Effects of Long-Term Caffeine Consumption on Renal Function in Spontaneously Hypertensive Heart Failure Prone Rats

Abstract

Our previous studies supported the hypothesis that prolonged administration of caffeine to animals with high-renin hypertension causes progressive deterioration of renal function. However, thus far this hypothesis has been tested with only a few animal models of hypertension. The aim of this study was to test this hypothesis further by investigating the effects of long-term caffeine consumption on renal function in adult spontaneously hypertensive heart failure (SHHF/Mcc-fa^cp) rats, another model of high-renin hypertension. Lean, male, 9-month-old SHHF/Mcc-fa^cp rats were randomized to receive either normal drinking water (control group) or drinking water containing 0.1% caffeine (caffeine group) for 20 weeks. No changes in body weight, food and fluid intake, urine volume, and sodium and potassium excretion were found in conscious SHHF/Mcc-fa^cp rats after 10 or 20 weeks of caffeine treatment. However, caffeine treatment accelerated the time-related decline in renal function and augmented urinary protein excretion. Ten weeks into the protocol, creatinine clearance was 3.6 ± 0.4 and 5.7 ± 0.9 L/kg/day in the caffeine group and control group, respectively (p < 0.02), whereas 20 weeks into the study, creatinine clearance was similarly diminished in both groups. Proteinuria was greater in the caffeine group compared with the control group at both 10 (928 ± 131 vs. 439 ± 21 mg/kg/day, respectively; p < 0.02) and 20 weeks (1,202 ± 196 vs. 603 + 30 mg/kg/day, respectively; p < 0.01) into the protocol. After 20 weeks, all animals were anesthetized and instrumented. Caffeine treatment for 20 weeks had no effects on blood pressure, heart rate, or vascular resistance in four examined vascular beds (abdominal aorta and renal, carotid, and mesenteric arteries). No changes in renal hemodynamics and electrolyte excretion were found, whereas significantly lower glomerular filtration rate (GFR; inulin clearance) and creatinine clearance (p < 0.05) were observed in caffeine-treated animals. These data support our hypothesis that prolonged consumption of caffeine has adverse effects on renal function, in high-renin hypertension.

Previous studies demonstrated that caffeine consumption (6 weeks) exacerbates hypertension and has detrimental effects on renal function in two-kidney, one-clip (2K-1C) rats, a model of renin-dependent renovascular hypertension ^(1-5). In 2K-1C rats, caffeine treatment decreases creatinine clearance by ∼50%, and that change is accompanied by a severalfold increase in plasma renin activity (PRA) and by glomerular injury similar to that seen in malignant hypertension ⁽¹⁾. Blockade of the renin-angiotensin system (RAS) with an angiotensin-converting enzyme inhibitor prevents the adverse effects of caffeine in 2K-1C rats suggesting that the ill effects of caffeine in high-renin hypertension are mediated by activation of the RAS. This is further supported by the fact that in 1-kidney-1-clip renovascular hypertensive rats and in rats with genetic hypertension, animal models with normal or low-renin hypertension, caffeine treatment does not affect renal function and blood pressure ⁽¹⁾.

Furthermore, very recently, we performed studies in SHHF × ZDF (spontaneously hypertensive heart failure × Zucker Diabetic Fatty) hybrid rats, a new model of hypertension, obesity, hypertriglyceridemia, and insulin resistance, that also has compromised renal function and overt proteinuria ⁽⁶⁾. In that study (manuscript in preparation), long-term caffeine consumption (0.1% in drinking water over 8 weeks) significantly decreased creatinine clearance, tended to increase urinary protein excretion, and increased kidney weight.

Several mechanisms may be involved in the detrimental effects of caffeine on renal function in high-renin hypertension. It is well accepted that pharmacologically relevant concentrations (2-10 μg/ml; i.e., concentrations seen in humans after moderate intake of coffee) of caffeine block adenosine receptors ⁽⁷⁾, that endogenous adenosine inhibits renin release ^(8-11), and that caffeine, by blocking adenosine receptors, increases basal renin release as well renin release in response to various stimuli in both animals and humans ^(12-16). Adenosine and angiotensin II play an important role in regulating vascular tone of afferent and efferent arterioles and thereby modify the intraglomerular pressure. Adenosine at low doses activates A₁ receptors and constricts predominantly the afferent arteriole, whereas higher doses of adenosine dilate the efferent arteriole via A₂ receptors ^(17,18). Both effects result in a marked decrease in intraglomerular pressure. In contrast, angiotensin II preferentially constricts the efferent arteriole, and thereby increases intraglomerular pressure ^(19,20). Therefore, by blocking adenosine receptors, caffeine may simultaneously increase intrarenal levels of angiotensin II and decrease the renovascular effects of adenosine, thus causing a greater percentage of the arterial blood pressure to be transmitted to the glomerular capillaries. Glomerular hypertension would than result in renal injury.

We previously demonstrated ⁽²¹⁾ that caffeine increases renin release in another model of hypertension with high PRA (i.e., the lean male SHHF/Mcc-fa^cp rat; ^22,23). If our hypothesis is correct that caffeine causes renal dysfunction in high-renin hypertension, then caffeine should also diminish renal function in SHHF/Mcc-fa^cp rats. The purpose of this study was to test this prediction.

MATERIALS AND METHODS

Animals

This study examined the effects of long-term caffeine consumption on renal function and hemodynamics in the SHHF/Mcc-fa^cp (abbreviated SHHF) rats. SHHF rats were selectively bred for spontaneous hypertension and heart failure and are allelic for fa and cp obesity genes. All SHHF rats, a relatively new model of spontaneous hypertension and heart failure, spontaneously, irrespective of the presence or absence of obesity and without any pharmacologic treatment or surgical intervention, develop dilated cardiomyopathy. The cardiac hypertrophy is noticeable as early as age 12 weeks and is fully developed at age 5-6 months ^(24-26), and the lean male SHHF rats die of overt congestive heart failure at the age of 15-18 months. The development of heart failure in SHHF rats is associated with increased blood pressure in a range ^(25,26) similar to that observed in the spontaneously hypertensive rat (SHR; ²⁷). However, the SHHF/Mcc-fa^cp rats are genetically predisposed to develop hypertensive heart failure at an incidence of 100%, whereas only some aged SHRs die of heart failure ⁽²⁷⁾. With the onset of severe heart failure in SHHF/Mcc-fa^cp rats, blood pressure tends to return to normotensive levels ^(25,26). There is strong gender (male vs. female) and genotype (obese vs. lean) influence on progression of heart failure, renal function, and glucose homeostasis. Obese male SHHF rats develop heart failure and die earlier, have more compromised renal function, and develop overt non-insulin dependent diabetes mellitus. In contrast to obese male SHHF rats, lean counterparts develop heart failure later, have less compromised renal function, and demonstrate modest insulin resistance (no increase in fasting glucose, insulin, or lipids) that is commonly seen in other hypertensive models ⁽²⁶⁾.

All animals used in this study were lean; however, because of lack of a genetic marker, it was not known if animals were homozygous lean (+/+) or heterozygous lean (+/fa^cp). A total of 19, 8-month-old, male, SHHF rats (body weight, 428.9 ± 7.6 g), obtained from Genetic Model Inc. (Indianapolis, IN, U.S.A.), were used in this study. Animals were housed in the University of Pittsburgh Animal Care Facility for 4 weeks before being used in this study. Temperature, relative humidity, and the light cycle were kept at 22°C, 55%, and 12 h (7:00 a.m. to 7:00 p.m.), respectively. Animals were fed Wayne Rodent Blox 8604 (sodium, 135 mEq/kg and potassium 254 mEq/kg; Madison, WI, U.S.A.) and water ad libitum. Institutional guidelines for animal welfare were followed.

Long-term study (metabolic cages)

Before initiating treatment, baseline 24-h urine samples were collected. Urine samples also were collected after 10 weeks of treatment and at the end of the experiment (after 20 weeks of treatment). In this regard, animals were placed in standard metabolic cages for 2 days before 24-h food and water intake and urine volume were measured. Urine samples were analyzed for creatinine, protein, sodium, and potassium concentrations. A volume of blood (1.0 ml) was drawn from the tail vein, and plasma obtained from those samples was used for determination of electrolyte, creatinine, and renin activity levels. Electrolytes and creatinine were measured with a flame photometry (model IL943 flame photometer; Instrumentation Laboratory, Lexington, MA, U.S.A.) and creatinine analyzer (Creatinine Analyzer 2; Beckman Instruments, Inc., Fullerton, CA, U.S.A.), respectively. Urine protein was determined by using the Sigma Diagnostics Total Protein kit (Sigma, St. Louis, MO, U.S.A.).

After the initial 24-h urine collection, animals were randomized to either continue drinking tap water (control group, n = 9) or to receive drinking water containing 0.1% of caffeine (caffeine group, n = 10). In rats, prolonged administration of 0.1% of caffeine in drinking water results in plasma levels of caffeine (5-10 μg/ml) that are seen in humans after moderate (two to three cups of coffee) caffeine consumption, and those levels of caffeine block the effects of exogenous adenosine on blood pressure ⁽¹⁾. Body weight was determined once a week, for the entire study. During the treatment, one animal in the control group and two animals in caffeine group died. Only one of the rats in the caffeine group developed overt heart failure (rapid increase in body weight due to fluid retention, ascites, cyanosis, labored breathing) and died.

Acute experiments

After 20 weeks of treatment, at age 13 months, animals underwent surgical instrumentation, and acute experiments were carried out. In brief, rats were anesthetized with pentobarbital (50 mg/kg, i.p.), and a heat lamp was positioned above the animal. The body temperature was maintained at 37 ± 0.5°C by adjusting the position of the lamp above the animal. Body temperature was monitored by a rectal temperature probe (Electro-therm, TM99A; Cooper Instrument Corp., Middlefield, CT, U.S.A.). The animal's trachea was cannulated (PE-240) to maintain a patent airway, and two polyethylene catheters (PE-50) were inserted into the left jugular vein. One catheter was used for administration of supplementary boluses of anesthetic, and the second was used for infusion of [¹⁴C]inulin.

An additional polyethylene cannula (PE-50) was inserted into the left femoral artery and connected to a Micro-Med digital blood pressure analyzer (Micro-Med, Inc., Louisville, KY, U.S.A.) for continuous monitoring of mean arterial blood pressure (MABP) and heart rate (HR). The blood pressure analyzer was set to time-average MABP and HR at 1-min or 5-min intervals (printer) and 5-s intervals (digital display).

Next, a transit-time blood flow probe (model T206; Transonic System Inc., Ithaca, NY, U.S.A.) was placed around the left carotid artery to monitor blood flow continuously. Later, the animal's abdominal cavity was opened with a midline incision, and the right kidney removed. The abdominal aorta and superior mesenteric artery were cleaned, and flow probes were placed on those arteries for measurement of hindlimb and mesenteric blood flows. The left ureter was then cannulated (PE-10) to collect urine continuously. A transit-time blood flow probe was placed around the left renal artery to monitor renal blood flow (RBF) continuously. A 22-gauge needle was placed into the left renal vein for sampling of renal venous blood, and an i.v. infusion of inulin [carboxyl-¹⁴C] (0.5 μCi bolus followed by 0.035 μCi/min infusion) was initiated. Animals were allowed to stabilize for 45 min before 30-min urine samples were collected into a preweighed microfuge tubes. Twenty microliters of urine from these samples was placed in a scintillation vial to measure ¹⁴C radioactivity with a liquid scintillation analyzer (model 2500TR; Packard Instrument Company, Downers Grove, IL, U.S.A.). The remaining urine was used to measure the urinary concentration of creatinine, sodium, and potassium, as described earlier. At the midpoint of the clearance period, a 0.5-ml arterial blood sample was collected for measurement of hematocrit, plasma ¹⁴C radioactivity, and plasma sodium, potassium, and creatinine. At the end of the urine collection, 1.0 ml of arterial (carotid artery) and renal venous blood was withdrawn for measurement of PRA and catecholamines.

Plasma renin activity analysis

PRA was determined by using the Rainen Angiotensin I [125-I] Radioimmunoassay Kit (Du Pont NEN Research Products, Boston, MA, U.S.A.) with appropriate modifications and validations ⁽²⁸⁾. PRA was expressed as nanograms angiotensin I generated/ml/h and was calculated by the RIASmart Software Package (Packard Instrument Company, Meridan, CT, U.S.A.) by using a weighted LOGIT analysis program.

Catecholamine analysis

Catecholamines were assayed by high-performance liquid chromatography with electrochemical detection. For sample analysis, a commercial ESA Plasma Catecholamine Analysis Kit (ESA Inc., Bedford, MA, U.S.A.) was used. To monitor recovery, an internal standard (DHBA) was added to each sample. The chromatographic system was composed of an Isco pump (model 2350) and gradient programmer (model 2360), and ChemResearch Data Management System (Isco, Lincoln, NE, U.S.A.), along with an ESA Catecholamine HR-80 Column and Coulochem Electrochemical Detector (model 5200).

Statistical analysis

Results are presented as mean ± SEM. Statistical analyses were performed by using the Number Crunchers Statistical System (Kaysville, UT, U.S.A.). Group comparisons were done by one- (1F) or two-factor (2F) nested (hierarchic) analysis of variance (ANOVA), or by Student's t test, as appropriate. If ANOVA indicated a significant difference among the means, specific comparisons were made with a Fisher's LSD test.

RESULTS

Studies in metabolic cages

Table 1 summarizes metabolic cage data obtained before the beginning of caffeine treatment and shows that there were no significant differences in baseline values for the examined parameters between the two groups. No changes in body weight, food and fluid intake, urine volume, and sodium and potassium excretion were found after 10 and 20 weeks of treatment (data not shown). Long-term caffeine consumption accelerated the rate of the decline in renal function (Fig. 1). After 10 weeks of treatment, significantly lower creatinine clearances (p < 0.02, expressed both as absolute value and as a percentage of initial value, Fig. 1, middle and bottom, respectively) were observed in caffeine-treated animals as compared with the control animals. After 20 weeks of treatment, the decline in creatinine clearance was similar in both groups, reaching 40% reduction as compared with the baseline values.

TABLE 1: Baseline values from experiments in metabolic cages

FIG. 1: Time-course ot the effects of prolonged caffeine consumption on plasma creatinine (top) and creatinine clearance expressed in absolute values (middle) and as percentage changes from baseline values (bottom). a, p < 0.05, compared with control baseline: b, p < 0.05, compared with caffeine baseline.

As shown in Table 1 and Fig. 2, at the age of 9 months, animals already excreted significant amounts of proteins. Caffeine treatment augmented urinary protein excretion (Fig. 2), and after 10 weeks of treatment, significantly higher values for urinary protein excretion (p = 0.02) were determined in the caffeine group compared with the control group. This effect was even more prominent after 20 weeks of treatment (p = 0.01, two-factor ANOVA).

FIG. 2: Time-course of the effects of long-term caffeine consumption on urinary creatinine excretion (top), urinary protein excretion (middle), and protein/creatinine ratio (bottom). a, p < 0.05, compared with control baseline; b, p < 0.05, compared with caffeine baseline.

Hemodynamic effects

The effects of 20-week treatment with caffeine on blood pressure and blood flow in different vascular beds in anesthetized SHHF rats are reported in Table 2. Treatment with caffeine did not alter either blood pressure or blood flows in the four examined vascular beds (abdominal aorta, and carotid, mesenteric, and renal arteries) that account for >80% of total blood flow in rats. There were no significant differences in the sum of vascular resistances in the four examined vascular beds.

TABLE 2: Hemodynamic parameters after 20 weeks of treatment with caffeine

Renal function

Renal hemodynamic and excretory function in anesthetized SHHF rats after 20 weeks of treatment are shown in Table 3. Prolonged caffeine consumption did not alter renal hemodynamics (RBF, RVR, RPF), and no changes were found in urine volume, sodium and potassium excretion, and filtration fraction. However, significantly lower GFRs (inulin clearance) and creatinine clearances (p < 0.05) were observed in caffeine-treated animals.

TABLE 3: Renal hemodynamic and excretory function after 20 weeks of treatment with caffeine

Neurohumoral status

The effects of long-term caffeine consumption on neurohumoral status are shown in Figs. 3 and 4. After 20 weeks of treatment, both in metabolic studies (tail-vein samples) and acute experiments (renal vein and carotid artery), PRA tended to be higher in caffeine-treated animals (Fig. 3), but these changes did not reach statistical significance. No differences in plasma norepinephrine levels in carotid artery blood samples were found, whereas significantly increased norepinephrine concentrations (p < 0.03) in renal vein plasma samples were found at the end of the treatment (Fig. 4).

FIG. 3: Plasma renin activity, after 20 weeks of caffeine consumption, in conscious (tail-vein samples) and anesthetized (carotid artery and renal vein samples) spontaneously hypertensive and heart failure (SHHF) rats.

FIG. 4: Norepinephrine levels after 20 weeks of caffeine consumption in anesthetized spontaneously hypertensive and heart failure (SHHF) rats.

DISCUSSION

This study demonstrates that prolonged caffeine consumption has adverse effects on renal function in SHHF/Mcc-fa^cp rats, a genetic model of hypertensive cardiomyopathy, high renin levels, and renal dysfunction. Compared with age-matched normotensive and spontaneously hypertensive rats, lean male SHHF rats have increased PRA ^(22,23). Moreover, PRA, atrial natriuretic peptide, and aldosterone levels progressively increase with age and progression of heart failure ⁽²³⁾, and the changes in the neurohumoral status are accompanied by increased urinary protein excretion ⁽²⁶⁾. In this study we used old lean SHHF rats, 9-13 months, an age at which animals still have compensated heart function as compared with age-matched SHRs (unpublished data; ²¹). Long-term caffeine consumption had no effects on blood pressure and blood flow in the four examined vascular beds (carotid, mesenteric, and renal arteries and abdominal aorta) that account for >80% of blood flow in rats. Despite no changes in systemic and renal hemodynamics, prolonged caffeine ingestion accelerated the time-related decline in renal function (i.e., decrease in creatinine clearance) and augmented urinary protein excretion. Accelerated decline in renal function and augmented proteinuria may be the result of accelerated development of heart failure due to caffeine treatment. However, in this study we also assessed heart function in situ (left ventricular catheterization and measurement of time-pressure parameters, data not shown) and found no differences in heart performance in vivo between control and caffeine-treated animals.

PRA levels determined in this study are comparable with those reported previously ⁽²³⁾ and confirm that in this animal model, an increase in PRA occurs before overt heart failure. Therefore, in the period preceding overt heart failure, this animal model is a high-renin hypertensive model that may be used to study the effects of different stimuli on renin secretion. In addition, in a previous study, we showed that short-term caffeine consumption increases basal renal renin secretion ⁽²¹⁾ in SHHF rats. In this study, after 20 weeks of treatment, caffeine tended to increase PRA levels, but this effect did not reach statistical significance. However, an increase in the activity of the intrarenal RAS cannot be ruled out.

In this study, after 20 weeks of caffeine treatment, a significant increase in renal vein norepinephrine plasma levels was observed. These findings are consistent with our previous demonstration that during baroreceptor activation, short-term caffeine administration augments renal sympathetic nerve activity (i.e., renal norepinephrine spillover; ²⁹). Whether enchancement of renal norepinephrine release by caffeine contributes to caffeine-induced renal dysfunction needs to be examined in future studies.

The fact that caffeine accelerated the decline in renal function in this animal model of high-renin hypertension with preexisting renal dysfunction causes concern about the effects of prolonged caffeine consumption on renal function, and the question about the possible mechanisms of the nephrotoxic effects of caffeine must be addressed.

Although caffeine is the most widely use drug in the world, the effects of caffeine on renal function have received little attention. This is surprising, particularly because caffeine is present in most analgesic combination drugs in which long-term use may lead to analgesic nephropathy. Limited experimental and clinical data ^(30-32) suggest that caffeine may be a direct nephrotoxin. However, in those studies, high bolus doses of caffeine were used (2.4 g in healthy humans and 200-300 mg/kg in rats), making questionable the clinical relevance of these findings. In rats with normal renal function, short-term (10 days) caffeine consumption augments urinary protein excretion ⁽³³⁾; however, this effect occurs only with near-lethal doses. Therefore, it seems very unlikely that in pharmacologically relevant doses, caffeine has direct nephrotoxic effects. More important, in doses present in various analgesic combinations, caffeine has been shown to potentiate the nephrotoxicity of mefenamic acid, a nonsteroidal antiinflammatory drug. Mefenamic acid-induced histological changes in the rat renal medulla (papillary necrosis) were much more severe with coadministration of caffeine ^(34,35). In mice with chronic nephritis and azotemia induced by prolonged exposure to psychological stress, caffeine consumption increased the weight of the adrenal glands, plasma corticosterone concentration, PRA, and blood pressure ⁽³⁶⁾. Moreover, in this same animal model, long-term caffeine consumption (5 months) increased blood urea nitrogen and induced more severe morphologic changes of chronic interstitial nephritis ⁽³⁷⁾.

Adenosine plays an important role in controlling glomerular hemodynamics. Adenosine via activation of high-affinity A₁ receptors constricts afferent arterioles, whereas in higher concentrations via A₂ receptors, adenosine dilates the efferent arteriole ^(17,18), and both effects result in decreased intraglomerular pressure. In addition, adenosine may affect intraglomerular pressure indirectly through activation of the RAS. Angiotensin II predominantly constricts the efferent arteriole and thereby increases intraglomerular pressure. In this regard, the detrimental effects of activation of the RAS in chronic renal failure have been well described. Endogenous adenosine tonically inhibits basal renin secretion, as well as the renin-release response induced by various physiological or pharmacologic stimuli ^(8-11). Therefore by inhibiting renin release, adenosine may attenuate the adverse effects of prolonged activation of the intrarenal RAS on renal hemodynamics and function. Blockade of this "adenosine brake" on renin release may in part explain the adverse effects of caffeine on renal function in high-renin hypertension.

Although there are no data regarding the effects of long-term adenosine-receptor blockade on renal function, experimental and clinical data suggest that prolonged blockade of adenosine receptors with caffeine may be disadvantageous in nephropathy. In patients with mild to moderate chronic renal failure, short-term administration of the A₁-receptor antagonist KF 453 augments renin release ⁽³⁸⁾. In rats, short-term blockade of A₁ receptors with DPCPX, a highly selective A₁-receptor antagonist, causes an increase in glomerular capillary pressure ⁽³⁹⁾, and long-term administration of caffeine enhances the ability of angiotensin II to increases filtration fraction ⁽⁴⁰⁾. These data suggest that blockade of renal adenosine receptors may augment angiotensin II-induced glomerular hypertension.

In contrast, pharmacologic manipulations that increase interstitial adenosine concentration (i.e., blockade of adenosine uptake by dipyridamole; ⁴¹) have beneficial effects on renal function in different experimental models of nephropathy and in humans. For example, in rats with puromycin-induced nephropathy, dipyridamole decreases urinary protein excretion ⁽⁴²⁾, and in streptozotocin-diabetic rats, characterized by increased urinary protein excretion, increased GFR, increased kidney weight, and decreased tubuloglomerular feedback, early daily treatment with dipyridamole corrects these changes in diabetic kidneys ⁽⁴³⁾. In humans with either diabetic nephropathy ⁽⁴⁴⁾ or nephrotic syndrome of different etiology ⁽⁴⁵⁾, dipyridamole decreases urinary protein excretion. More important, the decrease in proteinuria positively correlates with the decrease in filtration fraction ⁽⁴⁵⁾, suggesting that the antiproteinuric effects of dipyridamole may be the result of the decline in intraglomerular capillary pressure.

The results of this study, together with the available published data, suggest that long-term caffeine consumption may have adverse effects on renal function in high-renin hypertension with preexisting nephropathy. By blocking A₁ receptors at the level of the afferent arteriole, caffeine abolishes the vasoconstricting effects of endogenous adenosine and exposes the glomerulus to higher blood pressure. In addition, via A₁-receptor blockade, caffeine increases renin release and the activity of the circulating and intrarenal RAS. The predominant constrictor effects of angiotensin II on the efferent arteriole would result in further increased intraglomerular pressure. Thus caffeine may permit a larger percentage of systemic pressure to be transmitted to the glomerulus. Although this effect may be not important in normotensive subjects with healthy kidneys, in hypertensive patients with preexisting glomerular damage, caffeine consumption might have detrimental effects on renal function. Although further studies are required to determine the mechanisms by which caffeine affects renal function, these data provide strong evidence supporting an adverse effect of caffeine on kidney function in hypertensive nephropathy.

Acknowledgment: This work was supported by grants from the National Institutes of Health (HL55314 and HL35909).