Erika I. Boesen

Renal Collecting Duct NOS1 Maintains Fluid–Electrolyte Homeostasis and Blood Pressure

Nitric oxide is a pronatriuretic and prodiuretic factor. The highest renal NO synthase (NOS) activity is found in the inner medullary collecting duct. The collecting duct (CD) is the site of daily fine-tune regulation of sodium balance, and led us to hypothesize that a CD-specific deletion of NOS1 would result in an impaired ability to excrete a sodium load leading to a salt-sensitive blood pressure phenotype. We bred AQP2-CRE mice with NOS1 floxed mice to produce flox control and CD-specific NOS1 knockout (CDNOS1KO) littermates. CDs from CDNOS1KO mice produced 75% less nitrite, and urinary nitrite+nitrate (NOx) excretion was significantly blunted in the knockout genotype. When challenged with high dietary sodium, CDNOS1KO mice showed significantly reduced urine output, sodium, chloride, and NOx excretion, and increased mean arterial pressure relative to flox control mice. In humans, urinary NOx is a newly identified biomarker for the progression of hypertension. These findings reveal that NOS1 in the CD is critical in the regulation of fluid–electrolyte balance, and this new genetic model of CD NOS1 gene deletion will be a valuable tool to study salt-dependent blood pressure mechanisms.

Endothelin receptors, renal effects and blood pressure.

The endothelin system has emerged as a key player in the renal control of salt and water homeostasis, exerting profound effects on both the renal vasculature and tubular epithelial cells. Recent advances include new actions of endothelins in the glomerulus, an emerging role for the ETA receptor in chronic kidney disease (CKD) progression and in tubular function, and a more detailed understanding of the tubular response to high salt intake. A large body of evidence also implicates dysfunction of the endothelin system in hypertension, particularly salt-sensitive hypertension, although recent data suggests important sex-differences may exist. Finally, clinical trials indicate that antagonists of endothelin receptors hold great promise in treating resistant hypertension and proteinuric renal disease.

Estradiol regulates AQP2 expression in the collecting duct: a novel inhibitory role for estrogen receptor α

While there is evidence that sex hormones influence multiple systems involved in salt and water homeostasis, the question of whether sex hormones regulate aquaporin-2 (AQP2) and thus water handling by the collecting duct has been largely ignored. Accordingly, the present study investigated AQP2 expression, localization and renal water handling in intact and ovariectomized (OVX) female rats, with and without estradiol or progesterone replacement. OVX resulted in a significant increase in urine osmolality and increase in p256-AQP2 in the renal cortex at 7 days post-OVX, as well as induced body weight changes. Relative to OVX alone, estradiol repletion produced a significant increase in urine output, normalized urinary osmolality and reduced both total AQP2 (protein and mRNA) and p256-AQP2 expression, whereas progesterone repletion had little effect. Direct effects of estradiol on AQP2 mRNA and protein levels were further tested in vitro using the mpkCCD principal cell line. Estradiol treatment of mpkCCD cells reduced AQP2 at both the mRNA and protein level in the absence of deamino-8-d-AVP (dDAVP) and significantly blunted the dDAVP-induced increase in AQP2 at the protein level only. We determined that mpkCCD and native mouse collecting ducts express both estrogen receptor (ER)α and ERβ and that female mice lacking ERα displayed significant increases in AQP2 protein compared with wild-type littermates, implicating ERα in mediating the inhibitory effect of estradiol on AQP2 expression. These findings suggest that changes in estradiol levels, such as during menopause or following reproductive surgeries, may contribute to dysregulation of water homeostasis in women.

Chronic elevation of IL-1β induces diuresis via a cyclooxygenase 2-mediated mechanism

Chronic renal inflammation is an increasingly recognized phenomenon in multiple disease states, but the impact of specific cytokines on renal function is unclear. Previously, we found that 14-day interleukin-1β (IL-1β) infusion increased urine flow in mice. To determine the mechanism by which this occurs, the current study tested the possible involvement of three classical prodiuretic pathways. Chronic IL-1β infusion significantly increased urine flow (6.5 ± 1 ml/day at day 14 vs. 2.3 ± 0.3 ml/day in vehicle group; P < 0.05) and expression of cyclooxygenase (COX)-2, all three nitric oxide synthase (NOS) isoforms, and endothelin (ET)-1 in the kidney (P < 0.05 in all cases). Urinary prostaglandin E metabolite (PGEM) excretion was also significantly increased at day 14 of IL-1β infusion (1.21 ± 0.26 vs. 0.29 ± 0.06 ng/day in vehicle-infused mice; P = 0.001). The selective COX-2 inhibitor celecoxib markedly attenuated urinary PGEM excretion and abolished the diuretic response to chronic IL-1β infusion. In contrast, deletion of NOS3, or inhibition of NOS1 with L-VNIO, did not blunt the diuretic effect of IL-1β, nor did pharmacological blockade of endothelin ETA and ETB receptors with A-182086. Consistent with a primary effect on water transport, IL-1β infusion markedly reduced inner medullary aquaporin-2 expression (P < 0.05) and did not alter urinary Na⁺ or K⁺ excretion. These data indicate a critical role for COX-2 in mediating the effects of chronic IL-1β elevation on the kidney.

Interleukin-1β as a driver of renal NGAL production

ABSTRACT Neutrophil gelatinase‐associated lipocalin (NGAL) is increasingly regarded as a biomarker of acute kidney injury, or kidney injury in general, but the stimuli responsible for its production are incompletely understood. This study tested the relationship between the pro‐inflammatory cytokine interleukin‐1&bgr; (IL‐1&bgr;) and both circulating and renal NGAL, using chronic subcutaneous infusion of IL‐1&bgr; in mice and tissue culture of renal cell lines. Following a 14‐day subcutaneous infusion of vehicle or IL‐1&bgr; (10 ng/h) in male C57Bl/6 mice, a striking positive correlation (r2 = 0.94; P < 0.01) was observed between plasma IL‐1&bgr; and NGAL concentrations. NGAL was markedly increased in the kidneys of IL‐1&bgr;‐infused mice compared with vehicle‐treated mice, both at the protein and mRNA level, indicating increased local as well as systemic production of NGAL. Immunohistochemical staining revealed prominent increases of NGAL in the proximal tubular epithelium of IL‐1&bgr; infused mice. These effects occurred in the absence of overt renal injury, with plasma creatinine concentration not significantly different between groups. Further showing that IL‐1&bgr; has a direct effect on NGAL production by tubular epithelial cells, exposure of a proximal tubular cell line (HK‐2 cells) and a cortical collecting duct principal cell line (mpkCCD cells) to IL‐1&bgr; for 24 h produced a significant increase of NGAL mRNA levels (>30‐fold). These data indicate IL‐1&bgr; serves as a powerful stimulus for renal production of NGAL.

Lack of an apparent role for endothelin-1 in the prolonged reduction in renal perfusion following severe unilateral ischemia-reperfusion injury in the mouse

Therapeutic approaches to block the progression from acute kidney injury to chronic kidney disease are currently lacking. Endothelin‐1 (ET‐1) is a powerful vasoconstrictor, induced by hypoxia, and previously implicated in renal ischemia‐reperfusion (IR) injury. This study tested the hypothesis that blunting the vascular influence of ET‐1, either through endothelin ETA receptor blockade (ABT‐627) or vascular endothelial cell deletion of ET‐1 (VEET KO), would improve recovery of renal perfusion and repair of injury following a severe ischemic insult in mice (45 min unilateral renal ischemia). Male C57Bl/6 mice receiving vehicle or ABT‐627 commencing 2 days prior to surgery, and VEET KO mice and wild‐type littermates (WT) underwent 45 min unilateral renal IR surgery followed by 28 days recovery. Renal blood velocity was measured by pulsed‐wave Doppler ultrasound before and after surgery. Renal blood velocity was not significantly different between pairs of groups before surgery. Unilateral IR induced a marked reduction in renal blood velocity of the IR kidney at 24 h postsurgery in all groups, which partially recovered but remained below baseline at 28 days post‐IR. Despite the lack of effect on renal blood velocity, ETA receptor blockade significantly attenuated the atrophy of the post‐IR kidney, whereas this was not significantly affected by lack of endothelial ET‐1 expression. These data suggest that although blockade of the ETA receptor is mildly beneficial in preserving renal mass following a severe ischemic insult, this protective effect does not appear to involve improved recovery of renal perfusion.

Outside the mainstream: novel collecting duct proteins regulating water balance

Body water balance is critical to survival and, therefore, very tightly regulated by the hypothalamus and kidney. A key mechanism involved in this process, the arginine vasopressin-mediated phosphorylation and apical membrane insertion of aquaporin 2 in the collecting duct, has been extensively studied; however, with the increased availability of conditional knockout animals, several novel collecting duct proteins have recently been implicated in water homeostasis. In this Mini-Review, we briefly discuss these novel proteins and their roles in the regulation of water homeostasis.

Indoleamine 2,3-dioxygenase inhibition alters the non-coding RNA transcriptome following renal ischemia-reperfusion injury.

BACKGROUNDnIndoleamine 2,3 dioxygenase (IDO) degrades the essential amino acid tryptophan and has been shown to minimize rejection in animal models of renal transplantation. Ischemia-reperfusion injury (IRI) is unavoidable in renal transplantation and correlates with shorter graft survival times. Despite its favorable effects on rejection, there is evidence that IDO may facilitate renal IRI. Differentiating the negative impact of IDO on IRI from its pro-tolerant effects in allograft rejection is of clinical relevance. In these studies we hypothesized that constitutive IDO activity may influence renal genes associated with recovery from IRI, and that IDO inhibition may unmask these effects.nnnMETHODSnWe examined the renal transcriptome in a rat model of IRI with and without IDO inhibition with 1-methyl-d-tryptophan (1-MT), and assessed for alterations in the gene expression signature.nnnRESULTSnThese studies demonstrated that during recovery from renal IRI, pre-treatment with 1-MT alleviated alterations in 105 coding sequences associated with IRI, and in turn triggered new changes in 66 non-coding transcripts, the majority of which were represented by small nucleolar RNA.nnnCONCLUSIONnThese results suggest a biologic role for non-coding, IDO-dependent genes in regulating the early response to IRI.

ETA receptor activation contributes to T cell accumulation in the kidney following ischemia-reperfusion injury

Renal ischemia‐reperfusion (IR) injury and acute kidney injury (AKI) increase the risk of developing hypertension, with T cells suspected as a possible mechanistic link. Endothelin promotes renal T cell infiltration in several diseases, predominantly via the ETA receptor, but its contribution to renal T cell infiltration following renal IR injury is poorly understood. To test whether ETA receptor activation promotes T cell infiltration of the kidney following IR injury, male C57BL/6 mice were treated with the ETA receptor antagonist ABT‐627 or vehicle, commencing 2 days prior to unilateral renal IR injury. Mice were sacrificed at 24 h or 10 days post‐IR for assessment of the initial renal injury and subsequent infiltration of T cells. Vehicle and ABT‐627‐treated mice displayed significant upregulation of endothelin‐1 (ET‐1) in the IR compared to contralateral kidney at both 24 h and 10 days post‐IR (P < 0.001). Renal CD3+ T cell numbers were increased in the IR compared to contralateral kidneys at 10 days, but ABT‐627‐treated mice displayed a 35% reduction in this effect in the outer medulla (P < 0.05 vs. vehicle) and a nonsignificant 23% reduction in the cortex compared to vehicle‐treated mice. Whether specific T cell subsets were affected awaits confirmation by flow cytometry, but outer medullary expression of the T helper 17 transcription factor RORγt was reduced by ABT‐627 (P = 0.06). These data indicate that ET‐1 acting via the ETA receptor contributes to renal T cell infiltration post‐IR injury. This may have important implications for immune system‐mediated long‐term consequences of AKI, an area which awaits further investigation.

EHD4 is a novel regulator of urinary water homeostasis

The Eps15‐homology domain–containing (EHD) protein family comprises 4 members that regulate endocytic recycling. Although the kidney expresses all 4 EHD proteins, their physiologic roles are largely unknown. This study focused on EHD4, which we found to be expressed differentially across nephron segments with the highest expression in the inner medullary collecting duct. Under baseline conditions, Ehd4−/− [EHD4‐knockout (KO)] mice on a C57Bl/6 background excreted a higher volume of more dilute urine than control C57Bl/6 wild‐type (WT) mice while maintaining a similar plasma osmolality. Urine excretion after an acute intraperitoneal water load was significantly increased in EHD4‐KO mice compared to WT mice, and although EHD4‐KO mice concentrated their urine during 24‐h water restriction, urinary osmolality remained significantly lower than in WT mice, suggesting that EHD4 plays a role in renal water handling. Total aquaporin 2 (AQP2) and phospho‐S256‐AQP2 (pAQP2) protein expression in the inner medulla was similar in the two groups in baseline conditions. However, localization of both AQP2 and pAQP2 in the renal inner medullary principal cells appeared more dispersed, and the intensity of apical membrane staining for AQP2 was reduced significantly (by ~20%) in EHD4‐KO mice compared to WT mice in baseline conditions, suggesting an important role of EHD4 in trafficking of AQP2. Together, these data indicate that EHD4 play important roles in the regulation of water homeostasis.—Rahman, S. S., Moffitt, A. E. J., Trease, A. J., Foster, K. W., Storck, M. D., Band, H., Boesen, E.I. EHD4 is a novel regulator of urinary water homeostasis. FASEB J. 31, 5217–5233 (2017). www.fasebj.org