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Worldwide, more than one in ten adults are estimated to have chronic kidney disease (CKD). As CKD progresses, both the cost of treatment and associated risk of morbidity and mortality increase exponentially. As such, there is a great need for therapies that effectively slow CKD progression. Evidence from several small clinical trials indicates that alkali therapy may slow the rate of CKD progression. The biological mechanisms underlying this protective effect, however, remain unknown. In their recently published manuscript, Pastor Arroyo et al. (Clin Sci (Lond) (2022) 136(8): https://doi.org/10.1042/CS20220095) demonstrate that the alkali sodium bicarbonate protects against loss of renal function in a crystal nephropathy model in mice. Using unbiased approaches in both mice and human tissue, the authors go on to identify two novel mechanisms that may underly this protection. The first pathway is through promoting pathways of cell metabolism, which they speculate helps the remaining functional nephrons adapt to the greater metabolic needs required to maintain kidney filtration. The second pathway is by restoration of α-Klotho levels, which may limit the expression of adhesion molecules in the injured kidney. This, the authors speculate, may prevent inflammation from driving the functional decline of the kidney. Identifying these novel pathways represents an important step forward harnessing the potential benefits of alkali therapy in CKD.

Impairments in insulin sensitivity can occur in patients with chronic kidney disease (CKD). Correction of metabolic acidosis has been associated with improved insulin sensitivity in CKD, suggesting that metabolic acidosis may directly promote insulin resistance. Despite this, the effect of acid or alkali loading on insulin sensitivity in a rodent model of CKD (remnant kidney) has not been directly investigated. Such studies could better define the relationship between blood pH and insulin sensitivity. We hypothesized that in remnant kidney rats, acid or alkali loading would promote loss of pH homeostasis and consequently decrease insulin sensitivity. To test this hypothesis, we determined the impact of alkali (2 wk) or acid (5–7 days) loading on plasma electrolytes, acid-base balance, and insulin sensitivity in either sham control rats, 2/3 nephrectomized rats, or 5/6 nephrectomized rats. Rats with 5/6 nephrectomy had the greatest response to insulin followed by rats with 2/3 nephrectomy and sham control rats. We found that treatment with 0.1 M sodium bicarbonate solution in drinking water had no effect on insulin sensitivity. Acid loading with 0.1 M ammonium chloride resulted in significant reductions in pH and plasma bicarbonate. However, acidosis did not significantly impair insulin sensitivity. Similar effects were observed in Zucker obese rats with 5/6 nephrectomy. The effect of renal mass reduction on insulin sensitivity could not be explained by reduced insulin clearance or increased plasma insulin levels. We found that renal mass reduction alone increases sensitivity to exogenous insulin in rats and that this is not acutely reversed by the development of acidosis. NEW & NOTEWORTHY Impairments in insulin sensitivity can occur in patients with chronic kidney disease, and previous work has suggested that metabolic acidosis may be the underlying cause. Our study investigated the effect of acid or alkali loading on insulin sensitivity in a rodent model of chronic kidney disease. We found that renal mass reduction increases the blood glucose response to insulin and that this is not acutely reversed by the development of acidosis.

Acute kidney injury (AKI) represents a sudden reduction in renal function and is a major clinical problem with a high mortality rate. Despite decades of research, there are currently no direct therapies for AKI. The failure of therapeutic approaches identified in rodents to translate to human beings has led to questions regarding the appropriateness of these models. Our recent data indicate that there are two distinct processes driving tubular injury in the commonly used rat model of warm bilateral renal ischemia reperfusion injury, which often is used to mimic ischemic AKI. One results from the period of warm ischemia, manifesting as sublethal injury and coagulative necrosis of the proximal tubules in the renal cortex. This is the predominate type of injury observed 24 hours after reperfusion and the most well studied. The other results from red blood cell congestion of the outer medullary vasculature. This type of injury manifests as cell sloughing, along with the later formation of heme casts that fill distal nephron segments. Cell sloughing from congestion is most prominent in the early hours after reperfusion and often is masked by regeneration of the tubular epithelium by 24 hours postischemia. In this review, we argue that injury from outer medullary red blood cell congestion reflects the pathology observed in human kidneys and likely is representative of injury in most cases of ischemic AKI after shock. Greater focus on this congestive injury is likely to lead to improved translation in AKI.

Vascular congestion is common in ischemic acute kidney injury (AKI) and represents densely packed red blood cells (RBC) in the kidney circulation. In this study we tested the hypothesis that ‘vascular congestion directly promotes tubular injury’.Studies were performed in male and female Wistar-Kyoto rats. Vascular congestion and tubular injury were examined between renal venous clamping, arterial clamping and venous clamping of blood perfused and blood free kidneys. Vessels were occluded for either 15 or 45 minutes without reperfusion.We found that venous clamping resulted in greater vascular congestion than arterial clamping, particularly in the outer-medullary region (POur data demonstrate that congestion of the kidney results in the rapid, mass extravasation and uptake of RBCs by tubular cells causing toxic injury to the tubules. Tubular toxicity from extravasation of RBCs appears to be a major component of tubular injury in ischemic AKI which has not previously been recognized.

Noninvasive determination of the severity of parenchymal injury in acute kidney injury remains challenging. Edema is an early pathological process following injury, which may correlate with changes in kidney volume. The goal of the present study was to test the hypothesis that “increases in kidney volume measured in vivo using ultrasound correlate with the degree of renal parenchymal injury.” Ischemia-reperfusion (IR) of varying length was used to produce graded tissue injury. We first determined 1) whether regional kidney volume in rats varied with the severity (0, 15, 30, and 45 min) of warm bilateral IR and 2) whether this correlated with tubular injury score. We then determined whether these changes could be measured in vivo using three-dimensional ultrasound. Finally, we evaluated cumulative changes in kidney volume up to 14 days post-IR in rats to determine whether changes in renal volume were predictive of latent tubular injury following recovery of filtration. Experiments concluded that noninvasive ultrasound measurements of change in kidney volume over 2 wk are predictive of tubular injury following IR even in animals in which plasma creatinine was not elevated. We conclude that ultrasound measurements of volume are a sensitive, noninvasive marker of tissue injury in rats and that the use of three-dimensional ultrasound measurements may provide useful information regarding the timing, severity, and recovery from renal tissue injury in experimental studies.

Acute kidney injury (AKI) due to ischemia is a serious and frequent clinical complication with mortality rates as high as 80%. Vascular congestion in the renal outer medulla occurs early after ischemia reperfusion (IR) injury, and congestion has been linked to worsened outcomes following IR. There is evidence implicating both male sex and preexisting hypertension as risk factors for poor outcomes following IR. The present study tested the hypothesis that male spontaneously hypertensive rats (SHR) have greater vascular congestion and impaired renal recovery following renal IR vs. female SHR and normotensive male Sprague-Dawley rats (SD). Thirteen-week-old male and female SHR and SD were subjected to sham surgery or 30 min of warm bilateral ischemia followed by reperfusion. Rats were euthanized 24 h or 7 days post-IR. IR increased renal injury in all groups vs. sham controls at 24 h. At 7 days post-IR, injury remained elevated only in male SHR. Histological examination of SD and SHR kidneys 24 h post-IR showed vascular congestion in males and females. Vascular congestion was sustained only in male SHR 7 days post-IR. To assess the role of vascular congestion on impaired recovery following IR, additional male and female SHR were pretreated with heparin (200 U/kg) prior to IR. Heparin pretreatment reduced IR-induced vascular congestion and improved renal function in male SHR 7 days post-IR. Interestingly, preventing increases in blood pressure (BP) in male SHR did not alter sustained vascular congestion. Our data demonstrate that IR-induced vascular congestion is a major driving factor for impaired renal recovery in male SHR.

The kidneys receive approximately 20% of cardiac output and have a low fractional oxygen extraction. Quite paradoxically, however, the kidneys are highly susceptible to ischemic injury (injury associated with inadequate blood supply), which is most evident in the renal medulla. The predominant proposal to explain this susceptibility has been a mismatch between oxygen supply and metabolic demand. It has been proposed that unlike the well-perfused renal cortex, the renal medulla normally operates just above the threshold for hypoxia and that further reductions in renal perfusion cause hypoxic injury in this metabolically active region. An alternative proposal is that the true cause of ischemic injury is not a simple mismatch between medullary metabolic demand and oxygen supply, but rather the susceptibility of the outer medulla to vascular congestion. The capillary plexus of the renal outer medullary region is especially prone to vascular congestion during periods of ischemia. It is the failure to restore the circulation to the outer medulla that mediates complete and prolonged ischemia to much of this region, leading to injury and tubular cell death. We suggest that greater emphasis on developing clinically useful methods to help prevent or reverse the congestion of the renal medullary vasculature may provide a means to reduce the incidence and cost of acute kidney injury.

In mice, the initial stage of nephrotoxic serum-induced nephritis (NTN) mimics antibody-mediated human glomerulonephritis. Local immune deposits generate tumor necrosis factor (TNF), which activates pro-inflammatory pathways in glomerular endothelial cells (GEC) and podocytes. Because TNF receptors mediate antibacterial defense, existing anti-TNF therapies can promote infection; however, we have previously demonstrated that different functional domains of TNF may have opposing effects. The TIP peptide mimics the lectin-like domain of TNF, and has been shown to blunt inflammation in acute lung injury without impairing TNF receptor-mediated antibacterial activity. We evaluated the impact of TIP peptide in NTN. Intraperitoneal administration of TIP peptide reduced inflammation, proteinuria, and blood urea nitrogen. The protective effect was blocked by the cyclooxygenase inhibitor indomethacin, indicating involvement of prostaglandins. Targeted glomerular delivery of TIP peptide improved pathology in moderate NTN and reduced mortality in severe NTN, indicating a local protective effect. We show that TIP peptide activates the epithelial sodium channel (ENaC), which is expressed by GEC, upon binding to the channel’s α subunit. In vitro, TNF treatment of GEC activated pro-inflammatory pathways and decreased the generation of prostaglandin E(2) and nitric oxide, which promote recovery from NTN. TIP peptide counteracted these effects. Despite the capacity of TIP peptide to activate ENaC, it did not increase mean arterial blood pressure in mice. In the later autologous phase of NTN, TIP peptide blunted the infiltration of Th17 cells. By countering the deleterious effects of TNF through direct actions in GEC, TIP peptide could provide a novel strategy to treat glomerular inflammation.

IL-18 (interleukin-18) is elevated in hypertensive patients, but its contribution to high blood pressure and end-organ damage is unknown. We examined the role of IL-18 in the development of renal inflammation and injury in a mouse model of low-renin hypertension. Hypertension was induced in male C57BL6/J (WT) and IL-18−/−mice by uninephrectomy, deoxycorticosterone acetate (2.4 mg/d, s.c.) and 0.9% drinking saline (1K/DOCA/salt). Normotensive controls received uninephrectomy and placebo (1K/placebo). Blood pressure was measured via tail cuff or radiotelemetry. After 21 days, kidneys were harvested for (immuno)histochemical, quantitative-PCR and flow cytometric analyses of fibrosis, inflammation, and immune cell infiltration. 1K/DOCA/salt-treated WT mice developed hypertension, renal fibrosis, upregulation of proinflammatory genes, and accumulation of CD3+T cells in the kidneys. They also displayed increased expression of IL-18 on tubular epithelial cells. IL-18−/−mice were profoundly protected from hypertension, renal fibrosis, and inflammation. Bone marrow transplantation between WT and IL-18−/−mice revealed that IL-18-deficiency in non-bone marrow-derived cells alone afforded equivalent protection against hypertension and renal injury as global IL-18 deficiency. IL-18 receptor subunits—interleukin-18 receptor 1 and IL-18R accessory protein—were upregulated in kidneys of 1K/DOCA/salt-treated WT mice and localized to T cells and tubular epithelial cells. T cells from kidneys of 1K/DOCA/salt-treated mice produced interferon-γ upon ex vivo stimulation with IL-18, whereas those from 1K/placebo mice did not. In conclusion, IL-18 production by tubular epithelial cells contributes to elevated blood pressure, renal inflammation, and fibrosis in 1K/DOCA/salt-treated mice, highlighting it as a promising therapeutic target for hypertension and kidney disease.

Sodium bicarbonate (NaHCO3) has been recognized as a possible therapy to target chronic kidney disease (CKD) progression. Several small clinical trials have demonstrated that supplementation with NaHCO3or other alkalizing agents slows renal functional decline in patients with CKD. While the benefits of NaHCO3treatment have been thought to result from restoring pH homeostasis, a number of studies have now indicated that NaHCO3or other alkalis may provide benefit regardless of the presence of metabolic acidosis. These data have raised questions as to how NaHCO3protects the kidneys. To date, the physiological mechanism(s) that mediates the reported protective effect of NaHCO3in CKD remain unclear. In this review, we first examine the evidence from clinical trials in support of a beneficial effect of NaHCO3and other alkali in slowing kidney disease progression and their relationship to acid-base status. Then, we discuss the physiological pathways that have been proposed to underlie these renoprotective effects and highlight strengths and weaknesses in the data supporting each pathway. Finally, we discuss how answering key questions regarding the physiological mechanism(s) mediating the beneficial actions of NaHCO3therapy in CKD is likely to be important in the design of future clinical trials. We conclude that basic research in animal models is likely to be critical in identifying the physiological mechanisms underlying the benefits of NaHCO3treatment in CKD. Gaining an understanding of these pathways may lead to the improved implementation of NaHCO3as a therapy in CKD and perhaps other disease states.

Renal ischemia is the most common cause of acute kidney injury. Damage-associated molecular patterns (DAMPs) initiate an inflammatory response and contribute to ischemia–reperfusion (IR) injury in males, yet the contribution of DAMPs to IR injury in females is unknown. The goal of the current study was to test the hypothesis that males have greater increases in the DAMP high-mobility group box 1 (HMGB1), worsening injury compared with females. Thirteen-week-old male and female spontaneously hypertensive rats (SHR) were subjected to sham or 45-min warm bilateral ischemia followed by 24 h of reperfusion before measurement of HMGB1 and renal function. Additional SHR were pre-treated with control (IgG) or HMGB1 neutralizing antibody (300 µg/rat) 1 h prior to renal ischemia. Blood, urine and kidneys were harvested 24 h post-IR for histological and Western blot analyses. Initial studies confirmed that IR resulted in greater increases in renal HMGB1 in male SHR compared with females. Greater renal HMGB1 in male SHR post-IR resulted in greater increases in serum TNF-α and renal IL-1β, neutrophil infiltration and tubular cell death. Neutralization of HMGB1 attenuated IR-induced increases in plasma creatinine, blood urea nitrogen (BUN), inflammation, tubular damage and tubular cell death only in male SHR. In conclusion, our data demonstrate that there is a sex difference in the contribution of HMGB1 to IR-induced injury, where males exhibit greater increases in HMGB1-mediated renal injury in response to IR compared with females.

To investigate the role of protein kinase C-α (PKC-α) in glomerulonephritis, the capacity of PKC-α inhibition to reverse the course of established nephrotoxic nephritis (NTN) was evaluated. Nephritis was induced by a single injection of nephrotoxic serum and after its onset, a PKC-α inhibitor was administered either systemically or by targeted glomerular delivery. By day seven, all mice with NTN had severe nephritis, whereas mice that received PKC-α inhibitors in either form had minimal evidence of disease. To further understand the underlying mechanism, label-free shotgun proteomic analysis of the kidney cortexes were performed, using quantitative mass spectrometry. Ingenuity pathway analysis revealed 157 differentially expressed proteins and mitochondrial dysfunction as the most modulated pathway. Functional protein groups most affected by NTN were mitochondrial proteins associated with respiratory processes. These proteins were down-regulated in the mice with NTN, while their expression was restored with PKC-α inhibition. This suggests a role for proteins that regulate oxidative phosphorylation in recovery. In cultured glomerular endothelial cells, nephrotoxic serum caused a decrease in mitochondrial respiration and membrane potential, mitochondrial morphologic changes and an increase in glycolytic lactic acid production; all normalized by PKC-α inhibition. Thus, PKC-α has a critical role in NTN progression, and the results implicate mitochondrial processes through restoring oxidative phosphorylation, as an essential mechanism underlying recovery. Importantly, our study provides additional support for targeted therapy to glomeruli to reverse the course of progressive disease.

Background and purpose Macropinocytosis is involved in many pathologies, including cardiovascular disorders, cancer, allergic diseases, viral and bacterial infections. Unfortunately, the currently available pharmacological inhibitors of macropinocytosis interrupt other endocytic processes and have non-specific endocytosis-independent effects. Here we have sought to identify new, clinically relevant inhibitors of macropinocytosis, using an FDA-approved drug library. Experimental approach In the present study, 640 FDA-approved compounds were tested for their ability to inhibit macropinocytosis. A series of secondary assays were performed to confirm inhibitory activity, determine IC50 values and investigate cell toxicity. The ability of identified hits to inhibit phagocytosis and clathrin-mediated and caveolin-mediated endocytosis was also investigated. Scanning electron microscopy and molecular biology techniques were utilized to examine the mechanisms by which selected compounds inhibit macropinocytosis. Key results The primary screen identified 14 compounds that at ~10 μM concentration inhibit >95% of macropinocytotic solute internalization. Three compounds - imipramine, phenoxybenzamine and vinblastine - potently inhibited (IC50 ≤ 131 nM) macropinocytosis without exerting cytotoxic effects or inhibiting other endocytic pathways. Scanning electron microscopy imaging indicated that imipramine inhibits membrane ruffle formation, a critical early step leading to initiation of macropinocytosis. Finally, imipramine has been shown to inhibit macropinocytosis in several cell types, including cancer cells, dendritic cells and macrophages. Conclusions and implications Our results identify imipramine as a new pharmacological tool to study macropinocytosis in cellular and biological systems. This study also suggests that imipramine could be a good candidate for repurposing as a therapeutic agent in pathological processes involving macropinocytosis.

We tested the hypothesis that oral NaHCO3 intake stimulates splenic anti-inflammatory pathways. Following oral NaHCO3 loading, macrophage polarization was shifted from predominantly M1 (inflammatory) to M2 (regulatory) phenotypes, and FOXP3+CD4+ T-lymphocytes increased in the spleen, blood, and kidneys of rats. Similar anti-inflammatory changes in macrophage polarization were observed in the blood of human subjects following NaHCO3 ingestion. Surprisingly, we found that gentle manipulation to visualize the spleen at midline during surgical laparotomy (sham splenectomy) was sufficient to abolish the response in rats and resulted in hypertrophy/hyperplasia of the capsular mesothelial cells. Thin collagenous connections lined by mesothelial cells were found to connect to the capsular mesothelium. Mesothelial cells in these connections stained positive for the pan-neuronal marker PGP9.5 and acetylcholine esterase and contained many ultrastructural elements, which visually resembled neuronal structures. Both disruption of the fragile mesothelial connections or transection of the vagal nerves resulted in the loss of capsular mesothelial acetylcholine esterase staining and reduced splenic mass. Our data indicate that oral NaHCO3 activates a splenic anti-inflammatory pathway and provides evidence that the signals that mediate this response are transmitted to the spleen via a novel neuronal-like function of mesothelial cells.

Necrosis is a pathological form of cell death that induces an inflammatory response, and immune cell activation contributes to the development and maintenance of hypertension. Necrosis was measured in kidney, spleen, and aorta of 12- to 13-week-old male and female SHRs (spontaneously hypertensive rats); male SHRs had greater renal necrotic cell death than female SHRs. Because male SHRs have a higher blood pressure (BP) and a more proinflammatory T-cell profile than female SHRs, the current studies tested the hypothesis that greater necrotic cell death in male SHRs exacerbates increases in BP and contributes to the proinflammatory T-cell profile. Male and female SHRs were randomized to receive vehicle or Necrox-5—a cell permeable inhibitor of necrosis—from 6 to 12 weeks of age or from 11 to 13 weeks of age. In both studies, Necrox-5 decreased renal necrosis and abolished the sex difference. Treatment with Necrox-5 beginning at 6 weeks of age attenuated maturation-induced increases in BP in male SHR; BP in female SHR was not altered by Necrox-5 treatment. Necrox-5 decreased proinflammatory renal T cells in both sexes, although sex differences were maintained. Administration of Necrox-5 for 2 weeks in SHR with established hypertension resulted in a small but significant decrease in BP in males with no effect in females. These results suggest that greater necrotic cell death in male SHR exacerbates maturation-induced increases in BP with age contributing to sex differences in BP. Moreover, although necrosis is proinflammatory, it is unlikely to explain sex differences in the renal T-cell profile.

Experimental studies have demonstrated protective effects of NHE‐1 inhibition on cardiac function; however, clinical trials utilizing NHE‐1 antagonists found an increase in overall mortality attributed to thromboembolic strokes. NADPH oxidase‐derived reactive oxygen species (ROS) from microglial cells have been shown to contribute to injury following stroke. We have recently demonstrated that NHE‐1 inhibition enhances ROS in macrophages in a Hv1‐dependent manner. As Hv1 protein is highly expressed in microglia, we hypothesized that “NHE‐1 inhibition may augment neurovascular injury by activating Hv1,” providing a potential mechanism for the deleterious effects of NHE‐1. The goal of this study was to determine whether neurovascular injury and functional outcomes after experimental stroke differed in wild‐type and Hv1 mutant Dahl salt‐sensitive rats treated with an NHE‐1 inhibitor. Stroke was induced using both transient and permanent of middle cerebral artery occlusion (MCAO). Animals received vehicle or NHE‐1 inhibitor KR32568 (2 mg/kg, iv) either 30 min after the start of MCAO or were pretreated (2 mg/kg, iv, day) for 3 days and then subjected to MCAO. Our data indicate that Hv1 deletion confers both neuronal and vascular protection after ischemia. In contrast to our hypothesis, inhibition of NHE‐1 provided further protection from ischemic stroke, and the beneficial effects of both pre‐ and post‐treatment with KR32568 were similar in wild‐type and Hv1−/− rats. These data indicate that Hv1 activation is unlikely to be responsible for the increased incidence of cerebrovascular events observed in the heart disease patients after NHE‐1 inhibition treatment.

Much research now indicates that vagal nerve stimulation results in a systemic reduction in inflammatory cytokine production and an increase in anti-inflammatory cell populations that originates from the spleen. Termed the 'cholinergic anti-inflammatory pathway', therapeutic activation of this innate physiological response holds enormous promise for the treatment of inflammatory disease. Much controversy remains however, regarding the underlying physiological pathways mediating this response. This controversy is anchored in the fact that the vagal nerve itself does not innervate the spleen. Recent research from our own laboratory indicating that oral intake of sodium bicarbonate stimulates splenic anti-inflammatory pathways, and that this effect may require transmission of signals to the spleen through the mesothelium, provide new insight into the physiological pathways mediating the cholinergic anti-inflammatory pathway. In this review, we examine proposed models of the cholinergic anti-inflammatory pathway and attempt to frame our recent results in relation to these hypotheses. Following this discussion, we then provide an alternative model of the cholinergic anti-inflammatory pathway which is consistent both with our recent findings and the published literature. We then discuss experimental approaches that may be useful to delineate these hypotheses. We believe the outcome of these experiments will be critical in identifying the most appropriate methods to harness the therapeutic potential of the cholinergic anti-inflammatory pathway for the treatment of disease and may also shed light on the etiology of other pathologies, such as idiopathic fibrosis.

Aims: Macropinocytosis has been implicated in atherosclerosis, cancer, allergic disorders, and other pathologies. Unfortunately, most currently available pharmacological inhibitors of macropinocytosis interrupt other endocytic processes and have non-specific endocytosis-independent effects. The goal of the present study was to perform a high-throughput screen (HTS) of FDA-approved drugs to identify new, clinically relevant inhibitors of macropinocytosis. Methods: In the present study, 640 FDA-approved compounds were tested for their ability to inhibit macropinocytosis. A series of secondary assays were performed to confirm inhibitory activity, determine IC 50 values, and investigate cell toxicity. The ability of identified hits to inhibit phagocytosis, clathrin- and caveolin-mediated endocytosis was also investigated. Scanning electron microscopy and molecular biology techniques were utilized to investigate the mechanisms by which selected compounds inhibit macropinocytosis. Results: The HTS campaign identified 14 compounds that at ~10 μM concentration inhibit >95% of macropinocytotic solute internalization. Our results demonstrated that three lead compounds, namely imipramine, phenoxybenzamine, and vinblastine, potently inhibit (IC 50 ≤ 130 nM) macropinocytosis without exerting cytotoxic effects or inhibiting other endocytic pathways. Mechanistically, we found that imipramine inhibits translocation of ADP-ribosylation factor 6 to the plasma membrane and prevents membrane ruffle formation, a critical early step leading to macropinocytosis. Imipramine inhibited macropinocytosis in multiple cell types, including cancer cells, dendritic cells, and macrophages. Finally, incubation of macrophages with imipramine inhibited nLDL macropinocytosis and foam cell formation in vitro . Innovation and Conclusion: The identified macropinocytosis inhibitors may prove useful as new pharmacological tools to more fully discern the role of macropinocytosis in pathological processes and as therapeutic agents in various disorders involving macropinocytosis.

The physiological evidence linking the production of superoxide, hydrogen peroxide, and nitric oxide in the renal medullary thick ascending limb of Henle (mTAL) to regulation of medullary blood flow, sodium homeostasis, and long-term control of blood pressure is summarized in this review. Data obtained largely from rats indicate that experimentally induced elevations of either superoxide or hydrogen peroxide in the renal medulla result in reduction of medullary blood flow, enhanced Na+reabsorption, and hypertension. A shift in the redox balance between nitric oxide and reactive oxygen species (ROS) is found to occur naturally in the Dahl salt-sensitive (SS) rat model, where selective reduction of ROS production in the renal medulla reduces salt-induced hypertension. Excess medullary production of ROS in SS rats emanates from the medullary thick ascending limbs of Henle [from both the mitochondria and membrane NAD(P)H oxidases] in response to increased delivery and reabsorption of excess sodium and water. There is evidence that ROS and perhaps other mediators such as ATP diffuse from the mTAL to surrounding vasa recta capillaries, resulting in medullary ischemia, which thereby contributes to hypertension.

Sodium bicarbonate (NaHCO3) slows the decline in kidney function in patients with chronic kidney disease (CKD), yet the mechanisms mediating this effect remain unclear. The Dahl salt-sensitive (SS) rat develops hypertension and progressive renal injury when fed a high salt diet; however, the effect of alkali loading on kidney injury has never been investigated in this model. We hypothesized that NaHCO3 protects from the development of renal injury in Dahl salt-sensitive rats via luminal alkalization which limits the formation of tubular casts, which are a prominent pathological feature in this model. To examine this hypothesis, we determined blood pressure and renal injury responses in Dahl SS rats drinking vehicle (0.1 M NaCl) or NaHCO3 (0.1 M) solutions as well as in Dahl SS rats lacking the voltage-gated proton channel (Hv1). We found that oral NaHCO3 reduced tubular NH4+ production, tubular cast formation, and interstitial fibrosis in rats fed a high salt diet for 2 weeks. This effect was independent of changes in blood pressure, glomerular injury, or proteinuria and did not associate with changes in renal inflammatory status. We found that null mutation of Hv1 also limited cast formation in Dahl SS rats independent of proteinuria or glomerular injury. As Hv1 is localized to the luminal membrane of TAL, our data suggest that alkalization of the luminal fluid within this segment limits cast formation in this model. Reduced cast formation, secondary to luminal alkalization within TAL segments may mediate some of the protective effects of alkali loading observed in CKD patients.

We tested the hypothesis that 'Oral NaHCO(3) intake stimulates splenic anti-inflammatory pathways. Following oral NaHCO(3) loading, macrophage polarization was shifted from predominantly M1 (inflammatory) to M2 (regulatory) phenotypes and FOXP3(+)CD4(+) T-lymphocytes increased in the spleen, blood and kidneys of rats. Similar anti-inflammatory changes in macrophage polarization were observed in the blood of human subjects following NaHCO(3) ingestion. Surprisingly, we found that gentle manipulation to visualize the spleen at midline during surgical laparotomy (sham splenectomy) was sufficient to abolish the response in rats and resulted in hypertrophy/hyperplasia of the capsular mesothelial cells. Thin collagenous connections lined by mesothelial cells were found to connect to the capsular mesothelium. Mesothelial cells in these connections stained positive for the pan-neuronal marker PGP9.5 and acetylcholine esterase and contained many ultrastructural elements which visually resembled neuronal structures. Both disruption of the fragile mesothelial connections or transection of the vagal nerves, resulted in loss of capsular mesothelial acetylcholine esterase staining and reduced splenic mass. Our data indicate that oral NaHCO(3) activates a splenic anti-inflammatory pathway and provides evidence that the signals that mediate this response are transmitted to the spleen via a novel neuronal like function of mesothelial cells.

Recent evidence suggests that a greater density of pericytes in renal cadaveric allografts is associated with better recovery following transplant. The physiological mechanism(s) through which pericyte density may be beneficial is not well understood. The goal of this study was to test the hypothesis that lower medullary pericyte density is associated with greater renal injury following ischemia reperfusion (IR) in a rat model, providing a basis for future studies to better understand pericytes in a pathological environment. To test our hypothesis, we determined the association between medullary pericyte density and renal injury in spontaneously hypertensive rats (SHR) following 45 min of warm bilateral IR. We found that there was a significant negative relationship between pericyte density and plasma creatinine (slope = −0.03, P = 0.02) and blood urea nitrogen (slope = −0.5, P = 0.01) in female but not male SHR. Pericyte density was negatively associated with medullary peritubular capillary (PT) congestion in both sexes following IR (male: slope = −0.04, P = 0.009; female: slope = −0.03, P = 0.0001). To further test this relationship, we used a previously reported method to reduce pericyte density in SHR. Medullary erythrocyte congestion in vasa recta (VR) and PT significantly increased following IR in both sexes when pericyte density was pharmacologically decreased (VR: P = 0.03; PT: P = 0.03). Our data support the hypothesis that pericyte density is negatively associated with the development of IR injury in SHR, which may be mediated by erythrocyte congestion in the medullary vasculature.

Aim This study was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high-salt diet and/or hypoxia. Methods C57BL6/J control mice or vascular endothelial cell ET-1 knockout (VEET KO) mice were subjected to 3-h exposure to hypoxia (8% O2) and/or 2 weeks of high-salt diet (4% NaCl) prior to metabolic cage assessment of renal function and isolation of glomeruli for the determination of reactive oxygen species (ROS). Results In control mice, hypoxia significantly increased urinary protein excretion during the initial 24 h, but only in animals on a high-salt diet. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Under normoxic conditions, mice on a high-salt diet had approx. 150% higher glomerular ET-1 mRNA expression compared with a normal-salt diet (P

We examined how the presence of a fixed level of basal renal O2consumption (V̇o2basal; O2used for processes independent of Na+transport) confounds the utility of the ratio of Na+reabsorption (TNa+) to total renal V̇o2(V̇o2total) as an index of the efficiency of O2utilization for TNa+. We performed a systematic review and additional experiments in anesthetized rabbits to obtain the best possible estimate of the fractional contribution of V̇o2basalto V̇o2totalunder physiological conditions (basal percent renal V̇o2). Estimates of basal percent renal V̇o2from 24 studies varied from 0% to 81.5%. Basal percent renal V̇o2varied with the fractional excretion of Na+(FENa+) in the 14 studies in which FENa+was measured under control conditions. Linear regression analysis predicted a basal percent renal V̇o2of 12.7–16.5% when FENa+= 1% ( r2= 0.48, P = 0.001). Experimentally induced changes in TNa+altered TNa+/V̇o2totalin a manner consistent with theoretical predictions. We conclude that, because V̇o2basalrepresents a significant proportion of V̇o2total, TNa+/V̇o2totalcan change markedly when TNa+itself changes. Therefore, caution should be taken when TNa+/V̇o2totalis interpreted as a measure of the efficiency of O2utilization for TNa+, particularly under experimental conditions where TNa+or V̇o2totalchanges.

Various stimuli, including hormones and growth factors, modulate epithelial sodium channels (ENaCs), which fine-tune Na(+) absorption in the kidney. Members of the EGF family are important for maintaining transepithelial Na(+) transport, but whether EGF influences ENaC, perhaps mediating salt-sensitive hypertension, is not well understood. Here, the ENaC inhibitor benzamil attenuated the development of hypertension in Dahl salt-sensitive rats. Feeding these salt-sensitive rats a high-salt diet led to lower levels of EGF in the kidney cortex and enhanced the expression and activity of ENaC compared with feeding a low-salt diet. To directly evaluate the role of EGF in the development of hypertension and its effect on ENaC activity, we infused EGF intravenously while continuously monitoring BP of the salt-sensitive rats. Infusion of EGF decreased ENaC activity, prevented the development of hypertension, and attenuated glomerular and renal tubular damage. Taken together, these findings indicate that cortical EGF levels decrease with a high-salt diet in salt-sensitive rats, promoting ENaC-mediated Na(+) reabsorption in the collecting duct and the development of hypertension.

Despite the high prevalence and major negative impact of uterine fibroids (UFs) on women's health, their pathogenesis remains largely unknown. While tumor-initiating cells have been previously isolated from UFs, the cell of origin for these tumors in normal myometrium has not been identified. We isolated cells with Stro1/CD44 surface markers from normal myometrium expressing stem cell markers Oct-4/c-kit/nanog that exhibited the properties of myometrial stem/progenitor-like cells (MSCs). Using a murine model for UFs, we showed that the cervix was a hypoxic “niche” and primary site (96%) for fibroid development in these animals. The pool size of these MSCs also responded to environmental cues, contracting with age and expanding in response to developmental environmental exposures that promote fibroid development. Translating these findings to women, the number of MSCs in unaffected human myometrium correlated with risk for developing UFs. Caucasian (CC) women with fibroids had increased numbers of MSCs relative to CC women without fibroids, and African-American (AA) women at highest risk for these tumors had the highest number of MSCs: AA-with fibroids > CC-with fibroids > AA-without fibroids > CC-without fibroids. These data identify Stro1+/CD44+ MSCs as MSC/progenitor cell for UFs, and a target for ethnic and environmental factors that increase UF risk.

Hv1 is a voltage-gated proton channel highly expressed in phagocytic cells, where it participates in the NADPH oxidase-dependent respiratory burst. We have recently identified Hv1 as a novel renal channel, expressed in the renal medullary thick ascending limb that appears to importantly contribute to the pathogenesis of renal hypertensive injury in the Dahl salt-sensitive rat model. The purpose of this review is to describe the experimental approaches that we have undertaken to identify the source of excess reactive oxygen species production in the renal outer medulla of Dahl salt-sensitive rats and the resulting evidence that the voltage-gated proton channel Hv1 mediates augmented superoxide production and contributes to renal medullary oxidative stress and renal injury. In addition, we will attempt to point out areas of current controversy, as well as propose areas in which further experimental studies are likely to move the field forward. The content of the following review was presented as part of the Water and Electrolyte Homeostasis Section New Investigator Award talk at Experimental Biology 2014.

The adult kidney plays a central role in erythropoiesis and is the main source of erythropoietin (EPO), an oxygen-sensitive glycoprotein that is essential for red blood cell production. Decreases of renal pO2 promote hypoxia-inducible factor 2-mediated (HIF-2-mediated) induction of EPO in peritubular interstitial fibroblast-like cells, which serve as the cellular site of EPO synthesis in the kidney. It is not clear whether HIF signaling in other renal cell types also contributes to the regulation of EPO production. Here, we used a genetic approach in mice to investigate the role of renal epithelial HIF in erythropoiesis. Specifically, we found that HIF activation in the proximal nephron via induced inactivation of the von Hippel-Lindau tumor suppressor, which targets the HIF-α subunit for proteasomal degradation, led to rapid development of hypoproliferative anemia that was associated with a reduction in the number of EPO-producing renal interstitial cells. Moreover, suppression of renal EPO production was associated with increased glucose uptake, enhanced glycolysis, reduced mitochondrial mass, diminished O2 consumption, and elevated renal tissue pO2. Our genetic analysis suggests that tubulointerstitial cellular crosstalk modulates renal EPO production under conditions of epithelial HIF activation in the kidney.

We have demonstrated previously that paracrine signaling occurs between medullary thick ascending limb (mTAL) and the contractile pericytes of outer-medullary vasa recta (VR), termed “tubular-vascular cross-talk.” The aim of the current study was to determine whether tubular-vascular cross-talk has a functional effect on vasoconstrictor responses to angiotensin II and to determine whether this is altered in the Dahl salt-sensitive (SS) rat. Studies were performed on salt-resistant consomic SS.13 Brown Norway (BN) and SS rats using a novel outer medullary tissue strip preparation in which freshly isolated VRs within VR bundles were perfused either alone or in combination with nearby mTAL. In VRs from SS.13 bn rats, angiotensin II (1 µmol/L) increased VR bundle intracellular Ca 2+ concentration 19 ± 9 nmol/L (n=8) and reduced focal diameter in perfused VRs by −20 ± 7% (n=5). In the presence of nearby mTAL, however, VR bundle intracellular Ca 2+ concentration (−9 ± 8 nmol/L; n=8) and VR diameter (−1 ± 4%, n=7) in SS.13 bn rats were unchanged by angiotensin II. In contrast, in Dahl SS rats, angiotensin II resulted in rapid and sustained increase in VR bundle intracellular Ca 2+ concentration (89 ± 48 nmol/L, n=7; 50 ± 24%, n=8) and a reduction in VR diameter of (−17 ± 7%, n=7; −11 ± 4%, n=5) in both isolated VRs and VRs with nearby mTAL, respectively. In VRs with mTAL from SS13 bn rats, inhibiton of purinergic receptors resulted in an increase in VR bundle intracellular Ca 2+ concentration, indicating that purinergic signaling buffers vasoconstriction. Importantly, our in vitro data were able to predict medullary blood flow responses to angiotensin II in SS and SS.13 bn rats in vivo. We conclude that paracrine signaling from mTAL buffers angiotensin II vasoconstriction in Dahl salt-resistant SS.13 bn rats but not SS rats.

A fundamental requirement for cellular vitality is the maintenance of plasma ion concentration within strict ranges. It is the function of the kidney to match urinary excretion of ions with daily ion intake and nonrenal losses to maintain a stable ionic milieu. NADPH oxidase is a source of reactive oxygen species (ROS) within many cell types, including the transporting renal epithelia. The focus of this review is to describe the role of NADPH oxidase-derived ROS toward local renal tubular ion transport in each nephron segment and to discuss how NADPH oxidase-derived ROS signaling within the nephron may mediate ion homeostasis. In each case, we will attempt to identify the various subunits of NADPH oxidase and reactive oxygen species involved and the ion transporters, which these affect. We will first review the role of NADPH oxidase on renal Na+ and K+ transport. Finally, we will review the relationship between tubular H+ efflux and NADPH oxidase activity.

To better understand what makes the kidney susceptible to tissue hypoxia, we compared, in the rabbit kidney and hindlimb, the ability of feedback mechanisms governing oxygen consumption (V̇o2) and oxygen delivery (Do2) to attenuate tissue hypoxia during hypoxemia. In the kidney (cortex and medulla) and hindlimb (biceps femoris muscle), we determined responses of whole organ blood flow and V̇o2, and local perfusion and tissue Po2, to reductions in Do2mediated by graded systemic hypoxemia. Progressive hypoxemia reduced tissue Po2similarly in the renal cortex, renal medulla, and biceps femoris. Falls in tissue Po2could be detected when arterial oxygen content was reduced by as little as 4–8%. V̇o2remained stable during progressive hypoxemia, only tending to fall once arterial oxygen content was reduced by 55% for the kidney or 42% for the hindlimb. Even then, the fall in renal V̇o2could be accounted for by reduced oxygen demand for sodium transport rather than limited oxygen availability. Hindlimb blood flow and local biceps femoris perfusion increased progressively during graded hypoxia. In contrast, neither total renal blood flow nor cortical or medullary perfusion was altered by hypoxemia. Our data suggest that the absence in the kidney of hyperemic responses to hypoxia, and the insensitivity of renal V̇o2to limited oxygen availability, contribute to kidney hypoxia during hypoxemia. The susceptibility of the kidney to tissue hypoxia, even in relatively mild hypoxemia, may have important implications for the progression of kidney disease, particularly in patients at high altitude or with chronic obstructive pulmonary disease.

Summary 1. Oxygen tension (PO2) in renal cortical tissue can remain relatively constant when renal blood flow changes in the physiological range, even when changes in renal oxygen delivery (DO2) and oxygen consumption (VO2) are mismatched. In the current study, we examined whether this also occurs in the renal medulla and skeletal muscle, or if it is an unusual property of the renal cortex. We also examined the potential for dysfunction of the mechanisms underlying this phenomenon to contribute to kidney hypoxia in disease states associated with increased renal VO2. 2. In both the kidney and hindlimb of pentobarbitone anaesthetized rabbits, whole organ blood flow was reduced by intra-arterial infusion of angiotensin-II and increased by acetylcholine infusion. In the kidney, this was carried out before and during renal arterial infusion of the mitochondrial uncoupler, 2,4-dinitrophenol (DNP), or its vehicle. 3. Angiotensin-II reduced renal (−34%) and hindlimb (−25%) DO2, whereas acetylcholine increased renal (+38%) and hindlimb (+66%) DO2. However, neither renal nor hindlimb VO2 were altered. Tissue PO2 varied with local perfusion in the renal medulla and biceps femoris, but not the renal cortex. DNP increased renal VO2 (+38%) and reduced cortical tissue PO2 (−44%), but both still remained stable during subsequent infusion of angiotensin-II and acetylcholine. 4. We conclude that maintenance of tissue PO2 in the face of mismatched changes in local perfusion and VO2 is an unusual property of the renal cortex. The underlying mechanisms remain unknown, but our current findings suggest they are not compromised when resting renal VO2 is increased.

We examined the mechanisms that maintain stable renal tissue Po2 during moderate renal ischemia, when changes in renal oxygen delivery (Ḋo2) and consumption (V̇o2) are mismatched. When renal artery pressure (RAP) was reduced progressively from 80 to 40 mmHg, V̇o2 (−38 ± 7%) was reduced more than Ḋo2 (−26 ± 4%). Electrical stimulation of the renal nerves (RNS) reduced Ḋo2 (−49 ± 4% at 2 Hz) more than V̇o2 (−30 ± 7% at 2 Hz). Renal arterial infusion of angiotensin II reduced Ḋo2 (−38 ± 3%) but not V̇o2 (+10 ± 10%). Despite mismatched changes in Ḋo2 and V̇o2, renal tissue Po2 remained remarkably stable at ≥40 mmHg RAP, during RNS at ≤2 Hz, and during angiotensin II infusion. The ratio of sodium reabsorption to V̇o2 was reduced by all three ischemic stimuli. None of the stimuli significantly altered the gradients in Pco2 or pH across the kidney. Fractional oxygen extraction increased and renal venous Po2 fell during 2-Hz RNS and angiotensin II infusion, but not when RAP was reduced to 40 mmHg. Thus reduced renal V̇o2 can help prevent tissue hypoxia during mild renal ischemia, but when renal V̇o2 is reduced less than Ḋo2, other mechanisms prevent a fall in renal Po2. These mechanisms do not include increased efficiency of renal oxygen utilization for sodium reabsorption or reduced washout of carbon dioxide from the kidney, leading to increased oxygen extraction. However, increased oxygen extraction could be driven by altered countercurrent exchange of carbon dioxide and/or oxygen between renal arteries and veins.