An improved reporter identifies ruxolitinib as a potent and cardioprotective CaMKII inhibitor.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) hyperactivity causes cardiac arrhythmias, a major source of morbidity and mortality worldwide. Despite proven benefits of CaMKII inhibition in numerous preclinical models of heart disease, translation of CaMKII antagonists into humans has been stymied by low potency, toxicity, and an enduring concern for adverse effects on cognition due to an established role of CaMKII in learning and memory. To address these challenges, we asked whether any clinically approved drugs, developed for other purposes, were potent CaMKII inhibitors. For this, we engineered an improved fluorescent reporter, CaMKAR (CaMKII activity reporter), which features superior sensitivity, kinetics, and tractability for high-throughput screening. Using this tool, we carried out a drug repurposing screen (4475 compounds in clinical use) in human cells expressing constitutively active CaMKII. This yielded five previously unrecognized CaMKII inhibitors with clinically relevant potency: ruxolitinib, baricitinib, silmitasertib, crenolanib, and abemaciclib. We found that ruxolitinib, an orally bioavailable and U.S. Food and Drug Administration-approved medication, inhibited CaMKII in cultured cardiomyocytes and in mice. Ruxolitinib abolished arrhythmogenesis in mouse and patient-derived models of CaMKII-driven arrhythmias. A 10-min pretreatment in vivo was sufficient to prevent catecholaminergic polymorphic ventricular tachycardia, a congenital source of pediatric cardiac arrest, and rescue atrial fibrillation, the most common clinical arrhythmia. At cardioprotective doses, ruxolitinib-treated mice did not show any adverse effects in established cognitive assays. Our results support further clinical investigation of ruxolitinib as a potential treatment for cardiac indications.

CaMKII as a Therapeutic Target in Cardiovascular Disease.

CaMKII (the multifunctional Ca2+ and calmodulin-dependent protein kinase II) is a highly validated signal for promoting a variety of common diseases, particularly in the cardiovascular system. Despite substantial amounts of convincing preclinical data, CaMKII inhibitors have yet to emerge in clinical practice. Therapeutic inhibition is challenged by the diversity of CaMKII isoforms and splice variants and by physiological CaMKII activity that contributes to learning and memory. Thus, uncoupling the harmful and beneficial aspects of CaMKII will be paramount to developing effective therapies. In the last decade, several targeting strategies have emerged, including small molecules, peptides, and nucleotides, which hold promise in discriminating pathological from physiological CaMKII activity. Here we review the cellular and molecular biology of CaMKII, discuss its role in physiological and pathological signaling, and consider new findings and approaches for developing CaMKII therapeutics.

WNK kinases sense molecular crowding and rescue cell volume via phase separation.

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.

The molecular chaperone GRP170 protects against ER stress and acute kidney injury in mice.

Molecular chaperones are responsible for maintaining cellular homeostasis, and one such chaperone, GRP170, is an endoplasmic reticulum (ER) resident that oversees both protein biogenesis and quality control. We previously discovered that GRP170 regulates the degradation and assembly of the epithelial sodium channel (ENaC), which reabsorbs sodium in the distal nephron and thereby regulates salt-water homeostasis and blood pressure. To define the role of GRP170 - and, more generally, molecular chaperones in kidney physiology - we developed an inducible, nephron-specific GRP170-KO mouse. Here, we show that GRP170 deficiency causes a dramatic phenotype: profound hypovolemia, hyperaldosteronemia, and dysregulation of ion homeostasis, all of which are associated with the loss of ENaC. Additionally, the GRP170-KO mouse exhibits hallmarks of acute kidney injury (AKI). We further demonstrate that the unfolded protein response (UPR) is activated in the GRP170-deficient mouse. Notably, the UPR is also activated in AKI when originating from various other etiologies, including ischemia, sepsis, glomerulonephritis, nephrotic syndrome, and transplant rejection. Our work establishes the central role of GRP170 in kidney homeostasis and directly links molecular chaperone function to kidney injury.

L-WNK1 is required for BK channel activation in intercalated cells.

Large-conductance K+ (BK) channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high-K+ diet. In cell culture, the long isoform of with-no-lysine kinase 1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high-K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high-K+ diet. Mice with IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high-K+ (5% K+, as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical + subapical α-subunit expression and BK channel-dependent whole cell currents compared with controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high-K+ diet have higher blood K+ concentration and reduced IC BK channel activity are consistent with impaired urinary K+ secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high-K+ diet.NEW & NOTEWORTHY When mice are placed on a high-K+ diet, genetic disruption of the long form of with no lysine kinase 1 (L-WNK1) in intercalated cells reduced relative apical + subapical localization of the large-conductance K+ channel, blunted large-conductance K+ channel currents in intercalated cells, and increased blood K+ concentration. These data confirm an in vivo role of L-WNK1 in intercalated cells in adaptation to a high-K+ diet.

Effects of extreme potassium stress on blood pressure and renal tubular sodium transport.

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.

Intercalated cell BKα subunit is required for flow-induced K+ secretion.

BK channels are expressed in intercalated cells (ICs) and principal cells (PCs) in the cortical collecting duct (CCD) of the mammalian kidney and have been proposed to be responsible for flow-induced K+ secretion (FIKS) and K+ adaptation. To examine the IC-specific role of BK channels, we generated a mouse with targeted disruption of the pore-forming BK α subunit (BKα) in ICs (IC-BKα-KO). Whole cell charybdotoxin-sensitive (ChTX-sensitive) K+ currents were readily detected in control ICs but largely absent in ICs of IC-BKα-KO mice. When placed on a high K+ (HK) diet for 13 days, blood [K+] was significantly greater in IC-BKα-KO mice versus controls in males only, although urinary K+ excretion rates following isotonic volume expansion were similar in males and females. FIKS was present in microperfused CCDs isolated from controls but was absent in IC-BKα-KO CCDs of both sexes. Also, flow-stimulated epithelial Na+ channel-mediated (ENaC-mediated) Na+ absorption was greater in CCDs from female IC-BKα-KO mice than in CCDs from males. Our results confirm a critical role of IC BK channels in FIKS. Sex contributes to the capacity for adaptation to a HK diet in IC-BKα-KO mice.

Potassium-regulated distal tubule WNK bodies are kidney-specific WNK1 dependent.

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.