Kidney collecting duct-derived vasopressin is not essential for appropriate concentration or dilution of urine.

We have previously shown that kidney collecting ducts make vasopressin. However, the physiological role of collecting duct-derived vasopressin is uncertain. We hypothesized that collecting duct-derived vasopressin is required for the appropriate concentration of urine. We developed a vasopressin conditional knockout (KO) mouse model wherein Cre recombinase expression induces deletion of arginine vasopressin (Avp) exon 1 in the distal nephron. We then used age-matched 8- to 12-wk-old Avp fl/fl;Ksp-Cre(-) [wild type (WT)] and Avp fl/fl;Ksp-Cre(+) mice for all experiments. We collected urine, serum, and kidney lysates at baseline. We then challenged both WT and knockout (KO) mice with 24-h water restriction, water loading, and administration of the vasopressin type 2 receptor agonist desmopressin (1 µg/kg ip) followed by the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). We performed immunofluorescence and immunoblot analysis at baseline and confirmed vasopressin KO in the collecting duct. We found that urinary osmolality (UOsm), plasma Na+, K+, Cl-, blood urea nitrogen, and copeptin were similar in WT vs. KO mice at baseline. Immunoblots of the vasopressin-regulated proteins Na+-K+-2Cl- cotransporter, NaCl cotransporter, and water channel aquaporin-2 showed no difference in expression or phosphorylation at baseline. Following 24-h water restriction, WT and KO mice had no differences in UOsm, plasma Na+, K+, Cl-, blood urea nitrogen, or copeptin. In addition, there were no differences in the rate of urinary concentration or dilution as in WT and KO mice UOsm was nearly identical after desmopressin and OPC-31260 administration. We conclude that collecting duct-derived vasopressin is not essential to appropriately concentrate or dilute urine.NEW & NOTEWORTHY Hypothalamic vasopressin is required for appropriate urinary concentration. However, whether collecting duct-derived vasopressin is involved remains unknown. We developed a novel transgenic mouse model to induce tissue-specific deletion of vasopressin and showed that collecting duct-derived vasopressin is not required to concentrate or dilute urine.

Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.

Prolonged obstruction of the ureter, which leads to injury of the kidney collecting ducts, results in permanent structural damage, while early reversal allows for repair. Cell structure is defined by the actin cytoskeleton, which is dynamically organized by small Rho guanosine triphosphatases (GTPases). In this study, we identified the Rho GTPase, Rac1, as a driver of postobstructive kidney collecting duct repair. After the relief of ureteric obstruction, Rac1 promoted actin cytoskeletal reconstitution, which was required to maintain normal mitotic morphology allowing for successful cell division. Mechanistically, Rac1 restricted excessive actomyosin activity that stabilized the negative mitotic entry kinase Wee1. This mechanism ensured mechanical G2-M checkpoint stability and prevented premature mitotic entry. The repair defects following injury could be rescued by direct myosin inhibition. Thus, Rac1-dependent control of the actin cytoskeleton integrates with the cell cycle to mediate kidney tubular repair by preventing dysmorphic cells from entering cell division.

Pharmacology of Compounds Targeting Cation-Chloride Cotransporter Physiology.

Transporters of the solute carrier family 12 (SLC12) carry inorganic cations such as Na+ and/or K+ alongside Cl across the plasma membrane of cells. These tightly coupled, electroneutral, transporters are expressed in almost all tissues/organs in the body where they fulfil many critical functions. The family includes two key transporters participating in salt reabsorption in the kidney: the Na-K-2Cl cotransporter-2 (NKCC2), expressed in the loop of Henle, and the Na-Cl cotransporter (NCC), expressed in the distal convoluted tubule. NCC and NKCC2 are the targets of thiazides and "loop" diuretics, respectively, drugs that are widely used in clinical medicine to treat hypertension and edema. Bumetanide, in addition to its effect as a loop diuretic, has recently received increasing attention as a possible therapeutic agent for neurodevelopmental disorders. This chapter also describes how over the past two decades, the pharmacology of Na+ independent transporters has expanded significantly to provide novel tools for research. This work has indeed led to the identification of compounds that are 100-fold to 1000-fold more potent than furosemide, the first described inhibitor of K-Cl cotransport, and identified compounds that possibly directly stimulate the function of the K-Cl cotransporter. Finally, the recent cryo-electron microscopy revolution has begun providing answers as to where and how pharmacological agents bind to and affect the function of the transporters.

Epidermal growth factor receptor activation is essential for kidney fibrosis development.

Fibrosis is the progressive accumulation of excess extracellular matrix and can cause organ failure. Fibrosis can affect nearly every organ including kidney and there is no specific treatment currently. Although Epidermal Growth Factor Receptor (EGFR) signaling pathway has been implicated in development of kidney fibrosis, underlying mechanisms by which EGFR itself mediates kidney fibrosis have not been elucidated. We find that EGFR expression increases in interstitial myofibroblasts in human and mouse fibrotic kidneys. Selective EGFR deletion in the fibroblast/pericyte population inhibits interstitial fibrosis in response to unilateral ureteral obstruction, ischemia or nephrotoxins. In vivo and in vitro studies and single-nucleus RNA sequencing analysis demonstrate that EGFR activation does not induce myofibroblast transformation but is necessary for the initial pericyte/fibroblast migration and proliferation prior to subsequent myofibroblast transformation by TGF-ß or other profibrotic factors. These findings may also provide insight into development of fibrosis in other organs and in other conditions.

Randomized Trial on the Effect of Oral Potassium Chloride Supplementation on the Thiazide-Sensitive Sodium Chloride Cotransporter in Healthy Adults.

Introduction: The putative "renal-K switch" mechanism links dietary potassium intake with sodium retention and involves activation of the sodium chloride (NaCl) cotransporter (NCC) in the distal convoluted tubule in response to low potassium intake, and suppression in response to high potassium intake. This study examined NCC abundance and phosphorylation (phosphorylated NCC [pNCC]) in urinary extracellular vesicles (uEVs) isolated from healthy adults on a high sodium diet to determine tubular responses to alteration in potassium chloride (KCl) intake. Methods: Healthy adults maintained on a high sodium (∼4.5 g [200 mmol]/d) low potassium (∼2.3 g [60 mmol]/d) diet underwent a 5-day run-in period followed by a crossover study, with 5-day supplementary KCl (active phase, Span-K 3 tablets (potassium 24 mmol) thrice daily) or 5-day placebo administrated in random order and separated by 2-day washout. Ambulatory blood pressure (BP) and biochemistries were assessed, and uEVs were analyzed by western blotting. Results: Among the 18 participants who met analysis criteria, supplementary KCl administration (vs. placebo) was associated with markedly higher levels of plasma potassium and 24-hour urine excretion of potassium, chloride, and aldosterone. KCl supplementation was associated with lower uEV levels of NCC (median fold change (KCl/Placebo) = 0.74 [0.30-1.69], P < 0.01) and pNCC (fold change (KCl/Placebo) = 0.81 [0.19-1.75], P < 0.05). Plasma potassium inversely correlated with uEV NCC (R2 = 0.11, P = 0.05). Conclusions: The lower NCC and pNCC in uEVs in response to oral KCl supplementation provide evidence to support the hypothesis of a functional "renal-K switch" in healthy human subjects.

Bicarbonate is the primary inducer of KCC3a expression in renal cortical B-type intercalated cells.

A primary function of intercalated cells in the distal tubule of the kidney is to maintain pH homeostasis. For example, type B intercalated cells secrete bicarbonate largely through the action of the apical Cl-/HCO3- exchanger, pendrin, which helps correct metabolic alkalosis. Since both the K-Cl cotransporter, KCC3a and pendrin colocalize to the apical region of type B and non-A, non-B intercalated cells and since both are upregulated in models of metabolic alkalosis, such as with dietary NaHCO3 loading, we raised the possibility that apical KCC3a facilitates pendrin-mediated bicarbonate secretion, such as through apical Cl- recycling. The purpose of this study was to determine if KCC3a abundance changes through intake of bicarbonate alone or through bicarbonate plus its accompanying cation, and if it requires a direct interaction with pendrin or the renin-angiotensin-aldosterone system. We observed that KCC3a protein abundance, but not mRNA, increases in a mouse model of metabolic alkalosis, achieved with dietary NaHCO3 or KHCO3 intake. Bicarbonate ion increases KCC3a abundance, both in vivo and in vitro, independently of the accompanying cation. Moreover, bicarbonate intake upregulates KCC3a independently of aldosterone or angiotensin II. Since NaHCO3 intake increased KCC3a abundance in wild-type as well as in pendrin knockout mice, this KCC3a upregulation by bicarbonate does not depend on a direct interaction with pendrin. We conclude that increased extracellular bicarbonate, as observed in models of metabolic alkalosis, directly raises KCC3a abundance independently of angiotensin II, aldosterone, or changes in KCC3a transcription and does not involve a direct interaction with pendrin.NEW & NOTEWORTHY KCC3a expression is stimulated in alkalemia. This paper shows that bicarbonate itself is mediating this effect through a posttranscriptional mechanism. The paper also shows that this phenomenon is not mediated by aldosterone or angiotensin II.

High sodium intake does not worsen low potassium-induced kidney damage.

High sodium and low potassium intake have both been linked to poor cardiovascular health outcomes and increased mortality rates. A combination of the two is thought to be particularly detrimental. While mechanisms are multiple, the kidney is an important target of harmful effects and low potassium influences on both proximal and distal nephron segments are especially potent. We recently reported that a combined high sodium/low potassium diet causes kidney injury and that low potassium in isolation can have similar effects. However, how sodium intake alters this process is not well-understood. Here we tested the hypothesis that a high sodium intake amplifies effects of low dietary potassium on kidney injury. We observed adding high sodium to low potassium caused an expected increase in blood pressure, but did not worsen markers of kidney injury, inflammation, and fibrosis. It also did not increase abundance or phosphorylation of the sodium chloride cotransporter or its regulatory kinases, SPAK and OxSR1, known renal targets of low potassium. Findings support the claim that dietary potassium deficiency, and not high sodium, is a dominant factor affecting kidney injury in animal models of high sodium/low potassium intake. This suggests further investigation is required to identify optimal ranges of sodium and potassium intake in both healthy populations and in those with kidney disease.

Insulin-regulated aminopeptidase is required for water excretion in response to acute hypotonic stress.

The objective of this study was to understand the response of mice lacking insulin-regulated aminopeptidase (IRAP) to an acute water load. For mammals to respond appropriately to acute water loading, vasopressin activity needs to decrease. IRAP degrades vasopressin in vivo. Therefore, we hypothesized that mice lacking IRAP have an impaired ability to degrade vasopressin and, thus, have persistent urinary concentration. Age-matched 8- to 12-wk-old IRAP wild-type (WT) and knockout (KO) male mice were used for all experiments. Blood electrolytes and urine osmolality were measured before and 1 h after water load (∼2 mL sterile water via intraperitoneal injection). Urine was collected from IRAP WT and KO mice for urine osmolality measurements at baseline and after 1 h administration of the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). Immunofluorescence and immunoblot analysis were performed on kidneys at baseline and after 1 h acute water load. IRAP was expressed in the glomerulus, thick ascending loop of Henle, distal tubule, connecting duct, and collecting duct. IRAP KO mice had elevated urine osmolality compared with WT mice due to higher membrane expression of aquaporin 2 (AQP2), which was restored to that of controls after administration of OPC-31260. IRAP KO mice developed hyponatremia after an acute water load because they were unable to increase free water excretion due to increased surface expression of AQP2. In conclusion, IRAP is required to increase water excretion in response to an acute water load due to persistent vasopressin stimulation of AQP2.NEW & NOTEWORTHY Insulin-regulated aminopeptidase (IRAP) degrades vasopressin, but its role in urinary concentration and dilution is unknown. Here, we show that IRAP-deficient mice have a high urinary osmolality at baseline and are unable to excrete free water in response to water loading. These results reveal a novel regulatory role for IRAP in urine concentration and dilution.

Kir4.2 mediates proximal potassium effects on glutaminase activity and kidney injury.

Inadequate potassium (K+) consumption correlates with increased mortality and poor cardiovascular outcomes. Potassium effects on blood pressure have been described previously; however, whether or not low K+ independently affects kidney disease progression remains unclear. Here, we demonstrate that dietary K+ deficiency causes direct kidney injury. Effects depend on reduced blood K+ and are kidney specific. In response to reduced K+, the channel Kir4.2 mediates altered proximal tubule (PT) basolateral K+ flux, causing intracellular acidosis and activation of the enzyme glutaminase and the ammoniagenesis pathway. Deletion of either Kir4.2 or glutaminase protects from low-K+ injury. Reduced K+ also mediates injury and fibrosis in a model of aldosteronism. These results demonstrate that the PT epithelium, like the distal nephron, is K+ sensitive, with reduced blood K+ causing direct PT injury. Kir4.2 and glutaminase are essential mediators of this injury process, and we identify their potential for future targeting in the treatment of chronic kidney disease.

Kidney collecting duct cells make vasopressin in response to NaCl-induced hypertonicity.

Vasopressin has traditionally been thought to be produced by the neurohypophyseal system and then released into the circulation where it regulates water homeostasis. The questions of whether vasopressin could be produced outside of the brain and if the kidney could be a source of vasopressin are raised by the syndrome of inappropriate antidiuretic hormone secretion (vasopressin). We found that mouse and human kidneys expressed vasopressin mRNA. Using an antibody that detects preprovasopressin, we found that immunoreactive preprovasopressin protein was found in mouse and human kidneys. Moreover, we found that murine collecting duct cells made biologically active vasopressin, which increased in response to NaCl-mediated hypertonicity, and that water restriction increased the abundance of kidney-derived vasopressin mRNA and protein expression in mouse kidneys. Thus, we provide evidence of biologically active production of kidney-derived vasopressin in kidney tubular epithelial cells.

EGFR-mediated activation of adipose tissue macrophages promotes obesity and insulin resistance.

Obesity and obesity-related health complications are increasing in prevalence. Adipose tissue from obese subjects has low-grade, chronic inflammation, leading to insulin resistance. Adipose tissue macrophages (ATMs) are a source of proinflammatory cytokines that further aggravate adipocyte dysfunction. In response to a high fat diet (HFD), ATM numbers initially increase by proliferation of resident macrophages, but subsequent increases also result from infiltration in response to chemotactic signals from inflamed adipose tissue. To elucidate the underlying mechanisms regulating the increases in ATMs and their proinflammatory phenotype, we investigated the role of activation of ATM epidermal growth factor receptor (EGFR). A high fat diet increased expression of EGFR and its ligand amphiregulin in ATMs. Selective deletion of EGFR in ATMs inhibited both resident ATM proliferation and monocyte infiltration into adipose tissue and decreased obesity and development of insulin resistance. Therefore, ATM EGFR activation plays an important role in adipose tissue dysfunction.

KCC3a, a Strong Candidate Pathway for K+ Loss in Alkalemia.

Loss-of-function mutations in the human potassium chloride cotransporter-3 (KCC3) cause a hereditary motor sensory neuropathy associated with agenesis of the corpus callosum. While recapitulating the neuropathy, KCC3-knockout mice also exhibit high blood pressure. This phenotype is believed to have neurogenic and/or vascular origins. The role of KCC3 in the kidney is poorly understood. KCC3 is encoded by two major isoforms originating from alternative promoters: KCC3a and KCC3b, with KCC3b being the predominant transcript in the kidney. Although the transporter has previously been localized to the proximal tubule, we show here the unique expression of the KCC3a isoform in the connecting tubule. Using a KCC3a-specific polyclonal antibody validated for both immunofluorescence and immunoblotting, we showed an intense KCC3a signal restricted to cortical intercalated cells. No overlap is detected between KCC3a and sodium chloride cotransporter (NCC), a distal convoluted tubule (DCT) marker; or between KCC3a and ENaC or calbindin, which are both principal cell markers. KCC3a signal was observed in cells expressing the apical V-ATPase and pendrin, establishing a unique expression pattern characteristic of intercalated cells of type-B or type-nonA/nonB. We further show that treatment of wild-type mice with hydrochlorothiazide, amiloride, or fed a K+-deficient diet up-regulates KCC3a level, suggesting that volume depletion increases KCC3a abundance. This hypothesis was confirmed by showing a higher abundance of KCC3a protein after 23-h water restriction or after placing the mice on a low-salt diet. More importantly, abundance of the Cl-/HCO3 - exchanger, pendrin, which is known to secrete bicarbonate in alkalotic conditions, was significantly diminished in KCC3-knockout mice. In addition, KCC3a abundance increased significantly alongside pendrin abundance in bicarbonate-treated alkalotic mice, providing a credible mechanism for K+ loss in metabolic alkalosis.

Cyclooxygenase-2 in adipose tissue macrophages limits adipose tissue dysfunction in obese mice.

Obesity-associated complications are causing increasing morbidity and mortality worldwide. Expansion of adipose tissue in obesity leads to a state of low-grade chronic inflammation and dysregulated metabolism, resulting in insulin resistance and metabolic syndrome. Adipose tissue macrophages (ATMs) accumulate in obesity and are a source of proinflammatory cytokines that further aggravate adipocyte dysfunction. Macrophages are rich sources of cyclooxygenase (COX), the rate limiting enzyme for prostaglandin E2 (PGE2) production. When mice were fed a high-fat diet (HFD), ATMs increased expression of COX-2. Selective myeloid cell COX-2 deletion resulted in increased monocyte recruitment and proliferation of ATMs, leading to increased proinflammatory ATMs with decreased phagocytic ability. There were increased weight gain and adiposity, decreased peripheral insulin sensitivity and glucose utilization, increased adipose tissue inflammation and fibrosis, and abnormal adipose tissue angiogenesis. HFD pair-feeding led to similar increases in body weight, but mice with selective myeloid cell COX-2 still exhibited decreased peripheral insulin sensitivity and glucose utilization. Selective myeloid deletion of the macrophage PGE2 receptor subtype, EP4, produced a similar phenotype, and a selective EP4 agonist ameliorated the metabolic abnormalities seen with ATM COX-2 deletion. Therefore, these studies demonstrated that an ATM COX-2/PGE2/EP4 axis plays an important role in inhibiting adipose tissue dysfunction.

VU6036720: The First Potent and Selective In Vitro Inhibitor of Heteromeric Kir4.1/5.1 Inward Rectifier Potassium Channels.

Heteromeric Kir4.1/Kir5.1 (KCNJ10/KCNJ16) inward rectifier potassium (Kir) channels play key roles in the brain and kidney, but pharmacological tools for probing their physiology and therapeutic potential have not been developed. Here, we report the discovery, in a high-throughput screening of 80,475 compounds, of the moderately potent and selective inhibitor VU0493690, which we selected for characterization and chemical optimization. VU0493690 concentration-dependently inhibits Kir4.1/5.1 with an IC50 of 0.96 µM and exhibits at least 10-fold selectivity over Kir4.1 and ten other Kir channels. Multidimensional chemical optimization of VU0493690 led to the development of VU6036720, the most potent (IC50 = 0.24 µM) and selective (>40-fold over Kir4.1) Kir4.1/5.1 inhibitor reported to date. Cell-attached patch single-channel recordings revealed that VU6036720 inhibits Kir4.1/5.1 activity through a reduction of channel open-state probability and single-channel current amplitude. Elevating extracellular potassium ion by 20 mM shifted the IC50 6.8-fold, suggesting that VU6036720 is a pore blocker that binds in the ion-conduction pathway. Mutation of the "rectification controller" asparagine 161 to glutamate (N161E), which is equivalent to small-molecule binding sites in other Kir channels, led to a strong reduction of inhibition by VU6036720. Renal clearance studies in mice failed to show a diuretic response that would be consistent with inhibition of Kir4.1/5.1 in the renal tubule. Drug metabolism and pharmacokinetics profiling revealed that high VU6036720 clearance and plasma protein binding may prevent target engagement in vivo. In conclusion, VU6036720 represents the current state-of-the-art Kir4.1/5.1 inhibitor that should be useful for probing the functions of Kir4.1/5.1 in vitro and ex vivo. SIGNIFICANCE STATEMENT: Heteromeric inward rectifier potassium (Kir) channels comprising Kir4.1 and Kir5.1 subunits play important roles in renal and neural physiology and may represent inhibitory drug targets for hypertension and edema. Herein, we employ high-throughput compound library screening, patch clamp electrophysiology, and medicinal chemistry to develop and characterize the first potent and specific in vitro inhibitor of Kir4.1/5.1, VU6036720, which provides proof-of-concept that drug-like inhibitors of this channel may be developed.

Macrophage interferon regulatory factor 4 deletion ameliorates aristolochic acid nephropathy via reduced migration and increased apoptosis.

Aristolochic acid (AA) is the causative nephrotoxic alkaloid in AA nephropathy, which results in a tubulointerstitial fibrosis. AA causes direct proximal tubule damage as well as an influx of macrophages, although the role of macrophages in pathogenesis is poorly understood. Here, we demonstrate that AA directly stimulates migration, inflammation, and ROS production in macrophages ex vivo. Cells lacking interferon regulatory factor 4 (IRF4), a known regulator of macrophage migration and phenotype, had a reduced migratory response, though effects on ROS production and inflammation were preserved or increased relative to WT cells. Macrophage-specific IRF4-knockout mice were protected from both acute and chronic kidney effects of AA administration based on functional and histological analysis. Renal macrophages from kidneys of AA-treated macrophage-specific IRF4-knockout mice demonstrated increased apoptosis and ROS production compared with WT controls, indicating that AA directly polarizes macrophages to a promigratory and proinflammatory phenotype. However, knockout mice had reduced renal macrophage abundance following AA administration. While macrophages lacking IRF4 can adopt a proinflammatory phenotype upon AA exposure, their inability to migrate to the kidney and increased rates of apoptosis upon infiltration provide protection from AA in vivo. These results provide evidence of direct AA effects on macrophages in AA nephropathy and add to the growing body of evidence that supports a key role of IRF4 in modulating macrophage function in kidney injury.

Myeloid cyclooxygenase-2/prostaglandin E2/E-type prostanoid receptor 4 promotes transcription factor MafB-dependent inflammatory resolution in acute kidney injury.

Following acute injury to the kidney, macrophages play an important role in recovery of functional and structural integrity, but organ fibrosis and progressive functional decline occur with incomplete recovery. Pro-resolving macrophages are characterized by increased cyclooxygenase 2 (COX-2) expression and this expression was selectively increased in kidney macrophages following injury and myeloid-specific COX-2 deletion inhibited recovery. Deletion of the myeloid prostaglandin E2 (PGE2) receptor, E-type prostanoid receptor 4 (EP4), mimicked effects seen with myeloid COX-2-/- deletion. PGE2-mediated EP4 activation induced expression of the transcription factor MafB in kidney macrophages, which upregulated anti-inflammatory genes and suppressed pro-inflammatory genes. Myeloid Mafb deletion recapitulated the effects seen with either myeloid COX-2 or EP4 deletion following acute kidney injury, with delayed recovery, persistent presence of pro-inflammatory kidney macrophages, and increased kidney fibrosis. Thus, our studies identified a previously unknown mechanism by which prostaglandins modulate macrophage phenotype following acute organ injury and provide new insight into mechanisms underlying detrimental kidney effects of non-steroidal anti-inflammatory drugs that inhibit cyclooxygenase activity.