Dynamics of Renal Electrolyte Excretion in Growing Mice

Genetically modified mice represent important models for elucidating renal pathophysiology, but gene deletions frequently cause severe failure to thrive. In such cases, the analysis of the phenotype is often limited to the first weeks of life when renal excretory function undergoes dramatic physiological changes. Here, we investigated the postnatal dynamics of urinary ion excretion in mice. The profiles of urinary electrolyte excretion of mice were examined from birth until after weaning using an automated ion chromatography system. Postnatally, mice grew about 0.4 g/day, except during two phases with slower weight gain: (i) directly after birth during adaptation to extrauterine conditions (P0-P2) and (ii) during the weaning period (P15-P21), when nutrition changed from mother's milk to solid chow and water. During the first 3 days after birth, remarkable changes in urinary Na+, Ca2+, Mg2+, and phosphate concentrations occurred, whereas K+ and Cl- concentrations hardly changed. From days 4-14 after birth, Na+, Ca2+, Mg2+, K+, and Cl- concentrations remained relatively stable at low levels. Urinary concentrations of creatinine, NH4+, phosphate, and sulfate constantly increased from birth until after weaning. Profiles of salt excretion in KCNJ10-/- mice exemplified the relevance of age-dependent analysis of urinary excretion. In conclusion, the most critical phases for analysis of renal ion excretion during the first weeks of life are directly after birth and during the weaning period. The age dependence of urinary excretion varies for the different ions. This should be taken into consideration when the renal phenotype of mice is investigated during the first weeks of life

A missense mutation in Ehd1 associated with defective spermatogenesis and male infertility

Normal function of the C-terminal Eps15 homology domain-containing protein 1 (EHD1) has previously been associated with endocytic vesicle trafficking, shaping of intracellular membranes, and ciliogenesis. We recently identified an autosomal recessive missense mutation c.1192C>T (p.R398W) of EHD1 in patients who had low molecular weight proteinuria (0.7–2.1 g/d) and high-frequency hearing loss. It was already known from Ehd1 knockout mice that inactivation of Ehd1 can lead to male infertility. However, the exact role of the EHD1 protein and its p.R398W mutant during spermatogenesis remained still unclear. Here, we report the testicular phenotype of a knockin mouse model carrying the p.R398W mutation in the EHD1 protein. Male homozygous knockin mice were infertile, whereas the mutation had no effect on female fertility. Testes and epididymes were significantly reduced in size and weight. The testicular epithelium appeared profoundly damaged and had a disorganized architecture. The composition of developing cell types was altered. Malformed acrosomes covered underdeveloped and misshaped sperm heads. In the sperm tail, midpieces were largely missing indicating disturbed assembly of the sperm tail. Defective structures, i.e., nuclei, acrosomes, and sperm tail midpieces, were observed in large vacuoles scattered throughout the epithelium. Interestingly, cilia formation itself did not appear to be affected, as the axoneme and other parts of the sperm tails except the midpieces appeared to be intact. In wildtype mice, EHD1 co-localized with acrosomal granules on round spermatids, suggesting a role of the EHD1 protein during acrosomal development. Wildtype EHD1 also co-localized with the VPS35 component of the retromer complex, whereas the p.R398W mutant did not. The testicular pathologies appeared very early during the first spermatogenic wave in young mice (starting at 14 dpp) and tubular destruction worsened with age. Taken together, EHD1 plays an important and probably multifaceted role in spermatogenesis in mice. Therefore, EHD1 may also be a hitherto underestimated infertility gene in humans

A missense mutation in Ehd1 associated with defective spermatogenesis and male infertility

Normal function of the C-terminal Eps15 homology domain-containing protein 1 (EHD1) has previously been associated with endocytic vesicle trafficking, shaping of intracellular membranes, and ciliogenesis. We recently identified an autosomal recessive missense mutation c.1192C>T (p.R398W) of EHD1 in patients who had low molecular weight proteinuria (0.7–2.1 g/d) and high-frequency hearing loss. It was already known from Ehd1 knockout mice that inactivation of Ehd1 can lead to male infertility. However, the exact role of the EHD1 protein and its p.R398W mutant during spermatogenesis remained still unclear. Here, we report the testicular phenotype of a knockin mouse model carrying the p.R398W mutation in the EHD1 protein. Male homozygous knockin mice were infertile, whereas the mutation had no effect on female fertility. Testes and epididymes were significantly reduced in size and weight. The testicular epithelium appeared profoundly damaged and had a disorganized architecture. The composition of developing cell types was altered. Malformed acrosomes covered underdeveloped and misshaped sperm heads. In the sperm tail, midpieces were largely missing indicating disturbed assembly of the sperm tail. Defective structures, i.e., nuclei, acrosomes, and sperm tail midpieces, were observed in large vacuoles scattered throughout the epithelium. Interestingly, cilia formation itself did not appear to be affected, as the axoneme and other parts of the sperm tails except the midpieces appeared to be intact. In wildtype mice, EHD1 co-localized with acrosomal granules on round spermatids, suggesting a role of the EHD1 protein during acrosomal development. Wildtype EHD1 also co-localized with the VPS35 component of the retromer complex, whereas the p.R398W mutant did not. The testicular pathologies appeared very early during the first spermatogenic wave in young mice (starting at 14 dpp) and tubular destruction worsened with age. Taken together, EHD1 plays an important and probably multifaceted role in spermatogenesis in mice. Therefore, EHD1 may also be a hitherto underestimated infertility gene in humans

CLN7/MFSD8 may be an important factor for SARS-CoV-2 cell entry

The SARS-CoV-2 virus has triggered a worldwide pandemic. According to the BioGrid database, CLN7 (MFSD8) is thought to interact with several viral proteins. The aim of this work was to investigate a possible involvement of CLN7 in the infection process. Experiments on a CLN7-deficient HEK293T cell line exhibited a 90% reduced viral load compared to wild-type cells. This observation may be linked to the finding that CLN7 ko cells have a significantly reduced GM1 content in their cell membrane. GM1 is found highly enriched in lipid rafts, which are thought to play an important role in SARS-CoV-2 infection. In contrast, overexpression of CLN7 led to an increase in viral load. This study provides evidence that CLN7 is involved in SARS-CoV-2 infection. This makes it a potential pharmacological target for drug development against COVID-19. Furthermore, it provides insights into the physiological function of CLN7 where still only little is known about

The ex vivo perfused mouse adrenal gland—a new model to study aldosterone secretion

Aldosterone is a steroid hormone that is important for maintaining the volume and ionic composition of extracellular fluids and is produced in the zona glomerulosa of the adrenal cortex. The basic mechanisms controlling aldosterone secretion are known. However, more detailed studies on the regulation of aldosterone secretion often fail due to the lack of suitable models: although secretion can be studied in cultured adrenocortical cells under defined conditions, the differentiation status of the cells is difficult to control and the complex anatomy of the adrenal cortex is lost. In living animals, the physiological context is intact, but the influences are manifold and the examination conditions cannot be sufficiently controlled. One method that closes the gap between cell models and studies in living animals is the isolated perfused adrenal gland. In the past, this method has provided important data on the pathophysiology of adrenal glands from larger animals, but the technique was not used in mice. Here, we developed a method for isolation and perfusion of the mouse adrenal gland to study aldosterone secretion. This technique preserves the complex anatomical and functional context of the mouse adrenal cortex, to ensure defined experimental conditions and to minimize extra-adrenal influences. Initial series of experiments with the ex vivo perfused mouse adrenal gland show that this model offers the possibility for unique insights into pathophysiological regulatory principles and is suitable for the use of genetically modified mouse models

Cellular Pathophysiology of Mutant Voltage-Dependent Ca2+ Channel CACNA1H in Primary Aldosteronism

The physiological stimulation of aldosterone production in adrenocortical glomerulosa cells by angiotensin II and high plasma K+ depends on the depolarization of the cell membrane potential and the subsequent Ca2+ influx via voltage-activated Ca2+ channels. Germline mutations of the low-voltage activated T-type Ca2+ channel CACNA1H (Cav3.2) have been found in patients with primary aldosteronism. Here, we investigated the electrophysiology and Ca2+ signaling of adrenal NCI-H295R cells overexpressing CACNA1H wildtype and mutant M1549V in order to understand how mutant CACNA1H alters adrenal cell function. Whole-cell patch-clamp measurements revealed a strong activation of mutant CACNA1H at the resting membrane potential of adrenal cells. Both the expression of wildtype and mutant CACNA1H led to a depolarized membrane potential. In addition, cells expressing mutant CACNA1H developed pronounced action potential-like membrane voltage oscillations. Ca2+ measurements showed an increased basal Ca2+ activity, an altered K+ sensitivity, and abnormal oscillating Ca2+ changes in cells with mutant CACNA1H. In addition, removal of extracellular Na+ reduced CACNA1H current, voltage oscillations, and Ca(2+ )levels in mutant cells, suggesting a role of the partial Na+ conductance of CACNA1H in cellular pathology. In conclusion, the pathogenesis of stimulus-independent aldosterone production in patients with CACNA1H mutations involves several factors: i) a loss of normal control of the membrane potential, ii) an increasedCa(2+) influx at basal conditions, and iii) alterations in sensitivity to extracellular K+ and Na+. Finally, our findings underline the importance of CACNA1H in the control of aldosterone production and support the concept of the glomerulosa cell as an electrical oscillator

Abnormal respiration under hyperoxia in TASK-1/3 potassium channel double knockout mice

Despite intensive research, the exact function of TASK potassium channels in central and peripheral chemoreception is still under debate. In this study, we investigated the respiration of unrestrained TASK-3 (TASK-3(-/-)) and TASK-1/TASK-3 double knockout (TASK-1/3(-/-)) adult male mice in vivo using a plethysmographic device. Ventilation parameters of TASK-3/mice were normal under control condition (21% 02) and upon hypoxia and hypercapnia they displayed the physiological increase of ventilation. TASK-1/3(-/-) mice showed increased ventilation under control conditions. This increase of ventilation was caused by increased tidal volumes (V-T), a phenomenon similarly observed in TASK-1(-/-) mice. Under acute hypoxia, TASK-1/3(-/-) mice displayed the physiological increase of the minute volume. Interestingly, this increase was not related to an increase of the respiratory frequency (f(R)), as observed in wild-type mice, but was caused by a strong increase of V-T. This particular respiratory phenotype is reminiscent of the respiratory phenotype of carotid body-denervated rodents in the compensated state. Acute hypercapnia (5% CO2) stimulated ventilation in TASK-1/3 i and wild-type mice to a similar extent; however, at higher CO2 concentrations ( > 5% CO2) the stimulation of ventilation was more pronounced in TASK-1/3(-/-) mice. At hyperoxia (100% O-2), TASK-1(-/-) , TASK-3(-/-) and wild-type mice showed the physiological small decrease of ventilation. In sharp contrast, TASK-1/3(-/-) mice exhibited an abnormal increase of ventilation under hyperoxia. In summary, these measurements showed a grossly normal respiration of TASK-3(-/-) mice and a respiratory phenotype of TASK-1/3(-/-) mice that was characterized by a markedly enhanced tidal volume, similar to the one observed in TASK-1(-/-) mice. The abnormal hyperoxia response, exclusively found in TASK-1/3(-/-) double mutant mice, indicates that both TASK-1(-/-) and TASK-3 are essential for the hyperoxia-induced hypoventilation. The peculiar respiratory phenotype of TASK-1/3(-/-) knockout mice is reminiscent of the respiration of animals with long-term carotid body dysfunction. Taken together, TASK-1 and TASK-3 appear to serve specific and distinct roles in the complex processes underlying chemoreception and respiratory control

Sex-dependent differences in the in vivo respiratory phenotype of the TASK-1 potassium channel knockout mouse

TASK-1 potassium channels have been implicated in central and peripheral chemoreception; however, the precise contribution of TASK-1 for the control of respiration is still under debate. Here, we investigated the respiration of unrestrained adult and neonatal TASK-1 knockout mice (TASK-1−/−) using a plethysmographic device. Respiration in adult female TASK-1−/− mice under control (21% O2), hypoxia and hypercapnia was unaffected. Under acute hypoxia male TASK-1−/− mice exhibited a reduced increase of the respiratory frequency (fR) compared to wildtypes. However, the tidal volume (VT) of male TASK-1−/− mice was strongly enhanced. The volatile anesthetic isoflurane induced in male TASK-1−/− and male wild type mice (TASK-1+/+) a similar respiratory depression. Neonatal TASK-1−/− mice demonstrated a 30–40% decrease of the minute volume, caused by a reduction of the fR under control condition (21% O2). Under hypoxia, neonatal TASK-1−/− mice more frequently stopped breathing (apnea > 3s) suggesting an increased hypoxia-sensitivity. As reported before, this increased hypoxia sensitivity had no influence on the survival rate of neonatal TASK-1−/− mice. In adult and neonatal mice, TASK-1 gene deletion induced a significant prolongation of the relaxation time (RT), which is a parameter for expiration kinetics. Additionally, screening for mutations in the human TASK-1 gene in 155 cases of sudden infant death syndrome (SIDS) was inconclusive. In conclusion, these data are suggestive for an increased hypoxia-sensitivity of neonatal TASK-1−/− mice, however, without causing an increase in neonatal lethality. In adult female TASK-1−/− mice respiration was unaffected, whereas adult male TASK-1−/− mice showed a modified breathing pattern. These results are suggestive for sex-specific mechanisms for compensating the inactivation of TASK-1 in mice

DataSheet1_A missense mutation in Ehd1 associated with defective spermatogenesis and male infertility.PDF

Normal function of the C-terminal Eps15 homology domain-containing protein 1 (EHD1) has previously been associated with endocytic vesicle trafficking, shaping of intracellular membranes, and ciliogenesis. We recently identified an autosomal recessive missense mutation c.1192C>T (p.R398W) of EHD1 in patients who had low molecular weight proteinuria (0.7–2.1 g/d) and high-frequency hearing loss. It was already known from Ehd1 knockout mice that inactivation of Ehd1 can lead to male infertility. However, the exact role of the EHD1 protein and its p.R398W mutant during spermatogenesis remained still unclear. Here, we report the testicular phenotype of a knockin mouse model carrying the p.R398W mutation in the EHD1 protein. Male homozygous knockin mice were infertile, whereas the mutation had no effect on female fertility. Testes and epididymes were significantly reduced in size and weight. The testicular epithelium appeared profoundly damaged and had a disorganized architecture. The composition of developing cell types was altered. Malformed acrosomes covered underdeveloped and misshaped sperm heads. In the sperm tail, midpieces were largely missing indicating disturbed assembly of the sperm tail. Defective structures, i.e., nuclei, acrosomes, and sperm tail midpieces, were observed in large vacuoles scattered throughout the epithelium. Interestingly, cilia formation itself did not appear to be affected, as the axoneme and other parts of the sperm tails except the midpieces appeared to be intact. In wildtype mice, EHD1 co-localized with acrosomal granules on round spermatids, suggesting a role of the EHD1 protein during acrosomal development. Wildtype EHD1 also co-localized with the VPS35 component of the retromer complex, whereas the p.R398W mutant did not. The testicular pathologies appeared very early during the first spermatogenic wave in young mice (starting at 14 dpp) and tubular destruction worsened with age. Taken together, EHD1 plays an important and probably multifaceted role in spermatogenesis in mice. Therefore, EHD1 may also be a hitherto underestimated infertility gene in humans.</p

Task2 potassium channels set central respiratory CO2 and O2 sensitivity

Task2 K+ channel expression in the central nervous system is surprisingly restricted to a few brainstem nuclei, including the retrotrapezoid (RTN) region. All Task2-positive RTN neurons were lost in mice bearing a Phox2b mutation that causes the human congenital central hypoventilation syndrome. In plethysmography, Task2−/− mice showed disturbed chemosensory function with hypersensitivity to low CO2 concentrations, leading to hyperventilation. Task2 probably is needed to stabilize the membrane potential of chemoreceptive cells. In addition, Task2−/− mice lost the long-term hypoxia-induced respiratory decrease whereas the acute carotid-body-mediated increase was maintained. The lack of anoxia-induced respiratory depression in the isolated brainstem–spinal cord preparation suggested a central origin of the phenotype. Task2 activation by reactive oxygen species generated during hypoxia could silence RTN neurons, thus contributing to respiratory depression. These data identify Task2 as a determinant of central O2 chemoreception and demonstrate that this phenomenon is due to the activity of a small number of neurons located at the ventral medullary surface