Genomic Fabrics of the Excretory System's Functional Pathways Remodeled in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most frequent form of kidney cancer. Metastatic stages of ccRCC reduce the five-year survival rate to 15%. In this report, we analyze the ccRCC-induced remodeling of the five KEGG-constructed excretory functional pathways in a surgically removed right kidney and its metastasis in the chest wall from the perspective of the Genomic Fabric Paradigm (GFP). The GFP characterizes every single gene in each region by these independent variables: the average expression level (AVE), relative expression variability (REV), and expression correlation (COR) with each other gene. While the traditional approach is limited to only AVE analysis, the novel REV analysis identifies the genes whose correct expression level is critical for cell survival and proliferation. The COR analysis determines the real gene networks responsible for functional pathways. The analyses covered the pathways for aldosterone-regulated sodium reabsorption, collecting duct acid secretion, endocrine and other factor-regulated sodium reabsorption, proximal tubule bicarbonate reclamation, and vasopressin-regulated water reabsorption. The present study confirms the conclusion of our previously published articles on prostate and kidney cancers that even equally graded cancer nodules from the same tumor have different transcriptomic topologies. Therefore, the personalization of anti-cancer therapy should go beyond the individual, to his/her major cancer nodules.

Antioxidant, antihyperglycemic, and antihyperlipidemic properties of Chimonanthus salicifolius S. Y. Hu leaves in experimental animals: modulation of thioredoxin and glutathione systems, renal water reabsorption, and gut microbiota.

Introduction: Excessive calorie intake and physical inactivity have dramatically increased nutrient overload-associated disease, becoming a global public health issue. Chimonanthus salicifolius S. Y. Hu (CHI) is a homology plant of food and medicine in China and shows several health benefits. Methods: This work investigated the antioxidant activity, the alleviating effects, and the mechanism of action on diabetes and hyperlipidemia of CHI leaves. Results and discussion: Results showed that CHI leaves infusion displayed in vitro antioxidant activity measured by ABTS and ferric reducing antioxidant power methods. In wild-type Kunming mice, CHI leaves infusion consumption activated the hepatic antioxidant enzymes, including glutathione reductase, glutathione S-transferase, glutathione peroxidase and thioredoxin reductase as well as thioredoxin reductase 1. In alloxan-induced type 1 diabetic mice, CHI leaves infusion ameliorated diabetic symptoms, including polyuria, polydipsia, polyphagia and hyperglycemia, in a dose-dependent and time-course manners. The mechanism involved CHI leaves up-regulating renal water reabsorption associated protein - urine transporter A1-and promoting the trafficking of urine transporter A1 and aquaporin 2 to the apical plasma membrane. Despite this, in high-fat diet-induced hyperlipidemic golden hamsters, CHI leaves powder did not significantly effect on hyperlipidemia and body weight gain. This might be attributed to CHI leaves powder increasing the calorie intake. Interestingly, we found that CHI leaves extract containing a lower dose of total flavonoid than CHI leaves powder pronouncedly reduced the levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol in serum in golden hamsters fed a high-fat diet. Furthermore, CHI leaves extract elevated the diversity of gut microbiota and the abundance of Bifidobacterium and Ruminococcaceae_UCG-014. It also decreased the abundance of Lactobacillus at the genus level in golden hamsters fed a high-fat diet. Overall, CHI leaves benefit oxidative stress prevention and metabolic syndrome amelioration in vivo.

Expression Regulation of Water Reabsorption Genes and Transcription Factors in the Kidneys of Lepus yarkandensis.

Lepus yarkandensis is a desert-dwelling animal that has various adaptations to cope with drought. The kidney maintains water and acid-base balance mainly through the vasopressin-regulated water reabsorption pathway and proximal tubular bicarbonate reabsorption pathway. In this study, we compared the differentially expressed genes (DEGs) and transcription factors in the kidneys of L. yarkandensis and Oryctolagus cuniculus to explore the relationship between the DEGs in kidneys and the animals' adaptations. Transcriptome sequencing data were used to predict the differentially-expressed water reabsorption genes and their transcription factors. Quantitative real-time PCR, immunohistochemistry, and western blotting were used to detect and verify the expression of DEGs in the kidney at mRNA and protein levels. Transcriptome analysis of the kidney of L. yarkandensis and O. cuniculus showed that 6,610 genes were up-regulated and 5,727 genes down-regulated in data shared by both species. According to the data, 232 transcription factors and their corresponding target genes were predicted, from which genes and transcription factors related to renal water reabsorption were screened. Quantitative RT-PCR results showed AQP1, AQP2, ADCY3, HIF1A, CREB3, and NFATc1 had higher expression in the L. yarkandensis kidney; in comparison, FXYD2 mRNA expression levels were lower. In western blotting, transcription factors HIF1A, NFATc1, NF-κB1, and critical genes ADCY3, ATPA1, and SLC4A4, were highly expressed in the kidneys of L. yarkandensis. Immunohistochemical results showed that the ADCY3 protein was in the basolateral membrane of the collecting duct, the ATP1A1 protein was in the basolateral membrane and medulla of proximal tubules, and the SLC4A4 protein was in the basolateral membrane of proximal tubules. According to these results can be inferred that HIF1A, NFATc1, and NF-κB1 play a certain role in regulating the expression of genes related to water reabsorption in the kidney of L. yarkandensis, thus improving the water reclamation efficiency of L. yarkandensis, so as to adapt to the arid desert environment.

On the emerging of thawing drip: Role of myofibrillar protein renaturation.

This study aimed to facilitate the understanding on the origin of thawing drip under different freezing rate. Eventually we observed significantly greater thaw loss produced by slow freezing (8.58%) as compared to fast freezing (6.41%) after 24 h of thawing. Back to the freezing, ice crystallization induced decline in pH and the cold denaturation of myofibrillar protein. However, independent of freezing rate, we noticed protein renaturation with pH restoring during thawing, evidenced by the decreasing surface hydrophobicity, increasing solubility and thermal stability, and gradually stabilized secondary structure. Meanwhile, the water-holding of myofibrils increased with thawing process along with the rising water mobility. Under fast freezing, the results indicated less extensive protein cold denaturation and lower water mobility during thawing. Besides, we proposed that the microenvironment of lower ionic strength in fast freezing should benefit the protein renaturation and water re-absorption, ultimately contributed to lower thaw loss.

Molecular Characterization of an Aquaporin-2 Mutation Causing Nephrogenic Diabetes Insipidus.

The aquaporin 2 (AQP2) plays a critical role in water reabsorption to maintain water homeostasis. AQP2 mutation leads to nephrogenic diabetes insipidus (NDI), characterized by polyuria, polydipsia, and hypernatremia. We previously reported that a novel AQP2 mutation (G215S) caused NDI in a boy. In this study, we aimed to elucidate the cell biological consequences of this mutation on AQP2 function and clarify the molecular pathogenic mechanism for NDI in this patient. First, we analyzed AQP2 expression in Madin-Darby canine kidney (MDCK) cells by AQP2-G215S or AQP2-WT plasmid transfection and found significantly decreased AQP2-G215S expression in cytoplasmic membrane compared with AQP2-WT, independent of forskolin treatment. Further, we found co-localization of endoplasmic reticulum (ER) marker (Calnexin) with AQP2-G215S rather than AQP2-WT in MDCK cells by immunocytochemistry. The functional analysis showed that MDCK cells transfected with AQP2-G215S displayed reduced water permeability compared with AQP2-WT. Visualization of AQP2 structure implied that AQP2-G215S mutation might interrupt the folding of the sixth transmembrane α-helix and/or the packing of α-helices, resulting in the misfolding of monomer and further impaired formation of tetramer. Taken together, these findings suggested that AQP2-G215S was misfolded and retained in the ER and could not be translocated to the apical membrane to function as a water channel, which revealed the molecular pathogenic mechanism of AQP2-G215S mutation and explained for the phenotype of NDI in this patient.

Case Report: A Case of Congenital Nephrogenic Diabetes Insipidus Caused by Thr273Met Mutation in Arginine Vasopressin Receptor 2.

Congenital nephrogenic diabetes insipidus (CNDI) is a rare hereditary tubular dysfunction caused mainly by X-linked recessive inheritance of AVPR2 gene mutations. Pathogenic genes are a result of mutations in AVPR2 on chromosome Xq28 and in AQP2 on chromosome 12q13. The clinical manifestations of CNDI include polyuria, compensatory polydipsia, thirst, irritability, constipation, developmental delay, mental retardation, persistent decrease in the specific gravity of urine, dehydration, and electrolyte disorders (hypernatremia and hyperchloremia). Herein, we report a rare case of CNDI caused by an AVPR2 mutation in a 2-year-old Chinese boy who had sustained polyuria, polydipsia, and irritability for more than 20 months. Laboratory examinations showed no obvious abnormality in blood sodium and chloride levels but decreased urine osmolality and specific gravity. Imaging findings were also normal. However, genetic analysis revealed a C > T transition leading to T273M missense mutations in AVPR2. We provided the boy a low-sodium diet and administered oral hydrochlorothiazide and indomethacin for 1 month, after which his clinical symptoms significantly improved. This case report suggests that CNDI is characterized by pathogenic T273M missense mutations alone and expands our understanding of the pathogenesis of CNDI.

Subtypes of Neurohypophyseal Nonapeptide Receptors and Their Functions in Rat Kidneys.

The nonapeptides of neurohypophysis, vasotocin and mesotocin, detected in most vertebrates, are replaced by vasopressin and oxytocin in mammals. Using bioinformatics methods, we determined the spectrum of receptor subtypes for these hormones in mammals and their physiological effects in the kidneys of rats. A search for sequences similar to the vertebrate vasotocin receptor by proteomes and transcriptomas of nine mammalian species and the rat genome revealed three subtypes of vasopressin receptors (V1a, V1b, and V2) and one type of oxytocin receptors. In the kidneys of non-anesthetized rats, which received a water load of 2 ml per 100 g of body weight, three effects of vasopressin were revealed: 1) increased reabsorption of water and sodium, 2) increased excretion of potassium ions, and 3) increased excretion of sodium ions. It has been suggested that each of the effects on the kidney is associated with selective stimulation of the vasopressin receptor subtypes V2, V1b, and V1a depending on the concentration of nonapeptide. In experiments on non-anaesthetized rats with a water load, the injection of oxytocin reduces the reabsorption of solute-free water in the kidneys and increases the excretion of sodium ions. The possible physiological mechanisms behind the realization of both effects with the participation of a single type of oxytocin receptors are being analyzed. Thus, the spectrum of activated receptor subtypes varies depending on the current concentration of neurohypophyseal hormones, as a result of which the predominant effect on renal function changes, which ensures precise regulation of water-salt homeostasis.

A Case of Congenital Nephrogenic Diabetes Insipidus Caused by Thr108Met Variant of Aquaporin 2.

Congenital nephrogenic diabetes insipidus (CNDI) is a rare renal disorder caused by mutations in arginine vasopressin receptor 2 (AVPR2) or aquaporin 2 (AQP2). The clinical signs of CNDI include polyuria, compensatory polydipsia, dehydration, electrolyte disorder, and developmental retardation without prompt treatment. In this study we report a rare case of CNDI caused by a single base transition in AQP2 gene. A 4.5 years old male patient suffered from oral dryness, polydipsia, and polyuria for more than 3 years. Laboratory examinations showed hypernatremia, hyperchloremia, and decreased urine osmolality and specific gravity. Ultrasound and MRI found bilateral upper ureteral dilatation and hydronephrosis. Furthermore, sequencing analysis found a C>T transition leading to a T108M missense mutation of AQP2. The patient was given low sodium diet and treated with hydrochlorothiazide followed by amiloride with indomethacin. The patient's clinical course improved remarkably after 1 year of treatment. This study reports the first case of CNDI featuring T108M missense mutation alone. These findings demonstrate a causative role of T108M mutation for CNDI and contribute to the mechanistic understanding of CNDI disease process.

AQP1 expression in the proximal tubule of diabetic rat kidney.

Polyuria is a hallmark symptom and the first clinical manifestation of diabetes mellitus (DM). The glucose that remains in renal tubules was proposed to produce an osmotic effect resulting in polyuria. Although water is reabsorbed in proximal tubules through an aquaporin-1 (AQP1) dependent mechanism, AQP1 role in the genesis of polyuria is unknown. AQP1 expression was studied in a rat model of Type-1 DM at 15-days and 5-months of evolution. A different AQP1 expression pattern was found in both experimental groups, with no changes in AQP1 localization, suggesting that changes in AQP1 may be involved in the development of polyuria.

Osmotic diuresis by SGLT2 inhibition stimulates vasopressin-induced water reabsorption to maintain body fluid volume.

Most of the filtered glucose is reabsorbed in the early proximal tubule by the sodium-glucose cotransporter SGLT2. The glycosuric effect of the SGLT2 inhibitor ipragliflozin is linked to a diuretic and natriuretic effect that activates compensatory increases in fluid and food intake to stabilize body fluid volume (BFV). However, the compensatory mechanisms that are activated on the level of renal tubules remain unclear. Type 2 diabetic Goto-Kakizaki (GK) rats were treated with vehicle or 0.01% (in diet) ipragliflozin with free access to fluid and food. After 8 weeks, GK rats were placed in metabolic cages for 24-hr. Ipragliflozin decreased body weight, serum glucose and systolic blood pressure, and increased fluid and food intake, urinary glucose and Na+ excretion, urine volume, and renal osmolar clearance, as well as urine vasopressin and solute-free water reabsorption (TcH2O). BFV, measured by bioimpedance spectroscopy, and fluid balance were similar among the two groups. Urine vasopressin in ipragliflozin-treated rats was negatively and positively associated with fluid balance and TcH2O, respectively. Ipragliflozin increased the renal membrane protein expression of SGLT2, aquaporin (AQP) 2 phosphorylated at Ser269 and vasopressin V2 receptor. The expression of SGLT1, GLUT2, AQP1, and AQP2 was similar between the groups. In conclusion, the SGLT2 inhibitor ipragliflozin induced a sustained glucosuria, diuresis, and natriuresis, with compensatory increases in fluid intake and vasopressin-induced TcH2O in proportion to the reduced fluid balance to maintain BFV. These results indicate that the osmotic diuresis induced by SGLT2 inhibition stimulates compensatory fluid intake and renal water reabsorption to maintain BFV.

Remote Ischemic Perconditioning Modulates Apelin Expression After Renal Ischemia-Reperfusion Injury.

BACKGROUND: Renal ischemia/reperfusion injury (IRI) can result in impaired ability of urine concentration and increased sodium fractional excretion. Apelin, a (neuro) vasoactive peptide, enhances diuresis by increasing the renal microcirculation and by counteracting the antidiuretic effect of arginine vasopressin on the tubules. However, changes in renal apelin expression in renal IRI rat model have not been elucidated. Remote ischemic perconditioning (RIPerC) improves renal sodium and water handling after IRI. Here, we investigated whether RIPerC prevents dysregulation of renal sodium and water handling in response to IRI by apelin signaling pathway in rats. MATERIALS AND METHODS: Renal IRI was induced by 45-min clamping of renal arteries followed by 24 h reperfusion. RIPerC was created by applying four cycles of 2-min ischemia of the left femoral artery followed by 3-min reperfusion at the start of renal ischemia. Rats were randomly divided into sham, ischemia/reperfusion, and RIPerC + ischemia/reperfusion groups. Urine and blood were sampled after reperfusion period. The kidney was harvested for mRNA isolation and histopathological study. RESULTS: IRI resulted in decreased clearance of creatinine, increased sodium fractional excretion, and reduced urine osmolality compared with sham animals. This occurred with an increase in mRNA expression levels of apelin and histological damages in both cortical and medullary regions of kidney tissues. RIPerC treatment ameliorated all these changes. CONCLUSIONS: This study showed that RIPerC has protective effects against dysregulation of renal sodium and water handling after renal IRI, which might be related with inhibition of apelin signaling pathway.

Dynamic Physiological Phenotyping of Drought-Stressed Pepper Plants Treated With "Productivity-Enhancing" and "Survivability-Enhancing" Biostimulants.

The improvement of crop productivity under abiotic stress is one of the biggest challenges faced by the agricultural scientific community. Despite extensive research, the research-to-commercial transfer rate of abiotic stress-resistant crops remains very low. This is mainly due to the complexity of genotype × environment interactions and in particular, the ability to quantify the dynamic plant physiological response profile to a dynamic environment. Most existing phenotyping facilities collect information using robotics and automated image acquisition and analysis. However, their ability to directly measure the physiological properties of the whole plant is limited. We demonstrate a high-throughput functional phenotyping system (HFPS) that enables comparing plants' dynamic responses to different ambient conditions in dynamic environments due to its direct and simultaneous measurement of yield-related physiological traits of plants under several treatments. The system is designed as one-to-one (1:1) plant-[sensors+controller] units, i.e., each individual plant has its own personalized sensor, controller and irrigation valves that enable (i) monitoring water-relation kinetics of each plant-environment response throughout the plant's life cycle with high spatiotemporal resolution, (ii) a truly randomized experimental design due to multiple independent treatment scenarios for every plant, and (iii) reduction of artificial ambient perturbations due to the immobility of the plants or other objects. In addition, we propose two new resilience-quantifying-related traits that can also be phenotyped using the HFPS: transpiration recovery rate and night water reabsorption. We use the HFPS to screen the effects of two commercial biostimulants (a seaweed extract -ICL-SW, and a metabolite formula - ICL-NewFo1) on Capsicum annuum under different irrigation regimes. Biostimulants are considered an alternative approach to improving crop productivity. However, their complex mode of action necessitates cost-effective pre-field phenotyping. The combination of two types of treatment (biostimulants and drought) enabled us to evaluate the precision and resolution of the system in investigating the effect of biostimulants on drought tolerance. We analyze and discuss plant behavior at different stages, and assess the penalty and trade-off between productivity and resilience. In this test case, we suggest a protocol for the screening of biostimulants' physiological mechanisms of action.

High dietary zinc and glutamine do not improve the performance or reduce excreta moisture of broiler chickens fed diets with and without magnesium supplementation.

An experiment was conducted to investigate the effect of supplemental L-glutamine (L-Gln) and a higher concentration of zinc (Zn) on excreta moisture under nutritionally induced wet droppings via decreased intestinal water reabsorption. A 2 × 2 × 2 factorial arrangement of treatments was used to investigate 3 dietary factors of L-Gln supplementation (0 or 10 g/kg), and added Zn concentration (80 and 160 mg/kg) with or without magnesium chloride (MgCl) (2 g/kg-only in grower diets). A total of 576 male day-old Ross 308 broiler chickens were assigned to the experimental diets. Each diet was replicated 6 times with 12 birds per replicate. Wheat-based diets were formulated to be isocaloric and isonitrogenous. Starter diets were given from day 0 to 9 followed by grower (day 10 to 23) and finisher diets (day 24 to 35). Excreta moisture was measured for all the growth phases. The moisture content of different segments of intestine was assessed for starter and grower phases of feeding. There was no significant effect of any of the 3 main treatments on body weight gain or feed intake of birds during the experiment. Birds fed higher Zn (160 mg/kg) tended (P = 0.09) to have higher weight gain only in the first 9 days of age. Feeding 10 g/kg L-Gln increased the feed conversion ratio of the birds only from hatch until day 9 after which there was no significant effect. No effect of experimental treatments was found on digesta or excreta moisture, except a reduction in ileal moisture at the starter phase resulting from higher Zn concentration in the diets. MgCl at 2 g/kg was not effective in inducing wet droppings in birds fed grower diets. Under the conditions of this study, no positive response was observed in terms of performance or reduction in excreta moisture when birds were fed diets containing 10 g/kg L-Gln or higher concentration of Zn.

Cold tolerance is linked to osmoregulatory function of the hindgut in Locusta migratoria.

There is growing evidence that maintenance of ion and water balance determines cold tolerance in many insects. The hindgut of terrestrial insects is critical for maintaining organismal homeostasis as it regulates solute and water balance of the haemolymph. Here, we used ex vivo everted gut sacs of Locustamigratoria to examine the effects of temperature (0-30°C), thermal acclimation, hypoxia, and ionic and osmotic forces on bulk water and ion (Na+, K+ and Cl-) movement across the rectal epithelium. These findings were related to simultaneous in vivo measurements of water and ion balance in locusts exposed to similar temperatures. As predicted, we observed a critical inhibition of net water and ion reabsorption at low temperature that was proportional to the in vivo loss of water and ion homeostasis. Further, cold-acclimated locusts, which are known to defend ion and water balance at low temperature, were characterised by improved reabsorptive capacity at low temperature. These findings strongly support the hypothesis that transport mechanisms in the hindgut at low temperature are essential for cold tolerance. The loss of osmoregulatory capacity at low temperature was primarily caused by reduced active transport, while rectal paracellular permeability to fluorescein isothiocyanate dextran was unchanged at 0 and 30°C. During cold exposure, water reabsorption was independent of major cation gradients across the epithelia, while a reduction in mucosal Cl- availability and an increase in mucosal osmolality markedly depressed water reabsorption. These findings are discussed in the context of existing knowledge and with suggestions for future physiological studies on cold acclimation and adaptation in insects.

Water transport through the intestinal epithelial barrier under different osmotic conditions is dependent on LI-cadherin trans-interaction.

In the intestine water has to be reabsorbed from the chymus across the intestinal epithelium. The osmolarity within the lumen is subjected to high variations meaning that water transport often has to take place against osmotic gradients. It has been hypothesized that LI-cadherin is important in this process by keeping the intercellular cleft narrow facilitating the buildup of an osmotic gradient allowing water reabsorption. LI-cadherin is exceptional among the cadherin superfamily with respect to its localization along the lateral plasma membrane of epithelial cells being excluded from adherens junction. Furthermore it has 7 but not 5 extracellular cadherin repeats (EC1-EC7) and a small cytosolic domain. In this study we identified the peptide VAALD as an inhibitor of LI-cadherin trans-interaction by modeling the structure of LI-cadherin and comparison with the known adhesive interfaces of E-cadherin. This inhibitory peptide was used to measure LI-cadherin dependency of water transport through a monolayer of epithelial CACO2 cells under various osmotic conditions. If LI-cadherin trans-interaction was inhibited by use of the peptide, water transport from the luminal to the basolateral side was impaired and even reversed in the case of hypertonic conditions whereas no effect could be observed at isotonic conditions. These data are in line with a recently published model predicting LI-cadherin to keep the width of the lateral intercellular cleft small. In this narrow cleft a high osmolarity can be achieved due to ion pumps yielding a standing osmotic gradient allowing water absorption from the gut even if the faeces is highly hypertonic.

Bacillus anthracis Edema Toxin Increases Fractional Free Water and Sodium Reabsorption in an Isolated Perfused Rat Kidney Model.

Bacillus anthracis edema toxin (ET) consists of protective antigen (PA), necessary for host cell toxin uptake, and edema factor (EF), the toxic moiety which increases host cell cyclic AMP (cAMP). Since vasopressin stimulates renal water and sodium reabsorption via increased tubular cell cAMP levels, we hypothesized the ET would also do so. To test this hypothesis, we employed an isolated perfused rat kidney model. Kidneys were isolated and perfused with modified Krebs-Henseleit buffer. Perfusate and urine samples were obtained at baseline and every 10 min over 150 min following the addition of challenges with or without treatments to the perfusate. In kidneys perfused under constant flow or constant pressure, compared to PA challenge (n = 14 or 15 kidneys, respectively), ET (13 or 15 kidneys, respectively) progressively increased urine cAMP levels, water and sodium reabsorption, and urine osmolality and decreased urine output (P ≤ 0.04, except for sodium reabsorption under constant pressure [P = 0.17]). In ET-challenged kidneys, compared to placebo treatment, adefovir, an EF inhibitor, decreased urine cAMP levels, water and sodium reabsorption, and urine osmolality and increased urine output, while raxibacumab, a PA-directed monoclonal antibody (MAb), decreased urine cAMP levels, free water reabsorption, and urine osmolality and increased urine output (P ≤ 0.03 except for urine output with raxibacumab [P = 0.17]). Upon immunohistochemistry, aquaporin 2 was concentrated along the apical membrane of tubular cells with ET but not PA, and urine aquaporin 2 levels were higher with ET (5.52 ± 1.06 ng/ml versus 1.51 ± 0.44 ng/ml [means ± standard errors of the means {SEM}; P = 0.0001). Edema toxin has renal effects that could contribute to extravascular fluid collection characterizing anthrax infection clinically.

Physiological insights into novel therapies for nephrogenic diabetes insipidus.

Fundamental kidney physiology research can provide important insight into how the kidney works and suggest novel therapeutic opportunities to treat human diseases. This is especially true for nephrogenic diabetes insipidus (NDI). Over the past decade, studies elucidating the molecular physiology and signaling pathways regulating water transport have suggested novel therapeutic possibilities. In patients with congenital NDI due to mutations in the type 2 vasopressin receptor (V2R) or acquired NDI due to lithium (or other medications), there are no functional abnormalities in the aquaporin-2 (AQP2) water channel, or in another key inner medullary transport protein, the UT-A1 urea transporter. If it is possible to phosphorylate and/or increase the apical membrane accumulation of these proteins, independent of vasopressin or cAMP, one may be able to treat NDI. Sildenifil (through cGMP), erlotinib, and simvastatin each stimulate AQP2 insertion into the apical plasma membrane. Some recent human data suggest that sildenafil and simvastatin may improve urine concentrating ability. ONO-AE1-329 (ONO) stimulates the EP4 prostanoid receptor (EP4), which stimulates kinases that in turn phosphorylate AQP2 and UT-A1. Clopidogrel is a P2Y12-R antagonist that potentiates the effect of vasopressin and increases AQP2 abundance. Metformin stimulates AMPK to phosphorylate and activate AQP2 and UT-A1, and it increases urine concentrating ability in two rodent models of NDI. Since metformin, sildenafil, and simvastatin are commercially available and have excellent safety records, the potential for rapidly advancing them into clinical trials is high.

Application of process analytical technology for monitoring freeze-drying of an amorphous protein formulation: use of complementary tools for real-time product temperature measurements and endpoint detection.

Process analytical technology (PAT) and quality by design have gained importance in all areas of pharmaceutical development and manufacturing. One important method for monitoring of critical product attributes and process optimization in laboratory scale freeze-drying is manometric temperature measurement (MTM). A drawback of this innovative technology is that problems are encountered when processing high-concentrated amorphous materials, particularly protein formulations. In this study, a model solution of bovine serum albumin and sucrose was lyophilized at both conservative and aggressive primary drying conditions. Different temperature sensors were employed to monitor product temperatures. The residual moisture content at primary drying endpoints as indicated by temperature sensors and batch PAT methods was quantified from extracted sample vials. The data from temperature probes were then used to recalculate critical product parameters, and the results were compared with MTM data. The drying endpoints indicated by the temperature sensors were not suitable for endpoint indication, in contrast to the batch methods endpoints. The accuracy of MTM Pice data was found to be influenced by water reabsorption. Recalculation of Rp and Pice values based on data from temperature sensors and weighed vials was possible. Overall, extensive information about critical product parameters could be obtained using data from complementary PAT tools.

Glutathionylation of the aquaporin-2 water channel: a novel post-translational modification modulated by the oxidative stress.

Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and urine concentration. AQP2 undergoes different regulated post-translational modifications, including phosphorylation and ubiquitylation, which are fundamental for controlling AQP2 cellular localization, stability, and function. The relationship between AQP2 and S-glutathionylation is of potential interest because reactive oxygen species (ROS), produced under renal failure or nephrotoxic drugs, may influence renal function as well as the expression and the activity of different transporters and channels, including aquaporins. Here, we show for the first time that AQP2 is subjected to S-glutathionylation in kidney and in HEK-293 cells stably expressing AQP2. S-Glutathionylation is a redox-dependent post-translational modification controlling several signal transduction pathways and displaying an acute effect on free cytosolic calcium concentration. Interestingly, we found that in fresh kidney slices, the increased AQP2 S-glutathionylation correlated with tert-butyl hydroperoxide-induced ROS generation. Moreover, we also found that cells expressing wild-type human calcium-sensing receptor (hCaSR-wt) and its gain of function (hCaSR-R990G; hCaSR-N124K) had a significant decrease in AQP2 S-glutathionylation secondary to reduced ROS levels and reduced basal intracellular calcium concentration compared with mock cells. Together, these new findings provide fundamental insight into cell biological aspects of AQP2 function and may be relevant to better understand and explain pathological states characterized by an oxidative stress and AQP2-dependent water reabsorption disturbs.

Expression of aquaporins in the lungs of mice with acute injury caused by LPS treatment.

The aim of the present study was to investigate the expression of aquaporin 1 (AQP1) and AQP5 in the lungs of mice with acute injury induced by LPS treatment. In the study, the concentrations of cytokines were all significantly increased in the BALF of mice received LPS at 12h and 24h (P<0.001). The lung wet/dry weight ratios (W/D) and total protein content in BALF were also increased in the mice treated with LPS (P<0.001). Interestingly the expression of AQP1 and AQP5 was significantly decreased (P<0.05) compared with these in the control mice, while TUNEL positive cells were increased. However, the AQP5 expression was significantly higher at 24h that it at 12h in the control mice. Our results showed that decreased AQP expression was associated with the increased inflammatory factors, as well as apoptotic cells. The increased expression of AQP5 at 24h in control mice might be due to its regulation in transcellular water reabsorption.