Urinary concentration of Cathepsin D as a relievable marker of preeclampsia.

BACKGROUND: The early and accurate diagnosis of preeclampsia is crucial to avoid serious complications for both the mother and baby. However, the current diagnostic methods are limited, and there is a need for new diagnostic biomarkers. Previous studies have shown that cathepsin D (CTD) participates in the pathophysiology of preeclampsia and is present in urine samples, making it a potential biomarker for the disease. This study aimed to compare urinary and serum levels of CTD in preeclamptic and normotensive women and analyze its potential role as a diagnostic biomarker in preeclampsia. METHODS: The study included thirty-nine patients with preeclampsia and twelve normotensive pregnant women as controls. Biomarkers were determined using Multiplex Assay kit, and serum prolactin (Prl) and urinary TNF-α levels were also evaluated. Statistical analysis was conducted using the Mann-Whitney U test. RESULTS: We found that urinary and serum CTD levels were significantly higher in the preeclampsia group than in the normotensive group, suggesting that CTD could be a diagnostic biomarker for preeclampsia. No significant differences were found in the levels of serum prolactin or urinary TNF-α between the two groups. CONCLUSIONS: The study provides evidence that non-invasive biological samples such as urine can be used to improve new therapeutic strategies for the early management of preeclampsia.

Impact of transplantation on neutrophil extracellular trap formation in patients with end-stage renal disease: A single-center, prospective cohort study.

ABSTRACT: Increased neutrophil extracellular trap (NET) formation associates with high cardiovascular risk and mortality in patients with end-stage renal disease (ESRD). However, the effect of transplantation on NETs and its associated markers remains unclear. This study aimed to characterize circulating citrullinated Histone H3 (H3cit) and Peptidyl Arginase Deiminase 4 (PAD4) in ESRD patients undergoing transplantation and evaluate the ability of their neutrophils to release NETs.This prospective cohort study included 80 healthy donors and 105 ESRD patients, out of which 95 received a transplant. H3cit and PAD4 circulating concentration was determined by enzyme-linked immunosorbent assay in healthy donors and ESRD patients at the time of enrollment. An additional measurement was carried out within the first 6 months after transplant surgery. In vitro NET formation assays were performed in neutrophils isolated from healthy donors, ESRD patients, and transplant recipients.H3cit and PAD4 levels were significantly higher in ESRD patients (H3cit, 14.38 ng/mL [5.78-27.13]; PAD4, 3.22 ng/mL [1.21-6.82]) than healthy donors (H3cit, 6.45 ng/mL [3.30-11.65], P < .0001; PAD4, 2.0 ng/mL [0.90-3.18], P = .0076). H3cit, but not PAD4, increased after transplantation, with 44.2% of post-transplant patients exhibiting high levels (≥ 27.1 ng/mL). In contrast, NET release triggered by phorbol 12-myristate 13-acetate was higher in neutrophils from ESRD patients (70.0% [52.7-94.6]) than healthy donors (32.2% [24.9-54.9], P < .001) and transplant recipients (19.5% [3.5-65.7], P < .05).The restoration of renal function due to transplantation could not reduce circulating levels of H3cit and PAD4 in ESRD patients. Furthermore, circulating H3cit levels were significantly increased after transplantation. Neutrophils from transplant recipients exhibit a reduced ability to form NETs.

SIRT7 modulates the stability and activity of the renal K-Cl cotransporter KCC4 through deacetylation.

SIRT7 is a NAD+ -dependent deacetylase that controls important aspects of metabolism, cancer, and bone formation. However, the molecular targets and functions of SIRT7 in the kidney are currently unknown. In silico analysis of kidney transcripts of the BXD murine genetic reference population revealed a positive correlation between Sirt7 and Slc12a7 mRNA expression, suggesting a link between the corresponding proteins that these transcripts encode, SIRT7, and the K-Cl cotransporter KCC4, respectively. Here, we find that protein levels and activity of heterologously expressed KCC4 are significantly modulated depending on its acetylation status in Xenopus laevis oocytes. Moreover, SIRT7 interacts with KCC4 in a NAD+ -dependent manner and increases its stability and activity in HEK293 cells. Interestingly, metabolic acidosis increases SIRT7 expression in kidney, as occurs with KCC4. In contrast, total SIRT7-deficient mice present lower KCC4 expression and an exacerbated metabolic acidosis than wild-type mice during an ammonium chloride challenge. Altogether, our data suggest that SIRT7 interacts with, stabilizes and modulates KCC4 activity through deacetylation, and reveals a novel role for SIRT7 in renal physiology.

Urinary expression of long non-coding RNA TUG1 in non-diabetic patients with glomerulonephritides.

Metabolic alterations serve a significant role in the pathogenesis of kidney disease. Long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) is a known regulator of podocyte health and mitochondrial biogenesis. Although TUG1 protects against podocyte loss in models of diabetic nephropathy, it is unknown if urinary TUG1 expression is associated with clinical and histopathological findings in non-diabetic patients diagnosed with glomerulonephritides. In the present study, the expression of TUG1, podocyte-specific markers (nephrin and podocin) and mitochondrial biogenesis-associated mRNAs (transcription factor A mitochondrial, cytochrome C oxidase subunit 5A and peroxisome proliferator-activated receptor γ coactivator 1α) were examined in urinary sediment of non-diabetic patients with biopsy-confirmed glomerulonephritides and healthy controls. Urinary expression of TUG1 was significantly lower in patients with glomerulonephritides, particularly those diagnosed with Focal Segmental Glomerulosclerosis (FSGS). Furthermore, TUG1 levels were associated with urinary expression of podocyte-specific markers and mRNAs associated with mitochondrial biogenesis. Loss of TUG1 expression in urinary sediment was strongly associated with FSGS, highlighting the potential of this lncRNA and its mitochondrial biogenesis-associated targets as non-invasive biomarkers of assessing podocytopathy.

Opioid Preconditioning Modulates Repair Responses to Prevent Renal Ischemia-Reperfusion Injury.

Progression to renal damage by ischemia-reperfusion injury (IRI) is the result of the dysregulation of various tissue damage repair mechanisms. Anesthetic preconditioning with opioids has been shown to be beneficial in myocardial IRI models. Our main objective was to analyze the influence of pharmacological preconditioning with opioids in renal function and expression of molecules involved in tissue repair and angiogenesis. Experimental protocol includes male rats with 45 min ischemia occluding the left renal hilum followed by 24 h of reperfusion with or without 60 min preconditioning with morphine/fentanyl. We analyzed serum creatinine and renal KIM-1 expression. We measured circulating and intrarenal VEGF. Immunohistochemistry for HIF-1 and Cathepsin D (CTD) and real-time PCR for angiogenic genes HIF-1α, VEGF, VEGF Receptor 2 (VEGF-R2), CTD, CD31 and IL-6 were performed. These molecules are considered important effectors of tissue repair responses mediated by the development of new blood vessels. We observed a decrease in acute renal injury mediated by pharmacological preconditioning with opioids. Renal function in opioid preconditioning groups was like in the sham control group. Both anesthetics modulated the expression of HIF-1, VEGF, VEGF-R2 and CD31. Preconditioning negatively regulated CTD. Opioid preconditioning decreased injury through modulation of angiogenic molecule expression. These are factors to consider when establishing strategies in pathophysiological and surgical processes.

Sex-dependent mechanisms involved in renal tolerance to ischemia-reperfusion: Role of inflammation and histone H3 citrullination.

Ischemia-reperfusion (I/R) injury, an inevitable result of kidney transplantation, triggers early inflammatory events that affect graft viability. Evidence from human transplantation and preclinical models of I/R suggests that a female hormonal environment positively influences the ability to recover from ischemic injury. However, the mechanisms behind these effects remain mostly unexplored. Here, we studied the influence of sex on pro-inflammatory mediators involved in the pathophysiology of acute I/R injury in male, female, and female ovariectomized (OVX) Wistar rats that underwent unilateral renal ischemia for 45 min, followed by 24 h of reperfusion. We found improved renal function, reduced cytokine expression, and decreased infiltration of myeloperoxidase-positive cells in females after I/R, when compared to their male and female OVX counterparts. Remarkably, citrullination of histone H3 was exacerbated in serum and renal tubules of females after I/R. In contrast, we observed lower levels of citrullinated histone H3 in male and female OVX rats in response to I/R, mostly in neutrophil extracellular traps. Our results demonstrate that female sex promotes renal I/R tolerance by attenuating pro-inflammatory mediators involved in I/R-induced damage.

Diagnostic, Prognostic, and Therapeutic Value of Non-Coding RNA Expression Profiles in Renal Transplantation.

End-stage renal disease is a public health problem responsible for millions of deaths worldwide each year. Although transplantation is the preferred treatment for patients in need of renal replacement therapy, long-term allograft survival remains challenging. Advances in high-throughput methods for large-scale molecular data generation and computational analysis are promising to overcome the current limitations posed by conventional diagnostic and disease classifications post-transplantation. Non-coding RNAs (ncRNAs) are RNA molecules that, despite lacking protein-coding potential, are essential in the regulation of epigenetic, transcriptional, and post-translational mechanisms involved in both health and disease. A large body of evidence suggests that ncRNAs can act as biomarkers of renal injury and graft loss after transplantation. Hence, the focus of this review is to discuss the existing molecular signatures of non-coding transcripts and their value to improve diagnosis, predict the risk of rejection, and guide therapeutic choices post-transplantation.

Anesthetic preconditioning increases sirtuin 2 gene expression in a renal ischemia reperfusion injury model.

BACKGROUND: Renal transplant surgical proceedings are known to elicit periods of hypoxia and consequent blood flow reestablishment triggering ischemia-reperfusion (I-R) injury. Kidney damage induced by I-R injury associates with a higher risk of graft dysfunction and rejection. Anesthetic preconditioning exerts a beneficial effect on I-R injury by reducing oxidative stress, inflammation and apoptosis. However, the degree of renoprotection stimulated by commonly used anesthetics, as well as their mechanisms of action, are largely unknown. Sirtuins are class III histone deacetylases that reduce cellular stress, promote genome stability and regulate senescence. So far, the relationship between sirtuins and anesthetic preconditioning in the context of renal I-R has not been studied. The main objective of the present work was to determine the renal expression of sirtuins after I-R damage in rats under different anesthetic preconditioning treatments. METHODS: Unilateral ischemia was performed via occlusion of the left renal hilum for 45 min and followed by 24 hours of reperfusion. Anesthetic preconditioning schemes (morphine 0.5 mg/kg, fentanyl 10 µg/kg, propofol 7.5 mg/kg, or dexmedetomidine 25 µg/kg) were administered 1 hour before ischemia. Creatinine levels were determined in serum, and expression of kidney injury molecule 1 and sirtuin 1, 2, 3 and 7 in kidney tissue was quantified by RT-PCR. RESULTS: Anesthetic preconditioning with morphine, fentanyl, propofol and dexmedetomidine reduced kidney injury markers after I-R and modulated sirtuin gene expression. Opioids or dexmedetomidine administration before ischemia increased sirtuin 2 expression and correlated with improved renal function. CONCLUSIONS: Anesthetic preconditioning is a promising strategy to prevent I-R injury associated with transplantation. Our results suggest that sirtuin 2 is involved in the protective mechanisms of some commonly used anesthetics against I-R damage.

Neutrophil Extracellular Traps in the Establishment and Progression of Renal Diseases.

Uncontrolled inflammatory and immune responses are often involved in the development of acute and chronic forms of renal injury. Neutrophils are innate immune cells recruited early to sites of inflammation, where they produce pro-inflammatory cytokines and release mesh-like structures comprised of DNA and granular proteins known as neutrophil extracellular traps (NETs). NETs are potentially toxic, contribute to glomerular injury, activate autoimmune processes, induce vascular damage, and promote kidney fibrosis. Evidence from multiple studies suggests that an imbalance between production and clearance of NETs is detrimental for renal health. Hence strategies aimed at modulating NET-associated processes could have a therapeutic impact on a myriad of inflammatory diseases that target the kidney. Here, we summarize the role of NETs in the pathogenesis of renal diseases and their mechanisms of tissue damage.

Opioids Preconditioning Upon Renal Function and Ischemia-Reperfusion Injury: A Narrative Review.

Kidneys have an important role in regulating water volume, blood pressure, secretion of hormones and acid-base and electrolyte balance. Kidney dysfunction derived from acute injury can, under certain conditions, progress to chronic kidney disease. In the late stages of kidney disease, treatment is limited to replacement therapy: Dialysis and transplantation. After renal transplant, grafts suffer from activation of immune cells and generation of oxidant molecules. Anesthetic preconditioning has emerged as a promising strategy to ameliorate ischemia reperfusion injury. This review compiles some significant aspects of renal physiology and discusses current understanding of the effects of anesthetic preconditioning upon renal function and ischemia reperfusion injury, focusing on opioids and its properties ameliorating renal injury. According to the available evidence, opioid preconditioning appears to reduce inflammation and reactive oxygen species generation after ischemia reperfusion. Therefore, opioid preconditioning represents a promising strategy to reduce renal ischemia reperfusion injury and, its application on current clinical practice could be beneficial in events such as acute renal injury and kidney transplantation.

Vasoinhibin Suppresses Nerve Growth Factor-Induced Differentiation and Survival of PC12 Pheochromocytoma Cells.

BACKGROUND: Vasoinhibin, a protein derived from prolactin, regulates various vascular functions including endothelial cell survival. Of note, vasoinhibin is present in the central nervous system, where it triggers neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin compromises nerve growth factor (NGF)-induced neurite outgrowth in primary sensory neurons of the peripheral nervous system. Nonetheless, information on the functions of vasoinhibin in developing neurons remains limited. The present study explored whether vasoinhibin affects the neurotrophic actions of NGF by measuring the cell differentiation and survival of PC12 pheochromocytoma cells. METHODS: The effects of recombinant or lentiviral vector-transduced human vasoinhibin were tested on differentiating PC12 cells. Neurite outgrowth was quantified by measuring their length and density. The MTT assay was employed to assess cell viability, and ELISA was used to quantify DNA fragmentation as an index of apoptosis. Phosphorylated Akt and ERK1/2 were analyzed by Western blotting. RESULTS: The addition of a human recombinant vasoinhibin, and the transduction of a lentiviral vector carrying a human vasoinhibin sequence, significantly reduced NGF-induced neurite outgrowth, cell survival, and phosphorylation of Akt and ERK1/2, and increased DNA fragmentation and caspase 3 activation in PC12 cells. CONCLUSIONS: Vasoinhibin downregulates NGF-induced differentiation and survival of PC12 cells, blocking tropomyosin receptor kinase A-triggered signaling pathways and increasing apoptosis. These results establish that vasoinhibin interaction with NGF and other neurotrophins may be critical in mediating pathways involved in neuronal survival and differentiation.

Novel Roles of Non-Coding RNAs in Opioid Signaling and Cardioprotection.

Cardiovascular disease (CVD) is a significant cause of morbidity and mortality across the world. A large proportion of CVD deaths are secondary to coronary artery disease (CAD) and myocardial infarction (MI). Even though prevention is the best strategy to reduce risk factors associated with MI, the use of cardioprotective interventions aimed at improving patient outcomes is of great interest. Opioid conditioning has been shown to be effective in reducing myocardial ischemia-reperfusion injury (IRI) and cardiomyocyte death. However, the molecular mechanisms behind these effects are under investigation and could provide the basis for the development of novel therapeutic approaches in the treatment of CVD. Non-coding RNAs (ncRNAs), which are functional RNA molecules that do not translate into proteins, are critical modulators of cardiac gene expression during heart development and disease. Moreover, ncRNAs such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are known to be induced by opioid receptor activation and regulate opioid signaling pathways. Recent advances in experimental and computational tools have accelerated the discovery and functional characterization of ncRNAs. In this study, we review the current understanding of the role of ncRNAs in opioid signaling and opioid-induced cardioprotection.

Vasoinhibin Suppresses the Neurotrophic Effects of VEGF and NGF in Newborn Rat Primary Sensory Neurons.

BACKGROUND/AIMS: Studies on the biological actions of vasoinhibins have focused mainly on endothelial cells. However, there is incipient knowledge about how vasoinhibins affect the nervous system, even if the target cells and mechanisms of action involved in these effects are unknown. METHODS: In order to determine if neurons are direct targets of vasoinhibins, we examined cellular outcomes and the intracellular pathways involved in the neuronal actions of vasoinhibins using newborn rat dorsal root ganglion (DRG) neurons as a model system. RESULTS: Vascular endothelial growth factor (VEGF) or nerve growth factor (NGF) treatment for 48 h resulted in neurite outgrowth stimulation in both DRG cultured explants and isolated primary sensory neurons. Interestingly, a recombinant vasoinhibin containing the first 123 amino acids of human prolactin antagonized the VEGF- and NGF-induced stimulation of neurite outgrowth. Vasoinhibin significantly reduced the density of neurites in DRG explants and obliterated neuritogenesis in isolated DRG neurons in primary culture, supporting a direct neuronal effect of vasoinhibin. In cultures of isolated DRG cells, virtually all ß3-tubulin-labeled cells express TrkA, and the majority of these cells also express VEGFR2. Short-term VEGF or NGF treatment of DRG explants resulted in increased ERK1/2 and AKT phosphorylation, whereas incubation of DRG with the combination of either VEGF or NGF together with vasoinhibin resulted in blunted VEGF- or NGF-induced phosphorylation of both ERK1/2 and AKT. CONCLUSION: Our results show that primary sensory neurons are direct targets of vasoinhibin, and suggest that vasoinhibin inhibition of neurite outgrowth involves the disruption of ERK and AKT phosphorylation cascades.

With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl- cotransporters.

The K(+)-Cl(-) cotransporters (KCC1-KCC4) encompass a branch of the SLC12 family of electroneutral cation-coupled chloride cotransporters that translocate ions out of the cell to regulate various factors, including cell volume and intracellular chloride concentration, among others. L-WNK1 is an ubiquitously expressed kinase that is activated in response to osmotic stress and intracellular chloride depletion, and it is implicated in two distinct hereditary syndromes: the renal disease pseudohypoaldosteronism type II (PHAII) and the neurological disease hereditary sensory neuropathy 2 (HSN2). The effect of L-WNK1 on KCC activity is unknown. Using Xenopus laevis oocytes and HEK-293 cells, we show that the activation of KCCs by cell swelling was prevented by L-WNK1 coexpression. In contrast, the activity of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 was remarkably increased with L-WNK1 coexpression. The negative effect of L-WNK1 on the KCCs is kinase dependent. Elimination of the STE20 proline-alanine rich kinase (SPAK)/oxidative stress-responsive kinase (OSR1) binding site or the HQ motif required for the WNK-WNK interaction prevented the effect of L-WNK1 on KCCs, suggesting a required interaction between L-WNK1 molecules and SPAK. Together, our data support that NKCC1 and KCCs are coordinately regulated by L-WNK1 isoforms.

The Effect of WNK4 on the Na+-Cl- Cotransporter Is Modulated by Intracellular Chloride.

It is widely recognized that the phenotype of familial hyperkalemic hypertension is mainly a consequence of increased activity of the renal Na(+)-Cl(-) cotransporter (NCC) because of altered regulation by with no-lysine-kinase 1 (WNK1) or WNK4. The effect of WNK4 on NCC, however, has been controversial because both inhibition and activation have been reported. It has been recently shown that the long isoform of WNK1 (L-WNK1) is a chloride-sensitive kinase activated by a low Cl(-) concentration. Therefore, we hypothesized that WNK4 effects on NCC could be modulated by intracellular chloride concentration ([Cl(-)]i), and we tested this hypothesis in oocytes injected with NCC cRNA with or without WNK4 cRNA. At baseline in oocytes, [Cl(-)]i was near 50 mM, autophosphorylation of WNK4 was undetectable, and NCC activity was either decreased or unaffected by WNK4. A reduction of [Cl(-)]i, either by low chloride hypotonic stress or coinjection of oocytes with the solute carrier family 26 (anion exchanger)-member 9 (SLC26A9) cRNA, promoted WNK4 autophosphorylation and increased NCC-dependent Na(+) transport in a WNK4-dependent manner. Substitution of the leucine with phenylalanine at residue 322 of WNK4, homologous to the chloride-binding pocket in L-WNK1, converted WNK4 into a constitutively autophosphorylated kinase that activated NCC, even without chloride depletion. Elimination of the catalytic activity (D321A or D321K-K186D) or the autophosphorylation site (S335A) in mutant WNK4-L322F abrogated the positive effect on NCC. These observations suggest that WNK4 can exert differential effects on NCC, depending on the intracellular chloride concentration.

[Pathophysiological aspects of K+: Cl- cotransporters]. / Aspectos fisiopatologicos de los cotransportaores de K+ :CI- .

The K+:Cl- cotransporters or KCCs are membrane proteins that move K+ and Cl- ions across the membrane without changing the transmembrane potential. KCCs belong to the SLC12 (Solute Carrier Family 12) family of electroneutral cation-chloride cotransporters (CCC), and they are secondary active ion transporters because use the established gradients from the primary active transporter through the Na+/K+- ATPase. Although there are nine members identify in this family, up today only seven genes had been characterized. Among them are two loop diuretics-sensitive Na+:K+:2Clcotransporters (NKCC1/NKCC2), the thiazide-sensitive Na+:Cl- cotransporter (NCC), and finally the K+:Cl- cotransporters (KCC), encoded for at least four homologous genes (KCC1-KCC4), and from which there are many isoforms due to alternative splicing. KCC1 is a ubiquitous isoform, KCC3 and KCC4 isoforms are widely expressed, particularly in epithelial cells, while KCC2 is restricted to the central nervous system (CNS). All these cotransporters play an essential role in many physiological processes such as cell volume regulation, transepithelial salt transport and regulation of the intraneuronal chloride concentration. This review has the purpose to show briefly the molecular characteristics as well as the physiological importance and roles of the KCCs in several pathologies.

Molecular evidence for a role for K(+)-Cl(-) cotransporters in the kidney.

K(+)-Cl(-) cotransporter (KCC) isoforms 3 (KCC3) and 4 (KCC4) are expressed at the basolateral membrane of proximal convoluted tubule cells, and KCC4 is present in the basolateral membrane of the thick ascending loop of Henle's limb and α-intercalated cells of the collecting duct. Little is known, however, about the physiological roles of these transporters in the kidney. We evaluated KCC3 and KCC4 mRNA and protein expression levels and intrarenal distribution in male Wistar rats or C57 mice under five experimental conditions: hyperglycemia after a single dose of streptozotocin, a low-salt diet, metabolic acidosis induced by ammonium chloride in drinking water, and low- or high-K(+) diets. Both KCC3 mRNA and protein expression were increased during hyperglycemia in the renal cortex and at the basolateral membrane of proximal tubule cells but not with a low-salt diet or acidosis. In contrast, KCC4 protein expression was increased by a low-sodium diet in the whole kidney and by metabolic acidosis in the renal outer medulla, specifically at the basolateral membrane of α-intercalated cells. The increased protein expression of KCC4 by a low-salt diet was also observed in WNK4 knockout mice, suggesting that upregulation of KCC4 in these circumstances is not WNK4 dependent. No change in KCC3 or KCC4 protein expression was observed under low- or high-K(+) diets. Our data are consistent with a role for KCC3 in the proximal tubule glucose reabsorption mechanism and for KCC4 in salt reabsorption of the thick ascending loop of Henle's loop and acid secretion of the collecting duct.

N-terminal serine dephosphorylation is required for KCC3 cotransporter full activation by cell swelling.

The K(+):Cl(-) cotransporter (KCC) activity is modulated by phosphorylation/dephosphorylation processes. In isotonic conditions, KCCs are inactive and phosphorylated, whereas hypotonicity promotes their dephosphorylation and activation. Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation. However, here we show that the double mutant KCC3-T991A/T1048A could be further activated by hypotonicity, suggesting that additional phosphorylation site(s) are involved. We observed that in vitro activated STE20/SPS1-related proline/alanine-rich kinase (SPAK) complexed to its regulatory MO25 subunit phosphorylated KCC3 at Ser-96 and that in Xenopus laevis oocytes Ser-96 of human KCC3 is phosphorylated in isotonic conditions and becomes dephosphorylated during incubation in hypotonicity, leading to a dramatic increase in KCC3 function. Additionally, WNK3, which inhibits the activity of KCC3, promoted phosphorylation of Ser-96 as well as Thr-991 and Thr-1048. These observations were corroborated in HEK293 cells stably transfected with WNK3. Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3. However, when compared with the double mutant KCC3-T991A/T1048A, the triple mutant KCC3-S96A/T991A/T1048A activity in isotonic conditions was significantly higher, and it was not further increased by hypotonicity or inhibited by WNK3. We conclude that serine residue 96 of human KCC3 is a third site that has to be dephosphorylated for full activation of the cotransporter during hypotonicity.

Similar effects of all WNK3 variants on SLC12 cotransporters.

With-no-lysine kinase 3 (WNK3) is a member of a subfamily of serine/threonine kinases that modulate the activity of the electroneutral cation-coupled chloride cotransporters. WNK3 activates NKCC1/2 and NCC and inhibits the KCCs. Four splice variants are generated from the WNK3 gene. Our previous studies focused on the WNK3-18a variant. However, it has been suggested that other variants could have different effects on the cotransporters. Thus, the present study was designed to define the effects of all WNK3 variants on members of the SLC12 family. By RT-PCR from a fetal brain library, exons 18b and 22 were separately amplified and subcloned into the original WNK3-18a or catalytically inactive WNK3-D294A to obtain all four potential combinations with and without catalytic activity (18a, 18a+22, 18b, and 18b+22). The basal activity of the cotransporters and the effects of WNK3 isoforms were assessed in Xenopus laevis oocytes coinjected with each of the WNK3 variant cRNAs. In isotonic conditions, the basal activity of NCC and NKCC1/2 were increased by coinjection with any of the WNK3. The positive effects occurred even in hypotonic conditions, in which the basal activity of NKCC1 is completely prevented. Consistent with these observations, when expressed in hypotonicity, all KCCs were active, but in the presence of any of the WNK3 variants, KCC activity was completely reduced. That is, NKCC1/2 and NCC were inhibited, even in hypertonicity, while KCCs were activated, even in isotonic conditions. We conclude that the effects of all WNK3 variants toward SLC12 proteins are similar.