https://orcid.org/0000-0002-1850-1781
Abstract
Increased circulating bilirubin attenuates angiotensin (Ang) II-induced hypertension and improves renal hemodynamics. However, the intrarenal mechanisms that mediate these effects are not known. The goal of the present study was to test the hypothesis that bilirubin generation in the renal medulla plays a protective role against Ang II-induced hypertension.
Twenty-week-old male C57Bl/6J mice were implanted with intrarenal medullary interstitial (IRMI) catheters following unilateral nephrectomy. After this time, biliverdin IXα was specifically infused into the kidney (3.6 mg/day) for 3 days before implantation with an osmotic minipump delivering Ang II (1,000 ng/kg/min). BP was recorded for 3 days, 1 week after minipump infusion, in conscious mice. To further explore the antihypertensive role of renal medullary bilirubin generation, mice with specific deletion of biliverdin reductase-A (Blvra) in the thick ascending loop of Henle were generated. At 20 weeks, BlvraTALHKO and control mice (Blvrafl/fl) were infused with Ang II for 2 weeks.
IRMI infusion of biliverdin significantly decreased blood pressure compared with mice infused with vehicle (118 ± 4 vs. 158 ± 2 mmHg, p < 0.05). Angiotensin-II infusion resulted in significantly higher blood pressure measured in conscious mice 7 days after implantation in BlvraTALHKO as compared to Blvrafl/fl mice (152 ± 2 vs. 140 ± 3 mmHg, P < 0.05).
Altogether, these findings show that medullary bilirubin and biliverdin reductase can improve hypertension and that mechanisms that increase bilirubin and biliverdin reductase in the renal medulla could be an effective approach to treat hypertension.
Hypertension remains the most significant risk factor for cardiovascular morbidity and mortality. The kidney plays a significant role in the development and maintenance of hypertension. Within the kidney, the renal medulla is critical to maintaining the pressure-natriuretic response and regulating blood pressure.1 Changes in renal medullary blood flow are essential for the normal function of the kidney, as unlike renal cortical blood flow, renal medullary blood flow is poorly autoregulated.1,2 Likewise, the thick ascending loop of Henle (TALH) found in the renal medulla plays an important role in sodium and water reabsorption.3,4 However, the factors that regulate renal medullary blood flow and sodium and water reabsorption have not been fully elucidated.
Heme oxygenase is the enzyme responsible for the breakdown of heme.5 The process of heme degradation results in the formation of carbon monoxide, biliverdin, and free iron. Biliverdin is then rapidly reduced to bilirubin. Previous studies have demonstrated the important role of renal heme oxygenase-1 (HO-1) in regulating blood pressure and kidney function. HO-1 inhibition in the renal medulla exacerbates both high-salt diet-induced as well as angiotensin II (Ang II)-dependent hypertension.6,7 In contrast, specific induction of HO-1 in the renal medulla and TALH-specific overexpression of HO-1 attenuate Ang II-dependent hypertension.8,9 These studies demonstrate the important role of renal medullary HO-1 in protecting against hypertension.
Previous studies have demonstrated the protective effects of increased plasma bilirubin levels against the development of cardiovascular disease.10,11 Likewise, lower levels of plasma bilirubin are linked to the development of cardiovascular disease.12,13 Moderate hyperbilirubinemia from antagonism of hepatic UDP glucuronosyltransferase family 1 member A1 (UGT1A1) or treatment with bilirubin prevents Ang II hypertension and normalizes renal hemodynamics.14,15 Despite these findings, the specific role of renal medullary bilirubin and biliverdin reductase in the protective actions of HO-1 induction is not currently known.
METHODS
Animals
The experimental procedures and protocols of this study conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All mice had free access to food and water ad libitum. Animals were housed in a temperature-controlled environment with a 12 ohurs dark-light cycle. C57BL/6J mice were obtained from Jackson Labs (Bar Harbor, ME, stock # 000664). Blvrafl/fl mice were previously described.16 To generate TALH-specific knockout mice, Blvrafl/fl mice were crossed with Tamm-Horsfall Protein (THP)-Cre mice to generate BlvraTALHKO mice.17 Mice were genotyped at weaning as previously described.16,18 All mice were housed under standard temperatures between 24 °C and 25 °C. The standard mouse chow consisted of 17% fat (Teklad 22/5 rodent diet, #8604, Harland Laboratories, Inc., Indianapolis, IN). Studies were performed on 20-week-old male mice housed under standard conditions with full access to standard mouse chow and water described above. Investigators were blinded to the genotypes of all mice at the time of experimentation. Mice were euthanized via isoflurane anesthesia overdose, and blood and tissues were collected and stored at −80 °C for further analysis.
Intrarenal medullary interstitial infusions (IRMI) and measurement of blood pressure
All mice underwent unilateral nephrectomy of the right kidney. After seven days, intramedullary interstitial catheters were implanted 1.5–2 mm into the left kidney as previously described.8,19 Saline was then infused through the catheter for a period of 3 days after which time the infusion was switched to either bilirubin (3.6 mg/day, in 0.1 M NaOH, pH 8.3) or biliverdin (3.6 mg/day, in saline). Two days after the switch to bilirubin or biliverdin, mice were implanted with osmotic minipumps, delivering Ang II at a rate of 1 μg/kg/min. Five days after implantation of the osmotic minipumps, carotid artery catheters were implanted into the mice as previously described.20 After a 2-day recovery period, blood pressures were measured in 3-hour periods over the next 3 days in conscious, freely moving mice. Blood pressure data were continuously collected by a PowerLab 8/sp polygraph (AD Instruments, Denver, Colorado) and acquired on a computer running Chart 4 software provided by the manufacturer. Blood pressure data is presented as the average of the three-day measurements.
Measurement of biliverdin reductase (BVR) activity
BVR activity was measured as previously described.16 Briefly, BVR activity was measured using a colorimetric reaction to measure bilirubin formation. Renal cortical and medullary tissue samples (50 mg) were lysed in 150 μL of extraction buffer, and the assay was performed using 150 μg of total protein. Bilirubin levels were measured at 450 nm at 37oC 18 minutes after the start of the reaction. BVR activity is expressed as units/ml with 1 unit of biliverdin reductase converting 1 nanomole of biliverdin to bilirubin in an NADPH-dependent fashion at pH 8.5 at 37oC.
Western blotting
Western blots were performed on kidney samples as previously described.21 Membranes were incubated overnight at 4 °C with the following antibodies: rabbit anti-BVRA (1:1000, Santa Cruz, #393385) and rabbit anti-HSP90 (1:1000, Santa Cruz, #13119), rabbit anti-NKCC2 (1:1000, Cell signaling, #38436), mouse anti-renal outer medullary potassium (ROMK) (1:1000, Santa Cruz, # 393189), and mouse anti-GAPDH (1:1000, Proteintech, #CL488-60004). After three washes in TBS + 0.1% Tween 20, the membrane was incubated with an infrared anti-mouse (IRDye 800, green) or anti-rabbit (IRDye 680, red) secondary antibody labeled with IRDye infrared dye (LI-COR Biosciences) (1:10,000 dilution in TBS) for 2 h at 4 °C. Immunoreactivity was visualized and quantified by infrared scanning in the Odyssey system (LI-COR Biosciences).
Immunofluorescence
Kidneys were fixed with formalin and then dehydrated in 30% sucrose solution. Kidneys were then placed in OCT media (Tissue-Tek, Torrance, CA) and frozen at -20oC. Frozen kidneys were then sectioned at 5 μm and placed on coated glass slides. Cells were then blocked in 5% normal donkey serum (NDS) for 1 hour at room temperature. Samples were then incubated with primary antibodies rabbit anti-BVR (1:200, StressGen, Vancouver, Canada), and a mouse anti-THP antibody (Accurate Chemical & Scientific Corp, Westbury, NY,1:500) in 5% NDS overnight at 4˚C, then rinsed in PBS. Samples were incubated with a donkey anti-rabbit Alexa488 (1:1000, Invitrogen, Carlsbad, CA) and a donkey anti-mouse Cy3 (1:200, Jackson Immunologicals, West Grove, PA) for 1 hour at 37˚C in 5% NDS. Following a final rinse in PBS, samples were covered with Gel Mount mounting media and cover-slipped. Antibody localization was visualized using confocal microscopy (Leica TCS SP2). All samples were collected, immunolabeled, and imaged side-by-side under identical conditions.
Statistical analysis
Data were analyzed with Prism 9 (GraphPad Software, San Diego, CA) using analysis of variance combined with Tukey’s post-test to compare pairs of group means or unpaired t-tests. Results are expressed as mean ± SEM. A two-tailed and a two-way ANOVA was utilized in multiple comparisons, followed by the Bonferroni post hoc analysis to identify interactions. P values of 0.05 or smaller were considered significant.
RESULTS
Intrarenal medullary interstitial infusion of bilirubin or biliverdin attenuates Ang II-dependent hypertension
To determine the role of increased renal medullary interstitial levels of bilirubin or biliverdin on the development of Ang II-dependent hypertension, we utilized intrarenal medullary interstitial (IRMI) infusion in the mouse kidney.7,8,19 IRMI infusion of bilirubin before Ang II administration significantly attenuated the development of hypertension (Figure 1a). Consistent with the lowering of blood pressure by IRMI infusion of bilirubin was decreases in the measures of cardiac hypertrophy by heart weight to body weight ratio (Figure 1b) and heart weight to body length ratio (Figure 1c). IRMI infusion of biliverdin also decreased blood pressure as compared with vehicle-infused mice but also had a significantly greater effect on lower blood pressure than observed in IRMI bilirubin-treated mice (Figure 1a). IRMI infusion of biliverdin decreased cardiac hypertrophy as indexed by heart weight to body weight ratio (Figure 1b) and heart weight to body length ratio (Figure 1c). Interestingly, biliverdin treatment reduced the heart weight to body weight ratio more than bilirubin treatment (Figure 1b) but not the heart weight to body length ratio (Figure 1c).
Intrarenal medullary interstitial infusion (IRMI) of bilirubin or biliverdin lowers blood pressure and indices of cardiac hypertrophy in Ang II-dependent hypertension. (a) Three-day average of blood pressure. (b) Heart weight to body weight ratio. (c) Heart weight to body length ratio. *P < 0.05 as compared with vehicle-treated. #P < 0.05 as compared with bilirubin-treated. n = 6/group.
Molecular characterization of BlvraTALHKO mice
To create a model in which the Blvra gene was specifically knocked out in the TALH, we crossed our Blvrafl/fl mice with THP Cre mice to generate BlvraTAHLKO mice.16–18 We examined the level of BVRA protein by western blot in several tissues from Blvrafl/fl and BlvraTAHLKO mice. BlvraTAHLKO mice exhibited a decrease in BVRA protein in the medulla and cortex compared with Blvrafl/fl mice (Figure 2a). Next, we utilized immunofluorescence to confirm TALH-specific loss of BVRA in BlvraTAHLKO mice. Kidney sections were labeled with antibodies to the Tamm-Horsfall protein, a specific marker of TALH cells (labeled in red) and BVRA (labeled in green). Co-localization of Tamm-Horsfall protein and BVRA (yellowish signal) was evident in the kidney of Blvrafl/fl mice (yellow arrows, Figure 2b). In contrast, co-localization of the two proteins was absent in the kidney of BlvraTAHLKO mice (red arrows, Figure 2b).
Characterization of BlvraTALHKO mice. (a) Western blot of renal cortex (C), renal medulla (M), liver (L), and spleen (S) in BlvraTALHKO and Blvrafl/fl mice. (b) Immunofluorescence of kidney sections using anti-BVRA antibody (green) and anti-Tamm-Horsfall protein antibody (Red). Co-localization of BVRA and THP can be seen in Blvrafl/fl mice (yellow arrows), while no co-localization was observed in BlvraTALHKO mice (red arrows).
BlvraTALHKO mice exhibit an enhanced blood pressure response to Ang II-dependent hypertension
To determine the effect of loss of TALH BVRA on blood pressure, Blvrafl/fl and BlvraTAHLKO mice were implanted with osmotic minipumps containing Ang II (1 μg/kg/min) for 7 days, after which arterial catheters were implanted for the measurement of blood pressure for 3 consecutive days in conscious, freely moving mice. No differences in mean arterial blood pressure were detected between Blvrafl/fl and BlvraTAHLKO mice under basal conditions (Figure 3a). Ang II administration increased mean arterial blood pressure in both groups; however, BlvraTAHLKO mice exhibited a significantly greater blood pressure response as compared with Blvrafl/fl mice (Figure 3a). Both groups exhibited significantly increased cardiac hypertrophy in response to Ang II infusion; however, BlvraTAHLKO mice exhibited a much greater increase in heart weight to body weight ratio (Figure 3b) and heart weight to body length ratio (Figure 3c) as compared with Blvrafl/fl mice.
TALH-specific knockout of BVRA augments blood pressure and indices of cardiac hypertrophy in Ang II-dependent hypertension. (a) Three-day average blood pressure. (b) Heart weight to body weight ratio. (c) Heart weight to body length ratio. *P < 0.05 as compared with control. #P < 0.05 as compared with Blvrafl/fl. n = 5-6/group.
BVRA activity is decreased and levels of the ROMK channel are increased in the medulla of BlvraTALHKO mice
Renal cortical and medullary BVRA activity was measured in control, and Ang II treated Blvrafl/fl and BlvraTAHLKO mice. Renal cortical BVRA activity was not different between Blvrafl/fl and BlvraTAHLKO mice; however, cortical BVRA activity was significantly decreased by Ang II treatment in each group (Figure 4a). In contrast, renal medullary BVRA activity was significantly reduced in BlvraTAHLKO compared with Blvrafl/fl mice, and Ang II treatment did not affect BVRA activity in either of these groups (Figure 4b). To assess if the protein levels of electrolyte transporters were altered in the medulla of BlvraTAHLKO mice, protein levels of the Na+-K+-2Cl cotransporter (NKCC2) and ROMK were assessed. NKCC2 protein levels were not different in the medulla between Blvrafl/fl and BlvraTAHLKO mice under basal conditions or after Ang II treatment (Figure 4c). However, protein levels of ROMK were significantly increased the medulla of Ang II-treated BlvraTAHLKO as compared with Blvrafl/fl mice (Figure 4d).
BVRA activity in the cortex and medulla of control and Ang II-treated Blvrafl/fl and BlvraTALHKO mice. (a) BVRA activity in the renal cortex. (b) BVRA activity in the renal medulla. (c) Representative western blot of NKCC in the medulla. (d) Representative western blot of ROMK in the medulla. *P < 0.05 as compared with control. #P < 0.05 as compared with Blvrafl/fl. n = 3-5/group.
DISCUSSION
The kidneys have long been known to play a major role in the long-term regulation of blood pressure.22,23 Previous studies have established the heme oxygenase system as a regulator of renal function and blood pressure.6,8 Increases in the plasma levels of the HO metabolite, bilirubin, lower the blood pressure and improve renal hemodynamics14,15; however, the role of renal medullary bilirubin in regulating blood pressure has not been previously studied. Bilirubin is generated via the breakdown of heme by heme oxygenase to produce biliverdin and then the rapid reduction of biliverdin to bilirubin by BVRA.5 The present study examined the specific role of renal medullary bilirubin in regulating blood pressure in a model of Ang II-dependent hypertension using two approaches. The first approach was direct infusion of bilirubin or biliverdin into the renal medullary interstitium. The second approach was via knockout of BVRA, specifically in TALH cells of the renal medulla, via a Cre-loxP approach. IRMI infusion of both bilirubin and biliverdin lowered blood pressure in Ang II-dependent hypertension. However, IRMI infusion of biliverdin was more effective in lowering blood pressure in this model than IRMI infusion of bilirubin. This increased effectiveness of biliverdin administration could be a result of the better solubility of biliverdin as compared with bilirubin, which makes it easier to gain intercellular access where it is reduced to bilirubin via BVRA. One limitation of the present study was the initiation of IRMI bilirubin or biliverdin before administering Ang II. Thus, it is not known if increased medullary levels of bilirubin or biliverdin would be able to lower blood pressure in established Ang II-dependent hypertension. This question will need to be addressed in future experiments.
TALH-specific knockout of BVRA resulted in an enhanced blood pressure response and increased markers of cardiac hypertrophy compared with control mice. The loss of BVRA in the kidney was recently demonstrated in a proteomic analysis between the spontaneously hypertensive rat and its normotensive Wistar–Kyoto control.24 Furthermore, a mutation in the human Blvra gene that decreases BVRA activity has been linked to hypertension in Asians.25 The results of the present study demonstrate an important role for TALH BVRA in protection against AngII-dependent hypertension. The TALH is an important nephron segment responsible for ~25% of sodium reabsorption.3,4 Thus, alterations in the regulation of sodium reabsorption in this nephron segment could have a significant impact on the regulation of blood pressure, especially in response to increases in angiotensin II levels.
There are several potential mechanisms by which increases in medullary interstitial bilirubin/biliverdin protect against and the loss of TALH BVRA enhances Ang II-dependent hypertension. The first potential mechanism is through effects on renal medullary oxidative stress through bilirubin’s effect on superoxide production. Bilirubin is one of the most potent antioxidants in the body.26 It can scavenge superoxide anion as well as directly inhibit its formation through inhibition of NADPH oxidase.27 Previous studies have demonstrated that the induction of HO-1 prevents the increase in renal medullary superoxide production in Ang II-dependent hypertension.28 Likewise, knockdown of BVRA in cultured TALH and medullary collecting duct cells results in the augmentation of Ang II-mediated superoxide production.29 Increased superoxide production can influence blood pressure through its effects on the renal vasculature and tubular transport. Superoxide, through its interactions with nitric oxide, regulates renal vascular resistance.30,31 Superoxide generation has been shown to play an important role in regulating sodium transport in the TALH.32,33
Another potential mechanism by which increases in medullary interstitial bilirubin/biliverdin can protect against Ang II-dependent is its interaction with the peroxisome proliferator-activated receptor α (PPARα) pathway. Recent studies have demonstrated that bilirubin acts as a PPARα ligand and that PPARα mediates the transcriptional effects of increased bilirubin levels.34,35 PPARα agonists have been demonstrated to protect against hypertension in preclinical animal models and in humans.36–38 PPARα null mice exhibit alterations in renal pressure-natriuresis and develop salt-sensitive hypertension.39 More recently, PPARα null mice exhibited an increased blood pressure response to Ang II infusion due to increased Na+/K+ ATPase activity.40
PPARα is also expressed in endothelial and vascular smooth muscle cells.41 Hyperglycemia has been reported to down-regulated PPARα levels in endothelial cells, and deficiency of vascular smooth muscle cell PPARα promotes migration.42,43 PPARα is also expressed in podocytes, where previous studies have demonstrated that treatment with PPARα agonists increases nephrin expression, promoting stability of the glomerular filtration barrier.44 Thus, IRMI infusion of bilirubin or biliverdin may improve renal vascular function in Ang II-infused mice through interaction with PPARα on either endothelial or vascular smooth muscle cells.
The increased ROMK protein levels observed in the medulla of BlvraTAHLKO mice in response to Ang II treatment could impact sodium retention in this strain because the ROMK channel can drive both active and passive sodium reabsorption in the TALH.45 The activity of the ROMK channel is important in the recycling of K+ in this segment of the nephron which is critical to maintain a sufficient concentration of K+ in the tubular lumen which helps the NKCC2 transporter reabsorb Na+. Recycling of K+ via the ROMK channel is also important to maintain the lumen-positive transepithelial potential that drives passive reabsorption of Na+ in the TALH via the paracellular pathway. Elevated levels of the ROMK channel have previously been reported in the outer medulla of the hypertensive, salt-sensitive Dahl S rat.46 The effects to potentially increase TALH Na+ reabsorption via this pathway are consistent with the observation that the levels of the NKCC2 protein were not changed in the medulla of BlvraTAHLKO mice.
This study demonstrates that increases in renal medullary interstitial levels of bilirubin, either by direct infusion or infusion of biliverdin, lower the blood pressure in Ang II-dependent hypertension. Furthermore, the loss of the bilirubin-producing enzyme BVRA, specifically in TALH cells, augments the blood pressure response to Ang II-dependent hypertension. These studies highlight the important role of renal medullary bilirubin in regulating blood pressure in response to stressors such as increased Ang II levels. Further studies are needed to dissect the specific mechanisms that are impacted by bilirubin in the renal medulla so that novel therapeutics targeting this pathway can be developed for the treatment of hypertension.
Author contributions
G.A. and D.E.S. conceived and designed research; G.A., A.R.W., R.E.S., H.A.D., and D.E.S. performed experiments; G.A. and D.E.S. prepared figures; G.A. and D.E.S. drafted the manuscript; G.A., A.R.W., R.E.S., H.A.D., and D.E.S. edited and revised the manuscript; all authors approved the final version of the manuscript.
Acknowledgment
The authors would like to acknowledge the histology core in the Department of Physiology and Biophysics at the University of Mississippi Medical Center.
Funding
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK121748-01A1 to D.E.S. and R01DK137167-01A1 to H.A.D.), the National Heart, Lung and Blood Institute (P01 HL05197-11 to D.E.S., H.A.D.), and the National Institute of General Medical Sciences (P20GM104357-02 to D.E.S., H.A.D., P30GM149404 to D.E.S., H.A.D., P20GM144041 to D.E.S., and P20GM121334 to H.A.D.). The content is solely the authors’ responsibility and does not necessarily represent the official views of the National Institutes of Health.
Conflict of Interest
No conflict of interest to declare.
Data Availability
The data underlying this article are available in the article
