RESUMO
When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.
Assuntos
Proteínas Serina-Treonina Quinases , Fosforilação , Tamanho CelularRESUMO
The cytoplasm is densely packed with molecules that contribute to its nonideal behavior. Cytosolic crowding influences chemical reaction rates, intracellular water mobility, and macromolecular complex formation. Overcrowding is potentially catastrophic; to counteract this problem, cells have evolved acute and chronic homeostatic mechanisms that optimize cellular crowdedness. Here, we provide a physiology-focused overview of molecular crowding, highlighting contemporary advances in our understanding of its sensing and control. Long hypothesized as a form of crowding-induced microcompartmentation, phase separation allows cells to detect and respond to intracellular crowding through the action of biomolecular condensates, as indicated by recent studies. Growing evidence indicates that crowding is closely tied to cell size and fluid volume, homeostatic responses to physical compression and desiccation, tissue architecture, circadian rhythm, aging, transepithelial transport, and total body electrolyte and water balance. Thus, molecular crowding is a fundamental physiologic parameter that impacts diverse functions extending from molecule to organism.
Assuntos
Equilíbrio Hidroeletrolítico , Água , HumanosRESUMO
The purpose of this review is to highlight transformative advances that have been made in the field of biomolecular condensates, with special emphasis on condensate material properties, physiology, and kinases, using the With-No-Lysine (WNK) kinases as a prototypical example. To convey how WNK kinases illustrate important concepts for biomolecular condensates, we start with a brief history, focus on defining features of biomolecular condensates, and delve into some examples of how condensates are implicated in cellular physiology (and pathophysiology). We then highlight how WNK kinases, through the action of "WNK droplets" that ubiquitously regulate intracellular volume and kidney-specific "WNK bodies" that are implicated in distal tubule salt reabsorption and potassium homeostasis, exemplify many of the defining features of condensates. Finally, this review addresses the controversies within this emerging field and questions to address.
Assuntos
Transdução de Sinais , Proteína Quinase 1 Deficiente de Lisina WNK , Animais , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismoRESUMO
The multiligand receptors megalin (Lrp2) and cubilin (Cubn) and their endocytic adaptor protein Dab2 (Dab2) play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule (PT) cells and have complex and poorly understood roles in the development of chronic kidney disease. Here, we used RNA-sequencing and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin, cubilin, or Dab2 expression. KO of Lrp2 had the greatest transcriptional effect, and nearly all genes whose expression was affected in Cubn KO and Dab2 KO cells were also changed in Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggested changes in pathways with immunomodulatory functions that might trigger the pathological changes observed in KO mice and patients with Donnai-Barrow syndrome. In addition, differences in transcription patterns between Lrp2 and Dab2 KO cells suggested the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon the disruption of PT endocytic function. A reduction in transcripts encoding sodium-glucose cotransporter isoform 2 was confirmed in Lrp2 KO mouse kidney lysates by quantitative PCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with the studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes.NEW & NOTEWORTHY Megalin and cubilin receptors together with their adaptor protein Dab2 represent major components of the endocytic machinery responsible for efficient uptake of filtered proteins by the proximal tubule (PT). Dab2 and megalin expression have been implicated as both positive and negative modulators of kidney disease. We used RNA sequencing to knock out CRISPR/Cas9 cubilin, megalin, and Dab2 in highly differentiated PT cells to identify PT-specific changes that are directly consequent to knockout of each component.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de Superfície Celular/metabolismo , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal/genética , Agenesia do Corpo Caloso/genética , Agenesia do Corpo Caloso/metabolismo , Agenesia do Corpo Caloso/patologia , Animais , Proteínas Reguladoras de Apoptose/genética , Células Cultivadas , Bases de Dados Genéticas , Redes Reguladoras de Genes , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Hérnias Diafragmáticas Congênitas/genética , Hérnias Diafragmáticas Congênitas/metabolismo , Hérnias Diafragmáticas Congênitas/patologia , Humanos , Túbulos Renais Proximais/patologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Camundongos Knockout , Monodelphis , Miopia/genética , Miopia/metabolismo , Miopia/patologia , Proteinúria/genética , Proteinúria/metabolismo , Proteinúria/patologia , Receptores de Superfície Celular/genética , Erros Inatos do Transporte Tubular Renal/genética , Erros Inatos do Transporte Tubular Renal/metabolismo , Erros Inatos do Transporte Tubular Renal/patologiaRESUMO
We characterized mouse blood pressure and ion transport in the setting of commonly used rodent diets that drive K+ intake to the extremes of deficiency and excess. Male 129S2/Sv mice were fed either K+-deficient, control, high-K+ basic, or high-KCl diets for 10 days. Mice maintained on a K+-deficient diet exhibited no change in blood pressure, whereas K+-loaded mice developed an ~10-mmHg blood pressure increase. Following challenge with NaCl, K+-deficient mice developed a salt-sensitive 8 mmHg increase in blood pressure, whereas blood pressure was unchanged in mice fed high-K+ diets. Notably, 10 days of K+ depletion induced diabetes insipidus and upregulation of phosphorylated NaCl cotransporter, proximal Na+ transporters, and pendrin, likely contributing to the K+-deficient NaCl sensitivity. While the anionic content with high-K+ diets had distinct effects on transporter expression along the nephron, both K+ basic and KCl diets had a similar increase in blood pressure. The blood pressure elevation on high-K+ diets correlated with increased Na+-K+-2Cl- cotransporter and γ-epithelial Na+ channel expression and increased urinary response to furosemide and amiloride. We conclude that the dietary K+ maneuvers used here did not recapitulate the inverse effects of K+ on blood pressure observed in human epidemiological studies. This may be due to the extreme degree of K+ stress, the low-Na+-to-K+ ratio, the duration of treatment, and the development of other coinciding events, such as diabetes insipidus. These factors must be taken into consideration when studying the physiological effects of dietary K+ loading and depletion.
Assuntos
Pressão Arterial , Hipertensão/metabolismo , Túbulos Renais/metabolismo , Deficiência de Potássio/metabolismo , Potássio na Dieta/metabolismo , Cloreto de Sódio na Dieta/metabolismo , Ração Animal , Animais , Diabetes Insípido/etiologia , Diabetes Insípido/metabolismo , Diabetes Insípido/fisiopatologia , Canais Epiteliais de Sódio/metabolismo , Hipertensão/etiologia , Hipertensão/fisiopatologia , Transporte de Íons , Túbulos Renais/fisiopatologia , Masculino , Camundongos da Linhagem 129 , Natriurese , Fosforilação , Deficiência de Potássio/etiologia , Deficiência de Potássio/fisiopatologia , Potássio na Dieta/administração & dosagem , Potássio na Dieta/toxicidade , Simportadores de Cloreto de Sódio/metabolismo , Cloreto de Sódio na Dieta/toxicidade , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Transportadores de Sulfato/metabolismoRESUMO
PURPOSE OF REVIEW: The current review combines past findings with recent advances in our understanding of the homeostatic response to potassium imbalance. RECENT FINDINGS: Following the ingestion of a dietary potassium load, a combination of extrarenal and renal mechanisms act to maintain extracellular K+ within a tight window. Through hormonal regulation and direct K+ sensing, the nephron is ideally suited to respond to wide shifts in external K+ balance. Current evidence indicates that dietary K+ loading triggers a coordinated kaliuretic response that appears to involve voltage-dependent changes in sodium transport across multiple nephron segments, including the proximal tubule, medullary loop of Henle, and distal tubule. Inhibition of sodium transport in these segments would accomplish the final goal of enhancing distal NaCl delivery, luminal flow, and K+ secretion in the aldosterone sensitive distal nephron (ASDN). SUMMARY: Ongoing research seeks to define the relationship between potassium and volume homeostasis by elucidating pathways that couple renal K+ sensing and tubular function during the potassium stress response.
Assuntos
Néfrons/metabolismo , Potássio na Dieta/administração & dosagem , Potássio/metabolismo , Animais , Homeostase , Humanos , Transporte de Íons , Sódio/metabolismo , Estresse FisiológicoRESUMO
Megalin (Lrp2) is a multiligand receptor that drives endocytic flux in the kidney proximal tubule (PT) and is necessary for the recovery of albumin and other filtered proteins that escape the glomerular filtration barrier. Studies in our lab have shown that knockout (KO) of Lrp2 in opossum PT cells leads to a dramatic reduction in sodium-glucose co-transporter 2 (SGLT2) transcript and protein levels, as well as differential expression of genes involved in mitochondrial and metabolic function. SGLT2 transcript levels are reduced more modestly in Lrp2 KO mice. Here, we investigated the effects of Lrp2 KO on kidney function and health in mice fed regular chow (RC) or a Western-style diet (WD) high in fat and refined sugar. Despite a modest reduction in SGLT2 expression, Lrp2 KO mice on either diet showed increased glucose tolerance compared to control mice. Moreover, Lrp2 KO mice were protected against WD-induced fat gain. Surprisingly, renal function in male Lrp2 KO mice on WD was compromised, and the mice exhibited significant kidney injury compared with control mice on WD. Female Lrp2 KO mice were less susceptible to WD-induced kidney injury than male Lrp2 KO. Together, our findings reveal both positive and negative contributions of megalin expression to metabolic health, and highlight a megalin-mediated sex-dependent response to injury following WD.
Assuntos
Dieta Ocidental , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos Knockout , Transportador 2 de Glucose-Sódio , Animais , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Dieta Ocidental/efeitos adversos , Masculino , Camundongos , Feminino , Transportador 2 de Glucose-Sódio/genética , Transportador 2 de Glucose-Sódio/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Camundongos Endogâmicos C57BL , Rim/metabolismo , Rim/patologiaRESUMO
In patients with urinary magnesium wasting, oral and intravenous supplementation often fail to adequately improve serum magnesium levels. Glucose intolerance and diabetes mellitus frequently accompany hypomagnesemia. Clinical trials examining inhibitors of the type 2 sodium glucose cotransporter (SGLT2) show small but significant increases in serum magnesium levels in diabetic patients. This report describes dramatic improvement in serum magnesium levels and associated symptoms after initiating SGLT2 inhibitor therapy in 3 patients with refractory hypomagnesemia and diabetes. Each patient received a different SGLT2 inhibitor: canagliflozin, empagliflozin, or dapagliflozin. One patient discontinued daily intravenous magnesium supplements and exhibited higher serum magnesium levels than had been achieved by magnesium infusion. 2 of the 3 patients exhibited reduced urinary fractional excretion of magnesium, suggesting enhanced tubular reabsorption of magnesium. These observations demonstrate that SGLT2 inhibitors can improve the management of patients with otherwise intractable hypomagnesemia, representing a new tool in this challenging clinical disorder.
RESUMO
With-no-lysine (WNK) kinases coordinate volume and potassium homeostasis by regulating renal tubular electrolyte transport. In the distal convoluted tubule (DCT), potassium imbalance causes WNK signaling complexes to concentrate into large discrete foci, which we call "WNK bodies." Although these structures have been reported previously, the mechanisms that drive their assembly remain obscure. Here, we show that kidney-specific WNK1 (KS-WNK1), a truncated kinase-defective WNK1 isoform that is highly expressed in the DCT, is critical for WNK body formation. While morphologically distinct WNK bodies were evident in the distal tubules of mice subjected to dietary potassium loading and restriction, KS-WNK1 knockout mice were deficient in these structures under identical conditions. Combining in vivo observations in kidney with reconstitution studies in cell culture, we found that WNK bodies are dynamic membraneless foci that are distinct from conventional organelles, colocalize with the ribosomal protein L22, and cluster the WNK signaling pathway. The formation of WNK bodies requires an evolutionarily conserved cysteine-rich hydrophobic motif harbored within a unique N-terminal exon of KS-WNK1. We propose that WNK bodies are not pathological aggregates, but rather are KS-WNK1-dependent microdomains of the DCT cytosol that modulate WNK signaling during physiological shifts in potassium balance.