American Society of Nephrology Kidney Tutored Research and Education for Kidney Scholars (TREKS) Program: A 10-Year Interim Analysis.

In response to decreasing numbers of individuals entering into nephrology fellowships, the American Society of Nephrology launched Kidney Tutored Research and Education for Kidney Scholars (TREKS) to stimulate interest in nephrology among medical students, graduate students, and postdoctoral fellows. The program combines a 1-week intensive exposure to kidney physiology with a longitudinal mentorship program at the participants' home institutions. Ten years in, an analysis was conducted to assess its effectiveness. We surveyed participants to assess their opinions regarding nephrology before and after the course and followed them longitudinally to determine their career choices. TREKS applicants who were not selected to participate were used as a comparison group. Three hundred eighty-one people participated in the program, and 242 completed the survey. After TREKS, both medical students and graduate students showed increased interest in nephrology, with rank scores of 5.6±0.2 before to 7.5±0.1 after the course for medical students (mean±SD, n =189, P = 0.001) and 7.3±0.3 to 8.7±0.3 ( n =53, P = 0.001) for graduate students. In long-term follow-up, TREKS medical students chose a nephrology pipeline residency at a higher rate than medical students overall (57% versus 31%, P = 0.01) and TREKS applicants who did not participate (47% versus 31%, P = 0.04). Nephrology fellowship rates for these groups exceeded the general population but did not significantly differ between TREKS participants and applicants. Doctor of Philosophy students and postdoctoral TREKS participants had a higher rate of participation in nephrology research compared with TREKS applicants (66% versus 30%, P = 0.01). In summary, the American Society of Nephrology Kidney TREKS program has demonstrated that it can increase interest in nephrology in the short term and increase the number of individuals going into nephrology careers. This long-term effect is most evident in Doctor of Philosophy students and postdoctoral participants. Further study is needed to assess the impact of TREKS on enrollment in nephrology fellowship programs.

The IFNγ-PDL1 Pathway Enhances CD8T-DCT Interaction to Promote Hypertension.

BACKGROUND: Renal T cells contribute importantly to hypertension, but the underlying mechanism is incompletely understood. We reported that CD8Ts directly stimulate distal convoluted tubule cells (DCTs) to increase NCC (sodium chloride co-transporter) expression and salt reabsorption. However, the mechanistic basis of this pathogenic pathway that promotes hypertension remains to be elucidated. METHODS: We used mouse models of DOCA+salt (DOCA) treatment and adoptive transfer of CD8+ T cells (CD8T) from hypertensive animals to normotensive animals in in vivo studies. Co-culture of mouse DCTs and CD8Ts was used as in vitro model to test the effect of CD8T activation in promoting NCC-mediated sodium retention and to identify critical molecular players contributing to the CD8T-DCT interaction. Interferon (IFNγ)-KO mice and mice receiving renal tubule-specific knockdown of PDL1 were used to verify in vitro findings. Blood pressure was continuously monitored via radio-biotelemetry, and kidney samples were saved at experimental end points for analysis. RESULTS: We identified critical molecular players and demonstrated their roles in augmenting the CD8T-DCT interaction leading to salt-sensitive hypertension. We found that activated CD8Ts exhibit enhanced interaction with DCTs via IFN-γ-induced upregulation of MHC-I and PDL1 in DCTs, thereby stimulating higher expression of NCC in DCTs to cause excessive salt retention and progressive elevation of blood pressure. Eliminating IFN-γ or renal tubule-specific knockdown of PDL1 prevented T cell homing into the kidney, thereby attenuating hypertension in 2 different mouse models. CONCLUSIONS: Our results identified the role of activated CD8Ts in contributing to increased sodium retention in DCTS through the IFNγ-PDL1 pathway. These findings provide a new mechanism for T cell involvement in the pathogenesis of hypertension and reveal novel therapeutic targets.

Contribution of thick ascending limb and distal convoluted tubule to glucose-induced hypercalciuria in healthy controls.

Carbohydrates increase kidney stone risk and increase urine calcium and magnesium. We hypothesize that the effects of glucose as an allosteric modulator of calcium-sensing receptors may mediate this effect. Six healthy subjects were on a low-sodium diet before consuming 100 g of glucose beverage. Timed fasting (3) and postglucose (6) urine and blood samples were collected every 30 min. Urine composition and serum markers were measured and microvesicular abundance of tubular transport proteins (NHE3, NKCC2, NCC, and TRPV5) were quantified. Postglucose, serum glucose, and insulin rose rapidly with a parallel increase in calcium and magnesium excretion and no change in fractional excretion of sodium. Both serum parathyroid hormone (PTH) and urine TRPV5 fell in the postglucose periods. The rise in the calcium and magnesium excretion likely occurred primarily in the thick ascending limb where they are both under control of the calcium-sensing receptor. The fall in PTH and TRPV5 support the role of glucose as an allosteric modulator of calcium-sensing receptor.NEW & NOTEWORTHY Sugar increases urine calcium and magnesium as well as kidney stone and bone disease risk. Our study provided new insights into the underlying mechanism as we gave healthy subjects an oral glucose load and used newer tools such as fractional excretion of lithium, serum parathyroid hormone, and microvesicular abundance of tubular transport proteins to characterize the mechanism and identify the thick ascending limb with possible calcium-sensing receptor mediation as a likely contributor to this mechanism.

Interleukin 6 mediated activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron.

Hypertension is characterized by increased sodium (Na+) reabsorption along the aldosterone-sensitive distal nephron (ASDN) as well as chronic systemic inflammation. Interleukin-6 (IL-6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na+ reabsorption within the ASDN does not always correlate with increases in aldosterone (Aldo), the primary hormone that modulates Na+ reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased ASDN Na+ reabsorption may occur independent of Aldo stimulation is critical. Here, we show that IL-6 can activate the MR by activating Rac1 and stimulating the generation of reactive oxygen species (ROS) with a consequent increase in thiazide-sensitive Na+ uptake. Using an in vitro model of the distal convoluted tubule (DCT2), mDCT15 cells, we observed nuclear translocation of eGFP-tagged MR after IL-6 treatment. To confirm the activation of downstream transcription factors, mDCT15 cells were transfected with mineralocorticoid response element (MRE)-luciferase reporter constructs; then treated with vehicle, Aldo, or IL-6. Aldosterone or IL-6 treatment increased luciferase activity that was reversed with MR antagonist cotreatment, but IL-6 treatment was reversed by Rac1 inhibition or ROS reduction. In both mDCT15 and mpkCCD cells, IL-6 increased amiloride-sensitive transepithelial Na+ current. ROS and IL-6 increased 22Na+ uptake via the thiazide-sensitive sodium chloride cotransporter (NCC). These results are the first to demonstrate that IL-6 can activate the MR resulting in MRE activation and that IL-6 increases NCC-mediated Na+ reabsorption, providing evidence for an alternative mechanism for stimulating ASDN Na+ uptake during conditions where Aldo-mediated MR stimulation may not occur.

Mechanisms for falling urine pH with age in stone formers.

One of the main functions of the kidney is to excrete an acid load derived from both dietary and endogenous sources, thus maintaining the pH of other fluids in the body. Urine pH is also of particular interest in stone formers, since it determines the presence of either calcium phosphate or uric acid content in stones. Others have noted in epidemiological studies a rise in incidence of low pH-dependent uric acid stones with age, coinciding with a decrease in the incidence of high pH-dependent phosphate stones. Taken together, these trends are suggestive of a longitudinal decline in urine pH in stone-forming patients, and, if true, this could explain the observed trends in stone incidence. We studied 7,891 stone formers, all of whom collected a 24-h urine sample and matching serum. Multivariate modeling revealed that urine pH did indeed fall with age and particularly between the ages of 20 and 50 yr old in both men and women. We sought to explain this trend through the inclusion of traditionally understood determinants of urine pH such as urinary buffers, estimates of glomerular filtration, and dietary acid load, but these, taken together, accounted for but a small fraction of the pH fall. Gastrointestinal anion absorption was the strongest predictor of urine pH in all age groups, as we have previously reported in middle-aged normal men and women. However, we found that, despite a decreasing urine pH, gastrointestinal anion absorption increased monotonically with age. In fact, after adjustment for gastrointestinal anion absorption, urine pH declined more markedly, suggesting that bicarbonate-producing anion absorption is regulated in a manner that offsets the decline of urine pH.

Zinc deficiency induces hypertension by promoting renal Na+ reabsorption.

Zn2+ deficiency (ZnD) is a common comorbidity of many chronic diseases. In these settings, ZnD exacerbates hypertension. Whether ZnD alone is sufficient to alter blood pressure (BP) is unknown. To explore the role of Zn2+ in BP regulation, adult mice were fed a Zn2+-adequate (ZnA) or a Zn2+-deficient (ZnD) diet. A subset of ZnD mice were either returned to the ZnA diet or treated with hydrochlorothiazide (HCTZ), a Na+-Cl- cotransporter (NCC) inhibitor. To reduce intracellular Zn2+ in vitro, mouse distal convoluted tubule cells were cultured in N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, a Zn2+ chelator)- or vehicle (DMSO)-containing medium. To replete intracellular Zn2+, TPEN-exposed cells were then cultured in Zn2+-supplemented medium. ZnD promoted a biphasic BP response, characterized by episodes of high BP. BP increases were accompanied by reduced renal Na+ excretion and NCC upregulation. These effects were reversed in Zn2+-replete mice. Likewise, HCTZ stimulated natriuresis and reversed BP increases. In vitro, Zn2+ depletion increased NCC expression. Furthermore, TPEN promoted NCC surface localization and Na+ uptake activity. Zn2+ repletion reversed TPEN effects on NCC. These data indicate that 1) Zn2+ contributes to BP regulation via modulation of renal Na+ transport, 2) renal NCC mediates ZnD-induced hypertension, and 3) NCC is a Zn2+-regulated transporter that is upregulated with ZnD. This study links dysregulated renal Na+ handling to ZnD-induced hypertension. Furthermore, NCC is identified as a novel mechanism by which Zn2+ regulates BP. Understanding the mechanisms of ZnD-induced BP dysregulation may have an important therapeutic impact on hypertension.

Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery.

Virus-like particles (VLPs) have been extensively explored as nanoparticle vehicles for many applications in biotechnology (e.g., vaccines, drug delivery, imaging agents, biocatalysts). However, amino acid sequence plasticity relative to subunit expression and nanoparticle assembly has not been explored. Whereas the hepatitis B core protein (HBc) VLP appears to be the most promising model for fundamental and applied studies; particle instability, antigen fusion limitations, and intrinsic immunogenicity have limited its development. Here, we apply Escherichia coli-based cell-free protein synthesis (CFPS) to rapidly produce and screen HBc protein variants that still self-assemble into VLPs. To improve nanoparticle stability, artificial covalent disulfide bridges were introduced throughout the VLP. Negative charges on the HBc VLP surface were then reduced to improve surface conjugation. However, removal of surface negative charges caused low subunit solubility and poor VLP assembly. Solubility and assembly as well as surface conjugation were greatly improved by transplanting a rare spike region onto the common shell structure. The newly stabilized and extensively modified HBc VLP had almost no immunogenicity in mice, demonstrating great promise for medical applications. This study introduces a general paradigm for functional improvement of complex protein assemblies such as VLPs. This is the first study, to our knowledge, to systematically explore the sequence plasticity of viral capsids as an approach to defining structure function relationships for viral capsid proteins. Our observations on the unexpected importance of the HBc spike tip charged state may also suggest new mechanistic routes toward viral therapeutics that block capsid assembly.

Parathyroid hormone and the regulation of renal tubular calcium transport.

PURPOSE OF REVIEW: Renal tubule excretion of calcium is carefully and complexly regulated. At the center of this process, parathyroid hormone (PTH) is the chief regulator of renal calcium reabsorption. This review outlines our current understanding of PTH's effects on renal tubular calcium transport, focusing on the more recent discoveries beyond its direct regulation of epithelial Ca channel transient receptor potential vanilloid 5 (TRPV5) and looking at its interaction with sodium transport and the hormones fibroblast growth factor 23 (FGF23) and klotho. RECENT FINDINGS: PTH affects not only TRPV5 directly but other intracellular calcium transport proteins as well. In addition, PTH alters sodium transport that indirectly leads to enhanced TRPV5 activity. FGF23 and klotho have their own effects on TRPV5 as well as effects on PTH secretion. SUMMARY: These discoveries and the interactions between these hormones have direct effects on our understanding of nephrolithiasis and have implications in the development of bone mineral disease in chronic kidney disease and may provide future treatment options.

The sodium chloride cotransporter (NCC) and epithelial sodium channel (ENaC) associate.

The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α- and γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.

PTH modulation of NCC activity regulates TRPV5 Ca2+ reabsorption.

Since parathyroid hormone (PTH) is known to increase transient receptor potential vanilloid (TRPV)5 activity and decrease Na(+)-Cl(-) cotransporter (NCC) activity, we hypothesized that decreased NCC-mediated Na(+) reabsorption contributes to the enhanced TRPV5 Ca(2+) reabsorption seen with PTH. To test this, we used mDCT15 cells expressing functional TRPV5 and ruthenium red-sensitive (45)Ca(2+) uptake. PTH increased (45)Ca(2+) uptake to 8.8 ± 0.7 nmol·mg(-1)·min(-1) (n = 4, P < 0.01) and decreased NCC activity from 75.4 ± 2.7 to 20.3 ± 1.3 nmol·mg(-1)·min(-1) (n = 4, P < 0.01). Knockdown of Ras guanyl-releasing protein (RasGRP)1 had no baseline effect on (45)Ca(2+) uptake but significantly attenuated the response to PTH from a 45% increase (6.0 ± 0.2 to 8.7 ± 0.4 nmol·mg(-1)·min(-1)) in control cells to only 20% in knockdown cells (6.1 ± 0.1 to 7.3 ± 0.2 nmol·mg(-1)·min(-1), n = 4, P < 0.01). Inhibition of PKC and PKA resulted in further attenuation of the PTH effect. RasGRP1 knockdown decreased the magnitude of the TRPV5 response to PTH (7.9 ± 0.1 nmol·mg(-1)·min(-1) for knockdown compared with 9.1 ± 0.1 nmol·mg(-1)·min(-1) in control), and the addition of thiazide eliminated this effect (a nearly identical 9.0 ± 0.1 nmol·mg(-1)·min(-1)). This indicates that functionally active NCC is required for RasGRP1 knockdown to impact the PTH effect on TRPV5 activity. Knockdown of with no lysine kinase (WNK)4 resulted in an attenuation of the increase in PTH-mediated TRPV5 activity. TRPV5 activity increased by 36% compared with 45% in control (n = 4, P < 0.01 between PTH-treated groups). PKC blockade further attenuated the PTH effect, whereas combined PKC and PKA blockade in WNK4KD cells abolished the effect. We conclude that modulation of NCC activity contributes to the response to PTH, implying a role for hormonal modulation of NCC activity in distal Ca(2+) handling.

A role for the circadian clock protein Per1 in the regulation of the NaCl co-transporter (NCC) and the with-no-lysine kinase (WNK) cascade in mouse distal convoluted tubule cells.

It has been well established that blood pressure and renal function undergo circadian fluctuations. We have demonstrated that the circadian protein Per1 regulates multiple genes involved in sodium transport in the collecting duct of the kidney. However, the role of Per1 in other parts of the nephron has not been investigated. The distal convoluted tubule (DCT) plays a critical role in renal sodium reabsorption. Sodium is reabsorbed in this segment through the actions of the NaCl co-transporter (NCC), which is regulated by the with-no-lysine kinases (WNKs). The goal of this study was to test if Per1 regulates sodium transport in the DCT through modulation of NCC and the WNK kinases, WNK1 and WNK4. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of NCC and WNK1 but increased expression of WNK4 in the renal cortex of mice. These findings were confirmed by using Per1 siRNA and pharmacological blockade of Per1 nuclear entry in mDCT15 cells, a model of the mouse distal convoluted tubule. Transcriptional regulation was demonstrated by changes in short lived heterogeneous nuclear RNA. Chromatin immunoprecipitation experiments demonstrated interaction of Per1 and CLOCK with the promoters of NCC, WNK1, and WNK4. This interaction was modulated by blockade of Per1 nuclear entry. Importantly, NCC protein expression and NCC activity, as measured by thiazide-sensitive, chloride-dependent (22)Na uptake, were decreased upon pharmacological inhibition of Per1 nuclear entry. Taken together, these data demonstrate a role for Per1 in the transcriptional regulation of NCC, WNK1, and WNK4.

Mechanisms of angiotensin II stimulation of NCC are time-dependent in mDCT15 cells.

Angiotensin II (ANG II) increases thiazide-sensitive sodium-chloride cotransporter (NCC) activity both acutely and chronically. ANG II has been implicated as a switch that turns WNK4 from an inhibitor of NCC into an activator of NCC, and ANG II's effect on NCC appears to require WNK4. Chronically, ANG II stimulation of NCC results in an increase in total and phosphorylated NCC, but the role of NCC phosphorylation in acute ANG II actions is unclear. Here, using a mammalian cell model with robust native NCC activity, we corroborate the role that ANG II plays in WNK4 regulation and clarify the role of Ste20-related proline alanine-rich kinase (SPAK)-induced NCC phosphorylation in ANG II action. ANG II was noted to have a biphasic effect on NCC, with a peak increase in NCC activity in the physiologic range of 10(-11) M ANG II. This effect was apparent as early as 15 min and remained sustained through 120 min. These changes correlated with significant increases in NCC surface protein expression. Knockdown of WNK4 expression sharply attenuated the effect of ANG II. SPAK knockdown did not affect ANG II action at early time points (15 and 30 min), but it did attenuate the response at 60 min. Correspondingly, NCC phosphorylation did not increase at 15 or 30 min, but increased significantly at 60 min. We therefore conclude that within minutes of an increase in ANG II, NCC is rapidly trafficked to the cell surface in a phosphorylation-independent but WNK4-dependent manner. Then, after 60 min, ANG II induces SPAK-dependent phosphorylation of NCC.

Sex differences in proximal and distal nephron function contribute to the mechanism of idiopathic hypercalcuria in calcium stone formers.

Idiopathic hypercalciuria (IH) is a common familial trait among patients with calcium nephrolithiasis. Previously, we have demonstrated that hypercalciuria is primarily due to reduced renal proximal and distal tubule calcium reabsorption. Here, using measurements of the clearances of sodium, calcium, and endogenous lithium taken from the General Clinical Research Center, we test the hypothesis that patterns of segmental nephron tubule calcium reabsorption differ between the sexes in IH and normal subjects. When the sexes are compared, we reconfirm the reduced proximal and distal calcium reabsorption. In IH women, distal nephron calcium reabsorption is decreased compared to normal women. In IH men, proximal tubule calcium reabsorption falls significantly, with a more modest reduction in distal calcium reabsorption compared to normal men. Additionally, we demonstrate that male IH patients have lower systolic blood pressures than normal males. We conclude that women and men differ in the way they produce the hypercalciuria of IH, with females reducing distal reabsorption and males primarily reducing proximal tubule function.

Aldosterone acutely stimulates NCC activity via a SPAK-mediated pathway.

Hypertension is a leading cause of morbidity and mortality worldwide, and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 h) of aldosterone on NCC in the rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. This effect was dependent upon the presence of the mineralocorticoid receptor and serum- and glucocorticoid-regulated kinase 1 (SGK1). Furthermore, STE20/SPS-1-related proline/alanine-rich kinase (SPAK) phosphorylation also increased, and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via a minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity.

Endotoxemia alters tight junction gene and protein expression in the kidney.

Intact tight junctional (TJ) proteins are required for tubular ion transport and waste excretion. Disruption of TJs may contribute to a decreased glomerular filtration rate in acute kidney injury (AKI) via tubular backleak. The effect of LPS-mediated AKI on murine TJs has not been studied extensively. We hypothesized LPS endotoxin administration to mice would disrupt tubular TJ proteins including zonula occludens-1 (ZO-1), occludin, and claudins. ZO-1 and occludin immunofluorescence 24 h post-LPS revealed a marked change in localization from the usual circumferential fencework pattern to one with substantial fragmentation. Renal ZO-1 expression was significantly reduced 24 h after LPS (decrease of 56.1 ± 7.4%, P < 0.001), with subsequent recovery. ZO-1 mRNA expression was increased 24 h post-LPS (4.34 ± 0.87-fold, P = 0.0019), suggesting disruption of ZO-1 protein is not mediated by transcriptional regulation, but rather by degradation or changes in translation. Similarly, claudin-4 protein expression was decreased despite elevated mRNA. LPS administration resulted in dephosphorylation of occludin and fragmented tubular redistribution. Protein expression of claudin-1, and -3 was increased after LPS. ZO-1, occludin, and claudin-1, -3, and -4 gene expression were increased 48 h after LPS, suggesting a renal response to strengthen TJs following injury. Interestingly, reduced mRNA expression was found only for claudin-8. This study provides further support that LPS-induced AKI is associated with structural injury and is not merely due to hemodynamic changes.

A new model of the distal convoluted tubule.

The Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney is a key determinant of Na(+) balance. Disturbances in NCC function are characterized by disordered volume and blood pressure regulation. However, many details concerning the mechanisms of NCC regulation remain controversial or undefined. This is partially due to the lack of a mammalian cell model of the DCT that is amenable to functional assessment of NCC activity. Previously reported investigations of NCC regulation in mammalian cells have either not attempted measurements of NCC function or have required perturbation of the critical without a lysine kinase (WNK)/STE20/SPS-1-related proline/alanine-rich kinase regulatory pathway before functional assessment. Here, we present a new mammalian model of the DCT, the mouse DCT15 (mDCT15) cell line. These cells display native NCC function as measured by thiazide-sensitive, Cl(-)-dependent (22)Na(+) uptake and allow for the separate assessment of NCC surface expression and activity. Knockdown by short interfering RNA confirmed that this function was dependent on NCC protein. Similar to the mammalian DCT, these cells express many of the known regulators of NCC and display significant baseline activity and dimerization of NCC. As described in previous models, NCC activity is inhibited by appropriate concentrations of thiazides, and phorbol esters strongly suppress function. Importantly, they display release of WNK4 inhibition of NCC by small hairpin RNA knockdown. We feel that this new model represents a critical tool for the study of NCC physiology. The work that can be accomplished in such a system represents a significant step forward toward unraveling the complex regulation of NCC.

Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.

The Pharmacological Inhibition of CaMKII Regulates Sodium Chloride Cotransporter Activity in mDCT15 Cells.

The thiazide-sensitive sodium chloride cotransporter (NCC) in the distal convoluted tubule is responsible for reabsorbing up to one-tenth of the total filtered load of sodium in the kidney. The actin cytoskeleton is thought to regulate various transport proteins in the kidney but the regulation of the NCC by the actin cytoskeleton is largely unknown. Here, we identify a direct interaction between the NCC and the cytoskeletal protein filamin A in mouse distal convoluted tubule (mDCT15) cells and in the native kidney. We show that the disruption of the actin cytoskeleton by two different mechanisms downregulates NCC activity. As filamin A is a substrate of the Ca2+/calmodulin-dependent protein kinase II (CaMKII), we investigate the physiological significance of CaMKII inhibition on NCC luminal membrane protein expression and NCC activity in mDCT15 cells. The pharmacological inhibition of CaMKII with the compound KN93 increases the active form of the NCC (phospho-NCC) at the luminal membrane and also increases NCC activity in mDCT15 cells. These data suggest that the interaction between the NCC and filamin A is dependent on CaMKII activity, which may serve as a feedback mechanism to maintain basal levels of NCC activity in the distal nephron.

RasGRP1 stimulation enhances ubiquitination and endocytosis of the sodium-chloride cotransporter.

The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the distal convoluted tubule. Recently, we described a novel pathway of NCC regulation in which phorbol esters (PE) stimulate Ras guanyl-releasing protein 1 (RasGRP1), triggering a cascade ultimately activating ERK1/2 MAPK and decreasing NCC cell surface expression (Ko B, Joshi LM, Cooke LL, Vazquez N, Musch MW, Hebert SC, Gamba G, Hoover RS. Proc Natl Acad Sci USA 104: 20120-20125, 2007). Little is known about the mechanisms which underlie these effects on NCC activity. Regulation of NCC via changes in NCC surface expression has been reported, but endocytosis of NCC has not been demonstrated. In this study, utilizing biotinylation, internalization assays, and a dynamin dominant-negative construct, we demonstrate that the regulation of NCC by PE occurs via an enhancement in internalization of NCC and is dynamin dependent. In addition, immunoprecipitation of NCC and subsequent immunoblotting for ubiquitin showed increased ubiquitination of NCC with phorbol ester treatment. MEK1/2 inhibitors and gene silencing of RasGRP1 indicated that this effect was dependent on RasGRP1 and ERK1/2 activation. Inhibition of ubiquitination prevents any PE-mediated decrease in NCC surface expression as measured by biotinylation or NCC activity as measured by radiotracer uptake. These findings confirmed that the PE effect on NCC is mediated by endocytosis of NCC. Furthermore, ubiquitination of NCC is essential for this process and this ubiquitination is dependent upon RasGRP1-mediated ERK1/2 activation.

Phorbol ester stimulation of RasGRP1 regulates the sodium-chloride cotransporter by a PKC-independent pathway.

The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of one of the most effective classes of antihypertensive medications, thiazide diuretics. We developed a cell model system to assess NCC function in a mammalian cell line that natively expresses NCC, the mouse DCT (mDCT) cell line. We used this system to study the complex regulation of NCC by the phorbol ester (PE) 12-O-tetradecanoylphorbol-13-acetate (TPA), a diacylglycerol (DAG) analog. It has generally been thought that PEs mediate their effects on transporters through the activation of PKC. However, there are at least five other DAG/PE targets. Here we describe how one of those alternate targets of DAG/PE effects, Ras guanyl-releasing protein 1 (RasGRP1), mediates the PE-induced suppression of function and the surface expression of NCC. Functional assessment of NCC by using thiazide-sensitive (22)Na(+) uptakes revealed that TPA completely suppresses NCC function. Biotinylation experiments demonstrated that this result was primarily because of decreased surface expression of NCC. Although inhibitors of PKC had no effect on this suppression, MAPK inhibitors completely prevented the TPA effect. RasGRP1 activates the MAPK pathway through activation of the small G protein Ras. Gene silencing of RasGRP1 prevented the PE-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of RasGRP1 in mediating the PE-induced suppression of NCC activity through the stimulation of the MAPK pathway.