Renal Implications of Kappa Opioid Receptor Signaling in Sprague-Dawley Rats

Abstract

Opioid use for pain management and illicit consumption has been associated with adverse cardiovascular and cardiorenal outcomes. Despite these associations, the mechanisms underlying opioid-induced kidney damage remain poorly understood. Recently, we demonstrated that stimulation of kappa opioid receptors (KOR) is implicated in the aggravation of salt-sensitive hypertension, glomerular injury, and podocyte damage through excessive podocyte calcium influx. This study aims to elucidate the KOR signaling and renal outcomes underlying opioid use in Sprague-Dawley (SD) rats. Here, we employed freshly isolated glomeruli from SD male rats and immortalized human podocyte cell cultures to investigate the role of KORs in podocyte calcium regulation and overall glomerular function. A glomerular permeability assay was used to evaluate the impact of KORs on glomerular filter integrity. Additionally, the long-term effects of KOR activation were assessed in vivo by chronic intravenous infusion of selective KOR agonist BRL 52537 in SD rats. We found that acute application of BRL 52537 resulted in increased plasma membrane ion channel activity in immortalized human podocytes. Significant calcium influx in response to BRL 52537 was detected in podocytes of the isolated SD rat glomeruli. Further, glomerular permeability analysis revealed increased permeability and impaired filter integrity, indicating altered glomerular function. Lastly, prolonged KOR activation in SD rats results in an increase in blood pressure, an elevation of basal calcium levels in podocytes, and albuminuria. In conclusion, this study identifies novel renal physiological mechanisms through which opioid-induced KOR activation contributes to podocyte injury and glomerular damage in SD rats.

Graphical Abstract

Graphical Abstract.

Introduction

The United States opioid crisis, consisting of the illicit use and improper prescribing of opioids, is responsible for approximately 130 deaths per day and produces an annual economic burden of half a trillion dollars.¹ Prescription opioid mismanagement serves as an initial pathway for subsequent illicit use, where individuals will transition from using prescribed opioids to abusing higher-potency substances such as fentanyl and heroin. Opioids are not recommended for chronic noncancer pain management. However, opioids are commonly prescribed analgesics for patients with chronic kidney disease (CKD).² The extensive misuse of opioid-based pain management correlates with poor cardiorenal outcomes, including increased albuminuria and reduced glomerular filtration rate (GFR).³ These observations link opioid use to progressive renal damage.⁴ Moreover, it is noteworthy that 50% of the opioid-prescribed patients develop CKD.⁵ To date, limited studies have been conducted to elucidate the mechanism by which opioids can induce kidney damage. Recently, our group has demonstrated that kappa opioid receptor (KOR) stimulation aggravates the progression of salt-sensitive hypertension and induces podocyte damage and glomeruli injury in Dahl salt-sensitive rats. Understanding the mechanisms by which opioids induce albuminuria and kidney damage in healthy rats could generate insight into the molecular events underlying several cardiorenal complications due to opioid use.

The overarching goal of this work is to determine the acute and long-term effects of KOR stimulation on podocytes, particularly the effects on glomerular filtration barrier (GFB) integrity and calcium homeostasis in podocytes from a human cell line or healthy Sprague Dawley (SD) rat glomeruli. To accomplish this, we performed a comparative evaluation of the acute and prolonged stimulation of KORs. We investigated several aspects of KOR signaling in the pathogenesis of albuminuria, kidney damage, and blood pressure. We tested the effect of KOR stimulation on glomerular albumin permeability and further explored the direct impact of opioids on kidney filter integrity. We found that long-term administration of BRL 52537 results in blood pressure elevation and declined renal function. This work expands upon the notion that stimulation of KORs in the kidney contributes to podocyte damage during short-term and chronic opioid exposure, which challenges the current norms regarding the safety of opioid use in CKD patients.

Material and Methods

Experimental Protocol and Animals

The use of animals and all welfare ethical procedures adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals following protocols reviewed and approved by the Medical College of Wisconsin/University of South Florida IACUC. Eight-week-old male SD rats were purchased from Charles River Laboratories. Rats were provided food (LabDiet Rodent 5001) and water ad libitum. For surgical procedures, rats were anesthetized with inhalation of 2.5% isoflurane in 0.5 L/min O₂ flow. Catheters (RPT080 Braintree, MA, USA) were implanted in the femoral artery and vein, tunneled subcutaneously, and exteriorized at the back of the neck in a lightweight tethering spring. Following recovery from anesthesia, rats were placed into individual cages. The catheters were connected to a pressure transducer (041516504A, Argon Medical Devices, TX, USA) via swivels (375/D/22, Instech, PA, USA) for arterial blood pressure acquisition and daily intravenous bolus drug infusion. This approach allows the collection of blood samples in conscious animals within the study. We allowed rats to recover for at least 7 days following surgery. BRL 52537 was used at a dose of 1.5 mg/kg for 2 weeks as determined by our previous studies. Rats were treated with the drug or a corresponding vehicle for the entirety of the experiment. Mean arterial pressure (MAP) was calculated as the average pressure value following 3 h of drug infusion.

Cell Culture and Electrophysiology

An immortalized human podocyte cell line was provided by Dr. Moin Saleem (University of Bristol, Bristol, UK). Cells were cultured as described previously.⁶ Cells were grown at 33°C with 5% CO₂ in an RPMI-1640 medium (11875093) supplemented with Insulin-Transferrin-Selenium (41400045) and 10% fetal bovine serum (10437028, Thermo Fischer Inc.). Human podocytes between passages 4 and 15 were used. To induce differentiation, cells were switched to 37°C with 5% CO₂ for 10–14 days. Cells were then incubated in serum-free media for 3–5 h before experimental use. For electrophysiology studies, cover glasses with attached podocytes were placed into a perfusion chamber and mounted on an inverted Nikon Eclipse Ti-2 microscope. After a high resistance seal was obtained (>1 giga-ohm), cell-attached recording was performed immediately in the solutions as previously described.⁷ The activity of the channels was first monitored in response to the voltage steps of 20 mV in the range of −80 mV to +60 mV to estimate the channel’s conductance and I–V relationship. Following this, the voltage was clamped at −Vp = −60 mV, and the channels’ activity was recorded for several minutes before the application of BRL 52537.

Kidney Isolation and Glomerular Analysis

At the conclusion of the study, kidney tissue was harvested and weighed. Rats were anesthetized, and kidneys were flushed with phosphate-buffered saline via aortic catheterization (3 mL/min/kidney until blanched). For each rat, one kidney was used for glomeruli isolation, and cortex sections from the other kidney were used for Western blotting. Rat kidneys were formalin-fixed, paraffin-embedded, sectioned, and mounted on slides. Slides were stained with Masson’s trichrome stain as previously described.⁸ A glomerular injury score was assigned using a 0−4 scale, where 0 = no damage. A cumulative distribution plot was generated (OriginPro 9.0) using obtained glomerular injury scores, and the probability for a corresponding score interval was calculated. Medullary protein cast analysis was performed using a color thresholding method involving Metamorph software (Molecular Devices, Sunnyvale, CA, USA). Cortical fibrosis was assessed using color deconvolution in the Fiji image application (ImageJ 1.51 u, NIH).

Western Blotting

Changes in protein expression in renal cortex homogenates were assessed using primary antibodies (Abcam, anti-KOR (OPRK1) 1:1000, AOR-012, Alomone, OPRK1 blocking peptide, 1:500, BLP-OR012, Nephrin G-8, Santa Cruz Biotechnology, 1:1000, cat# sc-376522). The membranes were blocked with 2% BSA in Tris-buffered saline (TBS) and 0.01% Tween 20 overnight at room temperature and then incubated with primary antibody overnight at room temperature. The secondary antibody (1:10 000) was diluted in 2% BSA in TBS and 0.01% Tween 20, and membranes were incubated at room temperature for 1 h. Protein loading was assessed by immunoblotting using rabbit anti-actin antibodies (1:1000, Cell Signaling Technology, MA, USA).

Calcium Imaging and Permeability to Albumin

The laser scanning confocal microscope system Nikon A1-R was used to detect changes in cytosolic calcium. Samples were imaged using 20× and 60× objective lenses: Plan Apo 20x/NA 0.75 and 60x/NA 1.4 Oil. Open-source software ImageJ was used for analysis as described previously.⁹ For both cultured and isolated glomeruli and podocytes, changes in calcium concentration were estimated by ratiometric principle following fluorophores: Fluo4 (ex. 488 em. 520/20 nm; 20 190 588, Invitrogen, OR, USA) and Fura Red (ex. 488 em. >600 nm; 21 046, AAT Bioquest, Inc, or Fura 2-TH, 51 419, Setareh Biotech, OR, USA). Glomeruli were mounted in poly-l-lysine-covered Mattek dishes and washed for ∼10 min with a bath solution containing the following (in mm): 145 NaCl, 4.5 KCl, 2 CaCl₂, 2 MgCl₂, and 10 HEPES pH 7.35 (adjusted with NaOH). After the fluorescence signal stabilized, podocytes were identified based on anatomic considerations, and fluorescence intensity ratios (Fluo4/FuraRed) were recorded as described previously.^10,11 Opioid receptors were stimulated with specific agonist BRL 52537 (Cat. No. 0699; Tocris Bioscience, MO, USA). Basal cytosolic calcium concentrations were measured as previously described.¹²

For the evaluation of glomerular filter integrity, we used a modified oncotic gradient change assay combining fluorescent dilution and glomerular volume restriction techniques.^13,14 We employed the fluorescence dilution technique for the measurement of the albumin reflection coefficient in isolated glomeruli as described previously.¹⁵ Briefly, rats were infused with FITC-dextran (150 kDa dissolved in 0.9% NaCL, 50 mg/mL, TdB Consultancy AB, Uppsala, Sweden) via the renal vein. Following, rat kidneys were harvested, and glomeruli were isolated by differential sieving and were stored in a 6% BSA/RPMI solution. For glomerular volume restriction BSA/RPMI solution was colored with 150 kDa TRITC labeled dextran. Glomeruli were attached to poly-l-lysine-covered glass coverslips for further confocal microscopy imaging. Fluorescence intensities detected with the FITC and TRITC filters were monitored by confocal laser scanning microscope system Nikon A1-R, z-stack of 26 consecutive focal planes (73.83 µm) was collected every 2 min, allowing for reconstructing of fluorescence within glomerular capillaries (FITC), and glomerular volume (TRITC). Changes in oncotic pressure induced by switching the surrounding medium from 6% to 1% BSA were monitored by 3D imaging throughout the experiment. For glomeruli volume reconstitution, TRITC signal was inverted, and a sum of z-stack slices was processed by the Analyze Particles module (ImageJ). Total glomeruli FITC fluorescence within a z-stack was calculated as a sum of slices’ intensities (ImageJ). Fiji image processing package (ImageJ 1.47v, NIH) was used for image processing and calculations.

Electrolyte Measurements and Albuminuria Assay

Urine samples were collected for 24 h to determine electrolytes, albumin, and creatinine levels. Bloodurine electrolytes, and creatinine were measured with a blood gas and electrolyte analyzer (ABL system 800 Flex, Radiometer, Copenhagen, Denmark). Kidney function was determined by measuring albuminuria using a fluorescent assay (Albumin Blue 580 dye, Molecular Probes, Eugene, OR, USA) read by a fluorescent plate reader (FL600, Bio-Tek, Winooski, VT, USA).

Quantification of the RAAS

Analysis of ANG I, ANG II, ANG-(1–7), ANG-(1–5), ANG-(2–8), ANG-(3–8), ANG-(2–10), ANG-(2–7), ANG-(1–9), ANG-(3–7), and aldosterone were carried out according to quantifications of steady-state levels in equilibrated heparin plasma samples by Attoquant Diagnostics (Vienna, Austria) according to the company’s protocol. Briefly, angiotensin peptide levels and aldosterone were measured in conditioned lithium-heparin plasma at 37°C. Stable isotope-labeled internal standards for each metabolite were added to stabilized plasma samples at a concentration of 200 pg/mL and subjected to liquid chromatography-tandem mass spectrometry based angiotensin quantification by Attoquant Diagnostics.⁸

Assessment of Podocin in Kidney Tissue

Kidneys were collected at the conclusion of the experiment and embedded in paraffin. Embedded kidneys were prepared and stained on the Leica Bond Rx platform using the NPHS2 Polyclonal antibody (Proteintech cat# 20384-1-AP) and counterstained with Hematoxylin. Subsequently, the stained slides were digitized using the Hamamatsu NanoZoomer slide scanner. Sections were then analyzed in Qpath version 0.5.1. 125 glomeruli were selected in one kidney per animal. The 125 glomeruli were merged and the average percent of podocin per all glomeruli was calculated and recorded. Single dots represent 125 glomeruli per kidney per animal.

Assessment of Endothelin-1 (ET-1) in Kidney Tissue

Kidney tissue at the conclusion of the experiment was prepared as follows: RIPA Buffer (ThermoScientific; J63306.AK) was mixed with 1X Protease and Phosphatase Inhibitor (ThermoScientific; 1861280). Ten milligram of tissue was mechanically digested in 400 µL of the buffer utilizing a bead homogenizer and then centrifuged. The supernatant was then collected. A BCA assay was performed and normalized tissue concentrations for the enzyme-linked immunosorbent assay (ELISA) were 1 mg in 100 µL of RIPA plus inhibitors buffer. ET-1 levels were measured at the end of experiment using the kidney samples in the ELISA (R&D Systems Inc., Minneapolis, MN, USA, cat # QET00B).

Statistics

Data are presented as means ± SEM. Statistical analysis consisted of one-way analysis of variance (ANOVA) or unpaired student t-test (GraphPad Prism), with P < 0.05 considered significant. In addition, when the ANOVA test was significant (P < 0.05), Holm-Sidak multiple-comparisons post-hoc adjustment was applied.

Results

Stimulation of KOR Has Acute Effects on Ionotropic Channel Activity in Human Podocytes

The expression of KORs (OPRK1) in kidney tissue has been previously demonstrated. Data from the Epigenomic Consortium indicate that the kidney cortex expresses OPRK1, with levels intermediate between tissues with high expression (eg, heart) and low expression (eg, adrenal gland and renal pelvis).¹⁶ Additionally, OPRK1 expression in human podocytes has been confirmed by other investigators, specifically in an immortalized human podocyte cell line and stem-cell-derived podocyte culture.^17,18 (Supplementary Figure 1). Therefore, our first experiments tested the effect of KOR stimulation on single-channel activity in immortalized podocyte cultures.

During cell-attached patch clamp experiments, the acute application of 100 µm BRL 52537 to the bath solution induced prolonged activation of single-channel activity of endogenous channels (Figure 1A). Collectively, these experiments demonstrate that KOR stimulation significantly increases the open probability (P_o) and overall ion channel activity (NP_o) (Figure 1B).

Figure 1.

Effect of BRL 52537 on plasma membrane ion channels in human immortalized podocytes. (A) Representative current traces of plasma membrane ion channel from a cell-attached patch of a human immortalized podocyte before and after application of 100 µm of BRL 52537. Expanded fragments are shown below. The patch was clamped to −V_p = −60 mV, and “c” shows a closed state of channels. (B) Summary graphs demonstrating the effect of 100 µm BRL 52537 on the open probability (P_o) and channel activity (NP_o).

KORs Mediate Calcium Influx in Podocytes of Freshly Isolated SD Rat Glomeruli

To analyze opioid-induced calcium influx in healthy podocytes, freshly isolated glomeruli from SD rats were loaded with calcium fluorescent indicators (Fluo4/FuraRed). The acute application of BRL 52537 resulted in a significant calcium influx response (Figure 2). The sustained calcium influx observed over time leads to significant dysregulation of cytoplasmic calcium levels. As previously reported by us and others, prolonged calcium influx in podocytes is known to trigger apoptotic signaling pathways or may result in podocyte detachment, ultimately contributing to glomerular damage.^12,19

Figure 2.

KOR activation causes deleterious changes in podocyte calcium levels in SD rats. A. Ratiometric calcium response to application of a specific KOR agonist BRL 52537 (100 µm) in podocytes from freshly isolated glomeruli from SD rats (N = 3 biological replicates, 8 technical replicates, 71 cells). (B) Calcium release ([Ca²⁺]) during the baseline conditions and after stimulation with BRL 52537, p = 0.0039, paired t-test. (C) Representative image of a glomerulus loaded with ratiometric calcium dyes (Fluo4/Fura Red; merged) before and after the application of BRL 52537. Scale bar is 50 µm. Arrowheads denote the regions of interest in which the fluorescence signal intensity was recorded.

KOR Activation Impacts Glomerular Filter Integrity

Increased albuminuria is a consequence of GFB impairment, which is further aggravated by tubular impairment or a combination of both.^20,21  Figure 3A illustrates the schematic method for simultaneous imaging of the glomerular volume (TRITC inverted channel) and fluorescence of green dextran in glomerular capillary loops (FITC).^13,14 In the healthy, intact glomerulus, the negative changes in oncotic pressure from 6% to 1% of BSA promote increased glomerular volume and fluorescent dilution of green dextran in capillaries due to water entrance to the glomerulus. Correspondingly, the lack of glomerular barrier integrity results in the absence of changes or the “flat tire effect” (Figure 3B). We pretreated freshly isolated glomeruli with BRL 52537 and performed the described assay to detect the integrity of the filter. As seen in Figure 3C, an increase in glomerular volume was seen in our vehicle-treated group due to an imposed oncotic gradient. In contrast, the BRL 52537-treated group exhibited a slight decrease in volume, suggesting that KOR activation exerts a complex effect, potentially targeting both podocytes and the mesangium, ultimately contributing to the loss of glomerular function. Fluorescent dilution of the FITC capillary dye indicated a similar pattern showing the impaired response to an oncotic gradient in the BRL 52537-treated group (Figure 3D).

Figure 3.

Changes in glomerular volume and permeability following BRL52537 application. (A) General steps of the glomerular permeability assay: loading glomeruli with 150 kDa FITC-dextran through the injection of the drug into circulation via renal vein; glomeruli isolation, incubation and addition of 150 kDa TRITC-dextran into the solution containing FITC-labeled glomeruli; fast xyzt confocal fluorescence microscopy scanning during the changes in oncotic gradient (BSA 6%–1%). (B) An example of glomerular volume changes in the control and BRL 52537-treated group. Changes in glomerular volume are denoted by blue lines (6% BSA) or red lines (1% BSA). Scale bar is 50 µm. (C) A summary of glomerular volumes (which reversibly correlates with the permeability of the GFB) in control and BRL 52537-treated groups. P values mentioned describe the statistical difference from time point 0 and time point 16 for either vehicle or BRL 52537 group. P < 0.05. (D) Relative fluorescence intensity in the vehicle and BRL 52537-treated group. Note a decrease in fluorescence intensity due to dilution of the dye in a vehicle group (normal albumin permeability) and minimal changes during stimulation of KORs (impaired albumin permeability). P values mentioned describe the statistical difference from time point 0 and time point 16 for either vehicle or BRL 52537 group. N ≥ 14 glomeruli (technical replicates) or N = 4 biological replicates in vehicle-treated group; N ≥ 13 gloms (technical replicates) or N = 4 biological replicates in BRL 52537-treated group. P < 0.05.

Chronic Administration of KOR Agonist Promotes an Elevation in Blood Pressure, Kidney Damage, and Augmented Basal Calcium Levels in SD Rats

The use of prescription opioid drugs is associated with altered blood pressure.^22–24 To test the contribution of prolonged stimulation of KORs on kidney damage and blood pressure, animals were administered 1.5 mg/kg/day intravenous bolus injections of BRL 52537 or vehicle for 2 weeks. As shown in Figure 4A, chronic treatment with KOR agonist leads to an initial increase in MAP, followed by a sustained higher overall MAP compared to vehicle throughout the study. Also, we observed GFB impairment in SD rats, reflected by increased albuminuria (Figure 4B). After 2 weeks of exposure, we observed a gradual increase in albuminuria and sustained elevated blood pressure which indicates ongoing pathological remodeling. However, due to the early stages of these pathological changes, no significant alterations were detected in medullary protein casts or cortical fibrosis (Figures 4C, D). In the cortex, KOR expression was higher in our BRL 52537-treated group (Figures 4E, F).

Figure 4.

Effect of chronic treatment with BRL 52537 on blood pressure and kidney damage in SD rats. (A) Changes in the development of MAP in SD rats chronically treated with KOR agonist or vehicle. Asterisks represent same-day comparisons between vehicle and BRL 52537 groups. (N ≥ 4 rat per group; ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001). (B) Urinary albumin (Alb/Cre, 24 h collection) (ACR) changes for the experiments described in A (P < 0.05). (C and D) Summary graphs of the medullary protein cast area (percent total kidney area) and cortex fibrosis. (E) Western blot of KOR expression (OPRK1) in the kidney cortex. (F) Summary graphs reflecting KOR expression in kidney cortex normalized to β-actin loading control. For statistical analysis, the normality (Shapiro–Wilk) test was performed followed by two-way ANOVA to determine significant effects in MAP and ACR from the BRL 52537 treatment, P < 0.05.

Cumulative probability distribution analysis of the glomerular injury score revealed a rightward shift toward a high glomerular injury score in the group treated with BRL 52537 (Figure 5A). Confirmatory histological kidney tissue images are shown in Figure 5B comparing vehicle glomerular score of 1 vs. BRL 52537 treated glomerular score of 4. Additional histological glomerular scoring images (scores 3 and 4) are provided in Supplementary Figure 2. Cortical nephrin and podocin measurements at the conclusion of the study revealed no significant difference in either nephrin or podocin levels in BRL 52537-treated group compared to controls (Supplementary Figure 3). Notably, BRL 52537-treated rats had a significant increase in podocyte basal calcium concentration (Figure 6). Chronic elevation of intracellular basal calcium levels are strongly associated with progressive glomerular disease and decline in renal functions.^25,26 Taken together, these data support the hypothesis that chronic stimulation of KORs leads to GFB impairment, increased glomerular damage, and blood pressure elevation in SD rats.

Figure 5.

Glomerular damage after chronic opioid exposure. (A) Glomerular injury score (0–4, where 0 = no damage) assessed by semiquantitative morphometric analysis for vehicle-treated and BRL 52537-treated rats (control, N = 5, BRL 52537, N = 7). Numbers of glomeruli per score are shown on the y-axes. (B) Representative images of kidney tissue stained with Masson’s trichrome (×40 magnification). Scale bar is 50 µm.

Figure 6.

Basal intracellular calcium is higher in BRL 52537 treated rats A. Representative calcium imaging traces showing a typical experiment designed to assess the intracellular calcium ([Ca²⁺]_i) level in the podocytes. To measure [Ca²⁺]_i, glomeruli were loaded with Fluo4, AM, and fluorescence intensity was recorded in the baseline and after the addition of ionomycin and MnCl₂. The graph demonstrates the fluorescence signal changes in response to ionomycin (producing the maximum of the Fluo4, AM fluorescence, F_max) and MnCl₂, which quenches the dye and results in the lowest fluorescence intensity (F_min). The intensity of fluorescence (left axis) for each time point was converted into the actual calcium concentration in nanomoles (right axis) according to the formula shown on the graph. The transients shown on the graph reflect the fluorescence intensity of representative ROIs selected from a BRL 52537 or vehicle; images were taken every 4 s. B. Bar graph summarizing the concentration of basal [Ca²⁺]_i in the podocytes of freshly isolated glomeruli of rats treated either with BRL 52537 or a corresponding vehicle for 14 days. N = 9 technical replicates (N = 4 biological replicates) in vehicle-treated group, N = 15 (N = 4 biological replicates) in BRL 52537-treated group. P < 0.05.

KOR Agonist Impact on RAAS, Cortical Renal ET-1, and Electrolyte Homeostasis in SD Rats

We further performed an analysis of blood and urine electrolytes and renin-angiotensin-aldosterone system (RAAS) changes to assess effects of KOR agonist on this system. Furthermore, recent studies revealed that ETA receptor antagonist BQ123 potentiates morphine analgesia and restores antinociceptive response in morphine-tolerant mice.²⁷ Given these findings, we assessed cortical renal ET-1 changes during 2 weeks of chronic BRL 52537 administration in SD rats. Our data indicates the significant increase of plasma sodium concentration by days 7 and 14 in our BRL 52537-treated group (Table 1). Rats, treated with the opioid receptor agonist demonstrated enhanced diuresis starting from the first day of intervention (Table 1). To further investigate the mechanism of opioid-induced blood pressure elevation, we performed a detailed analysis of equilibrium RAAS components and local renal cortex ET-1 levels (Supplementary Figures 4 and 5). Equilibrium levels of RAAS hormones, including Ang I, Ang II, aldosterone, and other RAAS components were simultaneously quantified in plasma samples. As shown in Supplementary Figure 4, no changes were found between the groups. Similarly, no changes in ET-1 levels between groups were observed (Supplementary Figure 5).

Table 1.

Summary table depicting the effect of treatment with BRL 52537 on plasma electrolyte homeostasis and diuresis in SD rats

Parameter		Day 0	Day 1	Day 7	Day 14
Plasma K+ mm	Control	3.99 ± 0.16	4.06 ± 0.08	4.10 ± 0.07	4.21 ± 0.16
	BRL 52 537	3.85 ± 0.08	3.65 ± 0.16	3.50 ± 0.11	3.71 ± 0.14
Plasma Na+ mM	Control	142 ± 0.42	143 ± 0.47	142 ± 0.44	142 ± 1.2
	BRL 52 537	142 ± 0.58	144 ± 1.1	149 ± 0.82 (P < 0.001)	150 ± 1.5 (P = 0.005)
K+/Cre ratio	Control	51.0 ± 2.6	50.9 ± 2.2	48.8 ± 1.6	45.4 ± 2.4
	BRL 52 537	45.2 ± 3.2	50.3 ± 3.1	54.6 ± 4.5	48.3 ± 2.6
Na+/Cre ratio	Control	33 ± 5.2	32 ± 2.0	33 ± 1.1	30 ± 1.8
	BRL 52 537	33 ± 2.0	33 ± 3.1	37 ± 3.6	29 ± 1.8
Diuresis, mL/day	Control	20 ± 1.7	17 ± 2.2	21 ± 2.8	19 ± 1.6
	BRL 52 537	21 ± 2.9	35 ± 3.1 (P < 0.001)	34 ± 3.6 (P = 0.04)	38 ± 4.5 (P = 0.007)

For all data sets, urine samples were collected for 24 h. Data are displayed as means ± SEM. Significant differences occurred between BRL 52537 treatment and controls in plasma potassium levels day 7, plasma sodium levels on days 7 and 14, diuresis rates on days 1, 7, and 14.

Discussion

The modern-day opioid crisis is considered one of the leading public health crises in the United States. Driven by increased production and decreased cost of illicit opioids, coupled with exponential increases in opioid prescriptions, the crisis is responsible for approximately 130 deaths per day.^28,29 Scientists have shown a correlation between opioid use, increased albuminuria, prevalence of CKD, and decreased GFR.³⁰ Opioid use is now associated with increased cardiorenal complications; however, the precise mechanism remains unclear. Our data suggest that the observed changes in MAP are most likely mediated by effects on the kidney. In particular, structural damage to the GFB and the subsequent induction of albuminuria brought our attention to one of the major cell types of the GFB, the podocyte.³¹ Consistent with our findings, previous studies revealed that morphine-receiving mice exhibit significant albuminuria along with podocyte foot effacement.³² Morphine has also been reported to increase oxidative stress in podocytes, which was been proposed as a contributing factor to the downregulation of slit diaphragm-associated proteins.³² Although our study centers on podocytes, it is important to recognize that other cell types, such as mesangial cells, express KORs and are essential for GFB maintenance.^33,34 These cell types may represent important targets for future studies on opioid-induced renal injury.

Interestingly, Meariman et al. recently revealed that KOR agonist treatment enhanced diuresis response and decreased MAP.³⁵ However, the authors administered nalfurafine acutely via intravenous injection over 90 min, whereas our study involved chronic BRL 52537 treatment for 2 weeks. The short-term exposure to nalfurafine likely induced transient effects, including diuresis and reduced MAP, potentially through inhibition of vasopressin. In contrast, chronic exposure to BRL 52537 in our model appears to have resulted in structural and functional renal changes, ultimately contributing to elevated MAP through distinct mechanisms. These pharmacological distinctions, combined with differences in treatment duration, likely account for the contrasting effects on blood pressure and renal function observed between the two studies.

Calcium signaling plays an essential role in podocyte pathophysiology.^25,26 In this study, we used human immortalized podocytes to record cell membrane ion channel activity after stimulation of KORs with BRL 52537 and determined an increase in ion channel activity. Potential types of channels mediating, KOR stimulated influx could include TRPC channels or store-operated calcium entry (SOCE)/Orai1 channels; however, further mechanistic studies into the precise channel and degree of involvement are required.^25,26 Prior work by our group have demonstrated that effects of KOR agonists are mediated via the TRPC6 channel in Dahl salt-sensitive (SS) rats.³⁶ However, in SD rats, other TRPC channels may functionally interact during KOR activation, thus contributing to effective calcium handling.^37,25 Studies of this nature require further elucidation into the communicative, interactive properties of the TRPC channel isoforms upon KOR stimulation. Calcium overload leads to dysregulation of the actin cytoskeleton, foot process retraction, podocyte detachment, increased oxidative stress, and apoptosis.^25,26 Upon increased calcium influx, downstream signaling pathways are subsequently activated. For example, increases in calcium have been demonstrated to activate calcineurin, which disrupts the podocyte actin cytoskeleton via the dephosphorylation of synaptopodin.³⁷ Further, increases in calcium influx can affect actin dynamics via activation of Rho GTPase enzymes, which also leads to podocyte cytoskeleton rearrangement and the effacement of podocyte foot processes.³⁸ Activation of these pathways is prevalent in diseases such as focal and segmental glomerulosclerosis, diabetic kidney disease, and hypertensive renal injury.³⁹ Targeting the restoration of calcium to homeostatic levels in the podocyte could be an effective approach in treating nephrotic syndromes. The current study provides further evidence that KORs mediate GFB impairment and alter glomerular permeability to albumin. This is a consequence of the increased calcium influx in the podocytes and decreased glomerular volume, as shown in our experiments, contributing to albuminuria in our rats chronically treated with BRL 52537. Our BRL 52537 group also developed increased diuresis from the initial day of treatment. KOR agonists have been known for their diuretic effect.^40–42 Clinical evidence suggests that opioid administration induced decreased plasma sodium as either a syndrome of inappropriate antidiuretic hormone secretion⁴³ or AVP-independent hyponatremia.⁴⁴ The paradoxical hypernatremia of the current study is not likely to be a cause or contributor to the higher MAP in our rats but rather a direct result of increased diuresis and dysregulated ion homeostasis, of which the mechanism needs to be further elucidated.

BRL 52537 provided a constrictive response when applied to isolated glomeruli potentially altering GFR. Glomerular hyperfiltration is a hallmark abnormality in diabetic kidney disease and is a marker of early damage in pre-hypertension.^45,46 In prior in vivo studies involving salt-sensitive hypertension, increased GFR has been demonstrated to be associated with declined renal function preceding the development of glomerulosclerosis.⁴⁷ BRL 52537 contributed to glomerular constriction, leading to alterations in glomerular hemodynamics. In the absence of therapeutic interventions, GFR could fall progressively in parallel with associated augmentations in albuminuria, which could lead to end-stage renal failure.

Buprenorphine displays partial agonist activity at KORs and is considered a first-line opioid intervention in the management of pain in CKD patients.^48,49 Moreover, difelikefalin, a peripheral KOR agonist administered to treat inflammatory pain, has been recently approved by the FDA for treating moderate to severe pruritus in hemodialysis patients.^50,51 Given the importance of KOR-activation and the subsequent glomeruli damage and blood pressure elevation after acute or chronic administration of KOR agonists, physicians should use caution when considering administering KOR opioids to patients with hypertension or CKD. This study expands our knowledge of the pathological role of KOR signaling in podocytes in healthy glomeruli. Moreover, the KOR agonist increased podocyte injurious calcium influx mechanism described in this study could serve as a potential therapeutic target in opioid-induced kidney damage, where a novel, peripherally restricted KOR antagonist could be developed.

Author Contributions

Steven Didik (Data curation, Formal Analysis, Writing – original draft, Writing – review & editing), Daria Golosova (Conceptualization, Data curation, Formal Analysis, Investigation, Writing – original draft, Writing – review & editing), Adrian Zietara (Formal Analysis, Investigation, Writing – review & editing), Ruslan Bohovyk (Formal Analysis, Investigation, Methodology, Writing – review & editing), Ameneh Ahrari (Formal Analysis, Investigation, Writing – review & editing), Vladislav Levchenko (Data curation, Formal Analysis, Writing – review & editing), Olha Kravtsova (Formal Analysis, Writing – review & editing), Krish Taneja (Formal Analysis, Writing – review & editing), Sherif Khedr (Formal Analysis, Investigation, Writing – review & editing), Marharyta Semenikhina (Data curation, Formal Analysis, Writing – review & editing), Oleg Palygin (Conceptualization, Project administration, Supervision, Writing – original draft, Writing – review & editing), Alexander Staruschenko (Conceptualization, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing)

Funding

Research in the author’s laboratory was supported by the National Institutes of Health grants R01 DK135644 (to A.S.) and R01 DK129227 (to A.S. and O.P.) and T32 HL160529 (to R.B. and A.Z.), U.S. Department of Veteran Affairs grants I01 BX004024 (to A.S.) and American Heart Association 20POST35180224 (to D.G.) and TPA3549003 (to A.S). The contents do not represent the views of the Department of Veterans Affairs or the United States Government.

Conflict of Interest

None declared.

Data Availability

All original data are available from the authors on request.