in nephron tubule progenitors. Concomitant kctd15b knockdown with tfap2a overexpression produced genetic synergy and uncontrolled DE/TAL differentiation. Our data indicates nephron segmentation is determined by a transcription factor‐repressor feedback module that employs tight regulation of Tfap2a‐Kctd15 kinetics during kidney development.

ARL15 Regulates CNNM2‐dependent Mg2+ Transport by Modulating its N‐linked Glycosylation

BackgroundA large Genome‐wide association study identified that ARL15, a small GTP‐binding protein, is associated with urinary Mg2+ excretion. Within the kidney, ARL15 is highly expressed in the thick ascending limb (TAL) and distal convoluted tubule (DCT), where Mg2+ reabsorption is tightly regulated. However, the exact function of ARL15 and the mechanism by which ARL15 regulates renal Mg2+ handling are still unknown.MethodTo identify protein‐interaction between ARL15 and Cyclin M (CNNM) proteins, proximity‐dependent biotin identification (BioID) and co‐immunoprecipitation were performed. Immunohistochemistry were used to investigate co‐localization in mouse kidney and human embryonic kidney (HEK293) cells. Furthermore, cell surface biotinylation and 25Mg2+ uptake assays were used to assess cell surface expression of CNNM2 and Mg2+ transport activity. The glycosylation pattern of CNNMs was determined by far lectin Western blot and glycosidase assays.ResultsWe identified members of the CNNM family as direct interaction partners of ARL15 by BioID. Immunoprecipitation with truncated CNNM2 proteins indicated that ARL15 interacts with CNNM2 at its carboxyl‐(C)‐terminal conserved CBS domain. CNNM2 and ARL15 co‐localize in the basolateral membrane of DCT. Interestingly, overexpression of ARL15 in HEK293 cells showed subcellular localization in the Golgi‐apparatus and resulted in an increased N‐glycosylation of CNNM proteins. This ARL15‐mediated glycosylation was Mg2+‐sensitive and encompassed hybrid and complex glycosylation. The functional consequences of ARL15‐dependent glycosylation were examined by 25Mg2+ uptake experiments. ARL15 increased 25Mg2+ uptake via CNNM2 by increasing its cell surface expression.ConclusionARL15 increases CNNM2 plasma membrane expression by regulating its N‐glycosylation pattern. Altogether, our results establish ARL15 as novel regulatory mechanism of Mg2+ transport within the DCT.Support or Funding InformationThis work was financially supported by grants from the Netherlands Organization for Scientific Research NWO Veni 016.186.012 and VICI 016.130.668).

New Insights into the Role of Intracellular Ca2+ in the Regulation of the Human Kidney Chloride Channel ClC‐Ka

It has been recognized that the physiological role of ClC‐Ka and its mouse homolog ClC‐K1 in the nephron is to confer high Cl− permeability in the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla in the countercurrent system. Accordingly ClC‐K1 KO mice display a severe polyuria resembling a Nephrogenic Diabetes Insipidus (NDI) phenotype. Thus, ClC‐Ka in human nephron could be an interesting target for diuretic and anti‐hypertensive drugs. Unfortunately the regulation of ClC‐Ka in and its electrophysiological properties are still not completely known. We analyzed, by whole‐cell voltage‐clamp, Cl− currents in HEK293 cells expressing GFP‐ClC‐Ka and the accessory m‐Cherry Barttin subunit upon intracellular Ca2+mobilization. The patch clamp protocol consists of a holding potential from 0 mV, which is stepped to various test potentials from −195 and +125 mV for 150 ms. Pulses ended with a tail pulse to −125 mV for 20 ms. In control conditions we found a linear current/voltage relationship over a broad range of membrane voltages except of hyperpolarization below −100 mV, where the channel open probability was reduced, providing the well‐known hook‐shaped appearance of the I‐V curve for ClC‐Ka channel. Interestingly, both 100 μM ATP and 400 μg/ml of dandelion root extract (DRE), both increasing intracellular Ca2+ in HEK293, significantly inhibited Cl− currents in a time dependent way. Pretreatment of cells with either the Ca2+ chelator BAPTA‐AM or the PCK inhibitor Calphostin C, reverted the inhibitory effects of both ATP and DRE. Interestingly, 1μM of Phorbol Meristate Acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of ATP and DRE on Cl− currents. We finally demonstrated that 30 μM Heclin, an inhibitor of E3 ubiquitin ligase, did not revert the DRE‐ and ATP‐ induced Cl− currents inhibition, thus suggesting that the ClC‐Ka inhibition is independent of channel ubiquitination. Accordingly, live confocal analysis showed that all the Ca2+mobilizing compounds tested did not induced neither ClC‐Ka nor Barttin internalization. We hypothesized that phosphorylation in the putative PKC consensus sites present in ClC‐Ka amino acidic sequence may be involved in the inhibition of channel activity. In conclusion, we demonstrated for the first time that the activity of ClC‐Ka in renal cells could by inhibited by either receptor agonists or natural molecules able to activate an intracellular Ca2+/PKC pathway, thus opening venues for the development of new diuretic drugs.Support or Funding InformationGrant n# ARS01_01081 to MC from The National Operational Program (PON) of the Ministry of Education, ITALYFigure 1

Multiomics Evaluation of Gastrointestinal and Other Clinical Characteristics of COVID-19

Since December 2019, coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has produced a worldwide panic. Beyond the principal human-to-human transmission method by droplet and contact, there is still limited knowledge about possible alternate transmission methods to guide clinical care. Recent clinical studies have observed digestive symptoms in patients with COVID-19,1 possibly because of the enrichment and infection of SARS-CoV-2 in the gastrointestinal tract, mediated by virus receptor of angiotensin converting enzyme 2 (ACE2),2 which suggests the potential for a fecal-oral route of SARS-CoV-2 transmission.3,4 However, there is still a large gap in the biological knowledge of COVID-19. In this study, via a bulk-to-cell strategy focusing on ACE2, we performed an integrated omics analysis at the genome, transcriptome, and proteome levels in bulk tissues and single cells across species to decipher the potential routes for SARS-CoV-2 infection in depth.

Claudin-12 Knockout Mice Demonstrate Reduced Proximal Tubule Calcium Permeability.

The renal proximal tubule (PT) is responsible for the reabsorption of approximately 65% of filtered calcium, primarily via a paracellular pathway. However, which protein(s) contribute this paracellular calcium pore is not known. The claudin family of tight junction proteins confers permeability properties to an epithelium. Claudin-12 is expressed in the kidney and when overexpressed in cell culture contributes paracellular calcium permeability (PCa). We therefore examined claudin-12 renal localization and its contribution to tubular paracellular calcium permeability. Claudin-12 null mice (KO) were generated by replacing the single coding exon with β-galactosidase from Escherichia coli. X-gal staining revealed that claudin-12 promoter activity colocalized with aquaporin-1, consistent with the expression in the PT. PTs were microperfused ex vivo and PCa was measured. PCa in PTs from KO mice was significantly reduced compared with WT mice. However, urinary calcium excretion was not different between genotypes, including those on different calcium containing diets. To assess downstream compensation, we examined renal mRNA expression. Claudin-14 expression, a blocker of PCa in the thick ascending limb (TAL), was reduced in the kidney of KO animals. Thus, claudin-12 is expressed in the PT, where it confers paracellular calcium permeability. In the absence of claudin-12, reduced claudin-14 expression in the TAL may compensate for reduced PT calcium reabsorption.

Prostaglandin E2 receptor EP1 (PGE2/EP1) deletion promotes glomerular podocyte and endothelial cell injury in hypertensive TTRhRen mice

Prostaglandin E2 receptor EP1 (PGE2/EP1) promotes diabetic renal injury, and EP1 receptor deletion improves hyperfiltration, albuminuria, and fibrosis. The role of EP1 receptors in hypertensive kidney disease (HKD) remains controversial. We examined the contribution of EP1 receptors to HKD. EP1 null (EP1−/−) mice were bred with hypertensive TTRhRen mice (Htn) to evaluate kidney function and injury at 24 weeks. EP1 deletion had no effect on elevation of systolic blood pressure in Htn mice (HtnEP1−/−) but resulted in pronounced albuminuria and reduced FITC-inulin clearance, compared with Htn or wild-type (WT) mice. Ultrastructural injury to podocytes and glomerular endothelium was prominent in HtnEP1−/− mice; including widened subendothelial space, subendothelial lucent zones and focal lifting of endothelium from basement membrane, with focal subendothelial cell debris. Cortex COX2 mRNA was increased by EP1 deletion. Glomerular EP3 mRNA was reduced by EP1 deletion, and EP4 by Htn and EP1 deletion. In WT mice, PGE2 increased chloride reabsorption via EP1 in isolated perfused thick ascending limb (TAL), but PGE2 or EP1 deletion did not affect vasopressin-mediated chloride reabsorption. In WT and Htn mouse inner medullary collecting duct (IMCD), PGE2 inhibited vasopressin-water transport, but not in EP1−/− or HtnEP1−/− mice. Overall, EP1 mediated TAL and IMCD transport in response to PGE2 is unaltered in Htn, and EP1 is protective in HKD.

Upregulation of renal Na–K–2Cl cotransporter 2 in obese diabetes mellitus via a vasopressin receptor 2-dependent pathway

Na–K–2Cl cotransporter 2 (NKCC2) in thick ascending limb (TAL) in the kidney plays a central role in tubuloglomerular feedback (TGF) system by sensing NaCl delivery to the distal tubules. Although accumulating data indicate that dysregulated TGF contributes to the progression of diabetic complications, the regulation of NKCC2 in diabetes mellitus (DM) remains unclear. We here show that NKCC2 is overactivated via a vasopressin receptor 2 (V2R)-dependent mechanism in db/db mice, a mouse model of obese DM. Compared with db/+ mice, we found that both aquaporin 2 and NKCC2 levels were significantly increased in the kidney in db/db mice. Immunohistochemical analysis of V2R and NKCC2 in the kidney demonstrated that V2R is present in the TAL, as well as in the collecting duct. Moreover, the administration of tolvaptan, a selective V2R antagonist, sharply decreased aquaporin 2 and NKCC2 in db/db mice, confirming the causal role of V2R signaling in NKCC2 induction in this model. Although tolvaptan reduced aquaporin 2 abundance also in db/+ mice, its effect on NKCC2 was modest compared with db/db mice. In total kidney lysates, uromodulin expression was not altered between db/+ and db/db mice, suggesting that V2R signaling alters NKCC2 without altering uromodulin levels. These data implicate the dysregulation of NKCC2 in the pathophysiology of type 2 DM, and underscore the complex nature of fluid volume disorders in diabetic kidney disease.