Mice lacking γENaC palmitoylation sites maintain benzamil-sensitive Na+ transport despite reduced channel activity.

Epithelial Na+ channels (ENaCs) control extracellular fluid volume by facilitating Na+ absorption across transporting epithelia. In vitro studies showed that Cys-palmitoylation of the γENaC subunit is a major regulator of channel activity. We tested whether γ subunit palmitoylation sites are necessary for channel function in vivo by generating mice lacking the palmitoylated cysteines (γC33A,C41A) using CRISPR/Cas9 technology. ENaCs in dissected kidney tubules from γC33A,C41A mice had reduced open probability compared with wild-type (WT) littermates maintained on either standard or Na+-deficient diets. Male mutant mice also had higher aldosterone levels than WT littermates following Na+ restriction. However, γC33A,C41A mice did not have reduced amiloride-sensitive Na+ currents in the distal colon or benzamil-induced natriuresis compared to WT mice. We identified a second, larger conductance cation channel in the distal nephron with biophysical properties distinct from ENaC. The activity of this channel was higher in Na+-restricted γC33A,C41A versus WT mice and was blocked by benzamil, providing a possible compensatory mechanism for reduced prototypic ENaC function. We conclude that γ subunit palmitoylation sites are required for prototypic ENaC activity in vivo but are not necessary for amiloride/benzamil-sensitive Na+ transport in the distal nephron or colon.

Influence of glycoprotein MUC1 on trafficking of the Ca2+-selective ion channels, TRPV5 and TRPV6, and on in vivo calcium homeostasis.

Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5, in cultured unpolarized cells. Using biotinylation of cell surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Our results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, we found that mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+. In summary, our results provide further insight into the role of MUC1 in Ca2+-selective TRP channel endocytosis and the overall effects on Ca concentrations.

Suppression of NRF2 Activity by HIF-1α Promotes Fibrosis after Ischemic Acute Kidney Injury.

Acute kidney injury (AKI) is a rapid decline in renal function and can occur after ischemia/reperfusion injury (IRI) to the tubular epithelia. The nuclear factor erythroid-2-related factor 2 (NRF2) pathway protects against AKI and AKI-to-chronic kidney disease (CKD) progression, but we previously demonstrated that severe IRI maladaptively reduced NRF2 activity in mice. To understand the mechanism of this response, we subjected C57BL/6J mice to unilateral kidney IRI with ischemia times that were titrated to induce mild to severe injury. Mild IRI increased NRF2 activity and was associated with renal recovery, whereas severe IRI decreased NRF2 activity and led to progressive CKD. Due to these effects of ischemia, we tested the hypothesis that hypoxia-inducible factor-1α (HIF-1α) mediates NRF2 activity. To mimic mild and severe ischemia, we activated HIF-1α in HK-2 cells in nutrient-replete or nutrient-deficient conditions. HIF-1α activation in nutrient-replete conditions enhanced NRF2 nuclear localization and activity. However, in nutrient-deficient conditions, HIF-1α activation suppressed NRF2 nuclear localization and activity. Nuclear localization was rescued with HIF-1α siRNA knockdown. Our results suggest that severe ischemic AKI leads to HIF-1α-mediated suppression of NRF2, leading to AKI-to-CKD progression.

Cloning, Expression, and Purification of Galectins for In Vitro Studies.

Galectins are best known for their ability to bind glycoconjugates containing ß-galactose, but classification of these small proteins within the galectin family is also defined by amino acid homology within structural domains and exon/intron junctions within genes. As galectins are expressed by organisms as diverse as some fungi, C. elegans, fish, birds and mammals, and biological activities attributed to galectins are equally diverse, it becomes essential to identify, clone, and characterize galectins from many sources. Glutathione S-transferase (GST) fused to the amino-terminus of galectin cDNAs has proven to be especially useful for the preparation of recombinant galectins in bacteria for use on glycan arrays, in experiments with cultured or isolated cells, and in pull-down assays with immunopurified glycoproteins. Many galectins are stabilized by reducing reagents, such that binding and elution of GST-galectins from glutathione-conjugated Sepharose with excess glutathione is both efficient and innocuous. The ability to bind and elute GST-galectins from lactose-conjugated Sepharose with excess lactose provides a relatively easy means to insure that galectins are competent for glycoconjugate binding prior to experimentation. This chapter focuses primarily on the varied approaches to use GST-galectin binding to glutathione- and lactose-conjugated Sepharose to purify recombinant galectins and then develop effective experimental protocols to characterize the specificity, interactions and function of galectins cloned from any source. We provide one example where a pull-down assay with all the GST-tagged canine galectins reveals that the C-terminal carbohydrate recognition domain of galectin-9 (Gal-9C) specifically recognizes the glycan-dependent apical targeting signal from the glycoprotein MUC1.

Plasma Mucin-1 (CA15-3) Levels in Autosomal Dominant Tubulointerstitial Kidney Disease due to MUC1 Mutations.

INTRODUCTION: Patients with ADTKD-MUC1 have one allele producing normal mucin-1 (MUC1) and one allele producing mutant MUC1, which remains intracellular. We hypothesized that ADTKD-MUC1 patients, who have only 1 secretory-competent wild-type MUC1 allele, should exhibit decreased plasma mucin-1 (MUC1) levels. To test this hypothesis, we repurposed the serum CA15-3 assay used to measure MUC1 in breast cancer to measure plasma MUC1 levels in ADTKD-MUC1. METHODS: This cross-sectional study analyzed CA15-3 levels in a reference population of 6,850 individuals, in 85 individuals with ADTKD-MUC1, and in a control population including 135 individuals with ADTKD-UMOD and 114 healthy individuals. RESULTS: Plasma CA15-3 levels (mean ± standard deviation) were 8.6 ± 4.3 U/mL in individuals with ADTKD-MUC1 and 14.6 ± 5.6 U/mL in controls (p < 0.001). While there was a significant difference in mean CA15-3 levels, there was substantial overlap between the 2 groups. Plasma CA15-3 levels were <5 U/mL in 22% of ADTKD-MUC1 patients, in 0/249 controls, and in 1% of the reference population. Plasma CA15-3 levels were >20 U/mL in 1/85 ADTKD-MUC1 patients, in 18% of control individuals, and in 25% of the reference population. Segregation of plasma CA15-3 levels by the rs4072037 genotype did not significantly improve differentiation between affected and unaffected individuals. CA15-3 levels were minimally affected by gender and estimated glomerular filtration rate. DISCUSSION/CONCLUSIONS: Plasma CA15-3 levels in ADTKD-MUC1 patients are approximately 40% lower than levels in healthy individuals, though there is significant overlap between groups. Further investigations need to be performed to see if plasma CA15-3 levels would be useful in diagnosis, prognosis, or assessing response to new therapies in this disorder.

KIM-1-mediated anti-inflammatory activity is preserved by MUC1 induction in the proximal tubule during ischemia-reperfusion injury.

Cell-associated kidney injury molecule-1 (KIM-1) exerts an anti-inflammatory role following kidney injury by mediating efferocytosis and downregulating the NF-κB pathway. KIM-1 cleavage blunts its anti-inflammatory activities. We reported that mucin 1 (MUC1) is protective in a mouse model of ischemia-reperfusion injury (IRI). As both KIM-1 and MUC1 are induced in the proximal tubule (PT) during IRI and are a disintegrin and metalloprotease 17 (ADAM17) substrates, we tested the hypothesis that MUC1 protects KIM-1 activity. Muc1 knockout (KO) mice and wild-type (WT) littermates were subjected to IRI. KIM-1, MUC1, and ADAM17 levels (and signaling pathways) were assessed by immunoblot analysis. PT localization was assessed by confocal microscopy and an in situ proximity ligation assay. Findings were extended using human kidneys and urine as well as KIM-1-mediated efferocytosis assays in mouse PT cultures. In response to tubular injury in mouse and human kidneys, we observed induction and coexpression of KIM-1 and MUC1 in the PT. Compared with WT mice, Muc1 KO mice had higher urinary KIM-1 and lower kidney KIM-1. KIM-1 was apical in the PT of WT kidneys but predominately with luminal debris in Muc1 KO mice. Efferocytosis was reduced in Muc1 KO PT cultures compared with WT cultures, whereas inflammation was increased in Muc1 KO kidneys compared with WT kidneys. MUC1 was cleaved by ADAM17 in PT cultures and blocked KIM-1 shedding in Madin-Darby canine kidney cells. We conclude that KIM-1-mediated efferocytosis and thus anti-inflammatory activity during IRI is preserved in the injured kidney by MUC1 inhibition of KIM-1 shedding.NEW & NOTEWORTHY KIM-1 plays a key role in the recovery of the tubule epithelium during renal IRI by mediating efferocytosis and associated signaling that suppresses inflammation. Excessive cleavage of KIM-1 by ADAM17 provides a decoy receptor that aggravates efferocytosis and subsequent signaling. Our data from experiments in mice, patients, and cultured cells show that MUC1 is also induced during IRI and competes with KIM-1 for cleavage by ADAM17. Consequently, MUC1 protects KIM-1 anti-inflammatory activity in the damaged kidney.

Small Molecule Targets TMED9 and Promotes Lysosomal Degradation to Reverse Proteinopathy.

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.

Sialylation of MUC4ß N-glycans by ST6GAL1 orchestrates human airway epithelial cell differentiation associated with type-2 inflammation.

Although type-2-induced (T2-induced) epithelial dysfunction is likely to profoundly alter epithelial differentiation and repair in asthma, the mechanisms for these effects are poorly understood. A role for specific mucins, heavily N-glycosylated epithelial glycoproteins, in orchestrating epithelial cell fate in response to T2 stimuli has not previously been investigated. Levels of a sialylated MUC4ß isoform were found to be increased in airway specimens from asthmatic patients in association with T2 inflammation. We hypothesized that IL-13 would increase sialylation of MUC4ß, thereby altering its function and that the ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) would regulate the sialylation. Using human biologic specimens and cultured primary human airway epithelial cells (HAECs),we demonstrated that IL-13 increases ST6GAL1-mediated sialylation of MUC4ß and that both were increased in asthma, particularly in sputum supernatant and/or fresh isolated HAECs with elevated T2 biomarkers. ST6GAL1-induced sialylation of MUC4ß altered its lectin binding and secretion. Both ST6GAL1 and MUC4ß inhibited epithelial cell proliferation while promoting goblet cell differentiation. These in vivo and in vitro data provide strong evidence for a critical role for ST6GAL1-induced sialylation of MUC4ß in epithelial dysfunction associated with T2-high asthma, thereby identifying specific sialylation pathways as potential targets in asthma.

Thumb domains of the three epithelial Na+ channel subunits have distinct functions.

The epithelial Na+ channel (ENaC) possesses a large extracellular domain formed by a ß-strand core enclosed by three peripheral α-helical subdomains, which have been dubbed thumb, finger, and knuckle. Here we asked whether the ENaC thumb domains play specific roles in channel function. To this end, we examined the characteristics of channels lacking a thumb domain in an individual ENaC subunit (α, ß, or γ). Removing the γ subunit thumb domain had no effect on Na+ currents when expressed in Xenopus oocytes, but moderately reduced channel surface expression. In contrast, ENaCs lacking the α or ß subunit thumb domain exhibited significantly reduced Na+ currents along with a large reduction in channel surface expression. Moreover, channels lacking an α or γ thumb domain exhibited a diminished Na+ self-inhibition response, whereas this response was retained in channels lacking a ß thumb domain. In turn, deletion of the α thumb domain had no effect on the degradation rate of the immature α subunit as assessed by cycloheximide chase analysis. However, accelerated degradation of the immature ß subunit and mature γ subunit was observed when the ß or γ thumb domain was deleted, respectively. Our results suggest that the thumb domains in each ENaC subunit are required for optimal surface expression in oocytes and that the α and γ thumb domains both have important roles in the channel's inhibitory response to external Na+ Our findings support the notion that the extracellular helical domains serve as functional modules that regulate ENaC biogenesis and activity.

Epithelial Na+ Channel Regulation by Extracellular and Intracellular Factors.

Epithelial Na+ channels (ENaCs) are members of the ENaC/degenerin family of ion channels that evolved to respond to extracellular factors. In addition to being expressed in the distal aspects of the nephron, where ENaCs couple the absorption of filtered Na+ to K+ secretion, these channels are found in other epithelia as well as nonepithelial tissues. This review addresses mechanisms by which ENaC activity is regulated by extracellular factors, including proteases, Na+, and shear stress. It also addresses other factors, including acidic phospholipids and modification of ENaC cytoplasmic cysteine residues by palmitoylation, which enhance channel activity by altering interactions of the channel with the plasma membrane.

N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression.

Epithelial Na+ channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αßγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, ß-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αßγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na+. The lack of N-linked glycans on the ß-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the ß-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type ß-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the ß-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.

Trial of Amiloride in Type 2 Diabetes with Proteinuria.

INTRODUCTION: Renal Na+ retention and extracellular fluid volume expansion are hallmarks of nephrotic syndrome, which occurs even in the absence of activation of hormones that stimulate renal Na+ transporters. Plasmin-dependent activation of the epithelial Na+ channel (ENaC) has been proposed to have a role in renal Na+ retention in the setting of nephrotic syndrome. We hypothesized that the ENaC inhibitor amiloride would be an effective therapeutic agent in inducing a natriuresis and lowering blood pressure in individuals with macroscopic proteinuria. METHODS: We conducted a pilot double-blind randomized cross-over study comparing the effects of daily administration of either oral amiloride or hydrochlorothiazide (HCTZ) to patients with type 2 diabetes and macroscopic proteinuria. Safety and efficacy were assessed by monitoring systolic blood pressure (SBP), kidney function, adherence, weight, urinary Na+ excretion and serum electrolytes. Nine subjects were enrolled in the trial. RESULTS: No significant difference in SBP or weight was seen between HCTZ and amiloride (p≥0.15). Amiloride induced differences in serum K+ (p<0.001), with a 0.88±0.30 mmol/L greater acute increase observed. Two subjects developed acute kidney injury and hyperkalemia when treated with amiloride. Four subjects had readily detectable levels of urinary plasminogen plus plasmin (uPl), and five did not. Changes in SBP in response to amiloride did not differ between individuals with vs. those without detectable uPl. CONCLUSION: In summary, among patients with type 2 diabetes, normal renal function and proteinuria, there were reductions in SBP in groups treated with HCTZ or amiloride. Acute kidney injury and severe hyperkalemia were safety concerns with amiloride.

Regulation of the epithelial Na+ channel by paraoxonase-2.

Paraoxonase-2 (PON-2) is a membrane-bound lactonase with unique anti-oxidative and anti-atherosclerotic properties. PON-2 shares key structural elements with MEC-6, an endoplasmic reticulum-resident molecular chaperone in Caenorhabditis elegans MEC-6 modulates the expression of a mechanotransductive ion channel comprising MEC-4 and MEC-10 in touch-receptor neurons. Because pon-2 mRNA resides in multiple rat nephron segments, including the aldosterone-sensitive distal nephron where the epithelial Na+ channel (ENaC) is expressed, we hypothesized that PON-2 would similarly regulate ENaC expression. We observed PON-2 expression in aquaporin 2-positive principal cells of the distal nephron of adult human kidney. PON-2 also co-immunoprecipitated with ENaC when co-expressed in HEK293 cells. When PON-2 was co-expressed with ENaC in Xenopus oocytes, ENaC activity was reduced, reflecting a reduction in ENaC surface expression. MEC-6 also reduced ENaC activity when co-expressed in Xenopus oocytes. The PON-2 inhibitory effect was ENaC-specific, as PON-2 had no effect on functional expression of the renal outer medullary potassium channel. PON-2 did not alter the response of ENaC to extracellular Na+, mechanical shear stress, or α-chymotrypsin-mediated proteolysis, suggesting that PON-2 did not alter the regulation of ENaC by these factors. Together, our data suggest that PON-2 regulates ENaC activity by modulating its intracellular trafficking and surface expression.

Novel roles for mucin 1 in the kidney.

PURPOSE OF REVIEW: Recent studies in the kidney have revealed that the well characterized tumor antigen mucin 1 (MUC1/Muc1) also has numerous functions in the normal and injured kidney. RECENT FINDINGS: Mucin 1 is a transmembrane mucin with a robust glycan-dependent apical targeting signal and efficient recycling from endosomes. It was recently reported that the TRPV5 calcium channel is stabilized on the cell surface by galectin-dependent cross-linking to mucin 1, providing a novel mechanism for regulation of ion channels and normal electrolyte balance.Our recent studies in mice show that Muc 1 is induced after ischemia, stabilizing hypoxia-inducible factor 1 (HIF-1)α and ß-catenin levels, and transactivating the HIF-1 and ß-catenin protective pathways. However, prolonged induction of either pathway in the injured kidney can proceed from apparent full recovery to chronic kidney disease. A very recent report indicates that aberrant activation of mucin 1 signaling after ischemic injury in mice and humans is associated with development of chronic kidney disease and fibrosis. A frameshift mutation in MUC1 was recently identified as the genetic lesion causing medullary cystic kidney disease type 1, now appropriately renamed MUC1 Kidney Disease. SUMMARY: Studies of mucin 1 in the kidney now reveal significant functions for the extracellular mucin-like domain and signaling through the cytoplasmic tail.

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel.

The epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (α, ß, and γ). We previously reported that cytoplasmic residues on the ß (ßCys-43 and ßCys-557) and γ (γCys-33 and γCys-41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, γ subunit palmitoylation had a dominant role over ß subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in Xenopus oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7, and 14. ENaC activation by DHHCs was lost when γ subunit palmitoylation sites were mutated, whereas DHHCs 1, 2, and 14 still activated ENaC lacking ß subunit palmitoylation sites. ß subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking ß and γ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCDcl1 cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCDcl1 cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na+ currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.

Muc1 enhances the ß-catenin protective pathway during ischemia-reperfusion injury.

The hypoxia-inducible factor (HIF)-1 and ß-catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine mucin (Muc)1 protects kidney function and morphology in a mouse model of ischemia-reperfusion injury (IRI) by stabilizing HIF-1α, enhancing HIF-1 downstream signaling, and thereby preventing metabolic stress (Pastor-Soler et al. Muc1 is protective during kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 308: F1452-F1462, 2015). We asked if Muc1 regulates the ß-catenin protective pathway during IRI as 1) ß-catenin nuclear targeting is MUC1 dependent in cultured human cells, 2) ß-catenin is found in coimmunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, and 3) MUC1 prevents ß-catenin phosphorylation by glycogen synthase kinase (GSK)3ß and thereby ß-catenin degradation. Using the same mouse model of IRI, we found that levels of active GSK3ß were significantly lower in kidneys of control mice compared with Muc1 knockout (KO) mice. Consequently, ß-catenin was significantly upregulated at 24 and 72 h of recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both ß-catenin induction and nuclear targeting were absent in Muc1 KO mice. We also found downstream induction of ß-catenin prosurvival factors (activated Akt, survivin, transcription factor T cell factor 4 (TCF4), and its downstream target cyclin D1) and repression of proapoptotic factors (p53, active Bax, and cleaved caspase-3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during acute kidney injury proceeds by enhancing both the HIF-1 and ß-catenin protective pathways.

RNA-seq analysis of glycosylation related gene expression in STZ-induced diabetic rat kidney inner medulla.

UNLABELLED: The UT-A1 urea transporter is crucial to the kidney's ability to generate concentrated urine. Native UT-A1 from kidney inner medulla (IM) is a heavily glycosylated protein with two glycosylation forms of 97 and 117 kDa. In diabetes, UT-A1 protein abundance, particularly the 117 kD isoform, is significantly increased corresponding to an increased urea permeability in perfused IM collecting ducts, which plays an important role in preventing the osmotic diuresis caused by glucosuria. However, how the glycan carbohydrate structure change and the glycan related enzymes regulate kidney urea transport activity, particularly under diabetic condition, is largely unknown. In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions. These changes were accompanied by altered UT-A1 association with the galectin proteins, ß-galactoside glycan binding proteins. To explore the molecular basis of the alterations of glycan structures, the highly sensitive next generation sequencing (NGS) technology, Illumina RNA-seq, was employed to analyze genes involved in the process of UT-A1 glycosylation using streptozotocin (STZ)-induced diabetic rat kidney. Differential gene expression analysis combining with quantitative PCR revealed that expression of a number of important glycosylation related genes were changed under diabetic conditions. These genes include the glycosyltransferase genes Mgat4a, the sialylation enzymes St3gal1 and St3gal4 and glycan binding protein galectin-3, -5, -8, and -9. In contrast, although highly expressed in kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes. CONCLUSIONS: In diabetes, not only is UT-A1 protein abundance increased but the protein's glycan structure is also significantly changed. UT-A1 protein becomes highly sialylated, fucosylated and branched. Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions. The alteration of these genes may contribute to changes in the UT-A1 glycan structure and therefore modulate kidney urea transport activity and alleviate osmotic diuresis caused by glucosuria in diabetes.

Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain.

The extracellular regions of epithelial Na(+) channel subunits are highly ordered structures composed of domains formed by α helices and ß strands. Deletion of the peripheral knuckle domain of the α subunit in the αßγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na(+) (Na(+) self-inhibition). In contrast, deletion of either the ß or γ subunit knuckle domain within the αßγ trimer dramatically reduces epithelial Na(+) channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na(+) self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na(+) self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the ß or γ subunit knuckle domain resulted in little or no change in Na(+) self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na(+) self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.

Muc1 is protective during kidney ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1α, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells.

Intracellular Na+ regulates epithelial Na+ channel maturation.

Epithelial Na(+) channel (ENaC) function is regulated by the intracellular Na(+) concentration ([Na(+)]i) through a process known as Na(+) feedback inhibition. Although this process is known to decrease the expression of proteolytically processed active channels on the cell surface, it is unknown how [Na(+)]i alters ENaC cleavage. We show here that [Na(+)]i regulates the posttranslational processing of ENaC subunits during channel biogenesis. At times when [Na(+)]i is low, ENaC subunits develop mature N-glycans and are processed by proteases. Conversely, glycan maturation and sensitivity to proteolysis are reduced when [Na(+)]i is relatively high. Surface channels with immature N-glycans were not processed by endogenous channel activating proteases, nor were they sensitive to cleavage by exogenous trypsin. Biotin chase experiments revealed that the immature surface channels were not converted into mature cleaved channels following a reduction in [Na(+)]i. The hypothesis that [Na(+)]i regulates ENaC maturation within the biosynthetic pathways is further supported by the finding that Brefeldin A prevented the accumulation of processed surface channels following a reduction in [Na(+)]i. Therefore, increased [Na(+)]i interferes with ENaC N-glycan maturation and prevents the channel from entering a state that allows proteolytic processing.