An innate antiviral pathway acting before interferons at epithelial surfaces.

Mucosal surfaces are exposed to environmental substances and represent a major portal of entry for microorganisms. The innate immune system is responsible for early defense against infections and it is believed that the interferons (IFNs) constitute the first line of defense against viruses. Here we identify an innate antiviral pathway that works at epithelial surfaces before the IFNs. The pathway is activated independently of known innate sensors of viral infections through a mechanism dependent on viral O-linked glycans, which induce CXCR3 chemokines and stimulate antiviral activity in a manner dependent on neutrophils. This study therefore identifies a previously unknown layer of antiviral defense that exerts its action on epithelial surfaces before the classical IFN response is operative.

Animal models to study cognitive impairment of chronic kidney disease.

Mild cognitive impairment (MCI) is common in people with chronic kidney disease (CKD), and its prevalence increases with progressive loss of kidney function. MCI is characterized by a decline in cognitive performance greater than expected for an individual age and education level but with minimal impairment of instrumental activities of daily living. Deterioration can affect one or several cognitive domains (attention, memory, executive functions, language, and perceptual motor or social cognition). Given the increasing prevalence of kidney disease, more and more people with CKD will also develop MCI causing an enormous disease burden for these individuals, their relatives, and society. However, the underlying pathomechanisms are poorly understood, and current therapies mostly aim at supporting patients in their daily lives. This illustrates the urgent need to elucidate the pathogenesis and potential therapeutic targets and test novel therapies in appropriate preclinical models. Here, we will outline the necessary criteria for experimental modeling of cognitive disorders in CKD. We discuss the use of mice, rats, and zebrafish as model systems and present valuable techniques through which kidney function and cognitive impairment can be assessed in this setting. Our objective is to enable researchers to overcome hurdles and accelerate preclinical research aimed at improving the therapy of people with CKD and MCI.

Loss of the Secretin Receptor Impairs Renal Bicarbonate Excretion and Aggravates Metabolic Alkalosis in Mice during Acute Base-Loading.

SIGNIFICANCE STATEMENT: During acute base excess, the renal collecting duct ß -intercalated cells ( ß -ICs) become activated to increase urine base excretion. This process is dependent on pendrin and cystic fibrosis transmembrane regulator (CFTR) expressed in the apical membrane of ß -ICs. The signal that leads to activation of this process was unknown. Plasma secretin levels increase during acute alkalosis, and the secretin receptor (SCTR) is functionally expressed in ß -ICs. We find that mice with global knockout for the SCTR lose their ability to acutely increase renal base excretion. This forces the mice to lower their ventilation to cope with this challenge. Our findings suggest that secretin is a systemic bicarbonate-regulating hormone, likely being released from the small intestine during alkalosis. BACKGROUND: The secretin receptor (SCTR) is functionally expressed in the basolateral membrane of the ß -intercalated cells of the kidney cortical collecting duct and stimulates urine alkalization by activating the ß -intercalated cells. Interestingly, the plasma secretin level increases during acute metabolic alkalosis, but its role in systemic acid-base homeostasis was unclear. We hypothesized that the SCTR system is essential for renal base excretion during acute metabolic alkalosis. METHODS: We conducted bladder catheterization experiments, metabolic cage studies, blood gas analysis, barometric respirometry, perfusion of isolated cortical collecting ducts, immunoblotting, and immunohistochemistry in SCTR wild-type and knockout (KO) mice. We also perfused isolated rat small intestines to study secretin release. RESULTS: In wild-type mice, secretin acutely increased urine pH and pendrin function in isolated perfused cortical collecting ducts. These effects were absent in KO mice, which also did not sufficiently increase renal base excretion during acute base loading. In line with these findings, KO mice developed prolonged metabolic alkalosis when exposed to acute oral or intraperitoneal base loading. Furthermore, KO mice exhibited transient but marked hypoventilation after acute base loading. In rats, increased blood alkalinity of the perfused upper small intestine increased venous secretin release. CONCLUSIONS: Our results suggest that loss of SCTR impairs the appropriate increase of renal base excretion during acute base loading and that SCTR is necessary for acute correction of metabolic alkalosis. In addition, our findings suggest that blood alkalinity increases secretin release from the small intestine and that secretin action is critical for bicarbonate homeostasis.

Deletion of aquaporin-4 improves capillary blood flow distribution in brain edema.

Brain edema is a feared complication to disorders and insults affecting the brain. It can be fatal if the increase in intracranial pressure is sufficiently large to cause brain herniation. Moreover, accruing evidence suggests that even slight elevations of intracranial pressure have adverse effects, for instance on brain perfusion. The water channel aquaporin-4 (AQP4), densely expressed in perivascular astrocytic endfeet, plays a key role in brain edema formation. Using two-photon microscopy, we have studied AQP4-mediated swelling of astrocytes affects capillary blood flow and intracranial pressure (ICP) in unanesthetized mice using a mild brain edema model. We found improved regulation of capillary blood flow in mice devoid of AQP4, independently of the severity of ICP increase. Furthermore, we found brisk AQP4-dependent astrocytic Ca2+ signals in perivascular endfeet during edema that may play a role in the perturbed capillary blood flow dynamics. The study suggests that astrocytic endfoot swelling and pathological signaling disrupts microvascular flow regulation during brain edema formation.

Sulfate and acid-base balance.

It has been acknowledged for years that compounds containing sulfur (S) are an important source of endogenous acid production. In the metabolism, S is oxidized to sulfate, and therefore the mEq sulfate excreted in the urine is counted as acid retained in the body. In this study we show that pH in fluids with constant [Na] and [HEPES] declines as sulfate ions are added, and we show that titratable acidity increases exactly with the equivalents of sulfate. Therefore, sulfate excretion in urine is also acid excretion per se. This is in accordance with the down-regulation of proximal sulfate reabsorption under acidosis and the observation that children with distal renal tubular acidosis may be sulfate depleted. These results are well explained using charge-balance modeling, which is based only on the three fundamental principles of electroneutrality, conservation of mass, and rules of dissociation as devised from physical chemistry. In contrast, the findings are in contrast to expectations from conventional narratives. These are unable to understand the decreasing pH as sulfate is added since no conventional acid is present. The results may undermine the traditional notion of endogenous acid production since in the case of sulfur balance, S oxidation and its excretion as sulfate exactly balance each other. Possible clinical correlates with these findings are discussed.

Single nephron glomerular filtration rate measured by linescan multiphoton microscopy compared to conventional micropuncture.

Renal micropuncture, which requires the direct access to the renal tubules, has for long time been the technique of choice to measure the single nephron glomerular filtration rate (SNGFR) in animal models. This approach is challenging by virtue of complex animal preparation and numerous technically difficult steps. The introduction of intravital multiphoton microscopy (MPM) offers another approach to the measure of the SNGFR by mean of the high laser-tissue penetration and the optical sectioning capacity. Previous MPM studies measuring SNGFR in vivo relied on fast full-frame acquisition during the filtration process obtainable with high performance resonant scanners. In this study, we describe an innovative linescan-based MPM method. The new method can discriminate SNGFR variations both in conditions of low and high glomerular filtration, and shows results comparable to conventional micropuncture both for rats and mice. Moreover, this novel approach has improved spatial and time resolution and is faster than previous methods, thus enabling the investigation of SNGFR from more tubules and improving options for data-analysis.

The Probe for Renal Organic Cation Secretion (4-Dimethylaminostyryl)-N-Methylpyridinium (ASP+)) Shows Amplified Fluorescence by Binding to Albumin and Is Accumulated In Vivo.

Accumulation of uremic toxins may lead to the life-threatening condition "uremic syndrome" in patients with advanced chronic kidney disease (CKD) requiring renal replacement therapy. Clinical evaluation of proximal tubular secretion of organic cations (OC), of which some are uremic toxins, is desired, but difficult. The biomedical knowledge on OC secretion and cellular transport partly relies on studies using the fluorescent tracer 4-dimethylaminostyryl)-N-methylpyridinium (ASP+), which has been used in many studies of renal excretion mechanisms of organic ions and which could be a candidate as a PET tracer. This study is aimed at expanding the knowledge of the tracer characteristics of ASP+ by recording the distribution and intensity of ASP+ signals in vivo both by fluorescence and by positron emission tomography (PET) imaging and at investigating if the fluorescence signal of ASP+ is influenced by the presence of albumin. Two-photon in vivo microscopy of male Münich Wistar Frömter rats showed that a bolus injection of ASP+ conferred a fluorescence signal to the blood plasma lasting for about 30 minutes. In the renal proximal tubule, the bolus resulted in a complex pattern of fluorescence including a rapid and strong transient signal at the brush border, a very low signal in the luminal fluid, and a slow transient intracellular signal. PET imaging using 11C-labelled ASP+ showed accumulation in the liver, heart, and kidney. Fluorescence emission spectra recorded in vitro of ASP+ alone and in the presence of albumin using both 1-photon excitation and two-photon excitation showed that albumin strongly enhance the emission from ASP+ and induce a shift of the emission maximum from 600 to 570 nm. Conclusion. The renal pattern of fluorescence observed from ASP+ in vivo is likely affected by the local concentration of albumin, and quantification of ASP+ fluorescent signals in vivo cannot be directly translated to ASP+ concentrations.

Acid content and buffer-capacity: a charge-balance perspective.

Rational treatment and thorough diagnostic classification of acid-base disorders requires quantitative understanding of the mechanisms that generate and dissipate loads of acid and base. A natural precondition for this tallying is the ability to quantify the acid content in any specified fluid. Physical chemistry defines the pH-dependent charge on any buffer species, and also on strong ions on which, by definition, the charge is pH-invariant. Based, then, on the requirement of electroneutrality and conservation of mass, it was shown in 1914 that pH can be calculated and understood on the basis of the chemical composition of any fluid. Herein we first show that this specification for [H+] of the charge-balance model directly delivers the pH-dependent buffer-capacity as defined in the literature. Next, we show how the notion of acid transport as proposed in experimental physiology can be understood as a change in strong ion difference, ΔSID. Finally, based on Brønsted-Lowry theory we demonstrate that by defining the acid content as titratable acidity, this is equal to SIDref - SID, where SIDref is SID at pH 7.4. Thereby, any chemical situation is represented as a curve in a novel diagram with titratable acidity = SIDref - SID as a function of pH. For any specification of buffer chemistry, therefore, the change in acid content in the fluid is path invariant. Since constituents of SID and titratable acidity are additive, we thereby, based on first principles, have defined a new framework for modeling acid balance across a cell, a whole organ, or the whole-body.

SARS-CoV-2 prevalence and transmission in swimming activities: Results from a retrospective cohort study.

There is an urgent need for research on the epidemiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19), as the transmissibility differs between settings and populations. Here we report on a questionnaire-based retrospective cohort study of the prevalence and transmission of SARS-CoV-2 among participants in swimming activities in Denmark in the last 5 months of 2020 during the COVID-19 pandemic. Eight of 162 swimming activities with a SARS-CoV-2 positive participant led to transmission to 23 other participants. Overall, the percentage of episodes leading to transmission was 4.9% (competitive swimming 8.9%; recreational swimming 1.3%). Overall, the incidence rate of transmission was 19.5 participants per 100 000 pool activity hours (corresponding values: 43.5 and 4.7 for competitive and recreational swimming, respectively). Compliance with precautionary restrictions was highest regarding hand hygiene (98.1%) and lowest in distancing personal sports bags (69.9%). As a result of low statistical power, the study showed no significant effect of restrictions. Insight into the risk of transmission of SARS-CoV-2 during indoor swimming is needed to estimate the efficiency of restrictive measures on this and other sports and leisure activities. Only when we know how the virus spreads through various settings, optimal strategies to handle the COVID-19 pandemic can be developed.

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

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

H+-ATPase B1 subunit localizes to thick ascending limb and distal convoluted tubule of rodent and human kidney.

The vacuolar-type H+-ATPase B1 subunit is heavily expressed in the intercalated cells of the collecting system, where it contributes to H+ transport, but has also been described in other segments of the renal tubule. This study aimed to determine the localization of the B1 subunit of the vacuolar-type H+-ATPase in the early distal nephron, encompassing thick ascending limbs (TAL) and distal convoluted tubules (DCT), in human kidney and determine whether the localization differs between rodents and humans. Antibodies directed against the H+-ATPase B1 subunit were used to determine its localization in paraffin-embedded formalin-fixed mouse, rat, and human kidneys by light microscopy and in sections of Lowicryl-embedded rat kidneys by electron microscopy. Abundant H+-ATPase B1 subunit immunoreactivity was observed in the human kidney. As expected, intercalated cells showed the strongest signal, but significant signal was also observed in apical membrane domains of the distal nephron, including TAL, macula densa, and DCT. In mouse and rat, H+-ATPase B1 subunit expression could also be detected in apical membrane domains of these segments. In rat, electron microscopy revealed that the H+-ATPase B1 subunit was located in the apical membrane. Furthermore, the H+-ATPase B1 subunit colocalized with other H+-ATPase subunits in the TAL and DCT. In conclusion, the B1 subunit is expressed in the early distal nephron. The physiological importance of H+-ATPase expression in these segments remains to be delineated in detail. The phenotype of disease-causing mutations in the B1 subunit may also relate to its presence in the TAL and DCT.

Hemoglobin polymerization via disulfide bond formation in the hypoxia-tolerant turtle Trachemys scripta: implications for antioxidant defense and O2 transport.

The ability of many reptilian hemoglobins (Hbs) to form high-molecular weight polymers, albeit known for decades, has not been investigated in detail. Given that turtle Hbs often contain a high number of cysteine (Cys), potentially contributing to the red blood cell defense against reactive oxygen species, we have examined whether polymerization of Hb could occur via intermolecular disulfide bonds in red blood cells of freshwater turtle Trachemys scripta, a species that is highly tolerant of hypoxia and oxidative stress. We find that one of the two Hb isoforms of the hemolysate HbA is prone to polymerization in vitro into linear flexible chains of different size that are visible by electron microscopy but not the HbD isoform. Polymerization of purified HbA is favored by hydrogen peroxide, a main cellular reactive oxygen species and a thiol oxidant, and inhibited by thiol reduction and alkylation, indicating that HbA polymerization is due to disulfide bonds. By using mass spectrometry, we identify Cys5 of the αA-subunit of HbA as specifically responsible for forming disulfide bonds between adjacent HbA tetramers. Polymerization of HbA does not affect oxygen affinity, cooperativity, and sensitivity to the allosteric cofactor ATP, indicating that HbA is still fully functional. Polymers also form in T. scripta blood after exposure to anoxia but not normoxia, indicating that they are of physiological relevance. Taken together, these results show that HbA polymers may form during oxidative stress and that Cys5αA of HbA is a key element of the antioxidant capacity of turtle red blood cells.

Measurement of total CO2 in microliter samples of urine and other biological fluids using infrared detection of CO2.

The purpose of this study is to describe a low-cost and simply made instrument capable of measuring the total CO2 content of microliter volumes of biological fluids utilizing a commercially available CO2 sensor based on a NDIR detector. The described instrument is based on transformation of dissolved HCO3- to CO2 by acidification and subsequent measurement of the produced CO2. The instrument has a linear response in the range 0.025-10 µmol HCO3-, which enables measurements in fresh urine and plasma samples down to 5 µl. The values from plasma were compared to measurements made on 65 µl whole blood in an automatic blood gas analyzer and found not to differ significantly. Compared to currently commercially available instruments applying the same principles to measure total CO2, this study provides a simple and robust alternative which even can be used on smaller sample volumes.

Tubulovascular cross-talk by vascular endothelial growth factor a maintains peritubular microvasculature in kidney.

Vascular endothelial growth factor A (VEGFA) production by podocytes is critical for glomerular endothelial health. VEGFA is also expressed in tubular epithelial cells in kidney; however, its physiologic role in the tubule has not been established. Using targeted transgenic mouse models, we found that Vegfa is expressed by specific epithelial cells along the nephron, whereas expression of its receptor (Kdr/Vegfr2) is largely restricted to adjacent peritubular capillaries. Embryonic deletion of tubular Vegfa did not affect systemic Vegfa levels, whereas renal Vegfa abundance was markedly decreased. Excision of Vegfa from renal tubules resulted in the formation of a smaller kidney, with a striking reduction in the density of peritubular capillaries. Consequently, elimination of tubular Vegfa caused pronounced polycythemia because of increased renal erythropoietin (Epo) production. Reducing hematocrit to normal levels in tubular Vegfa-deficient mice resulted in a markedly augmented renal Epo production, comparable with that observed in anemic wild-type mice. Here, we show that tubulovascular cross-talk by Vegfa is essential for maintenance of peritubular capillary networks in kidney. Disruption of this communication leads to increased renal Epo production and resulting polycythemia, presumably to counterbalance microvascular losses.

Ultrastructural and immunohistochemical localization of plasma membrane Ca2+-ATPase 4 in Ca2+-transporting epithelia.

Plasma membrane Ca(2+)-ATPases (PMCAs) participate in epithelial Ca(2+) transport and intracellular Ca(2+) signaling. The Pmca4 isoform is enriched in distal nephron isolates and decreased in mice lacking the epithelial transient receptor potential vanilloid 5 Ca(2+) channel. We therefore hypothesized that Pmca4 plays a significant role in transcellular Ca(2+) flux and investigated the localization and regulation of Pmca4 in Ca(2+)-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestines, whereas pan-specific Pmca antibodies detected Pmca1 in lateral membranes of enterocytes. In the kidney, Pmca4 showed broad localization to the distal nephron. In the mouse, expression was most abundant in segments coexpressing the epithelial ransient receptor potential vanilloid 5 Ca(2+) channel. Significant, albeit lower, expression was also evident in the region encompassing the cortical thick ascending limbs, macula densa, and early distal tubules as well as smooth muscle layers surrounding renal vessels. In the human kidney, a similar pattern of distribution was observed, with the highest PMCA4 expression in Na(+)-Cl(-) cotransporter-positive tubules. Electron microscopy demonstrated Pmca4 localization in distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca(2+) balance, pointing to a housekeeping function of the pump in Ca(2+)-transporting epithelia. In conclusion, Pmca4 shows a divergent expression pattern in Ca(2+)-transporting epithelia, inferring diverse roles for this isoform not limited to transepithelial Ca(2+) transport.

Furosemide-induced urinary acidification is caused by pronounced H+ secretion in the thick ascending limb.

The loop diuretic furosemide inhibits NaCl reabsorption in the thick ascending limb (TAL). In addition, furosemide acidifies the urine, which is traditionally explained by increased Na+ loading to the distal tubule causing an activation of H+ secretion via H+-ATPase in α-intercalated cells. The inability to acidify urine in response to furosemide serves to diagnose distal renal tubular acidosis (dysfunction of α-intercalated cells). Since the TAL is important for acid/base regulation, we speculated that it is involved in furosemide-induced urinary acidification. Luminal furosemide (100 µM) caused major, stable, and reversible intracellular alkalization (7.27 ± 0.06 to 7.6 ± 0.04) in isolated perfused murine medullary TAL and pronounced H+ secretion. This H+ secretion was fully inhibited with luminal amiloride (1 mM) and the Na+/H+ exchanger (NHE)3-specific antagonist #4167 (1 µM). Moreover, furosemide triggered a substantial drop of intracellular Na+ concentration in the medullary TAL. These results suggest that the furosemide-induced H+ secretion is a consequence of a drop in intracellular Na+ concentration, increasing the driving force for NHE3. Intriguingly, in whole animal experiments, furosemide-induced urinary acidification and net acid excretion were markedly reduced by specific NHE3 inhibition. Furthermore, the furosemide-induced urinary acidification was partially preserved during epithelial Na+ channel inhibition with benzamil. These results provide new insights in the mechanism of furosemide-induced urinary acidification and emphasize the role of the TAL in renal acid/base handling.

Superparamagnetic iron oxide polyacrylic acid coated γ-Fe2O3 nanoparticles do not affect kidney function but cause acute effect on the cardiovascular function in healthy mice.

This study describes the distribution of intravenously injected polyacrylic acid (PAA) coated γ-Fe(2)O(3) NPs (10 mg kg(-1)) at the organ, cellular and subcellular levels in healthy BALB/cJ mice and in parallel addresses the effects of NP injection on kidney function, blood pressure and vascular contractility. Magnetic resonance imaging (MRI) and transmission electron microscopy (TEM) showed accumulation of NPs in the liver within 1h after intravenous infusion, accommodated by intracellular uptake in endothelial and Kupffer cells with subsequent intracellular uptake in renal cells, particularly the cytoplasm of the proximal tubule, in podocytes and mesangial cells. The renofunctional effects of NPs were evaluated by arterial acid-base status and measurements of glomerular filtration rate (GFR) after instrumentation with chronically indwelling catheters. Arterial pH was 7.46±0.02 and 7.41±0.02 in mice 0.5 h after injections of saline or NP, and did not change over the next 12 h. In addition, the injections of NP did not affect arterial PCO(2) or [HCO(3)(-)] either. Twenty-four and 96 h after NP injections, the GFR averaged 0.35±0.04 and 0.35±0.01 ml min(-1) g(-1), respectively, values which were statistically comparable with controls (0.29±0.02 and 0.33±0.1 ml(-1) min(-1) 25 g(-1)). Mean arterial blood pressure (MAP) decreased 12-24 h after NP injections (111.1±11.5 vs 123.0±6.1 min(-1)) associated with a decreased contractility of small mesenteric arteries revealed by myography to characterize endothelial function. In conclusion, our study demonstrates that accumulation of superparamagnetic iron oxide nanoparticles does not affect kidney function in healthy mice but temporarily decreases blood pressure.

Tissue kallikrein permits early renal adaptation to potassium load.

Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K+ intake, we hypothesized that this enzyme might regulate plasma K+ concentration ([K+]). We showed in wild-type mice that renal K+ and TK excretion increase in parallel after a single meal, representing an acute K+ load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption (TK-/-) were used to assess the impact of the enzyme on plasma [K+]. A single large feeding did not lead to any significant change in plasma [K+] in TK+/+, whereas TK-/- mice became hyperkalemic. We next examined the impact of TK disruption on K+ transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK-/- mice exhibit net transepithelial K+ absorption because of abnormal activation of the colonic H+,K+-ATPase in the intercalated cells. Finally, in CCDs isolated from TK-/- mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H+,K+-ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K+ load.