Renal Salt Handling Research Articles

Deletion of Kcnj16 altered transcriptomic and metabolomic profiles of Dahl salt-sensitive rats

The essential role of inwardly rectifying K+ Channel Kir5.1 (encoded by the Kcnj16 gene) in renal salt handling and blood pressure control has been demonstrated in both human and animal models. However, the underlying mechanisms are still not fully understood. Here, we aimed to integrate transcriptomics and metabolomics to profile the comprehensive changes of genes and metabolites under the deletion of Kcnj16 in the Dahl salt-sensitive (SS) rat background to identify novel mechanisms. For the transcriptomics profiling, we performed RNA-sequencing on kidney cortex tissue of Kcnj16 knockout (KO) and wild-type (WT) SS rats (N=5 per group). For the metabolomics profiling, we performed untargeted metabolomics of kidney cortex tissue, plasma, and urine of KO (N=6) and WT (N=5) rats. Transcriptomics analysis revealed over 6,000 differentially expressed genes in KO rats compared to WT rats, with renin Ren (the gene encoding renin) being the most upregulated gene. Consistent with the phenotype observed in the KO rats, ingenuity pathway analysis (IPA) based on the transcriptomic profile predicted reduced blood pressure and kidney damage and increased ion transport. Canonical pathway analysis suggested activation of metabolic-related pathways [e.g., xenobiotic metabolism, tricarboxylic acid (TCA) cycle, phenylalanine degradation, etc.] and suppression of immune responses-related pathways [e.g., pathogen-induced cytokine storm signaling pathway, macrophage classical activation signaling pathway, nuclear factor of activated T cells (NFAT) pathway, etc.] in KO rats. Untargeted metabolomic analysis suggested distinct metabolic profiles of plasma, urine, and kidneys between WT and KO rats, with 219, 790, and 31 differentially expressed metabolites identified in plasma, urine, and kidney in KO rats compared to WT rats. Canonical pathway analysis identified spermine and spermidine degradation pathways in both plasma and urine and taurine biosynthesis pathways in both urine and kidney. Integration analysis based on transcriptomic and metabolomic profiles suggested an altered TCA cycle, amino acid, and reactive oxygen species metabolism. In conclusion, besides significantly altered ion transport, our transcriptomics and metabolomics data suggest significant suppressed immune responses-related and altered metabolic-related pathways in SS rats lacking Kir5.1, which provides new insights into the underlying mechanisms of the regulation of blood pressure and renal function by Kir5.1. This work is supported by National Institutes of Health Grants R35 HL135749 and R01 DK135644 (to A.S.), Department of Veteran Affairs grant I01 BX004024 (to A.S.), the American Physiological Society Postdoctoral Fellowship (to B.X.) and the American Society of Nephrology Dimitrios G. Oreopoulos Research Fellowship Award (to L.V.D.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

#4127 MODULATION OF BLOOD PRESSURE THROUGH MICROBIOME EXCHANGE BETWEEN MILAN NORMOTENSIVE AND HYPERTENSIVE STRAINS OF RATS

Abstract Background and Aims Hypertension is one of the major health problems contributing to the development of cardiovascular diseases. In contrast to essential hypertension, which often displays complex pathogenesis due to multifactorial aetiology, several monogenic forms of hypertension are caused by mutations in genes associated with renal salt handling. Thus, studies on monogenic forms of hypertension could hold potential for novel therapeutic approaches for essential hypertension. Recently, increasing numbers of studies have demonstrated the alteration of the gut microbiome in pathological states including hypertension. These observations suggest the involvement of the intestinal microbiome in the development and/or maintenance of hypertension. A reciprocal congenic strain of Milan hypertensive rat (NA rat), harbouring a missense mutation in the gene encoding the alpha subunit of the membrane-skeletal protein adducin (Add1), develops hypertension starting from 3 months after birth. Preliminary studies on NA rats and its littermate Milan normotensive (MN) rats showed diverse expression patterns of renal sodium transporters/ion channels in two strains of rats, indicating a putative role of altered renal salt handling on the enhanced blood pressure in NA rats (unpublished data). Based on this background, the aim of this study was to investigate a possible connection between the gut microbiome and blood pressure phenotypes in these two strains. Furthermore, we evaluated whether the exchange of faeces between the two strains could transfer the phenotypes through the gut microbiome. Finally, mechanisms underlying the altered phenotype after microbiome exchange were investigated. Method Hypertensive NA rats and normotensive MN rats were subjected to ‘homogenization (HOM)’ of the microbiome after birth, which consisted of the exchange of bedding containing faeces from either strain, followed by co-housing of MN and NA rats in the same cage (MN-HOM and NA-HOM groups). For the baseline (BSL) condition, rats were homogenized within the same strain (MN-BSL and NA-BSL). At the age of 5 months, blood pressure measurement was performed using a non-invasive tail-cuff BP-2000 Blood Pressure Analysis system. Faeces from each rat were collected for microbiota metataxonomic analysis using next generation sequencing of 16S ribosome encoding DNA. At the end of the treatment, rats were sacrificed for the collections of blood and organs to assess the expressions of genes involved in renal salt reabsorption. Results Systolic blood pressure (SBP) in MN-BSL was averaged at 143.6 mmHg, while NA-BSL showed average SBP 163.3 mmHg, confirming the hypertensive phenotype in NA rats at baseline (P<0.001). Interestingly, MN-HOM showed a significantly enhanced SBP compared to MN-BSL, reaching 153.5 mmHg (P = 0.005). On the other hand, the average SBP of NA-HOM was163.7 mmHg, indicating that homogenization did not significantly impact blood pressure in NA rats. Although the MN-HOM group showed significantly increased SBP compared with MN-BSL group, the average SBP of MN-HOM was still significantly lower than both the NA-BSL and NA-HOM groups. Different gut microbiota compositions were observed in the two strains of rats at baseline, and HOM altered both. Conclusion We have demonstrated that the hypertensive phenotype in NA rats was transferred to normotensive MN rats through the exchange of gut microbiota, indicating that the intestinal microbiota and/or metabolites derived therefrom could eventually modulate blood pressure. Further studies including immunoblotting as well as uremic toxin analyses are required to unveil the molecular mechanisms by which the gut microbiome modulates blood pressure.

Vitamin D and hypertension: Is there any significant relation?

Vitamin D and hypertension: Is there any significant relation?

The acute pressure natriuresis response is suppressed by selective ETA receptor blockade.

Hypertension is a major risk factor for cardiovascular disease. In a significant minority of people, it develops when salt intake is increased (salt-sensitivity). It is not clear whether this represents impaired vascular function or disruption to the relationship between blood pressure (BP) and renal salt-handling (pressure natriuresis, PN). Endothelin-1 (ET-1) regulates BP via ETA and ETB receptor subtypes. Blockade of ETA receptors reduces BP but promotes sodium retention by an unknown mechanism. ETB blockade increases both BP and sodium retention. We hypothesized that ETA blockade promotes sodium and water retention by suppressing PN. We also investigated whether suppression of PN might reflect off-target ETB blockade. Acute PN was induced by sequential arterial ligation in male Sprague Dawley rats. Intravenous atrasentan (ETA antagonist, 5 mg/kg) halved the normal increase in medullary perfusion and reduced sodium and water excretion by >60%. This was not due to off-target ETB blockade because intravenous A-192621 (ETB antagonist, 10 mg/kg) increased natriuresis by 50% without modifying medullary perfusion. In a separate experiment in salt-loaded rats monitored by radiotelemetry, oral atrasentan reduced systolic and diastolic BP by ∼10 mmHg, but additional oral A-192621 reversed these effects. Endogenous ETA stimulation has natriuretic effects mediated by renal vascular dilation while endogenous ETB stimulation in the kidney has antinatriuretic effects via renal tubular mechanisms. Pharmacological manipulation of vascular function with ET antagonists modifies the BP set-point, but even highly selective ETA antagonists attenuate PN, which may be associated with salt and water retention.

MTOR-Activating Mutations in RRAGD Are Causative for Kidney Tubulopathy and Cardiomyopathy.

Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis. We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.

MTOR-activating mutations of RRAGD cause a novel syndrome combining kidney tubulopathy and cardiomyopathy

Advances in genetic techniques have resulted in the identification of rare inherited disorders of renal magnesium and salt handling. Nevertheless, ∼20% of all patients with primary kidney tubulopathy have no diagnosis. We explore a large multicentric cohort presenting with a novel inherited salt-losing tubulopathy mostly characterized by hypomagnesemia, combined with early-onset dilated cardiomyopathy (DCM). Whole exome and genome sequencings were performed with various subsequent functional analyses of identified RRAGD variants in vitro . In 8 children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt-wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo . Six of these patients additionally suffered from DCM, and a heart transplantation was performed in 3 of them before the age of 25y. A dominant variant in RRAGD was simultaneously identified in 8 members of a large family, which segregated with a similar renal magnesium-losing phenotype. No DCM was detected in this family. RRAGD encodes Rag GTPase D mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron includes the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro . Our findings establish a novel diseased phenotype combining kidney tubulopathy and cardiomyopathy caused by an activation of mTOR signaling suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.

β2-Adrenergic receptor agonism as a therapeutic strategy for kidney disease.

Approximately 14% of the general population suffer from chronic kidney disease that can lead to acute kidney injury (AKI), a condition with up to 50% mortality for which there is no effective treatment. Hypertension, diabetes, and cardiovascular disease are the main comorbidities, and more than 660,000 Americans have kidney failure. β2-Adrenergic receptors (β2ARs) have been extensively studied in association with lung and cardiovascular disease, but with limited scope in kidney and renal diseases. β2ARs are expressed in multiple parts of the kidney including proximal and distal convoluted tubules, glomeruli, and podocytes. Classical and noncanonical β2AR signaling pathways interface with other intracellular mechanisms in the kidney to regulate important cellular functions including renal blood flow, electrolyte balance and salt handling, and tubular function that in turn exert control over critical physiology and pathology such as blood pressure and inflammatory responses. Nephroprotection through activation of β2ARs has surfaced as a promising field of investigation; however, there is limited data on the pharmacology and potential side effects of renal β2AR modulation. Here, we provide updates on some of the major areas of preclinical kidney research involving β2AR signaling that have advanced to describe molecular pathways and identify potential drug targets some of which are currently under clinical development for the treatment of kidney-related diseases.

Regulation of renal pendrin activity by aldosterone.

Pendrin resides on the luminal membrane of type B intercalated cells in the renal collecting tubule system mediating the absorption of chloride in exchange for bicarbonate. In mice or humans lacking pendrin, blood pressure is lower, and pendrin knockout mice are resistant to aldosterone-induced hypertension. Here we discuss recent findings on the regulation of pendrin. Pendrin activity is stimulated during alkalosis partly mediated by secretin. Also, angiotensin II and aldosterone stimulate pendrin activity requiring the mineralocorticoid receptor in intercalated cells. Angiotensin II induces dephosphorylation of the mineralocorticoid receptor rendering the receptor susceptible for aldosterone binding. In the absence of the mineralocorticoid receptor in intercalated cells, angiotensin II does not stimulate pendrin. The effect of aldosterone on pendrin expression is in part mediated by the development of hypokalemic alkalosis and blunted by K-supplements or amiloride. Part of the blood pressure-increasing effect of pendrin is also mediated by its stimulatory effect on the epithelial Na-channel in neighbouring principal cells. These findings identify pendrin as a critical regulator of renal salt handling and blood pressure along with acid--base balance. A regulatory network of hormones fine-tuning activity is emerging. Drugs blocking pendrin are being developed.

Abstract P013: Role Of Kir4.1 ( Kcnj10 ) In The Regulation Of Salt-Induced Hypertension

In the kidney, K ir 4.1 ( Kcnj10 ) and K ir 5.1 ( Kcnj16 ) are highly expressed in the aldosterone sensitive distal nephron, a major target for hormones controlling blood pressure. These basolateral inwardly rectifying potassium channel subunits assemble to form both heteromeric K ir 4.1/K ir 5.1 and homomeric K ir 4.1 channels which control the transepithelial voltage, fine-tune renal electrolyte homeostasis, and contribute to long-term blood pressure control. To study the role of K ir 4.1 in the renal control of blood pressure, renin-angiotensin-aldosterone system (RAAS) balance, and salt-sensitive hypertension, we created a Kcnj10 knockout (SS Kcnj10-/- ) on the Dahl SS rat background using CRISPR/Cas9 mutagenesis. Homozygous SS Kcnj10-/- rats were hypokalemic, had reduced heart, kidney, and body weights, and did not survive more than 3 weeks from birth. Mass spectrometry-based quantification of RAAS peptides revealed increased Ang I, Ang 1-5, and Ang 1-7 levels in SS Kcnj10-/- rats compared to age-matched SS Kcnj10+/+ controls. There were no differences in Ang II, Ang III, Ang IV, or aldosterone levels. In addition, ACE activity was decreased in SS Kcnj10-/- rats. The observed increases in alternative RAS axis peptides and decrease in ACE activity in SS Kcnj10-/- rats are suggestive of a low blood pressure phenotype, however, limited survival precluded blood pressure measurement. Heterozygous (SS Kcnj10+/- ) rats developed normally and showed no changes in serum electrolytes. Survival rate of SS Kcnj10+/- rats was lower than wild-type rats but improved with dietary K + supplementation (1.41% K + ) allowing for telemetric blood pressure measurement. We found no differences in baseline blood pressure under control conditions, but salt-induced (4% NaCl) elevations in blood pressure were significantly attenuated in SS Kcnj10+/- rats. After 3 weeks on high salt, SS Kcnj10+/- rats showed decreased diuresis and improved albuminuria compared to wildtype. Our results demonstrate that renal K ir 4.1 containing channels mediate salt-sensitive increases in blood pressure through regulation of potassium homeostasis, modulation of alternative RAS axis hormones, and control of renal salt handling in the ASDN.

Calcium-Sensing Receptor and Regulation of WNK Kinases in the Kidney.

The kidney is essential for systemic calcium homeostasis. Urinary calcium excretion can be viewed as an integrative renal response to endocrine and local stimuli. The extracellular calcium-sensing receptor (CaSR) elicits a number of adaptive reactions to increased plasma Ca2+ levels including the control of parathyroid hormone release and regulation of the renal calcium handling. Calcium reabsorption in the distal nephron of the kidney is functionally coupled to sodium transport. Apart from Ca2+ transport systems, CaSR signaling affects relevant distal Na+-(K+)-2Cl− cotransporters, NKCC2 and NCC. NKCC2 and NCC are activated by a kinase cascade comprising with-no-lysine [K] kinases (WNKs) and two homologous Ste20-related kinases, SPAK and OSR1. Gain-of-function mutations within the WNK-SPAK/OSR1-NKCC2/NCC pathway lead to renal salt retention and hypertension, whereas loss-of-function mutations have been associated with salt-losing tubulopathies such as Bartter or Gitelman syndromes. A Bartter-like syndrome has been also described in patients carrying gain-of-function mutations in the CaSR gene. Recent work suggested that CaSR signals via the WNK-SPAK/OSR1 cascade to modulate salt reabsorption along the distal nephron. The review presented here summarizes the latest progress in understanding of functional interactions between CaSR and WNKs and their potential impact on the renal salt handling and blood pressure.

Impaired Renal Endothelin‐1 Excretion in Diet‐induced Obesity Contributes to Salt Sensitivity in Male Sprague‐Dawley Rats

The renal endothelin system is an important regulator of sodium excretion in response to high salt intake. Urinary Na+ excretion (UNaV) as well as endothelin‐1 (ET‐1) excretion follow a diurnal rhythm, which is controlled by central and peripheral clock genes. Decreased renal medullary ET‐1 secretion has been attributed to salt sensitive (SS) hypertension. Diet‐induced obesity leads to clock gene dysfunction and Na+ imbalance. However, it is not clear how a high fat (HF) diet could influence the diurnal regulation of these systems. Our overarching hypothesis is that HF diet contributes to the development of salt‐sensitive hypertension through renal ET‐1 dysfunction. Male Sprague‐Dawley rats were put on either high fat (45 Kcal% fat, HF) or normal fat (10 Kcal% fat, NF) diet for 8 weeks duration starting at 6 weeks of age. Half‐way through the feeding protocol, rats were implanted with telemetry transmitters for continuous blood pressure measurement. For the last 2 weeks of feeding, HF and NF groups were divided into 2 subgroups to receive an additional high salt (4% NaCl, HS) diet or continue on the normal salt (0.3% NaCl, NS) diet with continuous blood pressure monitoring. For the last 4 days of the feeding protocol, rats were put in metabolic cages for 2 days of acclimation, and then urine was collected and food and water intakes were monitored in 12‐hr increments (inactive‐lights on/active‐lights off) for the subsequent 2 days. Systolic blood pressure (BP) was significantly increased in HF compared to NF rats after 6 weeks of feeding (145 ± 1 vs 138 ± 2 mmHg, n =14 and 11 respectively) during their active period. Chronic HS diet led to a further increase in active‐time systolic BP in HF rats compared to their NS control (153 ± 2 vs 146 ± 2 mmHg, n =6/group), while addition of HS to the NF group showed no significant increase in systolic BP (145 ± 2 vs 140 ± 3 mmHg, n =6/group) during the active period. The NF group on HS diet had a significant increase in active period ET‐1 excretion compared to NF/NS (4.34 ± 0.35 vs 1.65 ± 0.38 pg/12hrs, n= 6/group). However, HS diet failed to increase active period ET‐1 excretion in the HF (2.56 ±0.29 vs 1.32 ± 0.12 pg/12 hrs, n=6/group) compared to their NS controls. Rats on HF/NS and NF/NS diets were given an acute salt load of 900 μEq NaCl in 1 ml water, i.p. to assess their acute natriuretic response. The natriuretic response to an acute salt load given at the beginning of the active period was significantly reduced in rats on HF vs. NF with ⊗UNaV of 629 ± 91 vs 985 ± 75 μEq/12 hrs in the first 12 hours post‐load, respectively, n= 6/group. This was associated with attenuated ET‐1 excretion in HF versus NF diet (0.93 ± 0.15 vs 1.69 ± 0.16 pg/12hrs, respectively, n=11/group). There were no significant differences in the natriuretic response when the salt load was given at the beginning of the inactive period. These data show that HF diet reduces renal ET‐1 and salt handling in a time of day dependent manner. These findings are also consistent with our hypothesis that timing of Na+ handling may contribute to salt‐sensitive hypertension in diet‐induced obesity.Support or Funding InformationFunded by P01 HL136267 to DMP

Role of Kir4.1 (Kcnj10) in the Regulation of Salt‐Induced Hypertension

In the kidney, Kir4.1 (Kcnj10) and Kir5.1 (Kcnj16) are highly expressed in the aldosterone sensitive distal nephron (ASDN), a major target for multiple hormones controlling blood pressure. These basolateral inwardly rectifying potassium channel subunits assemble to form both heteromeric Kir4.1/Kir5.1 channels and homomeric Kir4.1 channels. These channels control the transepithelial voltage, fine‐tune renal electrolyte homeostasis and contribute to long‐term blood pressure control, correspondingly. To study the role of Kir4.1 containing channels in the renal control of blood pressure, renin‐angiotensin‐aldosterone system (RAAS) balance, and salt‐sensitive (SS) hypertension, we created a Kcnj10 knockout (SSKcnj10−/−) on the Dahl SS rat background. The SSKcnj10−/− rat was created using CRISPR/Cas9 mutagenesis, resulting in a single base pair insertion in exon 2 of the Kcnj10 gene and producing a frameshift in Kir4.1 protein translation after only 26 amino acids. Homozygous SSKcnj10−/− rats were hypokalemic, had reduced heart, kidney, and body weights, and did not survive more than 3 weeks after birth. Mass spectrometry based quantification of serum RAAS peptides revealed increased Ang I (1631 vs. 560 pM), Ang 1–5 (499 vs. 69 pM), and Ang 1–7 (598 vs. 42 pM) levels in SSKcnj10−/− rats compared to age‐matched SSKcnj10+/+ controls. No differences were observed in serum Ang II, Ang III, Ang IV, or aldosterone levels. In addition, ACE activity represented by the Ang II to Ang I ratio was decreased in SSKcnj10−/− rats (0.58 vs. 1.1). The increases in alternative RAS axis peptides (Ang 1–5 and Ang 1–7) and decrease in ACE activity in SSKcnj10−/− rats are suggestive of a low blood pressure phenotype, however, their limited survival precluded blood pressure measurement. Heterozygous (SSKcnj10+/−) developed normally and showed no changes in serum electrolytes. Although survival rate of both male and female SSKcnj10+/− rats was dramatically lower compared to SSKcnj10+/+ rats, they were able to survive much longer than SSKcnj10−/− rats (21 vs. 83 days on average, respectively) with dietary K+ supplementation (HK; 1.41% K+), allowing for telemetric blood pressure measurement. We found no differences in baseline blood pressure under control conditions (LS+HK; 123±2 vs 125±4, mmHg), but salt‐induced (HS, 4% NaCl) elevations in blood pressure were significantly attenuated in SSKcnj10+/− rats fed a HS diet (149±6 vs 183±9 mmHg for SSKcnj10+/− and SSKcnj10+/+ rats, respectively). Furthermore, after 3 weeks on HS, SSKcnj10+/− rats showed decreased diuresis (25±5 vs 38±2, ml/day) and improved albuminuria (6.0±4.0 vs 17.2±7.6, Alb/Cre ratio) compared to SSKcnj10+/+ rats. Our results demonstrate that renal Kir4.1 containing channels mediate salt‐sensitive increases in blood pressure through regulation of potassium homeostasis, modulation of alternative RAS axis hormones, and control of renal salt handling in the ASDN.Support or Funding InformationSupported by the National Institutes of Health grants R35 HL135749, R56 DK121750, R24 HL114474, and F31 DK122647

Tyrosine phosphorylation modulates cell surface expression of chloride cotransporters NKCC2 and KCC3

Cellular chloride transport has a fundamental role in cell volume regulation and renal salt handling. Cellular chloride entry or exit are mediated at the plasma membrane by cotransporter proteins of the solute carrier 12 family. For example, NKCC2 resorbs chloride with sodium and potassium ions at the apical membrane of epithelial cells in the kidney, whereas KCC3 releases chloride with potassium ions at the basolateral membrane. Their ion transport activity is regulated by protein phosphorylation in response to signaling pathways. An additional regulatory mechanism concerns the amount of cotransporter molecules inserted into the plasma membrane. Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that spleen tyrosine kinase (SYK) phosphorylates a specific N-terminal tyrosine residue in each cotransporter. Experimental depletion of endogenous SYK or pharmacological inhibition of its kinase activity increased the abundance of NKCC2 at the plasma membrane of human embryonic kidney cells. In contrast, overexpression of a constitutively active SYK mutant decreased NKCC2 membrane abundance. Intriguingly, the same experimental approaches revealed the opposite effect on KCC3 abundance at the plasma membrane, compatible with the known antagonistic roles of NKCC and KCC cotransporters in cell volume regulation. Thus, we identified a novel pathway modulating the cell surface expression of NKCC2 and KCC3 and show that this same pathway has opposite functional outcomes for these two cotransporters. The findings have several biomedical implications considering the role of these cotransporters in regulating blood pressure and cell volume.

Inhibition of Sodium Glucose Cotransporter 2 Attenuates the Dysregulation of Kelch-Like 3 and NaCl Cotransporter in Obese Diabetic Mice.

Mechanisms underlying the frequent association between salt-sensitive hypertension and type 2 diabetes remain obscure. We previously found that protein kinase C (PKC) activation phosphorylates Kelch-like 3 (KLHL3), an E3 ubiquitin ligase component, at serine 433. We investigated whether impaired KLHL3 activity results in increased renal salt reabsorption via NaCl cotransporter (NCC). We used the db/db diabetes mouse model to explore KLHL3's role in renal salt handling in type 2 diabetes and evaluated mechanisms of KLHL3 dysregulation in cultured cells. We observed PKC activity in the db/db mouse kidney and phosphorylation of serine 433 in KLHL3 (KLHL3S433-P). This modification prevents binding of with-no-lysine (WNK) kinases; however, total KLHL3 levels were decreased, indicating severely impaired KLHL3 activity. This resulted in WNK accumulation, activating NCC in distal convoluted tubules. Ipragliflozin, a sodium glucose cotransporter 2 (SGLT2) inhibitor, lowered PKC activity in distal convoluted tubule cells and reduced KLHL3S433-P and NCC levels, whereas the thiazolidinedione pioglitazone did not, although the two agents similarly reduced in blood glucose levels. We found that, in human embryonic kidney cells expressing KLHL3 and distal convoluted tubule cells, cellular glucose accumulation increased KLHL3S433-P levels through PKC. Finally, the effect of PKC inhibition in the kidney of db/db mice confirmed PKC's causal role in KLHL3S433-P and NCC induction. Dysregulation of KLHL3 is involved in the pathophysiology of type 2 diabetes. These data offer a rationale for use of thiazide in individuals with diabetes and provide insights into the mechanism for cardiorenal protective effects of SGLT2 inhibitors.

The interplay of renal potassium and sodium handling in blood pressure regulation: critical role of the WNK-SPAK-NCC pathway.

Renal salt handling has a profound effect on body fluid and blood pressure (BP) maintenance as exemplified by the use of diuretic medications to treat states of volume expansion or hypertension. It has recently been proposed that a low potassium (K+) intake turns on a "renal K+ switch" which increases sodium (Na+) and chloride (Cl-) reabsorption, causing salt-retention, and in susceptible individuals, this causes hypertension. A signaling network, involving with-no-lysine (WNK) kinases, underpins the switch activity to coordinate aldosterone's two essential actions (K+ secretion and Na+ retention). A dysfunctional WNK kinase network drives excessive and inappropriate Na+, Cl- and urinary K+ retention in familial hyperkalemic hypertension (FHHt, also known as Gordon's syndrome). Mutations in genes encoding WNK1 and WNK4 or components of an ubiquitin ligase complex, cullin3, and kelch-like family member 3 (KLHL3), cause FHHt by upregulating the thiazide-sensitive sodium chloride cotransporter (NCC). Inhibition of NCC with thiazide diuretics corrects hypertension and hyperkalaemia in FHHt. These observations highlight the critical role of the NCC in the regulation of Na+ and K+ balance and of BP. Here we discuss the physiology of Na+ and K+ handling in the distal renal tubule with respect to BP regulation, with a focus on recent discoveries in the WNK- Ste20-related proline-alanine-rich kinase (SPAK)-NCC pathway.

Abstract P198: WNK-SPAK-NKCC1 Cascade Activation Contributes to Worsened Brain Damage in Mice With Hypertension Co-Morbidity after Ischemic Stroke

Objectives: The WNK-SPAK/OSR1 kinase complex plays an important role in renal salt handling and pathogenesis of hypertension by regulating ion transporters and channels. Hypertension is the most common risk factor for stroke and stroke patients with hypertension comorbidity have worsened outcome with an increased risk of dependency or death. However, the mechanisms underlying the worsened ischemic stroke pathophysiology with hypertension comorbidity remain poorly defined. In this study, we investigated roles of the WNK-SPAK-NKCC1 signaling pathway in ischemic brain damage in mice with hypertension comorbidity. Methods: Hypertension was induced in C57BL/6j male mouse (12-15 weeks) by subcutaneous infusion of 1000 ng/kg/min angiotensin II (AngII, mini-osmotic pump) for two weeks. Permanent ischemic stroke was induced by permanent occlusion of the distal branches of the left middle cerebral artery (pd-MCAO). Brain tissues were harvested for immunoblot assessment of expression levels of NKCC1, SPAK/OSR1 or WNK1-4. Infarct volume and hemisphere swelling were determined by TTC staining, and behavioral deficits were analyzed by foot fault test, cylinder test and adhesive tape removal test. Results: pd-MCAO stimulated expression of WNK proteins (isoforms 1, 2, 4), total and phosphorylated SPAK/OSR1 and NKCC1 proteins in ischemic brains of the AngII-infused hypertensive mice compared to normotensive saline controls. In parallel with the increased activation of WNK-SPAK-NKCC1 signaling, hypertensive mice displayed significantly larger infarct volume and hemispheric swelling at 24 h after pd-MCAO compared to normotensive controls. Moreover, hypertensive mice exhibited a slow recovery of neurological function after ischemic stroke compared to normotensive counterparts as assessed by sensory-motor sensitive tests. Conclusions: These results suggest that activation of the WNK-SPAK-NKCC1 complex in hypertensive ischemic brains associates, at least in part, with the worsened brain damage and neurological deficits. Pharmacological inhibition of WNK-SPAK complex has therapeutic potentials for stroke therapy with hypertension comorbidity.

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na+/Cl- cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4-/- mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2+-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

Contributions of rare coding variants in hypotension syndrome genes to population blood pressure variation.

Rare variants, in particular renal salt handling genes, contribute to monogenic forms of hypertension and hypotension syndromes with electrolyte abnormalities. A study by Ji et al (2008) demonstrated this effect for rare loss-of-function coding variants in SLC12A3 (NCCT), SLC12A1 (NKCC2), and KCNJ1 (ROMK) that led to reduction of ∼6 mm Hg for SBP and ∼3 mm Hg for DBP among carriers in 2492 European ancestry Framingham Heart Study (FHS) subjects. These findings support a potentially large role for these variants in interindividual variation in systolic and diastolic blood pressure (SBP, DBP) in the population. The present study focuses on replicating the analyses completed by Ji et al to identify effects of rare variants in the population-based Atherosclerosis Risk in Communities (ARIC) study.We attempted to replicate the findings by Ji et al by applying their criteria to identify putative loss-of-function variants with allele frequency <0.001 and complete conservation across a set of orthologs, to exome sequencing data from 7444 European ancestry participants of the ARIC study.Although we failed to replicate the previous findings when applying their methods to the ARIC study data, we observed a similar effect when we restricted analyses to the subset of variants they observed.These results simultaneously support the utility of exome sequencing data for studying extremely rare coding variants in hypertension and underscore the need for improved filtering methods for identifying functional variants in human sequences.

Angiotensin‐II‐Induced Increase in Blood Pressure in Female and Male Mice

The kidney regulates blood pressure by controlling urinary salt and water excretion. Angiotensin II (AngII), a key player in controlling blood pressure, constricts blood vessels, stimulates vasopressin and aldosterone release, stimulates thirst, and increases sodium absorption from the proximal tubule. All effects cause increased blood pressure. Sex differences in the regulation of arterial blood pressure involve events surrounding the renin‐angiotensin‐aldosterone system. Evidence generally supports a protective mechanism for females with AngII‐induced hypertension; although, conflicting evidence exists. The purpose of this study was to investigate sex differences in CD‐1 mice under AngII administration for a period of 10 days. We hypothesized that female mice would be protected from AngII‐induced increase in mean arterial pressure (MAP) compared to male mice and that the renal handling of salt and water contributes to this protection. Four‐week old CD‐1 mice were obtained from Envigo, Inc. (Indianapolis, IN). Two studies, one with females and one with males, were conducted. Mice were placed in metabolic cages and consumed normal diet and water, ad libitum, for 15 days. After the first 5 days, an Alzet™ minipump containing either AngII (n=5) or vehicle (n=5) was implanted subcutaneously in each mouse to ensure continuous AngII infusion (1 mg/kg/min). Daily measurements included MAP via the tail‐cuff method (Kent Scientific, CODA System), urinary output, and urinary sodium excretion. MAP (mmHg) was not different between females and males in the 5‐day control period (85.1 ± 3.7 vs 81.7 ± 4.0, respectively). AngII‐treatment increased MAP in both females and males (110.3 ± 4.4 and 105.8 ± 3.0, p&lt;0.001, compared to respective control period). Interestingly, AngII‐treated females showed higher urine output than AngII‐treated males (9.7 ± 0.8 vs 7.4 ± 0.4 ml/day, p&lt;0.05) and also had higher sodium excretion (242 mEq/day vs 166 mEq/day, p&lt;0.01). Absolute food intake in females and males was not different, however, females consumed more food per body weight. We conclude that female mice are not protected from AngII‐induced increase in MAP compared to male mice for a 10‐day AngII infusion period. However, based on the sodium excretion data, protection may be observed later from increased urine and sodium output in females. Results suggest that the renal handling of salt and water could be an important determinant of sex differences in blood pressure regulation.Support or Funding InformationNational Institute of General Medical Sciences of National Institutes of Health through Grant Number 8P20GM103447This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Co‐regulation of Renal Function Genes by the Circadian Clock Protein PER1 and Aldosterone

Hypertension is the leading cause of cardiovascular disease. A subset of hypertensive patients experience salt‐sensitive hypertension, which causes elevations in blood pressure (BP) after high salt intake. With large increases in dietary salt worldwide, the prevalence of hypertension has reached 30%. BP follows a circadian rhythm. The circadian clock is a highly conserved mechanism that allows organisms to adapt to the light/dark cycles of our environment. The molecular clock is comprised of core transcription proteins, such as the clock protein PER1. Further, the renin angiotensin aldosterone system (RAAS) is an important homeostatic mechanism to manage sodium and BP homeostasis. Previous results from our lab show that the circadian clock and RAAS both contribute to sodium and BP homeostasis. Based on these studies, we hypothesized that aldosterone and Per1 coordinately regulate genes that contribute to kidney function. We tested this hypothesis first by using a cell culture model from the renal cortical collecting duct. Cells were treated with aldosterone and/or siRNA to knockdown down PER1 expression. Using an unbiased microarray approach, we determined PER1 and aldosterone‐dependent changes in gene expression. Several hundred genes exhibited significant changes in expression in response to hormone treatment and PER1 knockdown. For the first time, we identified secreted frizzled related protein 1 (sFRP1), Claudin‐1, and Rhesus glycoprotein C (Rhcg) as target genes of aldosterone and PER1. The expression of sFRP1 demonstrated a 1.5‐fold increase in response to aldosterone (p&lt;0.05, n=4), which was not affected in the absence of PER1, indicating that aldosterone and PER1 independently regulate sFRP1. In contrast, the 2.9‐fold (p&lt;0.05, n=4) and 1.9‐fold (p&lt;0.05, n=3) induction of Rhcg and Claudin‐1, respectively, by aldosterone was decreased significantly in the absence of PER1, suggesting that PER1 contributes to aldosterone‐mediated regulation of these genes. The ET‐1 gene is of particular interest due to its known role in the regulation of renal salt handling and BP. In the microarray study, knockdown of PER1 augmented the aldosterone response of ET‐1 mRNA by 3‐fold (p&lt;0.05, n=4). Importantly, a similar effect was observed in the kidneys of Per1 knockout mice treated with a high salt diet plus an aldosterone analogue: loss of Per1 augmented the response of ET‐1 by 50% (p&lt;0.01, n=6) compared to no change in WT mice. Interrogation of the database CircaDB revealed that Claudin‐1 and ET‐1 are both expressed with a significant circadian rhythm in the kidney (p&lt;0.001). Together these data demonstrate that multiple genes critical to renal function are co‐regulated by the circadian clock PER1 and RAAS. These data may have important implications for understanding the underlying mechanisms of, and designing novel treatments for, salt‐sensitive hypertension in humans.Support or Funding InformationR01DK109570; R21AG052861; American Heart Association Grant‐in‐aid; Gatorade Trust Fund, Division of Nephrology, Department of Medicine, University of Florida; NF/SG VAMCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.