Patent Application
20210338630
Renal disease is a significant cause of adult mortality. Acute kidney injury (AKI) and chronic kidney disease (CKD) eventually progress to end-stage renal disease, requiring dialysis or kidney transplant. Unfortunately, no clinically proven therapies to prevent or to treat renal disease are available. A need in the art remains for methods to treat and to prevent renal disorders.
The invention provides store operated calcium entry (SOCE) inhibitors to treat a renal disorder in a mammal in need of such treatment, comprising administering to the mammal in need of treatment an effective amount of a SOCE inhibitor or a pharmaceutically acceptable salt thereof.
The invention further provides a pharmaceutical composition, comprising a SOCE inhibitor, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients.
The invention further provides a process for preparing a pharmaceutical composition, comprising admixing a SOCE inhibitor, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients.
This invention also provides a method to inhibit differentiation of a CD4+ cell to a T-helper 17 (TH17) cell, comprising administering to a mammal an effective amount of a store operated calcium entry SOCE inhibitor or a pharmaceutically acceptable salt thereof.
The invention additionally provides a method to modulate store operated calcium2+ entry into a cell, the method comprising administering to a mammal an effective amount of a store operated calcium entry SOCE inhibitor or a pharmaceutically acceptable salt thereof.
The invention further provides a method to decrease an amount of pro-inflammatory cytokine Interleuken 17 (IL-17), the method comprising administering to a mammal an effective amount of a store operated calcium entry SOCE inhibitor or a pharmaceutically acceptable salt thereof.
The invention also provides a method to decrease an amount of a Ca2+ release-activated Ca2+ channel pore forming subunit OraI1, the method comprising administering to a mammal an effective amount of a store operated calcium entry SOCE inhibitor or a pharmaceutically acceptable salt thereof.
The invention provides store operated calcium entry SOCE inhibitors to treat a renal disorder in a mammal in need of such treatment, comprising administering to the mammal in need of treatment an effective amount of a SOCE inhibitor or a pharmaceutically acceptable salt thereof. The SOCE inhibitors of the invention are any compound that interferes with the mechanism by which release of calcium ions from intracellular stores is coordinated with ion influx across the plasma membrane. In some embodiments, the SOCE inhibitor is selective for SOC channels and does not substantially affect the activity of other types of ion channels. In other embodiments, the SOCE inhibitor is selective for CRAC channels and does not substantially affect the activity of other types of ion channels and/or other SOC channels.
Exemplary SOCE inhibitors include, but are not limited to, 2-aminoethoxydiphenyl borate (2APB), (N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (YM58483/BTP2), AnCoA4, CM4620, GSK5948A, Synta66 and Oria1+Si RNA.
The SOCE inhibitors of the invention are preferably formulated as pharmaceutical compositions administered by any route which makes the compound bioavailable, including oral and transdermal routes. Most preferably, such compositions are for oral administration. Such pharmaceutical compositions and processes for preparing same are well known in the art (See, e.g., Remington: The Science and Practice of Pharmacy, L. V. Allen, Editor, 22nd Edition, Pharmaceutical Press, 2012). The SOCE inhibitors, or pharmaceutically acceptable salts thereof are particularly useful in the treatment methods of the invention.
The SOCE inhibitors of the invention can be provided as a pharmaceutically acceptable salt. A pharmaceutically acceptable salt of the SOCE inhibitors of the invention can be formed, for example, by reaction of an appropriate free base of a compound of the invention and an appropriate pharmaceutically acceptable acid in a suitable solvent under standard conditions well known in the art. The formation of such salts is well known and appreciated in the art. See, for example, Gould, P. L., “Salt selection for basic drugs,” International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R. J., et al. “Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,” Organic Process Research and Development, 4: 427-435 (2000); and Berge, S. M., et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66: 1-19, (1977).
The compounds of the present invention, or salts thereof, may be prepared by a variety of procedures known to one of ordinary skill in the art, some of which are illustrated in the schemes, preparations, and examples below. One of ordinary skill in the art recognizes that the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare compounds of the invention, or salts thereof. The products of each step in the schemes below can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art. Others may be made by standard techniques of organic and heterocyclic chemistry, which are analogous to the syntheses of known structurally similar compounds, and the procedures described herein which follow including any novel procedures. In addition, one of ordinary skill in the art appreciates that myriad SOCE inhibitors are commercially available and can therefore be readily obtained by one of skill in the art.
Renal disorders include, but are not limited to, acute kidney injury, chronic kidney disease and end stage renal disease. Acute kidney injury results from events such as renal ischemia, nephrotoxicity and/or sepsis. Renal disorders include renal inflammation, renal interstitial fibrosis, impaired renal function, proteinuria, and hypertension. In embodiments the renal disorder is renal inflammatory disorder and the disorder is ANCA associated vasculitis, crescentic glomerular nephritis, and nephrotic syndrome. In embodiments of the invention, the renal disorder is chronic allograft nephropathy in a kidney transplant recipient.
The terms “treating” or “to treat” include restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder. As used herein, the term “patient” refers to a human. The term “effective amount” refers to the amount or dose of compound of the invention, or a pharmaceutically acceptable salt thereof which, upon single or multiple dose administration to the patient, provides the desired effect in the patient under diagnosis or treatment.
An effective amount can be readily determined by one skilled in the art by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount for a patient, a number of factors are considered, including, but not limited to: the species of patient; its size, age, and general health; the specific disease or disorder involved; the degree of or involvement or the severity of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
The SOCE inhibitors are generally effective over a wide dosage range. For example, dosages per day normally fall within the range of about 0.5 μg/kg to about 30 mg/kg of body weight, preferably 0.5 mg/kg to about 10 mg/kg of body weight, more preferably 1.0 mg/kg to about 1.6 mg/kg of body weight. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed with acceptable side effects, and therefore the above dosage range is not intended to limit the scope of the invention in any way.
The term “inhibiting” refers to slowing, decreasing, delaying, preventing or abolishing.
The term “differentiation” refers to change from relatively generalized to specialized kinds during development.
Acute kidney injury (AKI) results from events such as renal ischemia, nephrotoxicity and/or sepsis. AKI increases the risk of death in the intensive care unit (ICU), and mortality rates in this setting range between 15-60%. Survival from AKI is dependent on recovery of renal function following injury, the success of which has been suggested to be dependent on the efficiency of adaptive repair processes. Progression of chronic kidney disease (CKD) and end-stage kidney disease is recognized as a possible outcome of AKI patients, and it has been suggested that incomplete or maladaptive repair may predispose progression of CKD following AKI.
Immune cell activity may contribute to renal injury or may enhance renal recovery. In the setting of renal ischemia reperfusion (I/R) injury, renal CD4+ T-helper 1 or 17 cells are thought to exacerbate renal injury while T-regulatory cells have been implicated in renal repair (6, 7). Following recovery from I/R injury in rats, subsequent exposure to high-salt diet was shown to hasten the development of interstitial fibrosis, inflammation, proteinuria and hypertension. These parameters of CKD progression were significantly attenuated by immunosuppression with mycophenolate, suggesting that lymphocyte activity also modulates the AKI-to-CKD transition.
Naïve CD4+ cells differentiate into effector T-helper cells in the ischemic milieu, where they are exposed to different antigens and pro-inflammatory cytokines. T-helper cells secrete various cytokines and are thought to orchestrate the adaptive immune response. It was demonstrated that T-helper 17 (Th17) cells, which secrete the cytokine IL17, are the prominent lymphocyte population found in rat kidney following I/R injury. These cells have been implicated in a variety of autoimmune diseases such as asthma, psoriasis, inflammatory bowel disease and lupus erythematosus. There is a significant expansion of Th17 cells in kidney within the first 3 days of I/R injury in rats, while Th17 levels resolve to near sham-operated control values within 7 days as renal function recovers. However, subsequent exposure of rats to high salt diet (4%) strongly reactivates Th17 cell expression in post-ischemic kidney. This re-activation may contribute to CKD, since an IL17R antagonist attenuated renal interstitial fibrosis and neutrophil infiltration in post I/R rats exposed to high salt diet.
The basis for activation of Th17 cells in response to renal injury and exposure to high-salt diet remains to be elucidated. Th17 cell differentiation is dependent on the activity of the transcription factor RORγT and inhibitors of this factor can alleviate the pathological activation of Th17 cells. Activation of these cells by high salt diet has also been demonstrated in a mouse model of autoimmune encephalitis and associated with the activity of serum and glucocorticoid regulated kinase (SGK-1) and nuclear factor of activated T-cells 5 (NFAT5). Elevation of extracellular Na+ to 170 mM enhanced differentiation from naïve CD4+ cells to Th17 cells in vitro in a process dependent SGK-1.
Using an in vitro stimulation assay, it was demonstrated that CD4+ T cells from post-ischemic kidney manifest enhanced expression of IL17 in response to Ang II and elevated extracellular Na+ in vitro, while no response was observed from sham-operated control derived CD4 cells. The mechanisms mediating the enhanced IL17 activation in CD4 cells post AKI are not known. Previous studies have also demonstrated that Orai1, the pore forming subunit of Ca2+release-activated Ca2+ channels (CRAC), is required for Th17 cell differentiation in vitro, due in part to NFAT activity. Interestingly, Orai1 mutant mice, or inhibitors of Orai1 show impaired T-cell receptor (TCR) activation, reduced IL17 production and are resistant to autoimmune disorders. Therefore, the hypothesis that renal I/R enhances lymphocyte Orai1 mediated Ca2+ signaling that drives Th17 cell expression and, in turn, modulates AKI and AKI-to-CKD progression was tested.
Previous studies demonstrated that Th17 cells are rapidly induced following renal I/R and that IL17 contributes to AKI. To investigate the potential that Orai1 participates in AKI, Orai1 expression was measured in Th17 cells from kidneys of rats 2 days following sham or I/R injury (Study I). Orai1 was detected in Th17 cells and the number of these cells was increased following I/R relative to sham (
Kidney Th17 levels return to sham-operated control values within ˜7 days of I/R. Despite the reduction of Th17 cells, Orai1 expression was maintained in CD4+ cells 7 days post I/R (
Kidney derived CD4+ cells were examined further for markers of effector memory T cells (CD44+/CD62L−) 7 days following I/R injury. There was a ˜4-fold increase in such cells from post I/R rats vs sham (1.85±0.01% vs 7.65±1.23%; p<0.05). Stimulation with Ang II and elevated Na+ did not affect the percentage CD44+ effector memory T cells suggesting this population is not responsive to stimulation that promotes IL17 expression (
To evaluate a potential role for Orai1 in the IL17 response, AKI-primed CD4+ cells were stimulated with Ang II and elevated Na+ in the presence or absence of different SOCE inhibitors. Both 2-ABP and YM58483/BPT2 completely blocked the increase of IL17 mRNA as well as the increase in IL17+ cells (
To evaluate Orai1 activity in AKI-primed CD4+ cells further, intracellular free Ca2+ responses were evaluated with the fluorescent indicator fura-2. Representative tracings of sham-operated and AKI-primed CD4+ cells are shown in (
To evaluate if SOCE influences Th17 cells in AKI, rats were fed YM58483/BPT2 approximately 2 hours prior to 40 min of I/R (Study II). YM58483/BPT2 significantly attenuated the level of renal injury 24 hours after reperfusion as indicated by the level of plasma creatinine (
Orai1 is present in immune cells, but may also be present in other cell types such as vascular cells. We sought to determine if the effect of YM58483/BPT on renal injury was primarily due to its effects on Th17 activation. To address this, rats were subjected to bilateral renal I/R and treated with either YM58483/BPT2, the IL17Rc receptor antagonist or both. YM58483/BPT2 treatment alone significantly reduced plasma creatinine as compared to vehicle controls in post ischemic rats, however IL-17Rc+YM58483/BPT2 treatment did not have any additional effect (Table 1). Levels of kidney CD4+ and CD4+/IL17+ cells were significantly reduced vs vehicle-treated rats by a similar degree in both YM58483/BPT and IL17Rc treated rats (Table 1). When rats were treated with a combination of YM58483/BPT and IL17Rc, the reduction in renal injury was similar to that observed with either YM58483/BPT or IL17Rc alone (Table 1). The lack of an additional effect of IL17Rc suggests that the primary effect of YM58483/BPT is due to inhibition of Th17 cells in the early post-ischemic period.
To explore further whether SOCE affects Th17 differentiation and renal injury, YM58483/BPT2 was tested in a different model of AKI associated with rhabdomyolysis. Intramuscular injection of glycerol into water deprived rats resulted in severe renal injury (Table 2). In this model, renal CD8+ cells including CD8+/IL17+ cells appeared to be the predominant lymphocyte population, as opposed to the more prevalent CD4+ lymphocyte response following I/R. Nevertheless, YM58483/BPT2 significantly attenuated the rise in serum creatinine as well as the total number of IL17+ expressing cells (Table 2).
Exposure of rats to high-salt diet at 5 weeks following recovery from I/R re-stimulates renal Th17 cell activity that is thought to contribute to CKD progression. The effect of SOCE on Th17 activation by high salt diet following AKI was evaluated in rats subjected to unilateral renal I/R followed by contralateral nephrectomy and transition to high salt diet (Study III). Post AKI rats treated with vehicle during high salt diet treatment manifested a significant infiltration of kidney CD4+ and CD8+ cells and IL-17 expressing cells. YM58483/BPT2 treatment significantly attenuated the increase in CD4+ and CD8+ cells and the increased expression of IL17+ cells (
It was previously demonstrated that increased circulating Th17 cells in rat blood following I/R, suggesting that blood may be used as source of activated lymphocytes in the setting of AKI. To investigate the potential that SOCE influences Th17 differentiation in human AKI, peripheral blood samples were obtained from critically ill patients with and without AKI. Samples were collected within 24-48 hours of AKI diagnosis for AKI cases or within 24-48 hours of ICU admission for frequency-matched (age, gender, baseline eGFR) controls without AKI (Table 3).
In isolated blood mononuclear cells, the percentages of total IL17+ cells and CD4+/IL17+ cells were significantly higher in AKI patients vs non-AKI patients (
Next, it was examined whether AKI enhanced IL-17 responses in circulating human CD4 cells and if these responses depend on SOCE activity. Human CD4+ cells responded in vitro to elevated extracellular sodium by increasing IL-17 expression in samples from AKI patients, but not in those of patients without AKI (
A novel regulatory pathway related to the activation of lymphocytes in the setting of acute and chronic kidney injury based on the role of intracellular calcium signaling and the differentiation of Th17 cells is proposed. It was demonstrated that the store-operated Ca2+ channel Orai1 is prominently induced in renal T-cells in the setting of kidney injury. Moreover, blockade of this channel attenuated Th17 cell induction and renal damage in response to ischemia/reperfusion injury as well as subsequent exposure to high salt diet. Thus, Orai1 may represent a therapeutic target to attenuate AKI or immune mediated renal fibrosis and hypertension, which may occur secondary to AKI.
Th17 cells were originally described as a distinct T-helper subset which secretes the cytokine IL-17 and is a major factor in autoimmune disorders. Th17 cells play an important role in host defense. However, in models of asthma, inflammatory bowel disease, psoriasis, or autoimmune encephalitis, Th17 cells aggravate inflammation by recruitment of other immune cells (such as neutrophils), which express the IL17RA receptor. Th17 cells have also received significant attention in the setting of renal inflammatory disorders including ANCA associated vasculitis, crescentic glomerular nephritis and nephrotic syndrome. Following renal transplant, there is an increased prevalence of Th17 cells in patients with chronic allograft nephropathy.
Th17 cells have recently been examined in the setting of AKI. Studies using Il17-null mice, suggest that Th17 cells contribute to the severity of renal injury in response to I/R or cisplatin. A biphasic Th17 response in rats was demonstrated, with an early transient phase of expression peaking between 1-3 days following injury and the second peak induced when rats are provided high-salt diet. Th17 cells were the predominant lymphocyte population activated by high salt diet while no significant effect was observed on Th1 or Th2 cells. The exposure to high salt diet exacerbates inflammation, fibrosis and hypertension and can be attenuated by mycophenolate or an IL17 antagonist. Therefore, the mechanisms mediating IL17 expression in response to I/R and high salt intake are of interest. An in vitro model using CD4+ cells from kidneys 7 days following I/R revealed that stimulation by Ang II and elevated extracellular sodium increased IL17 expression, while having no effect on Th1 or Th2 responses. This priming of CD4+ cells by AKI provided an opportunity to investigate altered lymphocyte signaling leading to Th17 differentiation in response to renal injury and was the basis of the current study.
Kleinewietfeld et al. investigated the potential mechanism by which Na+ may directly influence Th17 cell differentiation. These authors demonstrated that naïve T-cells, when cultured for 4 days with TGF-β and IL6, differentiate into Th17 cells and this response was potentiated elevating the concentration of Na+ in the culture media. In addition, the response was abrogated by inhibition of p38/MAPK, NFAT5 and SGK-1. It was suggested that the increase in Na+ concentration by up to an additional 40 mM might be observed in skin under high salt diet conditions and potentially influence T-cell function. Indeed, Th17 induction and severity of autoimmune encephalopathy was enhanced when mice were placed on a high salt diet.
TCR stimulation invokes an increase in intracellular Ca2+ via Ca2+-release activated Ca2+ channels (CRAC). The result of this activity is thought to be calcineurin mediated dephosphorylation of NFAT, which translocates to the nucleus and activates transcriptional programs. Genome wide RNAi screens helped to identify Orai1 as the pore forming subunit of CRAC channels. Activation of Orai1 is mediated by the activity of STIM1, an endoplasmic reticulum (ER) membrane spanning protein which senses Ca2+ depletion from the ER secondary to TCR stimulation. Interaction between STIM1 and Orai1 increases CRAC activity and results in sustained increases in intracellular Ca2+. Mutations in either ORAI1 or STIM1 result in a severe combined immunodeficiency (SCID) phenotype. For Th17 cell differentiation, TCR stimulation in combination with various other cytokines represents an important driver of differentiation, which is considered more inflammatory than Th1 or Th2 phenotypes. Interestingly, chemical library screening recently identified putative Orai1 inhibitors, which showed greater selectivity in abrogating Th17 differentiation vs Th1 or Th2 differentiation. Moreover, Orai1 inhibition reduced the nuclear accumulation of NFAT and RORγT, critical transcriptional regulators of Th17 differentiation.
Data from the current study provide compelling evidence for an essential role of Orai1 in the differentiation of Th17 cells following renal I/R. IL17+ expressing CD4+ cells are rapidly expanded following renal injury and, using FACS analysis, we show that Orai1 expression is also prominent in kidney CD4+ cells following renal injury. Measurements from circulating blood of critically ill patients with AKI also demonstrated a profound enhancement of Ora1+ IL17+ cells, indicating that this pathway is activated in human AKI.
Importantly, IL17 expression was almost exclusive to cells expressing Orai1, and was essentially absent in Oria1-negative cells. The data from the current study indicated that Orai1 is persistently expressed following the resolution of AKI (7 days post I/R). Given that the re-expression of IL17 following in vitro stimulation is inhibited by SOCE antagonist, the results suggest that Orai1 mediated SOCE channel is required for Th17 differentiation following I/R.
The potential ability to target IL17 has already shown promise in the setting of autoimmune disease and several therapeutic agents are in use to target this pathway in diseases such as psoriasis. IL17 could also represent a target for both acute and chronic kidney disease since both have been shown to be ameliorated by IL17 blockade or Il17 gene knockout strategies. Similarly, we confirmed that a SOCE antagonist showed a significant degree of protection using a standard model of AKI in rats induced by bilateral renal I/R and also extended this observation to a model of AKI secondary to rhabdomyolysis. The protection was associated with a clear reduction in the genesis of Th17 cells in response to injury. However, since Orai1 may be expressed in endothelial cells or smooth muscle cells, we cannot exclude the possibility that the effects observed may be independent of IL17 production. Nevertheless, in the current study, IL17Rc blockade provided no additional protection over YM58483/BPT2 alone suggesting that the primary activity of Orai1 in the early post I/R period is to promote Th17 activation. The transition from acute to chronic kidney disease has been the subject of significant research and maladaptive repair responses predispose CKD progression. Since immune suppression strongly attenuates the AKI to CKD transition, persistent inflammation in the kidney in response to AKI represents a potential maladaptive response. It is therefore noteworthy that Orai1 expression remains persistently elevated in CD4 cells despite the recovery of kidney function and the decline in IL17 expression. The degree and duration of sustained Orai1 expression following recovery from AKI remains unclear, but it worth noting that reactivation of Th17 cells by high salt diet was attenuated by SOCE inhibition between 35-63 days post injury. Whether Orai1 persists in activated T cells, memory T cells, or other leukocyte populations which secrete IL17 remains to be determined. An increase in effector memory T cells 7 days following I/R was demonstrated. However, this population was not affected by in vitro stimulation so it is not yet clear whether this population contributes directly to the IL17 response post injury. Nevertheless, we suggest that sustained Orai1 expression may represent the basis for susceptibility to re-activation of Th17 cells and therefore represents an important link predisposing to salt-sensitive CKD progression following AKI.
In addition to providing a potential link between AKI-to-CKD, there is also clear evidence that CKD predisposes to AKI. It could be suggested that sustained expression of Orai1 in CKD could enhance sensitivity to AKI, by promoting a greater inflammatory response to a given insult. It is also reasonable to suggest that increased Orai1 expression enhances the IL17 response to other inputs. For example, in the current study, IL17 expression was elevated in response to Ang II and elevated Na+ in vitro and was dependent on SOCE activity. Recent studies have shown that Ang II-dependent hypertension is, in part, dependent on IL17 activity; whether Orai1 modulates Ang II dependent Th17 responses in these models remains to be determined.
Taken together, the results from the current study suggest that Orai1 could be considered a novel pathway to target inflammatory renal disease associated with the Th17 phenotype. Novel inhibitors targeting this pathway are currently in development (38) and could represent therapies for inflammatory diseases associated with Th17 cells including autoimmune disease as well as AKI, CKD and salt-sensitive hypertension.
All studies used male Sprague-Dawley rats (250-300 g) that were purchased from Envigo (Indianapolis, Ind.). Rats were anesthetized with a cocktail of ketamine (100 mg·kg−1, KetoVed, Vedco Inc, St. Joseph, Mo.) and xylazine (50 mg·kg−1, AnaSed, Lloyd Inc, Shenandoah Iowa). Rats were placed on a heated surgical table to maintain body temperature. A midline incision and either unilateral (left) or bilateral renal ischemia (as indicated in Results) was induced by applying micro-aneurism clamps on the renal pedicles for a period of 40 min. Re-establishment of perfusion was verified by visual examination following removal of the clamps. Rats were provided post-operative analgesia using buprenorphine-SR (1 mg·kg−1).
Rats were allowed to recover for various periods of time as described in the Results. In Study I, lymphocytes were studied in vitro after recovery for either 2 or 7 days following I/R or sham surgery for evaluation of function in vitro or for FACS analysis. In Study II, the effect of SOCE on kidney injury was studied with the inhibitor YM58483/BPT2 (N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide; Tocris/Bio-Techne Minneapolis, Minn.). Rats were pretreated p.o. 2-3 hours prior to surgery with YM58483.BPT2 (1 mg·kg−1) in sugar-free chocolate pudding following dilution in 100% EtOH, at, a dose previously shown to affect T cell activation in rats. In some experiments, rats were also treated with the IL17Rc (150 ng/day i.p; R&D Systems). In addition, one experiment utilized a model of rhabdomyolysis, induced by injection of 50% glycerol (10 ml/kg) into the hindlimb muscle of rats following 16 hours of water restriction.
Study III was designed to investigate SOCE on progression of CKD induced by high-salt diet following recovery from I/R injury using a model of AKI-to-CKD described previously. Rats acclimated to a standard diet (AIN 76A, Dyets, Bethlehem, Pa.) containing 0.4% NaCl were subjected to left unilateral I/R or sham surgery and allowed to recover for ˜5 weeks. Rats were then subjected to right unilateral nephrectomy and subsequently exposed to elevated dietary Na+ (AIN76A plus 4% NaCl) for an additional 4 weeks (
A prospective case-control study was designed to examine peripheral blood from AKI patients (n=9) and matched-controls without AKI (n=8) admitted to the ICU at the University of Kentucky Hospital. AKI was defined by KDIGO criteria, using both serum creatinine (SCr) and urine output data. Only patients with AKI stage≥2 were included in the study as cases. Controls were frequency-matched by age (10-year intervals), gender and 2-category baseline estimated glomerular filtration rate (eGFR, calculated using CKD EPI equation, ≥90 and 60-89 ml/min/1.73 m2). Baseline SCr was defined as the most recent SCr within the 6-month period before ICU admission. Inclusion criteria included: adults≥18 years of age, admission to the ICU, and baseline eGFR≥60 ml/min/1.73 m2. Exclusion criteria consisted of prior kidney or any other solid organ transplant, end-stage kidney disease, evidence of AKI before ICU admission, or the presence of uroepithelial tumors.
Single-timepoint peripheral whole blood samples were obtained 24-48 hours after AKI diagnosis (cases) or ICU admission (controls). Standardized techniques for blood collection, transport and storage were employed.
To measure creatinine, tail blood was collected in heparin containing tubes and centrifuged to collect plasma. Plasma creatinine was measured using a Pointe Scientific Analyzer and Creatinine Assay reagents using methods outlined by the manufacturer (Pointe Scientific, Canton Mich.). Urine was collected for 24 hours by placing rats in metabolic cages and urine volume was determined gravimetrically. Urine creatinine was measured using a colorimetric assay adopted for microplate readers. Creatinine clearance was measured using Uc
At the time of tissue harvest, kidneys were bisected and one half was fixed by immersion in 10% formalin, embedded in paraffin and 5 μm sections stained with picrosirus red to assess fibrosis. For quantitative analysis, five random images of renal outer medulla were obtained using Leica DMLB (Scientific Instruments, Columbus, Ohio) microscope with a 20× objective. The percent area of picrosirus red stain was scored in a blinded fashion using Image J (NIH).
Total RNA was obtained from kidney using Trizol and the Zymogen RNA extraction kit and cDNA was prepared using MMLV enzyme (Invitrogen, Carlsburg, Calif.). Quantitative real time PCR (qPCR) using gene specific primers was performed using ABI 7500 (Applied Biosystems, Foster City, Calif.). mRNA values were calculated using 2−ΔΔCt. Specific primers sequences for IL-6 (catalog #Rn01410330_m1), Kim-1 (#Rn00597703_m1) and IL17 (Rn01757168_m1) were purchased from ThermoFisher (Waltham, Mass.).
Freshly harvested kidneys were minced and digested in liberase (2 μg/ml. Roche, Indianapolis Ind.) for 15 min at 37° C. using Gentle MACs (Miltenyli, San Diego, Calif.). The digested tissue was filtered through a 100-μm mesh and washed with RPMI containing 10% fetal bovine serum (Invitrogen). Mononuclear cells were isolated using Percol (Sigma) density gradient centrifugation.
All antibodies, their sources and concentrations used are listed in (Table 4a and 4b).
To evaluate protein expression of IL17 or Orai1, Orai2 or Orai3, cells were restimulated with PMA and ionomycin for 6 hours in the presence of monensin (Golgistop, 1 μg·ml, BD Biosciences) permeabilized with saponin (10%) and stained with relevant antibody. Cells were scanned using flow cytometry (FACSCalibur, BD Biosciences, San Jose, Calif.) and scans were analyzed using Flowjo software (Tree Star, Ashland, Oreg.). Lymphocyte gating strategy and a representative example of the gating strategy used in these studies is shown in
CD4+T cells were isolated using the MACS Pan-T cell microbead separation kit (Miltenyl, Glabach, Germany). T cells were stimulated with plate bound anti-CD3 (precoated with 2 μg/mL) and soluble anti-CD28 (1 μg/mL). Cells (2.5×105 in 0.25 ml) were incubated 12-14 hours at 37° C. in RPMI medium supplemented with 10% FBS (Invitrogen) in a 48-well plate. Cells were challenged with Ang II (Sigma, 10−7M) and raising the extracellular Na+ from 140 mM to 170 mM, using a 1 M NaCl solution. Calcium channel inhibitors 2ABP (10 μM, Sigma) AnCoA4 (10 μM, Millipore, Burlington, Mass.) and YM58483/BTP2 (10 μM) were included to evaluate effects on IL-17.
To assess Ca2+ responses, fura2 imaging was performed. Briefly, isolated CD4+ T cells were loaded with fura-2AM (2.5 μM, Sigma, St. Louis, Mo.) for 45 min, washed, placed on poly-lysine (Sigma) coated coverslips and placed in a superfusion chamber with physiological salt solution (PSS) containing 2 mM Ca2+. The chamber was mounted on an inverted epifluorescence microscope and signal measured with alternating excitation at 340 and 380 nm and emission at 510 nm using the InCa2+-imaging system (Intracellular Imaging Systems, Cincinnati, OH). Data were acquired at 1.5 Hz and representative tracings were smoothed to the 10 nearest neighbor points using GraphPad Prism. For analysis, an increase in signal intensity of >1 standard deviation from baseline was considered a positive response. Frequency of responding cells was determined from an average 53±19 cells for each animal. Cells that did not respond to the Ca2+ ionophore ionomycin (1 μM) at the conclusion of the study were excluded from analysis.
Fresh blood cells from patients were washed with PBS twice and stimulated with PMA, Iono and monensin for 4-6 hours. Cells were stained for antibodies against IL-17, CD4 and Orai-1 (Table 5).
The samples were initially blinded and diagnosis (AKI or non AKI) revealed after measurements of all samples were completed. For in vitro stimulation of human blood cells, primary T cells were isolated from fresh blood using Straight whole blood CD4 kit from Miltneyli Biotech (Miltenyli, San Diego, Calif.) according to manufactures' protocol. CD4+T cells were plated at a density of 1×106 cells/mL in RPMI medium supplemented with FBS. Cells were stimulated with human anti-CD3/CD28 dynabeads (Gibco; Catalogue no 11161D) along with labeled treatment overnight (˜12 hours). T cells were harvested and incubated with monensin for 6 hours, prior to staining for IL-17 (Table 4).
Lymphocytes were studied in vitro after recovery for either 2 or 7 days following I/R or sham surgery for evaluation of function in vitro or for FACS analysis. To investigate a potential role that Orai1 participates in AKI, Orai1 expression was measured in Th17 cells from kidneys of rats 2 days following sham or I/R injury. Orai1 was detected in Th17 cells and the number of these cells were increased following I/R relative to sham (
To evaluate a potential role for Orai1 in the IL17 response, AKI-primed CD4+ cells were stimulated with Ang II and elevated Na+ in the presence or absence of different SOCE inhibitors. Both 2-ABP and YM58483/BPT2 completely blocked the increase IL17 mRNA (
To evaluate Orai1 activity in AKI-primed CD4+ cells further, Ca2+ responses were evaluated after loading cells with Fura-2. Representative tracings of sham-operated and AKI-primed CD4+ cells are shown in (
The effect of SOCE on early ischemic kidney injury was studied with the inhibitor YM58483/BPT2 (N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide; Tocris/Bio-Techne Minneapolis, Minn.). Rats were pretreated 2-3 hours prior to surgery, p.o. in sugar-free chocolate pudding following dilution in 100% EtOH, at 1 mg·kg-1, a dose previously shown affect T cell activation in rats. To evaluate if SOCE influences Th17 cells in AKI, rats were fed YM58483/BPT2 approximately 2 hours prior to 40 of ischemia and reperfusion. YM58483/BPT2 significantly attenuated the level of renal injury 24 hours after reperfusion as indicated by the level of plasma creatinine (
As shown in
Study III was designed to investigate SOCE on progression of CKD induced by high-salt diet following recovery from I/R injury using a model of AKI-to-CKD described previously. Rats acclimated to a standard diet (AIN 76A, Dyets, Bethlehem, Pa.) containing 0.4% NaCl were subjected left unilateral I/R or sham surgery and allowed to recover for ˜5 weeks. Rats were then subjected to right unilateral nephrectomy and subsequently exposed to elevated dietary Na+ (AIN76A plus 4% NaCl) for an additional 4 weeks (
In post-AKI rats, exposure to high-salt diet at 5 weeks following recovery from I/R re-stimulates renal Th17 cell activity that is thought to contribute to CKD progression. The effect of SOCE on Th17 activation by high salt diet following AKI was evaluated in rats subjected to unilateral renal I/R followed by contralateral nephrectomy and transition to high salt diet. Post AKI rats treated with vehicle during high salt diet treatment manifested a significant infiltration of kidney CD4+ cells (
All data are expressed as means±SE or SD or median [IQ1-IQ3]. For experimental data, differences in means were established by 1-tailed Student's t-test or one-way ANOVA with Tukey's multiple comparison test as indicated in the figure legends. For clinical data, comparisons were done using Fisher's exact test for categorical variables and Mann-Whitney U test for continuous variables. Analysis was done with the aid of Graph Pad Prism software (La Jolla, Calif.) or SAS 9.4 (SAS institute, Cary, N.C.). P<0.05 was considered significant.
Rats were maintained in accordance with the policies of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All studies were approved by Institutional Animal Care and Use Committees at Indiana University School of Medicine, Indianapolis, Ind. For human studies, the protocol was approved by the Institutional Review Board of the University of Kentucky, Lexington, Ky. Informed consent was obtained for all study participants.
Various modifications and additions can be made to the embodiments disclosed herein without departing from the scope of the disclosure. For example, while the embodiments described above refer to particular features, the scope of this disclosure also includes embodiments having different combinations of features and embodiments that do not include all of the described features. Thus, the scope of the present disclosure is intended to embrace all such alternatives, modifications, and variations as fall within the scope of the claims, together with all equivalents.
All publications, patents and patent applications referenced herein are hereby incorporated by reference in their entirety for all purposes as if each such publication, patent or patent application had been individually indicated to be incorporated by reference.
This application is the National Stage of International Application No. PCT/US2019/054767, filed Oct. 4, 2019, which claims the benefit of U.S. Provisional Patent Application No. 62/741,302, filed Oct. 4, 2018, and U.S. Provisional Patent Application No. 62/876,186, filed Jul. 19, 2019 the entire disclosures of which are hereby incorporated by reference.
This invention was made with government support under DK063114 awarded by National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/054767 | 10/4/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62876186 | Jul 2019 | US | |
| 62741302 | Oct 2018 | US |
