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

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

Die physiologische Rolle des 2-P-Domänen Kaliumkanals TWIK1 in der Niere und im Pankreas

Die vorliegende Arbeit beschäftigte sich mit der physiologischen Rolle des 2-P-Domänen Kaliumkanals TWIK1 in der Niere und im Pankreas der Maus. In der Niere lag TWIK1 im Bürstensaum und in subapikalen Vesikeln des proximalen Tubulus. Interessanterweise waren Expression und Bürstensaumlokalisation im späten proximalen Tubulus (S3) bei weiblichen Mäusen stärker als bei den Männchen. Eine Behandlung von weiblichen Mäusen mit Testosteron reduzierte TWIK1 im S3-Segment, was auf eine Beteiligung von Androgenen bei der Regulation von TWIK1 hindeutet. Weiterhin konnte in diesem S3-Segment eine weitgehende Kolokalisation von TWIK1 mit dem natriumabhängigen Glucosetransporter SGLT1 beobachtet werden. Dieser zeigte in gleicher Weise wie TWIK1 eine geschlechtsabhängige Expression und Lokalisation. Unserer Hypothese nach dient TWIK1 im spätproximalen Tubulus der Repolarisation der apikalen Membran, wenn diese durch den elektrogenen Transport von Glucose depolarisiert wird. Tatsächlich zeigten TWIK1-/- Mäuse unter Glucosebelastung eine höhere renale Glucoseausscheidung. Im Pankreas der Maus war TWIK1 überwiegend in den Insulin produzierenden beta-Zellen lokalisiert. Sowohl in vivo als auch in vitro zeigten beta-Zellen von TWIK1-/- Mäusen eine verstärkte Insulinsekretion. Die Befunde von Ca2+-Messungen und Patch-Clamp Experimenten deuten darauf hin, dass TWIK1 in den beta-Zellen der Maus wahrscheinlich keine Funktion als membranständiger Kaliumkanal ausübt. Auch die hauptsächlich intrazelluläre Lokalisation von TWIK1 unterstreicht diese These. Bislang ist noch unklar, wie genau TWIK1 die Insulinsekretion beeinflusst. Allerdings wird vermutet, dass dabei die Interaktion von TWIK1 mit dem kleinen G-Protein ARF6 eine zentrale Rolle einnimmt. In Immunfluoreszenz-Experimenten wurde von uns eine Kolokalisation von TWIK1 mit ARF6 in beta-Zellen des Pankreas beobachtet. Es ist denkbar, dass eine veränderte Interaktion zwischen dem TWIK1 knockout Protein und ARF6 zu der verstärkten Insulinfreisetzung bei TWIK1-/- Mäusen führt

Distinct Mitochondrial Pathologies Caused by Mutations of the Proximal Tubular Enzymes EHHADH and GATM

The mitochondria of the proximal tubule are essential for providing energy in this nephron segment, whose ATP generation is almost exclusively oxygen dependent. In addition, mitochondria are involved in a variety of metabolic processes and complex signaling networks. Proximal tubular mitochondrial dysfunction can therefore affect renal function in very different ways. Two autosomal dominantly inherited forms of renal Fanconi syndrome illustrate how multifaceted mitochondrial pathology can be: Mutation of EHHADH, an enzyme in fatty acid metabolism, results in decreased ATP synthesis and a consecutive transport defect. In contrast, mutations of GATM, an enzyme in the creatine biosynthetic pathway, leave ATP synthesis unaffected but do lead to mitochondrial protein aggregates, inflammasome activation, and renal fibrosis with progressive renal failure. In this review article, the distinct pathophysiological mechanisms of these two diseases are presented, which are examples of the spectrum of proximal tubular mitochondrial diseases

Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure.Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations.Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death.Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis

TWIK1, a unique background channel with variable ion selectivity.

International audienceTWIK1 belongs to the family of background K(+) channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K(+) selective at neutral pH, it becomes permeable to Na(+) at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1(-/-) pancreatic β cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells

Does sumoylation control K2P1/TWIK1 background K+ channels?

A novel model for the regulation of cell excitability has recently been proposed. It originates from the observation that the background K(+) channel K2P1 (TWIK1) may be silenced by sumoylation in Xenopus oocytes and that inactivation of the putative sumoylation site (mutation K274E) gives rise to robust current expression in transfected COS-7 cells. Here, we show that only the mutation K274E, and not K274R, is associated with an increase of K2P1 current density, suggesting a charge effect of K274E. Furthermore, we failed to observe any band shift by western blot analysis that would confirm an eventual sumoylation of K2P1 in COS-7 cells and oocytes

Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs

Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain

Renal Fanconi Syndrome Is Caused by a Mistargeting-Based Mitochondriopathy

Summary: We recently reported an autosomal dominant form of renal Fanconi syndrome caused by a missense mutation in the third codon of the peroxisomal protein EHHADH. The mutation mistargets EHHADH to mitochondria, thereby impairing mitochondrial energy production and, consequently, reabsorption of electrolytes and low-molecular-weight nutrients in the proximal tubule. Here, we further elucidate the molecular mechanism underlying this pathology. We find that mutated EHHADH is incorporated into mitochondrial trifunctional protein (MTP), thereby disturbing β-oxidation of long-chain fatty acids. The resulting MTP deficiency leads to a characteristic accumulation of hydroxyacyl- and acylcarnitines. Mutated EHHADH also limits respiratory complex I and corresponding supercomplex formation, leading to decreases in oxidative phosphorylation capacity, mitochondrial membrane potential maintenance, and ATP generation. Activity of the Na+/K+-ATPase is thereby diminished, ultimately decreasing the transport activity of the proximal tubule cells. : Assmann et al. examine the molecular mechanism underlying a recently described Fanconi syndrome. Mistargeting of the peroxisomal protein EHHADH to mitochondria leads to impaired mitochondrial fatty acid β-oxidation and respiration, resulting in decreased ATP production. Diminished transport activity leads to the observed Fanconi syndrome. Keywords: Fanconi syndrome, mitochondriopathy, fatty acid oxidation, supercomplexe

Task3 Potassium Channel Gene Invalidation Causes Low Renin and Salt-Sensitive Arterial Hypertension.

International audienceTask1 and Task3 potassium channels (Task: tandem of P domains in a weak inward rectifying K(+) channel-related acid-sensitive K(+) channel) are believed to control the membrane voltage of aldosterone-producing adrenal glomerulosa cells. This study aimed at understanding the role of Task3 for the control of aldosterone secretion. The adrenal phenotype of Task3(-/-) mice was investigated using electrophysiology, adrenal slices, and blood pressure measurements. Primary adrenocortical cells of Task3(-/-) mice were strongly depolarized compared with wild-type (-52 vs. -79 mV), and in fresh adrenal slices Ca(2+) signaling of Task3(-/-) glomerulosa cells was abnormal. In living Task3(-/-) mice, the regulation of aldosterone secretion showed specific deficits: Under low Na(+) and high K(+) diets, protocols known to increase aldosterone, and under standard diet, Task3 inactivation was compensated and aldosterone was normal. However, high Na(+) and low K(+) diets, two protocols known to lower aldosterone, failed to lower aldosterone in Task3(-/-) mice. The physiological regulation of aldosterone was disturbed: aldosterone-renin ratio, an indicator of autonomous aldosterone secretion, was 3-fold elevated at standard and high Na(+) diets. Isolated adrenal glands of Task3(-/-) produced 2-fold more aldosterone. As a consequence, Task3(-/-) mice showed salt-sensitive arterial hypertension (plus 10 mm Hg). In conclusion, Task3 plays an important role in the adaptation of aldosterone secretion to dietary salt intake