Hemodynamic Renal Reserve Response in Conscious Normotensive and Hypertensive Mice

Glomerular filtration rate (GFR), urine and sodium excretion are considered critical indexes of kidney function [1‒3]. GFR is determined by measuring the clearance of certain exogenous markers or endogenous waste products. Nonetheless, GFR is not a constant value as the kidneys do not consistently operate at their maximum filtration capacity [4‒6]. They have the ability to adapt their performance in response to hemodynamic and metabolic demands [7, 8]. Renal function is often influenced by meals and diurnal cycles [9]. Thus, single-point assessments of renal function ignore varying GFR. Particularly, kidney function evaluation by GFR measurement may not be able to reveal the underlying dysfunctional portion under pathological situation. This is because in these situations, the remaining healthy nephrons may become stimulated and increase their filtration rate in order to compensate for the loss of function in the damaged nephrons. The undetected alteration in GFR due to the compensatory effect may mask the underlying lost function. Many investigators suspected that the functional recovery assessment might be clouded by the stimulated single-nephron GFR to compensate for nephron loss after kidney insults [10, 11]. Therefore, testing the renal functional response to stimuli in these recovered individuals could possibly unveil this undetected loss of functional units.

The concept of reserve forces of the kidney was proposed by Verney in 1930 and named renal functional reserve (RFR), which is defined as the difference between the stimulated GFR and basal GFR [11‒13]. The concept of RFR was subsequently refined to encompass the renal functional response assessed by administering a substantial liquid volume/substance to the animal and measuring the resulting hemodynamic changes, denoted as acute volume expansion (AVE).

Conventionally, the AVE was carried out either in anesthetized animals by intravenous (IV) bolus injection or in conscious animals by IV infusion of a larger volume of solution [14, 15]. Both strategies require anesthesia or surgery preparation. However, anesthesia or surgical stress can directly or indirectly affect renal hemodynamics and influence renal function and body fluid regulation, possibly through lowered blood pressure and cardiac output, increased sympathetic outflow, increased release of renin, angiotensin, and vasopressin, alterations in the autoregulation of renal blood flow, changes in the impact of antidiuretic hormone, and modifications to tubular transport of sodium and organic acids [16‒18]. Inhalational anesthetics generally reduce GFR and urine output mainly by extra-renal effects [19, 20]. Anesthetic management frequently has a physiologically significant impact on renal physiology [21, 22]. Furthermore, IV infusion via implanted catheters and urine collection using metabolic cages may introduce potential biases or confound the results, stemming from urine evaporation and sodium loss. Additionally, AVE conducted on anesthetized animals typically serves as a terminal experiment, constraining its applicability in repeated monitoring studies.

In this context, we have presented experimental data obtained through both conventional and modified protocols. The adjustments made to these protocols have demonstrated enhancements in the accuracy and precision of experimental outcomes, improved efficiency, and addressed various limitations associated with the conventional methods.

All procedures and experiments were performed with the approval of the Institutional Animal Care and Use Committee at the University of South Florida College of Medicine and in accordance with all federal guidelines. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated. Animals were euthanized as needed according to the guidelines set forth by the American Veterinary Medicine Association.

Male C57BL/6 J mice, aged 10–12 weeks (22–25 g), were obtained from a vendor (Jackson Lab, Indianapolis, IN, USA). The mice were randomly divided into 7 groups: normotensive anesthesia IV (NTN-A-IV), normotensive conscious IV (NTN-C-IV), normotensive conscious intraperitoneal (NTN-C-IP), hypertensive anesthesia IV (HTN-A-IV), hypertensive conscious IV (HTN-C-IV), hypertensive conscious IP (HTN-C-IP), and sham control groups (normotensive and hypertensive), which followed all the procedures but without acute saline load.

Radio-telemetry transmitters (PA-C10) were implanted subcutaneously in all groups of mice as we described previously [23‒25]. The mice were anesthetized via the inhalation of isoflurane (2% in air; flow 200 mL/min). A small incision was made in the middle of the neck, and the catheter of the telemetry transmitter was inserted into the left carotid artery, which was then guided down to the aortic arch. The body of the transmitter was placed subcutaneously in the right ventral flank of the animal. The wound in the middle of the neck was closed, and the mice were given a recovery period of 10 days. Basal mean artery blood pressure (MAP) was then measured for 5 days at an interval of 2 min with the measurement duration of 20 s.

After the basal MAP measurement, half of the mice were randomly selected and induced hypertension by ligating the left renal artery branch followed by right kidney nephrectomy as we described previously with modification [26]. Briefly, the mice implanted with transmitters were anesthetized with isoflurane. A midline abdominal incision was performed to expose two kidneys. The upper branch of the left renal artery was carefully dissected from the renal vein and ligated using a 7–0 braided silk suture (07C1500160; Teleflex Medical OEM, Gurnee, IL, USA). The right kidney was removed and the ischemic portion of the left kidney induced by upper branch ligation was left in place. The incision was closed and the mouse recovered in the normal cages. Plasma and urine were collected at 2 weeks to evaluate the kidney function and injury by plasma creatinine and blood urea nitrogen levels measured at Bioanalytical Core of O’Brien Center at University of Alabama at Birmingham as previously described [24, 27‒30].

Pure mouse urine was collected in transparent plastic-wrapped cages for qualitative analysis. Regular cages were cleaned and dried. The bottom of the cage was covered by white paper and then the whole cage was covered by Glad^® cling wrap. Mouse was housed individually. To minimize stress and anxiety, 3 days before the experiment, the mice were housed in the cages for 4 h every day and 2 h before the experiment on the same day to adapt to the new environment.

One hour before the experiment, the urine was voided from the experimental mouse’s bladder by holding the tail of the mouse with the thumb and index finger and pressing the root of the tail with the middle and fourth fingers. Press the bladder manually by the application of gentle trans-abdominal pressure over the bladder to overcome normal urethral pressure with a cotton swab to encourage the mouse to micturate. The mouse was then put in the AVE cage, and the timer was started. The voided urine was aspirated into micro-centrifuge tubes using a pipette. The total amount of urine collected in the first hour was combined and taken as a control sample. The mouse was then slightly anesthetized with isoflurane. The amount of 5% body weight (BW) sterile saline was administrated through retro-orbital or IP injections. After injection, the mouse was immediately returned to the prepared cage. The urine was collected immediately after micturated and centrifuged. All the urine samples collected within each hour were combined and taken as a single sample. The total collection period lasted for 3 h. The mice were euthanized and kidneys were collected at the end of experiment. The urine was stored under −20 °C for later analyses.

The natriuretic response to AVE was measured in anesthetized mice as previously described [23, 31]. The mice were anesthetized with ketamine (30 μg/g) and inactin (50 μg/g). Two catheters were placed in the femoral artery for the measurement of MAP and in the femoral vein for an IV infusion of 2% BSA and FITC-sinistrin (2 mg/mL) in a 0.9% NaCl solution at a rate of 0.5 mL/h. Two more catheters were inserted into the ureters for the collection of urine. After surgery, urine was collected during a 30-min period after a 30-min equilibration period. Then, infusion of 5% BW saline in a bolus was followed by the infusion of FITC-sinistrin at 0.5 mL/h. Urine and blood were collected during a 60-min period for 3 h after volume expansion. The urine Na+ concentration was measured with a Flame Photometer (BWB Technologies). The urine flow rates and Na+ excretion rates were calculated based on Na+ concentration and total urine volume. FITC-sinistrin concentration in the urine and plasma samples was measured using a plate reader (Cytation 3, BioTek). The calculation for GFR was performed using the formula: [Urine FITC-sinistrin] × Urine volume/[Plasma FITC-sinistrin].

GFR was measured in all the mice under conscious conditions using transdermal measurement of FITC-sinistrin clearance rate as previously reported [32‒34]. The right dorsal hair of the mice was removed by shaving and depilatory lotion on the day before measurement. The transdermal fluorescence detector (MB 0309 mini, MediBeacon Inc.) was affixed directly onto the mouse’s skin using a medical tape. FITC-sinistrin was then administered by retro-orbital injection at 0.05 mg/g body weight. The excitation kinetics of the exogenous GFR tracer were recorded and fitted to a three-compartment model with the software provided by the vendor (MB Studio Ver. 2.1). GFR was calculated based on the half-life (t1/2) of plasma FITC-sinistrin decay.

All values are presented as means ± SEM. Comparison of three or more datasets was performed using two-way ANOVA followed by Sidak’s multiple comparison test. A p value of <0.05 was considered to be statistically significant. Sample sizes are provided in the figure legends. All statistics were performed using GraphPad Prism, version 9.0 (GraphPad Software).

Experimental protocols for AVE and GFR measurements, as well as sample collection from the mice under both anesthetized and conscious conditions, are outlined in Figure 1.

The basal MAP values were similar for all mice in conscious normotensive groups (NTN-C-IV and NTN-C-IP). AVE was carried out with the bolus injection of saline in the mice under slight anesthesia with isoflurane. The MAPs decreased by about 10 mm Hg due to the anesthesia, which quickly returned to baseline in about 5 min. Thereafter, the MAP remained constant during the AVE for all the conscious groups (Fig. 2). For the anesthetized IV group (NTN-A-IV), the basal MAP was about 16 mm Hg lower than the conscious mice due to the anesthesia. The AVE on anesthetized mice was performed as described in the method section. The MAP maintained consistency during the whole AVE procedure.

GFR was measured using the transderminal measurement of the sinistrin clearance rates. Basal GFR was first measured. Another dose of FITC-sinistrin was administrated again. 20 min later, a 5% BW saline solution was administered via penis vein or IP injection to induce AVE. For the group of mice that underwent AVE in conscious conditions, GFR was enhanced by 62% and 53% for the IV- and IP-injected groups, respectively. However, the GFR only increased by 31% in the mice under anesthesia condition (Fig. 3).

Urine was collected from the normotensive mice under either conscious or anesthesia conditions every hour for 3 h after injection. The basal urine flow rates were similar for both NTN-C-IP and NTN-C-IV groups with the value of about 10.6 ± 1.7 μL/min.g kidney weight (KW). The IV bolus injection of 5% BW saline induced a dramatic increase in urine production rates with the maximum rate of 44.7 ± 5.8 μL/min.g KW, which was about 420% of baseline. The IP injection of the same amount of saline stimulated much less increase in the urine flow rates, which is about 178% of baseline, than the IV-injected conscious group within the experimental period (Fig. 4). Moreover, the NTN-C-IV mice displayed a significantly faster response in urine excretion to the AVE compared to the NTN-C-IP mice. In NTN-C-IV mice, the peak urinary flow rates were attained within 1 h after injection, whereas it took nearly 2 h for the NTN-C-IP mice to reach their peak urinary flow rate. By the end of 3 h following injection, NTN-C-IV mice had excreted the entire volume of injected saline, whereas NTN-C-IP mice had only micturated approximately 52% of the total injected saline (Table 1.).

The AVE in anesthetized mice (NTN-A-IV) was performed by the administration of 5% BW saline by bolus IV injection. The basal urine flow rate was 6.2 ± 1.3 μL/min.g KW, which was about 42% lower than that in conscious mice. AVE stimulated a 111% increase of urine flow rate with the maximum value of 13.1 ± 1.8 μL/min.g KW. The peak value of the urinary excretion in response to the saline injection in NTN-C-IV mice was about 3-fold and 2-fold of NTN-A-IV and NTN-C-IP mice, respectively. Additionally, compared with conscious animals, the response of the urinary excretion rates in the anesthetized mice failed to maintain and returned to baseline within 2 h after AVE. During the measurement period of 3 h, the anesthetized mice showed the least increase in urine output and only about 40% of injected saline was micturated.

The sodium excretion rates showed the similar pattern as the urinary flow rates (Fig. 4). The conscious mice exhibited quicker response to the stimulation of the bolus saline load and the maximal response maintained for longer period of time than the anesthetized mice.

These data indicated that anesthesia induced by isoflurane significantly decreased urine output and fractional sodium excretion during AVE. In response to the stressor of kidney overload, conscious mice demonstrated superior abilities in handling sodium excretion compared to anesthetized mice.

A low-nephron hypertensive model was successfully generated as described in the method and previously reported [35]. MAP was measured with a telemetry system. There were no significant differences between the different groups for basal conscious MAPs, which were about 96 mm Hg. Right kidney nephrectomy and left renal artery branch ligations induced drastic rise of blood pressure (Fig. 5a). The MAPs reached the maximum level in 2 weeks with a value of 136.4 ± 6.8 mm Hg, which increased about 40 mm Hg compared with baseline. The increased blood pressure was accompanied with impaired kidney function based on the plasma creatinine and urine ACR levels (Fig. 5b, c). The MAPs were consistent in the control mice.

We next tested the differences of hemodynamic response to a saline bolus challenge among the conscious hypertensive (HTN-C-IV and HTN-C-IP) mice and anesthetized hypertensive (HTN-A-IV) mice. AVE was conducted in the hypertensive mice by IV injection under conscious or anesthetized conditions or IP injection under conscious condition as it was carried out in the normotensive mice. MAPs were monitored during the whole AVE process with the telemetry system. The anesthesia by isoflurane induced about a decrease of 18 mm Hg of the basal MAP (from 136 to 118 mm Hg) in the anesthesia group. The MAP gradually and steadily rose throughout the AVE, with the maximum increase of about 14 mm Hg than the baseline during the monitoring period. In the conscious group, a mild anesthesia administered for the IV injection of saline led to a temporary drop of approximately 16 mm Hg in MAP during the injection phase. Subsequently, MAPs rebounded to around 10 mm Hg above the baseline, and within the initial hour post AVE, they returned to the baseline level and persisted consistently throughout the remainder of the AVE experiment. In the conscious IP group, there was a delayed yet steady increase in MAP, peaking with a maximum rise of approximately 6 mm Hg over the course of the AVE (Fig. 6).

Initially, basal GFR was determined. Subsequently, another dose of FITC-sinistrin was administered. 20 min later, a 5% BW saline solution was introduced via penile vein or IP injection to induce AVE. In the group of mice that underwent AVE under conscious conditions, GFR showed a respective enhancement of 25–28% for the IV and IP injected groups. However, under anesthesia conditions, the GFR only increased by 12% (Fig. 7).

The basal urine flow rates were similar for both HTN-C-IV and HTN-C-IP groups with the value of 16.2 ± 2.8 μL/min.g KW. The IV bolus injection of 5% BW saline in the conscious mice induced a dramatic increase in urine production rates. The urinary flow rate reached the maximum value of 38.7 ± 2.8 μL/min.g KW, which was about 238% of baseline, in 2 h. The urine excretion maintained the highest rate until 3 h after injection and decreased gradually after that. The IP injection of the same amount of saline also induced the similar levels of increase as the IV group; however, the increase of the urinary flow rate was significantly delayed (Fig. 8). The HTN-C-IV group showed much quicker response to the stimulation of massive saline load in urine excretion than HTN-C-IP injection.

Basal urinary flow for the anesthetized mice was suppressed compared with the conscious mice with a value of 10.42 ± 1.38 μL/min.g KW. The bolus injection of saline induced an increase of urinary flow rate up to 20.12 ± 1.86 μL/min.g KW for the HTN-A-IV mice. The peak value of the urinary excretion in response to saline injection in conscious mice HTN-C-IV was three times that of anesthetized mice HTN-A-IV. It is very interesting that the conscious animal showed more prolonged response to the stimulation of saline bolus load than the anesthetized mice based on the sustained peak urinary flow rates for up to 3 h for the HTN-C-IV verse 1 h for the HTN-A-IV. HTN-C-IP mice showed drastically delayed response to the saline loading challenge. Within 3 hours after injection, the HTN-C-IV group excreted 59% of the injected saline; however, IP-injected hypertensive mice and anesthetized hypertensive mice only excreted about 44% and 28% of the total injected saline, respectively.

The sodium excretion rates showed the similar pattern as the urinary flow rates (Fig. 8). The IV injection induced quicker response of urinary sodium excretion to the volume expansion than IP injection. Additionally, conscious mice exhibited faster and longer sustained stimulation in the excretion of sodium compared to the anesthetized mice.

Both the conscious and anesthetized mice that received IV injections showed higher urinary flow rates per KW in hypertensive mice compared to normotensive mice. This increase in flow rates may be attributed to hyperfiltration induced by kidney mass ablation. However, the delta of total urinary excretion in responding to AVE between hypertensive and normotensive mice is greater in conscious mice than that in anesthetized mice. In addition, normotensive mice excreted 47% more urine than hypertensive mice during the 3 h of conscious AVE period, whereas there was only a 30% difference in the urine output between normotensive and hypertensive mice when the mice were subjected to AVE under anesthetic conditions (Table 1).

Our research group has developed a new methodology to evaluate the hemodynamic response to AVE in unencumbered mice. This is accomplished through the administration of a bolus IV injection of a saline solution, with a volume equating to 5% of the animal’s BW.

This method has been validated in healthy normotensive and hypertensive animal models and compared with the conventional method under anesthesia conditions in this study. Our findings indicated that conscious animals, regardless of their health or hypertensive status, display a notably stronger renal hemodynamic response to a large volume infusion of saline compared to anesthetized animals. Therefore, we concluded that evaluating the full kidney functional capacity may be more accurate and informative in conscious animals compared to animals under anesthesia. This is because the state of consciousness appears to impact urinary excretion patterns in response to AVE, with anesthesia potentially concealing or reducing these distinctions. This method demonstrated that using conscious animals in the evaluation of RFR may provide a better strategy compared to using anesthetized mice, and that this approach may lead to improved diagnosis and prognosis of both acute and chronic kidney diseases.

The GFR and urine flow rates are common standards used in the evaluation of kidney function, and they can be used together to provide a more comprehensive assessment of kidney health [4‒6]. However, the GFR is not a fixed parameter, and it can vary in response to various factors such as changes in blood pressure, medications, and other physiological stimuli [36, 37]. Thus, baseline GFR does not describe the entire functional capacity of the kidneys and a stress test is required to quantify the functional reserve of the kidney, which has been described as RFR. Especially under the pathophysiological conditions, like acute kidney injury and hypertension induced by fewer nephrons, even if a normal basal kidney function is observed after recovery, a reduction in the functional reserve of the kidney due to the loss of nephrons and damage to the remaining nephrons may have occurred. Therefore, measurement of kidney function response to a stimulus can provide important information about the functional reserve of the kidney and can help identify early stages of kidney disease or dysfunction before significant damage occurs.

In clinical situations characterized by hyperfiltration, like during pregnancy, hypertension, or diabetic nephropathy, the baseline GFR can often stay within normal ranges or even exceed normal levels. This can persist until around 50% of nephrons are lost, or in cases involving patients with just one remaining kidney. This can obscure any underlying reduction in kidney functional reserve. The RFR test can be used to evaluate the ability of the kidney to increase its filtration rate in response to a stressor, such as an infusion of amino acids or glucose. This may represent a sensitive and early way to assess the functional decline in the kidney.

In animal studies, the RFR is usually determined by measuring the renal responses to ECVE with IV bolus injection under anesthesia condition or perfusion of isotonic saline in conscious animals [38]. As we and others did previously, IV bolus injection of 3–5% BW saline induced 40–60% increase in the GFR and urine flow rate [29, 39]. In the present study, we first confirmed that IV bolus injection of 5% BW saline did not significantly affect systemic blood pressure in conscious mice, which is similar to anesthetized mice. We then repeated the AVE experiments in anesthetized mice as we or other laboratories did before [29, 39]. The AVE in conscious mice showed greater and more sustained response to the saline load than the anesthetized mice with greater urinary excretion. The lower blood pressure due to anesthesia, the surgical stress, and nociceptive input followed by the loss of fluid during the operation can affect the renal function to different extents. It has been reported that most barbiturates and inhalational anesthetics tend to decrease the renal blood flow and GFR. The effects of anesthetics on the kidney go beyond a simple change in basal hemodynamics and include, for some drugs, an alteration in the ability for the kidney to autoregulate its blood flow and GFR [40, 41].

We also tested the AVE in conscious mice of a hypertensive kidney disease model that was generated by uninephrectomy combined with 2/3 renal infarction via a ligation of the upper renal artery branch on the contralateral kidney as reported previously [35]. We first applied the AVE with the traditional method under anesthetic conditions. The anesthetic AVE showed that the normotensive mice excreted about 30% more sodium than hypertensive mice, which is consistent with the results of previous studies of the GFR differences between the hypertensive and uninephrectomized mice [42‒44]. However, conscious AVE showed about 47% more sodium output in normotensive mice than hypertensive mice. This may be because the suppressed renal sodium excretion was further decreased due to the application of anesthesia. Although the sodium output was much less in the hypertensive mice than the normotensive mice, we did not test the increase in blood pressure during the AVE, which may be due to the short time of monitoring after AVE.

In summary, this study introduced and validated a novel approach for assessing renal functional capacities in conscious animals. This innovative method holds the potential to enhance diagnostic and prognostic capabilities in both acute and chronic kidney diseases. Furthermore, the new AVE procedure in conscious animals offers the advantage of repeated use in monitoring kidney functional capacities, in contrast to the AVE conducted in anesthetized animals, which is typically a terminal experiment.

All animal use and welfare adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This study followed the protocol approved by the Institutional Animal Care and Use Committee (IACUC, IS000011615R) at the University of South Florida.

The authors declare that they have no competing financial interests or personal relationships that could have potentially influenced the findings presented in this paper.

This work was supported by American Heart Association Career Development Grant (18CDA34110441) and National Institutes of Health Grant (R01DK122050).

M.T., C.L., and L.W. designed the research study and generated the draft of the manuscript. M.T. generated the hypertensive model, performed the acute volume expansion experiment, performed data analysis, and prepared figures. C.L. conducted radio-transmitter implantation, measured the blood pressure, and performed data analysis. M.N. performed GFR and sodium excretion measurement. C.P. measured plasma creatinine and urine ACR. L.W. participated in the design of the study and contributed to the interpretation of results and revision of the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Minh H. Tran and Catherine Y. Liu contributed equally to this work.

All the data and materials supporting the findings of this study are available within the article. Further inquiries can be directed to the corresponding author.