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Abstract Insulin-like growth factor (IGF) signaling is required for proper growth and skeletal development in vertebrates. Consequently, its dysregulation may lead to abnormalities of growth or skeletal structures. IGF is involved in the regulation of cell proliferation and differentiation of chondrocytes. However, the availability of bioactive IGF may be controlled by antagonizing IGF binding proteins (IGFBPs) in the circulation and tissues. As the metalloproteinase PAPP-A specifically cleaves members of the IGFBP family, we hypothesized that PAPP-A activity liberates bioactive IGF in cartilage. In PAPP-A knockout mice, the femur length was reduced and the mice showed a disorganized columnar organization of growth plate chondrocytes. Similarly, zebrafish lacking pappaa showed reduced length of Meckel’s cartilage and disorganized chondrocytes, reminiscent of the mouse knockout phenotype. Expression of chondrocyte differentiation markers (sox9a, ihha, and col10a1) was markedly affected in Meckel’s cartilage of pappaa knockout zebrafish, indicating that differentiation of chondrocytes was compromised. Additionally, the zebrafish pappaa knockout phenotype was mimicked by pharmacological inhibition of IGF signaling, and it could be rescued by treatment with exogenous recombinant IGF-I. In conclusion, our data suggests that IGF activity in the growing cartilage, and hence IGF signaling in chondrocytes, requires the presence of PAPP-A. The absence of PAPP-A causes aberrant chondrocyte organization and compromised growth in both mice and zebrafish.

Abstract DNA is a danger signal sensed by cGAS to engage signaling through STING to activate innate immune functions. The best-studied downstream responses to STING activation include expression of type I interferon and inflammatory genes, but STING also activates other pathways, including apoptosis. Here, we report that STING-dependent induction of apoptosis in macrophages occurs through the intrinsic mitochondrial pathway and is mediated via IRF3 but acts independently of gene transcription. By intersecting four mass spectrometry datasets, we identify SAM68 as crucial for the induction of apoptosis downstream of STING activation. SAM68 is essential for the full activation of apoptosis. Still, it is not required for STING-mediated activation of IFN expression or activation of NF-κB. Mechanistic studies reveal that protein trafficking is required and involves SAM68 recruitment to STING upon activation, with the two proteins associating at the Golgi or a post-Golgi compartment. Collectively, our work identifies SAM68 as a STING-interacting protein enabling induction of apoptosis through this DNA-activated innate immune pathway.

Background Inhibition of prostaglandin synthesis by nonsteroidal anti‐inflammatory drugs is associated with cardiovascular mortality and kidney disease. This study hypothesizes that urinary prostaglandin E2 (PGE2) and PGE2 metabolite (PGEM) excretions are markers of cardiovascular and kidney health, because they reflect both systemic and kidney‐derived PGE2 production. Methods and Results PGE2 and PGEM were measured in spot urine samples from 2291 participants (≥55 years old) of the population‐based Rotterdam Study. Urinary PGE2 and PGEM excretions were analyzed using linear regression analyses to identify cross‐sectional associations with cardiovascular risk factors and baseline estimated glomerular filtration rate (eGFR). Longitudinal associations with cardiovascular mortality and kidney outcomes (eGFR

Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine kinase/STE20/SPS1-related proline-alanine–rich protein kinase (WNK/SPAK) pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high-potassium “DASH-like” diets (dietary approaches to stop hypertension) inactivate the cotransporter and whether this decreases BP. A transcriptomics screen identified Ppp1Ca, encoding PP1A, as a potassium-upregulated gene, and its negative regulator Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK/SPAK kinase cascade, we confirmed that PP1A dephosphorylated NCC directly in a potassium-regulated manner. Prior adaptation to a high-potassium diet was required to maximally dephosphorylate NCC and lower BP in constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a and dephosphorylation of its cognate protein, inhibitory subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drove NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK/SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.

Abstract The corticomedullary osmotic gradient between renal cortex and medulla induces a specific spatial gene expression pattern. The factors that controls these differences are not fully addressed. Adaptation to hypertonic environment is mediated by the actions of the nuclear factor of activated T-cells 5 (NFAT5). NFAT5 induces the expression of genes that lead to intracellular accumulation of organic osmolytes. However, a systematical analysis of the NFAT5-dependent gene expression in the kidneys was missing. We used primary cultivated inner medullary collecting duct (IMCD) cells from control and NFAT5 deficient mice as well as renal cortex and inner medulla from principal cell specific NFAT5 deficient mice for gene expression profiling. In primary NFAT5 deficient IMCD cells, hyperosmolality induced changes in gene expression were abolished. The majority of the hyperosmolality induced transcripts in primary IMCD culture were determined to have the greatest expression in the inner medulla. Loss of NFAT5 altered the expression of more than 3000 genes in the renal cortex and more than 5000 genes in the inner medulla. Gene enrichment analysis indicated that loss of NFAT5 is associated with renal inflammation and increased expression of kidney injury marker genes, like lipocalin-2 or kidney injury molecule-1. In conclusion we show that NFAT5 is a master regulator of gene expression in the kidney collecting duct and in vivo loss of NFAT function induces a kidney injury like phenotype.

Dietary potassium (K+) supplementation is associated with a lowering effect in blood pressure (BP), but not all studies agree. Here, we examined the effects of short- and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) diet for 7 weeks or fed a K+-free diet for 2 weeks. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during the night) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels but in increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health.

Na+ and K+ balance is influenced by the activity of the sodium chloride cotransporter NCC in the distal convoluted tubule. NCC activity and abundance are reduced by high extracellular K+. The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4–2 (Nedd4-2) has been proposed as a modulator of NCC abundance. Here, we examined the functional role of Nedd4-2 on NCC regulation and whether Nedd4-2 is important for the effects of high extracellular K+ on NCC. Total and plasma membrane levels of ubiquitylated NCC were lower in NCC-expressing MDCKI cells after Nedd4-2 deletion. NCC and phosphorylated NCC (pT58-NCC) levels were higher after Nedd4-2 deletion, and NCC levels on the plasma membrane were elevated. No significant changes were seen after Nedd4-2 knockdown in the levels of SPAK and phosphorylated SPAK (pS373-SPAK), the major NCC regulatory kinase. Nedd4-2 deficiency had no effect on the internalization rate of NCC from the plasma membrane, but NCC protein half-life was increased. In ex vivo experiments with kidney tubule suspensions from Nedd4-2 knockout (KO) mice, high K+ reduced total and pT58-NCC regardless of genotype. We conclude that Nedd4-2 is involved in ubiquitylation of NCC and modulating its plasma membrane levels and degradation. However, Nedd4-2 does not appear to be important for K+ induced reductions in NCC abundance.

The thiazide-sensitive sodium chloride cotransporter (NCC), expressed in the renal distal convoluted tubule, plays a major role in Na+, Cl- and K+ homeostasis and blood pressure as exemplified by the symptoms of patients with non-functional NCC and Gitelman syndrome. NCC activity is modulated by a variety of hormones, but is also influenced by the extracellular K+ concentration. The putative “renal-K+ switch” mechanism is a relatively cohesive model that links dietary K+ intake to NCC activity, and may offer new targets for blood pressure control. However, a remaining hurdle for full acceptance of this model is the lack of human data to confirm molecular findings from animal models. Extracellular vesicles (EVs) have attracted attention from the scientific community due to their potential roles in intercellular communication, disease pathogenesis, drug delivery and as possible reservoirs of biomarkers. Urinary EVs (uEVs) are an excellent sample source for the study of physiology and pathology of renal, urothelial and prostate tissues, but the diverse origins of uEVs and their dynamic molecular composition present both methodological and data interpretation challenges. This review provides a brief overview of the state-of-the-art, challenges and knowledge gaps in current uEV-based analyses, with a focus on the application of uEVs to study the “renal-K+ switch” and NCC regulation. We also provide recommendations regarding biospecimen handling, processing and reporting requirements to improve experimental reproducibility and interoperability towards the realisation of the potential of uEV-derived biomarkers in hypertension and clinical practice.

Salmonella enterica is, globally, an important cause of human illness with beef being a significant attributable source. In the human patient, systemic Salmonella infection requires antibiotic therapy, and when strains are multidrug resistant (MDR), no effective treatment may be available. MDR in bacteria is often associated with the presence of mobile genetic elements (MGE) that mediate horizontal spread of antimicrobial resistance (AMR) genes. In this study, we sought to determine the potential relationship of MDR in bovine Salmonella isolates with MGE. The present study involved 111 bovine Salmonella isolates obtained collectively from specimens derived from healthy cattle or their environments at Midwestern U.S. feedyards (2000–2001, n = 19), or specimens from sick cattle submitted to the Nebraska Veterinary Diagnostic Center (2010–2020, n = 92). Phenotypically, 33/111 isolates (29.7%) were MDR (resistant to ≥3 drug classes). Based on whole-genome sequencing (WGS; n = 41) and PCR (n = 111), a MDR phenotype was strongly associated (OR = 186; p < 0.0001) with carriage of ISVsa3, an IS91-like Family transposase. In all 41 isolates analyzed by WGS ((31 MDR and 10 non-MDR (resistant to 0–2 antibiotic classes)), MDR genes were associated with carriage of ISVsa3, most often on an IncC type plasmid carrying blaCMY-2. The typical arrangement was floR, tet(A), aph(6)-Id, aph(3″)-Ib, and sul2 flanked by ISVsa3. These results suggest that AMR genes in MDR S. enterica isolates of cattle are frequently associated with ISVsa3 and carried on IncC plasmids. Further research is needed to better understand the role of ISVsa3 in dissemination of MDR Salmonella strains.

Primary hyperaldosteronism (PA) is characterized by aldosterone excess and hypertension. This may be linked to increased renal Na+ reabsorption via the epithelial Na+ channel (ENaC) and the NaCl cotransporter (NCC). The majority of PA patients have normal plasma K+ levels, but a subset of cases are associated with hypokalemia. High NCC levels observed in long-term studies with aldosterone-infused rodents have been attributed to direct effects of aldosterone. Aldosterone can also increase active phosphorylated NCC (pT58-NCC) acutely. However, direct effects of aldosterone on NCC have been contested by recent studies indicating that it is rather an indirect effect of hypokalemia. We therefore set out to determine isolated long-term aldosterone and K+ effects on ENaC and NCC using various in vivo and ex vivo approaches. In mice, aldosterone-induced hypokalemia was prevented by simultaneous amiloride infusion, coupled to increased cleavage of α- and γENaC but no effect on NCC. Regression analyses of in vivo data showed a positive correlation between aldosterone/K+ and αENaC but a negative correlation with NCC and pT58-NCC. Ex vivo, exposure of kidney tubules for 21 h to aldosterone increased cleavage of αENaC and γENaC, but no effects were observed on NCC or pT58-NCC. Exposure of tubules to low K+ media reduced αENaC but increased NCC and pT58-NCC. As hypokalemia can enhance cell proliferation markers in the distal convoluted tubule (DCT), we hypothesized that aldosterone infusion would increase proliferating cell nuclear antigen (PCNA) expression. Infusion of aldosterone in mice for 6 days greatly increased PCNA expression in the DCT. Collectively, in vivo and ex vivo data suggest that both aldosterone and K+ can increase ENaC directly. In contrast, the observed increase in abundance and phosphorylation of NCC in aldosterone-infused mice is likely an indirect effect of enhanced ENaC-mediated K+ secretion and subsequent hypokalemia. Thus, it is possible that NCC may only be increased in PA when the condition is associated with hypokalemia.

Abstract Background Genetic disruption of slc4a10, which encodes the sodium-dependent chloride/bicarbonate exchanger Ncbe, leads to a major decrease in Na+-dependent HCO3 − import into choroid plexus epithelial cells in mice and to a marked reduction in brain intraventricular fluid volume. This suggests that Ncbe functionally is a key element in vectorial Na+ transport and thereby for cerebrospinal fluid secretion in the choroid plexus. However, slc4a10 disruption results in severe changes in expression of Na+,K+-ATPase complexes and other major transport proteins, indicating that profound cellular changes accompany the genetic manipulation. Methods A tandem mass tag labeling strategy was chosen for quantitative mass spectrometry. Alterations in the broader patterns of protein expression in the choroid plexus in response to genetic disruption of Ncbe was validated by semi-quantitative immunoblotting, immunohistochemistry and morphometry. Results The abundance of 601 proteins were found significantly altered in the choroid plexus from Ncbe ko mice relative to Ncbe wt. In addition to a variety of transport proteins, particularly large changes in the abundance of proteins involved in cellular energy metabolism were detected in the Ncbe ko mice. In general, the abundance of rate limiting glycolytic enzymes and several mitochondrial enzymes were reduced following slc4a10 disruption. Surprisingly, this was accompanied by increased ATP levels in choroid plexus cells, indicating that the reduction in capacity for energy metabolism was adaptive to high ATP rather than causal for a decreased capacity for ion and water transport. Ncbe-deficient cells also had a reduced cell area and decreased K+ content. Conclusion Our findings suggest that the lack of effective Na+-entry into the epithelial cells of the choroid plexus leads to a profound change in the cellular phenotype, shifting from a high-rate secretory function towards a more dormant state; similar to what is observed during ageing or Alzheimer’s disease.

Arginine vasopressin (AVP) stimulates the concentration of renal urine by increasing the principal cell expression of aquaporin-2 (AQP2) water channels. Prostaglandin E2 (PGE2) and prostaglandin2α (PGF2α) increase the water absorption of the principal cell without AVP, but PGE2 decreases it in the presence of AVP. The underlying mechanism of this paradoxical response was investigated here. Mouse cortical collecting duct (mkpCCDc14) cells mimic principal cells as they endogenously express AQP2 in response to AVP. PGE2 increased AQP2 abundance without desmopressin (dDAVP), while in the presence of dDAVP, PGE2, and PGF2α reduced AQP2 abundance. dDAVP increased the cellular PGD2 and PGE2 release and decreased the PGF2α release. MpkCCD cells expressed mRNAs for the receptors of PGE2 (EP1/EP4), PGF2 (FP), and TxB2 (TP). Incubation with dDAVP increased the expression of EP1 and FP but decreased the expression of EP4. In the absence of dDAVP, incubation of mpkCCD cells with an EP4, but not EP1/3, agonist increased AQP2 abundance, and the PGE2-induced increase in AQP2 was blocked with an EP4 antagonist. Moreover, in the presence of dDAVP, an EP1/3, but not EP4, agonist decreased the AQP2 abundance, and the addition of EP1 antagonists prevented the PGE2-mediated downregulation of AQP2. Our study shows that in mpkCCDc14 cells, reduced EP4 receptor and increased EP1/FP receptor expression by dDAVP explains the differential effects of PGE2 and PGF2α on AQP2 abundance with or without dDAVP. As the V2R and EP4 receptor, but not the EP1 and FP receptor, can couple to Gs and stimulate the cyclic adenosine monophosphate (cAMP) pathway, our data support a view that cells can desensitize themselves for receptors activating the same pathway and sensitize themselves for receptors of alternative pathways.

Protein post-translational modification by the Small Ubiquitin-like MOdifier (SUMO) on lysine residues is a reversible process highly important for transcription and protein stability. In the kidney, SUMOylation appears to be important for the cellular response to aldosterone. Therefore, in this study, we generated a SUMOylation profile of the aldosterone-sensitive kidney distal convoluted tubule (DCT) as a basis for understanding SUMOylation events in this cell type. Using mass spectrometry-based proteomics, 1037 SUMO1 and 552 SUMO2 sites, corresponding to 546 SUMO1 and 356 SUMO2 proteins, were identified from a modified mouse kidney DCT cell line (mpkDCT). SUMOylation of the renal hydrogen-coupled oligopeptide and drug co-transporter (Pept2) at one site (K139) was found to be highly regulated by aldosterone. Using immunolabelling of mouse kidney sections Pept2 was localized to DCT cells in vivo. Aldosterone stimulation of mpkDCT cell lines expressing wild-type Pept2 or mutant K139R-Pept2, post-transcriptionally increased Pept2 expression up to four-fold. Aldosterone decreased wild-type Pept2 abundance in the apical membrane domain of mpkDCT cells, but this response was absent in K139R-Pept2 expressing cells. In summary, we have generated a SUMOylation landscape of the mouse DCT and determined that SUMOylation plays an important role in the physiological regulation of Pept2 trafficking by aldosterone.

Abstract Background Furosemide inhibits the sodium potassium chloride cotransporter (NKCC2) in the thick ascending limb of the loop of Henle and increases urinary water and sodium excretion. This study investigates the effect of furosemide on body composition estimated with multifrequency bioimpedance spectroscopy (BIS) technique and urinary proteins from NKCC2. Methods This study is a randomized, placebo‐controlled, crossover study where healthy subjects received either placebo or 40 mg furosemide on two separate occasions, where body composition with BIS, renal function, proteins from tubular proteins that mediate sodium and water transport, and plasma concentrations of vasoactive hormones were measured before and after intervention. Results We observed an expected increased diuresis with a subsequent reduction in bodyweight of (−1.51 ± 0.36 kg, p

The connexin 37 (Cx37) channel is clustered at gap junctions between cells in the renal vasculature or the renal tubule where it is abundant in basolateral cell interdigitations and infoldings of epithelial cells in the proximal tubule, thick ascending limb, distal convoluted tubule and collecting duct; however, physiological data regarding its role are limited. In this study, we investigated the role of Cx37 in fluid homeostasis using mice with a global deletion of Cx37 (Cx37-/- mice). Under baseline conditions, Cx37-/- had ~40% higher fluid intake associated with ~40% lower urine osmolality compared to wild-type (WT) mice. No differences were observed between genotypes in urinary adenosine triphosphate or prostaglandin E2, paracrine factors that alter renal water handling. After 18-hours of water deprivation, plasma aldosterone and urine osmolality increased significantly in Cx37-/- and WT mice; however, the latter remained ~375 mmol/kg lower in Cx37-/- mice, an effect associated with a more pronounced body weight loss despite higher urinary AVP/creatinine ratios compared to WT mice. Consistent with this, fluid intake in the first 3 hours after water deprivation was 37% greater in Cx37-/- vs WT mice. Cx37-/- mice showed significantly lower renal AQP2 abundance and AQP2 phosphorylation at serine 256 than WT mice in response to vehicle or dDAVP, suggesting a partial contribution of the kidney to the lower urine osmolality. The abundance and responses of the vasopressin V2 receptor, AQP3, NHE3, NKCC2, NCC, H+-ATPase, αENaC, γENaC or Na+/K+-ATPase were not significantly different between genotypes. In summary, these results demonstrate that Cx37 is important for body water handling.

The thiazide-sensitive sodium chloride cotransporter (NCC) plays a vital role in maintaining sodium (Na+) and potassium (K+) homeostasis. NCC activity is modulated by with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which is controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC (phosphorylated and total) and active (neddylated) forms of cullins (Cul1, 3, 4, and 5). High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/Δ9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect cullin neddylation levels in ex vivo renal tubules. In the short term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan-cullin inhibitor), but the response to low extracellular K+ was absent. In the long term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation, and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.

(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, β1, β2, β3, and phospholemman. The α1, α2, β1, and β2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.

Animal studies have shown that the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus can activate the thiazide-sensitive NaCl cotransporter (NCC). A common side effect of CNIs is hypertension. Renal salt transporters such as NCC are excreted in urinary extracellular vesicles (uEVs) after internalization into multivesicular bodies. Human studies indicate that CNIs also increase NCC abundance in uEVs, but results are conflicting and no relationship with NCC function has been shown. Therefore, we investigated the effects of CsA and Tac on the abundance of both total NCC (tNCC) and phosphorylated NCC at Thr60 phosphorylation site (pNCC) in uEVs, and assessed whether NCC abundance in uEVs predicts the blood pressure response to thiazide diuretics. Our results show that in kidney transplant recipients treated with cyclosporine (n = 9) or tacrolimus (n = 23), the abundance of both tNCC and pNCC in uEVs is 4-5 fold higher than in CNI-free kidney transplant recipients (n = 13) or healthy volunteers (n = 6). In hypertensive kidney transplant recipients, higher abundances of tNCC and pNCC prior to treatment with thiazides predicted the blood pressure response to thiazides. During thiazide treatment, the abundance of pNCC in uEVs increased in responders (n = 10), but markedly decreased in non-responders (n = 8). Thus, our results show that CNIs increase the abundance of both tNCC and pNCC in uEVs, and these increases correlate with the blood pressure response to thiazides. This implies that assessment of NCC in uEVs could represent an alternate method to guide anti-hypertensive therapy in kidney transplant recipients.

Aquaporin 2 (AQP2) mediates the osmotic water permeability of the kidney collecting duct in response to arginine vasopressin (VP) and is essential for body water homeostasis. VP effects on AQP2 occur via long-term alterations in AQP2 abundance and short-term changes in AQP2 localization. Several of the effects of VP on AQP2 are dependent on AQP2 phosphorylation and ubiquitylation; post-translational modifications (PTM) that modulate AQP2 subcellular distribution and function. Although several protein kinases, phosphatases, and ubiquitin E3 ligases have been implicated in AQP2 PTM, how AQP2 is deubiquitylated or the role of deubiquitylases (DUBS) in AQP2 function is unknown. Here, we report a novel role of the ubiquitin-specific protease USP4 in modulating AQP2 function. USP4 co-localized with AQP2 in the mouse kidney, and in mpkCCD14 cells USP4 and AQP2 abundance are increased by VP. AQP2 and USP4 co-immunoprecipitated from mpkCCD14 cells and mouse kidney, and in vitro, USP4 can deubiquitylate AQP2. In mpkCCD14 cells, shRNA mediated knockdown of USP4 decreased AQP2 protein abundance, whereas no changes in AQP2 mRNA levels or VP-induced cAMP production were detected. VP-induced AQP2 membrane accumulation in knockdown cells was significantly reduced, which was associated with higher levels of ubiquitylated AQP2. AQP2 protein half-life was also significantly reduced in USP4 knockdown cells. Taken together, the data suggest that USP4 is a key regulator of AQP2 deubiquitylation and that loss of USP4 leads to increased AQP2 ubiquitylation, decreased AQP2 levels, and decreased cell surface AQP2 accumulation upon VP treatment. These studies have implications for understanding body water homeostasis.

In normal individuals, the epithelium of the colon absorbs 1.5-2 L of water a day to generate dehydrated feces. However, in the condition of bile acid malabsorption (BAM), an excess of bile acids in the colon results in diarrhea. Several studies have attempted to address the mechanisms contributing to BAM induced by various bile acids. However, none have addressed a potential dysregulation of aquaporin water channels, which are responsible for the majority of transcellular water transport in epithelial cells, as a contributing factor to the onset of diarrhea and the pathogenesis of BAM. In this study we aimed to systematically analyze the expression of AQPs in colonic epithelia from rat, mouse and human and determine whether their expression is altered in a rat model of BAM. Mass spectrometry-based proteomics, RT-PCR, and western blotting identified various AQPs in isolated colonic epithelial cells from rats (AQP1, 3, 4, 7, 8) and mice (AQP1, 4, 8). Several AQPs were also detected in human colon (AQP1, 3, 4, 7-9). Immunohistochemistry localized AQP1 to the apical plasma membrane of epithelial cells in the bottom of the crypts, whereas AQP3 (rat, human) and AQP4 (mice, human) were localized predominantly in the basolateral plasma membrane. AQP8 was localized intracellularly and at the apical plasma membrane of epithelial cells. Rats fed sodium cholate for 72 h had significantly increased fecal water content, suggesting development of BAM associated diarrhea. Colonic epithelial cells isolated from this model had significantly altered levels of AQP3, 7, and 8, suggesting that these AQPs may be involved in the pathogenesis of bile acid induced diarrhea.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

Prolonged elevations of plasma aldosterone levels are associated with renal pathogenesis. We hypothesized that renal distress could be imposed by an augmented aldosterone-induced protein turnover challenging cellular protein degradation systems of the renal tubular cells. Cellular accumulation of specific protein aggregates in rat kidneys was assessed after 7 days of aldosterone administration. Aldosterone induced intracellular accumulation of 60 s ribosomal protein L22 in protein aggregates, specifically in the distal convoluted tubules. The mineralocorticoid receptor inhibitor spironolactone abolished aldosterone-induced accumulation of these aggregates. The aldosterone-induced protein aggregates also contained proteasome 20 s subunits. The partial de-ubiquitinase ataxin-3 was not localized to the distal renal tubule protein aggregates, and the aggregates only modestly colocalized with aggresome transfer proteins dynactin p62 and histone deacetylase 6. Intracellular protein aggregation in distal renal tubules did not lead to development of classical juxta-nuclear aggresomes or to autophagosome formation. Finally, aldosterone treatment induced foci in renal cortex of epithelial vimentin expression and a loss of E-cadherin expression, as signs of cellular stress. The cellular changes occurred within high, but physiological aldosterone concentrations. We conclude that aldosterone induces protein accumulation in distal renal tubules; these aggregates are not cleared by autophagy that may lead to early renal tubular damage.

The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC₃), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC₃ to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC₃ or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC₃ and MT (0.7 µM) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an EC₅₀ of 18.6 ± 12.2 nM was determined for binding of Alexa 488-PC₃ to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC₃/MT/Tf (0.7 µM). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC₃ and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.

Harnessing the RNA interference pathway offers a new therapeutic modality; however, solutions to overcome biological barriers to small interfering RNA (siRNA) delivery are required for clinical translation. This work demonstrates, by direct northern and quantitative PCR (qPCR) detection, stability, gastrointestinal (GI) deposition, and translocation into peripheral tissue of nonmodified siRNA after oral gavage of chitosan/siRNA nanoparticles in mice. In contrast to naked siRNA, retained structural integrity and deposition in the stomach, proximal and distal small intestine, and colon was observed at 1 and 5 hours for siRNA within nanoparticles. Furthermore, histological detection of fluorescent siRNA at the apical regions of the intestinal epithelium suggests mucoadhesion provided by chitosan. Detection of intact siRNA in the liver, spleen, and kidney was observed 1 hour after oral gavage, with an organ distribution pattern influenced by nanoparticle N:P ratio that could reflect differences in particle stability. This proof-of-concept work presents an oral delivery platform that could have the potential to treat local and systemic disorders by siRNA.