Tubular deficiency of ABCA1 augments cholesterol- and Na+-dependent effects on systemic blood pressure in male mice.

Dyslipidemia, with changes in plasma membrane (PM) composition, is associated with hypertension, while rising PM cholesterol induces Na+ channel activity. We hypothesize that ablation of renal tubular ABCA1, a cholesterol efflux protein, leads to cholesterol- and Na+-dependent changes in blood pressure (BP). Transgenic mice (TgPAX8rtTA;tetO-Cre/+) expressing a doxycycline (dox)-inducible CRE recombinase were bred with mice expressing floxed ABCA1 to generate renal tubules deficient in ABCA1 (ABCA1FF). Tail-cuff systolic BP (SBP) was measured in mice on specific diets. Immunoblotting was performed on whole and PM protein lysates of kidney from mice completing experimental diets. Cortical PM of ABCA1FF showed reduced ABCA1 (60 ± 28%; n = 10, P < 0.05) compared with wild-type littermates (WT; n = 9). Tail-cuff SBP of ABCA1FF (n = 11) was not only greater post dox, but also during cholesterol or high Na+ feeding (P < 0.05) compared with WT mice (n = 15). A Na+-deficient diet abolished the difference, while 6 wk of cholesterol diet raised SBP in ABCA1FF compared with mice before cholesterol feeding (P < 0.05). No difference in α-ENaC protein abundance was noted in kidney lysate; however, γ-ENaC increased in ABCA1FF mice versus WT mice. In kidney membranes, NKCC2 abundance was greater in ABCA1FF versus WT mice. Cortical lysates of ABCA1FF mouse kidneys expressed less renin and angiotensin I receptor than WT mouse kidneys. Furosemide injection induced a greater diuretic effect in ABCA1FF (n = 7; 45.2 ± 8.7 µL/g body wt) versus WT (n = 7; 33.1 ± 6.9 µL/g body wt; P < 0.05) but amiloride did not. Tubular ABCA1 deficiency induces cholesterol-dependent rise in SBP and modest Na+ sensitivity of SBP, which we speculate is partly related to Na+ transporters and channels.NEW & NOTEWORTHY Cholesterol has been linked to greater Na+ channel activity in kidney cells, which may predispose to systemic hypertension. We showed that when ABCA1, a protein that removes cholesterol from tissues, is ablated from mouse kidneys, systemic blood pressure is greater than normal mice. Dietary cholesterol further increases blood pressure in transgenic mice, whereas low dietary salt intake reduced blood pressure to that of normal mice. Thus, we speculate that diseases and pharmaceuticals that reduce renal ABCA1 expression, like diabetes and calcineurin inhibitors, respectively, contribute to the prominence of hypertension in their clinical presentation.

Initial mean arterial blood pressure (MABP) measurement is a risk factor for mortality in hypertensive COVID-19 positive hospitalized patients.

BACKGROUND: Hypertension (HTN) is associated with severe COVID-19 infection; however, it remains unknown if the level of blood pressure (BP) predicts mortality. We tested whether the initial BP in the emergency department of hospitalized patients portends mortality in COVID-19 positive(+) patients. METHODS: Data from COVID-19(+) and negative (-) hospitalized patients at Stony Brook University Hospital from March to July 2020 were included. The initial mean arterial BPs (MABPs) were categorized into tertiles (T) of MABP (65-85 [T1], 86-97 [T2] and ≥98 [T3] mmHg). Differences were evaluated using univariable (t-tests, chi-squared) tests. Multivariable (MV) logistic regression analyses were computed to assess links between MABP and mortality in hypertensive COVID-19 patients. RESULTS: 1549 adults were diagnosed with COVID-19 (+) and 2577 tested negative (-). Mortality of COVID-19(+) was 4.4-fold greater than COVID-19(-) patients. Though HTN prevalance did not differ between COVID-19 groups, the presenting systolic BP, diastolic BP, and MABP were lower in the COVID-19(+) vs (-) cohort. When subjects were categorized into tertiles of MABP, T2 tertile of MABP had the lowest mortality and the T1 tertile of MABP had greatest mortality compared to T2; however, no difference in mortality was noted across tertiles of MABP in COVID-19 (-). MV analysis of COVID-19 (+) subjects exposed death as a risk factor for T1 MABP. Next, the mortality of those with a historic diagnosis of hypertension or normotension were studied. On MV analysis, T1 MABP, gender, age, and first respiratory rate correlated with mortality while lymphocyte count inversely correlated with death in hypertensive COVID-19 (+) patients while neither T1 nor T3 categories of MABP predicted death in non-hypertensives. CONCLUSIONS: Low-normal admitting MABP in COVID-19 (+) subjects with a historical diagnosis of HTN is associated with mortality and may assist in identifying those at greatest mortality risk.

Unilateral Nephrectomy Stimulates ERK and Is Associated With Enhanced Na Transport.

Nephron loss initiates compensatory hemodynamic and cellular effects on the remaining nephrons. Increases in single nephron glomerular filtration rate and tubular flow rate exert higher fluid shear stress (FSS) on tubules. In principal cell (PC) culture models FSS induces ERK, and ERK is implicated in the regulation of transepithelial sodium (Na) transport, as well as, proliferation. Thus, we hypothesize that high tubular flow and FSS mediate ERK activation in the cortical collecting duct (CCD) of solitary kidney which regulates amiloride sensitive Na transport and affects CCD cell number. Immunoblotting of whole kidney protein lysate was performed to determine phospho-ERK (pERK) expression. Next, sham and unilateral nephrectomized mice were stained with anti-pERK antibodies, and dolichos biflorus agglutinin (DBA) to identify PCs with pERK. Murine PCs (mpkCCD) were grown on semi-permeable supports under static, FSS, and FSS with U0126 (a MEK1/2 inhibitor) conditions to measure the effects of FSS and ERK inhibition on amiloride sensitive Na short circuit current (Isc). pERK abundance was greater in kidney lysate of unilateral vs. sham nephrectomies. The total number of cells in CCD and pERK positive PCs increased in nephrectomized mice (9.3 ± 0.4 vs. 6.1 ± 0.2 and 5.1 ± 0.5 vs. 3.6 ± 0.3 cell per CCD nephrectomy vs. sham, respectively, n > 6 per group, p < 0.05). However, Ki67, a marker of proliferation, did not differ by immunoblot or immunohistochemistry in nephrectomy samples at 1 month compared to sham. Next, amiloride sensitive Isc in static mpkCCD cells was 25.3 ± 1.7 µA/cm2 (n = 21), but after exposure to 24 h of FSS the Isc increased to 41.4 ± 2.8 µA/cm2 (n = 22; p < 0.01) and returned to 19.1 ± 2.1 µA/cm2 (n = 18, p < 0.01) upon treatment with U0126. Though FSS did not alter α- or γ-ENaC expression in mpkCCD cells, γ-ENaC was reduced in U0126 treated cells. In conclusion, pERK increases in whole kidney and, specifically, CCD cells after nephrectomy, but pERK was not associated with active proliferation at 1-month post-nephrectomy. In vitro studies suggest high tubular flow induces ERK dependent ENaC Na absorption and may play a critical role in Na balance post-nephrectomy.

Telenephrology: An Emerging Platform for Delivering Renal Health Care.

Electronic-based health care delivery systems are gaining popularity among patients and clinicians because of convenience. Importantly, telemedicine, the delivery of health care and/or health information using electronic systems, can deliver primary and specialized health care to geographically isolated patients, who account for nearly 20% of the US population. In nephrology, where a growing discrepancy exists between the geographic location of nephrologists and patients with kidney disease, telenephrology can bridge distance and deliver renal care and education to the isolated. Large nationalized health care systems, for which incentives are aligned to innovate and implement new platforms to deliver cost-effective care, have been at the forefront of telenephrology. These systems include synchronous direct physician-patient care through clinical videoconferencing, and asynchronous modalities such as electronic consultation and video telehealth to educate internists about specialized clinical topics. Large health care organizations are adopting these platforms as standalone services; however, expansion into the private health care system has been limited by reimbursement, regulations, and other issues. Though telenephrology is patient centered, studies are needed to rigorously test its clinical efficacy and cost-effectiveness. Nonetheless, growing patient demand for patient-centric health care will continue to expand the telenephrology space.

Cellular cholesterol modifies flow-mediated gene expression.

Downregulation of heme oxygenase-1 (HO-1), cyclooxygenase-2 (COX2), and nitric oxide synthase-2 (NOS2) in the kidneys of Dahl rodents causes salt sensitivity, while restoring their expression aids in Na+ excretion and blood pressure reduction. Loading cholesterol into collecting duct (CD) cells represses fluid shear stress (FSS)-mediated COX2 activity. Thus, we hypothesized that cholesterol represses flow-responsive genes necessary to effectuate Na+ excretion. To this end, CD cells were used to test whether FSS induces these genes and if cholesterol loading represses them. Mice fed either 0% or 1% cholesterol diet were injected with saline, urine volume and electrolytes were measured, and renal gene expression determined. FSS-exposed CD cells demonstrated increases in HO-1 mRNA by 350-fold, COX2 by 25-fold, and NOS2 by 8-fold in sheared cells compared with static cells (P < 0.01). Immunoblot analysis of sheared cells showed increases in HO-1, COX2, and NOS2 protein, whereas conditioned media contained more HO-1 and PGE2 than static cells. Cholesterol loading repressed the sheared mediated protein abundance of HO-1 and NOS2 as well as HO-1 and PGE2 concentrations in media. In cholesterol-fed mice, urine volume was less at 6 h after injection of isotonic saline (P < 0.05). Urinary Na+ concentration, urinary K+ concentration, and osmolality were greater, whereas Na+ excretion was less, at the 6-h urine collection time point in cholesterol-fed versus control mice (P < 0.05). Renal cortical and medullary HO-1 (P < 0.05) and NOS2 (P < 0.05) mRNA were repressed in cholesterol-fed compared with control mice. Cholesterol acts to repress flow induced natriuretic gene expression, and this effect, in vivo, may contribute to renal Na+ avidity.

Telenephrology: Providing Healthcare to Remotely Located Patients with Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) patients who live far (>30 miles) from their nephrologist experience lower rates of clinic visit adherence, limited access to treatment, and higher rates of hospitalization and mortality than patients who live in close proximity to their nephrologist. Strategies to minimize disparities between urban and remotely located CKD patients are needed. The purpose of this study was to determine whether adherence to clinic visits and clinical outcomes in the remote management of CKD via telenephrology is comparable to in-person conventional care. METHODS: Renal clinic adherence and composite outcomes of death, end-stage renal disease (ESRD), or doubling of serum creatinine (Cr) were measured in geographically remote Hudson Valley VA Medical Center (HVVAMC) CKD patients enrolled in telenephrology (n = 112) and CKD patients enrolled in the Bronx VAMC renal clinic (n = 116). RESULTS: Prior to implementing the telenephrology service, 53.1% of scheduled visits of rural HVVAMC patients to the Bronx VAMC renal clinic were either cancelled or were "no-shows." This was reduced by nearly half (28.5%) after instituting telenephrology (p < 0.001). Moreover, the frequency of attending appointments was greater in the telenephrology (71.9%) vs. in-person Bronx VA cohort (61.0%). The incidence of the composite outcome of death, ESRD, or doubling of Cr was similar between both groups (p = 0.96) over 2 years of follow-up. CONCLUSIONS: Remote CKD care delivered through telenephrology improves renal clinic visit adherence while delivering comparable renal outcomes. Application of this technology is a promising method to provide access to care to rural CKD patients and to minimize the disparity between urban/rural patients.

Albuminuria confers renal resistance to loop diuretics via the stimulation of NLRP3 inflammasome/prostaglandin signaling in thick ascending limb.

Renal resistance to loop diuretics is a frequent complication in a number of kidney disease patients with elusive mechanism. Employing human renal biopsy specimens, albumin overload mouse model, and primary cultures of mouse renal tubular cells, albuminuria effect on NKCC2 expression and function and the underlying mechanisms were investigated. In the renal biopsy specimens of albuminuric patients, we found that NKCC2 was significantly downregulated with a negative correlation with albuminuria severity as examined by immunohistochemistry. Meanwhile, NLRP3 and mPGES-1 were stimulated in NKCC2 positive tubules (thick ascending limb, TAL) paralleled with increased urinary PGE2 excretion. To examine the role of albuminuria in the downregulation of NKCC2 and the potential role of NLRP3/prostaglandin signaling in NKCC2 downregulation, an albumin overload mouse model was employed. Interestingly, we discovered that albuminuria downregulated NKCC2 protein expression in murine kidney and impaired the renal response to loop diuretic furosemide. Specifically, albuminuria suppressed NKCC2 expression and function through NLRP3/prostaglandin dependent signaling in TAL. In primary cultures of renal tubular cells, albumin directly reduced NKCC2 but enhanced NLRP3, COX-2, and mPGES-1 expression. These novel findings demonstrated that albuminuria is of importance in mediating the renal resistance to loop diuretics via NLRP3/prostaglandin signaling-dependent NKCC2 downregulation in TAL. This may also offer novel, effective targets for dealing with the resistance of loop diuretics in proteinuric renal diseases.

Telenephrology: current perspectives and future directions.

There is increasing interest in telemedicine among physicians and patients; however, the evidence regarding the quality of care delivered by telemedicine, and telenephrology in particular, compared with in-person care is limited. In this review, different electronic modalities used to deliver nephrology care are reviewed and critiqued, with a focused analysis from the Australian and United States perspectives. Both countries are geographically expansive with significant rural populations where access to nephrology care is limited. However, their health care systems are organized differently. The Australian health care system is a mostly nonprofit, single-payer system, whereas the United States system is more fractured with a greater proportion of patients covered by for-profit private insurance or no insurance coverage. Videoconferencing is widely used in Australia to manage kidney disease including chronic kidney disease, dialysis, pediatric nephrology, and post-kidney transplantation care. In contrast, the United States telenephrology experience is limited, with most reports originating from the Veterans Health Administration, a single-payer system providing care for nearly 9 million veterans, ∼3 million of whom reside in rural communities. Preliminary reports from the Veterans Health Administration suggest that that delivery of nephrology care via videoconferencing results in clinical outcomes that are at least equivalent to in-person care and improved patient adherence to scheduled appointments. Nevertheless, large, adequately controlled studies are needed to identify patient populations that are most likely to benefit from telenephrology and to determine the optimal systems for the delivery of telenephrology care.

Targeting Access to Kidney Care Via Telehealth: The VA Experience.

The Veterans Affairs (VA) is the largest integrated health care system in the United States and is responsible for the care of a population with a disproportionately high rate of CKD. As such, ensuring access to kidney health services is a VA imperative. One facet of the VA's strategy to reduce CKD is to leverage the use of teletechnology to expand the VA's outreach to Veterans with kidney disease. A wide array of teletechnology services have been deployed to both pull in Veterans and push out kidney health services to Veterans in their preferred health care venue. Teletechnology, thus, expands Veteran choice, facilitates their access to care, and furthers the goal of delivering patient-centered kidney specialty care. The VA has demonstrated the feasibility of virtual delivery of kidney specialty care services and education via synchronous and asynchronous approaches. The challenges ahead include determining the relative health care value of kidney telehealth services, identifying Veterans most likely to benefit from specific technologies and optimizing the adoption of effective kidney telehealth services by both providers and patients alike to ensure optimal and timely kidney health care delivery.

Albuminuria enhances NHE3 and NCC via stimulation of mitochondrial oxidative stress/angiotensin II axis.

Imbalance of salt and water is a frequent and challenging complication of kidney disease, whose pathogenic mechanisms remain elusive. Employing an albumin overload mouse model, we discovered that albuminuria enhanced the expression of NHE3 and NCC but not other transporters in murine kidney in line with the stimulation of angiotensinogen (AGT)/angiotensin converting enzyme (ACE)/angiotensin (Ang) II cascade. In primary cultures of renal tubular cells, albumin directly stimulated AGT/ACE/Ang II and upregulated NHE3 and NCC expression. Blocking Ang II production with an ACE inhibitor normalized the upregulation of NHE3 and NCC in cells. Interestingly, albumin overload significantly reduced mitochondrial superoxide dismutase (SOD2), and administration of a SOD2 mimic (MnTBAP) normalized the expression of NHE3, NCC, and the components of AGT/ACE pathway affected by albuminuria, indicating a key role of mitochondria-derived oxidative stress in modulating renin-angiotensin system (RAS) and renal sodium transporters. In addition, the functional data showing the reduced urinary excretion of Na and Cl and enhanced response to specific NCC inhibitor further supported the regulatory results of sodium transporters following albumin overload. More importantly, the upregulation of NHE3 and NCC and activation of ACE/Ang II signaling pathway were also observed in albuminuric patient kidneys, suggesting that our animal model accurately replicates the human condition. Taken together, these novel findings demonstrated that albuminuria is of importance in resetting renal salt handling via mitochondrial oxidative stress-initiated stimulation of ACE/Ang II cascade. This may also offer novel, effective therapeutic targets for dealing with salt and water imbalance in proteinuric renal diseases.

Left ventricular outflow track obstruction and mitral valve regurgitation in a patient with takotsubo cardiomyopathy.

INTRODUCTION: Takotsubo cardiomyopathy (TCM) can be complicated by left ventricular outflow tract (LVOT) obstruction and severe acute mitral regurgitation (MR), leading to hemodynamic instability in an otherwise benign disorder. Despite the severity of these complications, there is a paucity of literature on the matter. Because up to 20-25% of TCM patients develop LVOT obstruction and/or MR, it is important to recognize the clinical manifestations of these complications and to adhere to specific management in order to reduce patient morbidity and mortality. We report the clinical history, imaging, treatment strategy, and clinical outcome of a patient with TCM that was complicated with severe MR and LVOT obstruction. We then discuss the pathophysiology, characteristic imaging, key clinical features, and current treatment strategy for this unique patient population. CASE REPORT: A postmenopausal woman with no clear risk factor for coronary artery disease (CAD) presented to the emergency department with chest pain after an episode of mental/physical stress. Physical examination revealed MR, mild hypotension, and pulmonary vascular congestion. Her troponins were mildly elevated. Cardiac catheterization excluded obstructive CAD, but revealed severe apical hypokinesia and ballooning. Notably, multiple diagnostic tests revealed the presence of severe acute MR and LVOT obstruction. The patient was diagnosed with TCM complicated by underlying MR and LVOT obstruction, and mild hemodynamic instability. The mechanism of her LVOT and MR was attributed to systolic anterior motion of the mitral valve (SAM), which the transesophageal echocardiogram clearly showed during workup. She was treated with beta-blocker, aspirin, and ACE-I with good outcome. Nitroglycerin and inotropes were discontinued and further avoided. CONCLUSIONS: Our case illustrated LVOT obstruction and MR associated with underlying SAM in a patient with TCM. LVOT obstruction and MR are severe complications of TCM and may result in heart failure and/or pulmonary edema. Timely and accurate identification of these complications is critical to achieve optimal clinical outcomes in patients with TCM.

Cholesterol affects flow-stimulated cyclooxygenase-2 expression and prostanoid secretion in the cortical collecting duct.

Essential hypertension (eHTN) is associated with hypercholesterolemia, but how cholesterol contributes to eHTN is unknown. Recent evidence demonstrates that short-term dietary cholesterol ingestion induces epithelial Na channel (ENaC)-dependent Na absorption with a subsequent rise in blood pressure (BP), implicating cholesterol in salt-sensitive HTN. Prostaglandin E2 (PGE2), an autocrine/paracrine molecule, is induced by flow in endothelia to vasodilate the vasculature and inhibit ENaC-dependent Na absorption in the renal collecting duct (CD), which reduce BP. We hypothesize that cholesterol suppresses flow-mediated cyclooxygenase-2 (COX-2) expression and PGE2 release in the CD, which, in turn, affects Na absorption. Cortical CDs (CCDs) were microperfused at 0, 1, and 5 nl·min(-1)·mm(-1), and PGE2 release was measured. Secreted PGE2 was similar between no- and low-flow (151 ± 28 vs. 121 ± 48 pg·ml(-1)·mm(-1)) CCDs, but PGE2 was greatest from high-flow (578 ± 146 pg·ml(-1)·mm(-1); P < 0.05) CCDs. Next, mice were fed either a 0 or 1% cholesterol diet, injected with saline to generate high urine flow rates, and CCDs were microdissected for PGE2 secretion. CCDs isolated from cholesterol-fed mice secreted less PGE2 and had a lower PGE2-generating capacity than CCDs isolated from control mice, implying cholesterol repressed flow-induced PGE2 synthesis. Next, cholesterol extraction in a CD cell line induced COX-2 expression and PGE2 release while cholesterol incorporation, conversely, suppressed their expression. Moreover, fluid shear stress (FSS) and cholesterol extraction induced COX-2 protein abundance via p38-dependent activation. Thus cellular cholesterol composition affects biomechanical signaling, which, in turn, affects FSS-mediated COX-2 expression and PGE2 release via a p38-dependent mechanism.

Flow regulation of endothelin-1 production in the inner medullary collecting duct.

Collecting duct-derived endothelin (ET)-1 is an autocrine inhibitor of Na(+) and water reabsorption; its deficiency causes hypertension and water retention. Extracellular fluid volume expansion increases collecting duct ET-1, thereby promoting natriuresis and diuresis; however, how this coupling between volume expansion and collecting duct ET-1 occurs is incompletely understood. One possibility is that volume expansion increases tubular fluid flow. To investigate this, cultured IMCD3 cells were subjected to static or flow conditions. Exposure to a shear stress of 2 dyn/cm(2) for 2 h increased ET-1 mRNA content by ∼2.3-fold. Absence of perfusate Ca(2+), chelation of intracellular Ca(2+), or inhibition of Ca(2+) signaling (calmodulin, Ca(2+)/calmodulin-dependent kinase, calcineurin, PKC, or phospholipase C) prevented the flow response. Evaluation of possible flow-activated Ca(2+) entry pathways revealed no role for transient receptor potential (TRP)C3, TRPC6, and TRPV4; however, cells with TRPP2 (polycystin-2) knockdown had no ET-1 flow response. Flow increased intracellular Ca(2+) was blunted in TRPP2 knockdown cells. Nonspecific blockade of P2 receptors, as well as specific inhibition of P2X7 and P2Y2 receptors, prevented the ET-1 flow response. The ET-1 flow response was not affected by inhibition of either epithelial Na(+) channels or the mitochondrial Na(+)/Ca(2+) exchanger. Taken together, these findings provide evidence that in IMCD3 cells, flow, via polycystin-2 and P2 receptors, engages Ca(2+)-dependent signaling pathways that stimulate ET-1 synthesis.

Angiotensin II type 2 receptor regulates ROMK-like K⁺ channel activity in the renal cortical collecting duct during high dietary K⁺ adaptation.

The kidney adjusts K⁺ excretion to match intake in part by regulation of the activity of apical K⁺ secretory channels, including renal outer medullary K⁺ (ROMK)-like K⁺ channels, in the cortical collecting duct (CCD). ANG II inhibits ROMK channels via the ANG II type 1 receptor (AT1R) during dietary K⁺ restriction. Because AT1Rs and ANG II type 2 receptors (AT2Rs) generally function in an antagonistic manner, we sought to characterize the regulation of ROMK channels by the AT2R. Patch-clamp experiments revealed that ANG II increased ROMK channel activity in CCDs isolated from high-K⁺ (HK)-fed but not normal K⁺ (NK)-fed rats. This response was blocked by PD-123319, an AT2R antagonist, but not by losartan, an AT1R antagonist, and was mimicked by the AT2R agonist CGP-42112. Nitric oxide (NO) synthase is present in CCD cells that express ROMK channels. Blockade of NO synthase with N-nitro-l-arginine methyl ester and free NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt completely abolished ANG II-stimulated ROMK channel activity. NO enhances the synthesis of cGMP, which inhibits phosphodiesterases (PDEs) that normally degrade cAMP; cAMP increases ROMK channel activity. Pretreatment of CCDs with IBMX, a broad-spectrum PDE inhibitor, or cilostamide, a PDE3 inhibitor, abolished the stimulatory effect of ANG II on ROMK channels. Furthermore, PKA inhibitor peptide, but not an activator of the exchange protein directly activated by cAMP (Epac), also prevented the stimulatory effect of ANG II. We conclude that ANG II acts at the AT2R to stimulate ROMK channel activity in CCDs from HK-fed rats, a response opposite to that mediated by the AT1R in dietary K⁺-restricted animals, via a NO/cGMP pathway linked to a cAMP-PKA pathway.

Effects of biomechanical forces on signaling in the cortical collecting duct (CCD).

An increase in tubular fluid flow rate (TFF) stimulates Na reabsorption and K secretion in the cortical collecting duct (CCD) and subjects cells therein to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular MAPK and extracellular autocrine/paracrine PGE2 signaling regulate cation transport in the CCD and, at least in other systems, are affected by biomechanical forces. We hypothesized that FSS and CS differentially affect MAPK signaling and PGE2 release to modulate cation transport in the CCD. To validate that CS is a physiological force in vivo, we applied the intravital microscopic approach to rodent kidneys in vivo to show that saline or furosemide injection led to a 46.5 ± 2.0 or 170 ± 32% increase, respectively, in distal tubular diameter. Next, murine CCD (mpkCCD) cells were grown on glass or silicone coated with collagen type IV and subjected to 0 or 0.4 dyne/cm(2) of FSS or 10% CS, respectively, forces chosen based on prior biomechanical modeling of ex vivo microperfused CCDs. Cells exposed to FSS expressed an approximately twofold greater abundance of phospho(p)-ERK and p-p38 vs. static cells, while CS did not alter p-p38 and p-ERK expression compared with unstretched controls. FSS induced whereas CS reduced PGE2 release by ∼40%. In conclusion, FSS and CS differentially affect ERK and p38 activation and PGE2 release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD.

Biomechanical regulation of cyclooxygenase-2 in the renal collecting duct.

High-dietary sodium (Na), a feature of the Western diet, requires the kidney to excrete ample Na to maintain homeostasis and prevent hypertension. High urinary flow rate, presumably, leads to an increase in fluid shear stress (FSS) and FSS-mediated release of prostaglandin E2 (PGE2) by the cortical collecting duct (CCD) that enhances renal Na excretion. The pathways by which tubular flow biomechanically regulates PGE2 release and cyclooxygenase-2 (COX-2) expression are limited. We hypothesized that FSS, through stimulation of neutral-sphingomyelinase (N-SM) activity, enhances COX-2 expression to boost Na excretion. To test this, inner medullary CD3 cells were exposed to FSS in vitro and mice were injected with isotonic saline in vivo to induce high tubular flow. In vitro, FSS induced N-SM activity and COX-2 protein expression in cells while inhibition of N-SM activity repressed FSS-induced COX-2 protein abundance. Moreover, the murine CCD expresses N-SM protein and, when mice are injected with isotonic saline to induce high tubular flow, renal immunodetectable COX-2 is induced. Urinary PGE2 (445 ± 91 vs. 205 ± 14 pg/ml; P < 0.05) and microdissected CCDs (135.8 ± 21.7 vs. 65.8 ± 11.0 pg·ml(-1)·mm(-1) CCD; P < 0.05) from saline-injected mice generate more PGE2 than sham-injected controls, respectively. Incubation of CCDs with arachidonic acid and subsequent measurement of secreted PGE2 are a reflection of the PGE2 generating potential of the epithelia. CCDs isolated from polyuric mice doubled their PGE2 generating potential and this was due to induction of COX-2 activity/protein. Thus, high tubular flow and FSS induce COX-2 protein/activity to enhance PGE2 release and, presumably, effectuate Na excretion.

Prostaglandin E(2) mediates proliferation and chloride secretion in ADPKD cystic renal epithelia.

Prostaglandin E(2) (PGE(2)) contributes to cystogenesis in genetically nonorthologous models of autosomal dominant polycystic kidney disease (ADPKD). However, it remains unknown whether PGE(2) induces the classic features of cystic epithelia in genetically orthologous models of ADPKD. We hypothesized that, in ADPKD epithelia, PGE(2) induces proliferation and chloride (Cl(-)) secretion, two archetypal phenotypic features of ADPKD. To test this hypothesis, proliferation and Cl(-) secretion were measured in renal epithelial cells deficient in polycystin-1 (PC-1). PC-1-deficient cells increased in cell number (proliferated) faster than PC-1-replete cells, and this proliferative advantage was abrogated by cyclooxygenase inhibition, indicating a role for PGE(2) in cell proliferation. Exogenous administration of PGE(2) increased proliferation of PC-1-deficient cells by 38.8 ± 5.2% (P < 0.05) but inhibited the growth of PC-1-replete control cells by 49.4 ± 1.9% (P < 0.05). Next, we tested whether PGE(2)-specific E prostanoid (EP) receptor agonists induce intracellular cAMP and downstream ß-catenin activation. PGE(2) and EP4 receptor agonism (TCS 2510) increased intracellular cAMP concentration and the abundance of active ß-catenin in PC-1-deficient cells, suggesting a mechanism for PGE(2)-mediated proliferation. Consistent with this hypothesis, antagonizing EP4 receptors reverted the growth advantage of PC-1-deficient cells, implicating a central role for the EP4 receptor in proliferation. To test whether PGE(2)-dependent Cl(-) secretion is also enhanced in PC-1-deficient cells, we used an Ussing chamber to measure short-circuit current (I(sc)). Addition of PGE(2) induced a fivefold higher increase in I(sc) in PC-1-deficient cells compared with PC-1-replete cells. This PGE(2)-induced increase in I(sc) in PC-1-deficient cells was blocked by CFTR-172 and flufenamic acid, indicating that PGE(2) activates CFTR and calcium-activated Cl(-) channels. In conclusion, PGE(2) activates aberrant signaling pathways in PC-1-deficient epithelia that contribute to the proliferative and secretory phenotype characteristic of ADPKD and suggests a therapeutic role for PGE(2) inhibition and EP4 receptor antagonism.

OCRL1 modulates cilia length in renal epithelial cells.

Lowe syndrome is an X-linked disorder characterized by cataracts at birth, mental retardation and progressive renal malfunction that results from loss of function of the OCRL1 (oculocerebrorenal syndrome of Lowe) protein. OCRL1 is a lipid phosphatase that converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The renal pathogenesis of Lowe syndrome patients has been suggested to result from alterations in membrane trafficking, but this cannot fully explain the disease progression. We found that knockdown of OCRL1 in zebrafish caused developmental defects consistent with disruption of ciliary function, including body axis curvature, pericardial edema, hydrocephaly and impaired renal clearance. In addition, cilia in the proximal tubule of the zebrafish pronephric kidney were longer in ocrl morphant embryos. We also found that knockdown of OCRL1 in polarized renal epithelial cells caused elongation of the primary cilium and disrupted formation of cysts in three-dimensional cultures. Calcium release in response to ATP was blunted in OCRL1 knockdown cells, suggesting changes in signaling that could lead to altered cell function. Our results suggest a new role for OCRL1 in renal epithelial cell function that could contribute to the pathogenesis of Lowe syndrome.

Flow-induced prostaglandin E2 release regulates Na and K transport in the collecting duct.

Fluid shear stress (FSS) is a critical regulator of cation transport in the collecting duct (CD). High-dietary sodium (Na) consumption increases urine flow, Na excretion, and prostaglandin E(2) (PGE(2)) excretion. We hypothesize that increases in FSS elicited by increasing tubular flow rate induce the release of PGE(2) from renal epithelial cells into the extracellular compartment and regulate ion transport. Media retrieved from CD cells exposed to physiologic levels of FSS reveal several fold higher concentration of PGE(2) compared with static controls. Treatment of CD cells with either cyclooxygenase-1 (COX-1) or COX-2 inhibitors during exposure to FSS limited the increase in PGE(2) concentration to an equal extent, suggesting COX-1 and COX-2 contribute equally to FSS-induced PGE(2) release. Cytosolic phospholipase A2 (cPLA2), the principal enzyme that generates the COX substrate arachidonic acid, is regulated by mitogen-activated protein-kinase-dependent phosphorylation and intracellular Ca(2+) concentration ([Ca(2+)](i)), both signaling processes, of which, are activated by FSS. Inhibition of the ERK and p38 pathways reduced PGE(2) release by 53.3 ± 8.4 and 32.6 ± 11.3%, respectively, while antagonizing the JNK pathway had no effect. In addition, chelation of [Ca(2+)](i) limited the FSS-mediated increase in PGE(2) concentration by 47.5 ± 7.5% of that observed in untreated sheared cells. Sheared cells expressed greater phospho-cPLA2 protein abundance than static cells; however, COX-2 protein expression was unaffected (P = 0.064) by FSS. In microperfused CDs, COX inhibition enhanced flow-stimulated Na reabsorption and abolished flow-stimulated potassium (K) secretion, but did not affect ion transport at a slow flow rate, implicating that high tubular flow activates autocrine/paracrine PGE(2) release and, in turn, regulates flow-stimulated cation transport. In conclusion, FSS activates cPLA2 to generate PGE(2) that regulates flow-mediated Na and K transport in the native CD. We speculate that dietary sodium intake modulates tubular flow rate to regulate paracrine PGE(2) release and cation transport in the CD.

Fluid shear stress induces renal epithelial gene expression through polycystin-2-dependent trafficking of extracellular regulated kinase.

BACKGROUND: The cilium and cilial proteins have emerged as principal mechanosensors of renal epithelial cells responsible for translating mechanical forces into intracellular signals. Polycystin-2 (PC-2), a cilial protein, regulates flow/shear-induced changes in intracellular Ca(2+) ([Ca(2+)](i)) and recently has been implicated in the regulation of mitogen-activated protein (MAP) kinases. We hypothesize that fluid shear stress (FSS) activates PC-2 which regulates MAP kinase and, in turn, induces MAP kinase-dependent gene expression, specifically, monocyte chemoattractant protein-1 (MCP-1). METHODS: To test this, PC-2 expression was constitutively reduced in a murine inner medullary collecting duct (IMCD3) cell line, and the expression of FSS-induced MCP-1 expression and MAP kinase signaling compared between the parental (PC-2-expressing) and PC-2-deficient IMCD3 cells. RESULTS: FSS induces MAP kinase signaling and downstream MCP-1 mRNA expression in wild-type IMCD3 cells, while inhibitors of MAP kinase prevented the FSS-induced MCP-1 mRNA response. In contradistinction, FSS did not induce MCP-1 mRNA expression in PC-2-deficient cells, but did increase activation of the upstream MAP kinases. Wild-type cells exposed to FSS augmented the nuclear abundance of activated MAP kinase while PC-2-deficient cells did not. CONCLUSIONS: PC-2 regulates FSS-induced MAP kinase trafficking into the nucleus of CD cells.