Neurourology and Urodynamics 20:287±295 (2001)

In¯uence of Gender on the DiurnalVariation of Urine Production andMicturition Characteristics of the RatFrank Schmidt,1 Yasukuni Yoshimura,2 Rong-Xiang Ni,3

Shawna Kneesel,3 and Christos E. Constantinou3*

1lnstitute of Experimental Clinical Research, Skejby, University of Aarhus, Aarhus, Denmark2Department of Urology, Fukushima Medical College, Fukushima, Japan3Spinal Cord Injury Center, Department of Veteran Affairs and Department of Urology,Stanford University, Stanford, California

The diurnal variation in the frequency/volume characteristics of male and female consciousrats was evaluated with reference to ¯uid consumption and urine production. Baseline valuesof the micturition volume and frequency of nine male and 10 female SD adult rats weremeasured over a 24-hour time period. The level of initial hydration conditions wasstandardized with 5 ml of water administered orally. With animals in a metabolism chamberhaving free access to water, the total volume of water consumed, the frequency/volumecharacteristics during micturition and the urine production rate were derived from themeasurements of voided volume as detected by a digital balance. To establish reliability ofmeasurements two separate micturition studies were done per rat at an interval of 1 week.Mean frequency of micturition and mean volume voided per micturition and urine productionrate were computed in 3-hour time bins and represented over the 24-hour period. In additionthe mean values of the number of micturitions and mean micturated volumes during theday/dark cycle were evaluated. The results show signi®cant gender speci®city in waterconsumption, urine production, and diurnal variations in micturition frequency/volumecharacteristics. Females consistently consume signi®cantly larger amounts of water (83%)than males while urine production rate was correspondingly higher in females. It is concludedthat water consumption and urine production are gender-speci®c. Because higher volumes ofwater are imbibed by females than males, the frequency/volume characteristic of micturitionin the rat is also gender-speci®c. Data suggest that the volume voided per micturition dependson the urine production rate. Neurourol. Urodynam. 20:287±295, 2001. ß 2001 Wiley-Liss, Inc.

Key words: urine production; micturition; metabolic; circadian rhythmicity; bladder

INTRODUCTION

Fluid intake, urine production, and urine storage by the bladder, like otherphysiologic processes, vary according to the time of day or night as well as physicalactivity. The storage function of the bladder is principally determined by its bio-

ß 2001 Wiley-Liss, Inc.

*Correspondence to: Christos E. Constantinou Ph.D., Department of Urology, Room S287, ClinicalSciences Building, Stanford University Medical School, Stanford, CA 94305. E-mail: ceconst@leland.stanford.edu

Received 19 November 1999; Accepted 12 July 2000

mechanical properties to expand as well as by inherent central re¯ex pathwayscontrolling the threshold volume for micturition [Vaughan and Satchell, 1995; de Groat,1998]. Functionally, the storage properties of the bladder can be de®ned in part by thefrequency/volume characteristics of micturition. Recent experimental evidenceindicates that diuresis increases the bladder's contractile strength and suggests thatthe rate of urine production plays an important role in regulating the mechanism ofmicturition. In view of these observations the present paper examines the storagecapability of the bladder in the context of ¯uid intake as well as urine production and thefrequency/volume characteristics of micturition.

Previous studies examined the age-dependent differences in the micturitioncharacteristics between male and female rats, and have shown that the micturitionfrequency/volume characteristics are dependant on the dark/light cycle [Longhurst etal., 1992a,b]. The present study was designed to evaluate the diurnal rhythm of urineproduction and micturition in order to identify the in¯uence of variations in urineproduction rate. The practical signi®cance of this approach rests on the fact that the newpharmacological agents, used clinically for the treatment of urinary urge incontinenceand other symptoms associated with an overactive bladder, or obstruction mayconfound the outcome of treatment [Andersson, 1997; de Groat, 1997]. This is becausepharmacological agents also modulate the urine production rate [Longhurst et al., 1991;Tillig and Constantinou, 1996; Lee et al., 1998]. For this reason it would be appropriateto identify the mechanism of interaction between the naturally occurring variation ofurine production and that which is drug-induced. Indeed the concept of overtlymodulating the urine production rate has been put into therapeutic use in the enureticpatient using DDAVP [Djurhuus and Rittig, 1998]. The possibility of establishing thetime in the diurnal cycle to apply pharmacological intervention for the suppression ofurine production is of critical importance in evaluating the symptoms of urinaryincontinence principally because urine production determines the amount of urine tobe stored by the bladder. Furthermore, since symptom scores are commonly usedin evaluating the effectiveness of pharmacological therapy for incontinence, it isappropriate to evaluate the pharmacological effects on the basis of the diurnalfrequency/volume characteristics.

In view of these considerations the protocol of the present study was designed toevaluate the contribution of gender on ¯uid consumption, urine production, and diurnalvariations in the volume/frequency parameters of micturition of the conscious animal.

MATERIALS AND METHODS

Animals

Male and female Sprague-Dawley rats, having the identical age of 66 days, werepurchased from Harlan Labs. Animals had free access to food and water, and each ratwas housed in a temperature-and ventilation-controlled environment and subject to12/12 dark/light cycle. All studies were carried with the approval of the protocol by theStanford University/VA animal care committee.

Micturition Studies of Conscious Rats

Before the initiation of any measurements 10 male (399� 15 g) and 10 female(249� 3 g) SD adult rats were acclimatized for 7 days in metabolic cages with 12/12-

288 Schmidt et al.

hour dark/light cycle and having free access to food and water. The baseline parametersof the micturition characteristics were obtained in terms of volume and frequency.To obtain a reliable measure of the diurnal variation in the volume and frequencyof micturition, ¯uid consumption, and urine production rate rats were monitoredcontinuously over a 24-hour time period in metabolism cages. To standardize the initialhydration conditions a 5 ml oral dose of water was administered and while in themetabolism cage rats had free access to water. Because of gender differences in the rats'body weight of the same age, data were normalized with respect to weight. Micturitionvolume/frequency characteristics were derived from the measurements of voidedvolume detected by a digital balance placed below the metabolism cage and connectedto a PC. Experiments were performed by placing animals in a (Nalgene) metabolic cageat 09:30 each morning. During the time of monitoring the animals had free access towater while urine from each rat was collected in a container that rested on an electronicscale (Sartorius, PT12O) located beneath each cage. The scales were connected to acentral PC computer via a RS232 interface and using a BASIC data acquisition programthe cumulative volume micturated was recorded for a period of 24 hours.

Statistics

Two studies were performed for each animal and the data collected from eachstudy were pooled and averaged. Data presented are expressed in terms of kg of animalbody weight, and frequency/volume parameters were averaged into 3-hour bin widths.Urine production rate was evaluated from the ratio of voided volume/period of timefrom last micturition for each 3-hour bin width. Statistical comparisons were madeusing the unpaired Student t-test; P values < 0:05 were considered signi®cant.

RESULTS

Water Consumption

Gender differences were observed in water consumption demonstrating thatfemales consistently imbibe larger amounts of water than males. Over the entire 24-hourperiod of observation females consistently consumed signi®cantly larger amounts ofwater, 61.0� 8.9 ml/kg vs. 33.2� 4.4 ml/kg (P < 0:001), in comparison to males.

Urine Production Rate

Diurnal variations in the amount of urine produced over a 24-hour period ofobservation were recorded. A typical 24-hour pattern of urine production, observed in amale and female animal is given by Fig. 1. As shown by this illustration, the diurnalgender-related variation in urine production rate by the females is characterized byconsistently larger volumes of urine voided throughout the entire 24-hour period ofobservation, while males void more frequently particularly in the early hours 02:00 to07:00.

Figure 2 shows the variation in the rate of the average urine production, calculatedfrom the cumulative volume voided over each 3-hour time bin. As indicated by Fig. 2,the qualitative diurnal pattern of urine production rate is approximately similar in bothmales and females. Quantitatively however, females produce more urine during both thedaylight and night time periods. Overall, during the 24-hour period of observation males

Gender and Micturition 289

produced 60.0� 5.7 ml/kg, which is signi®cantly lower (P < 0:01) than the femaleurine production which was 85� 8.7 ml/kg.

Frequency of Micturition

The diurnal pattern of the frequency of micturition, computed as the number ofmicturitions per 3-hour time period, is given by Fig. 3. As illustrated by Fig. 3, there is nosigni®cant gender-related difference in the number of micturitions between malesduring the ®rst 6 hours of daytime observation. However, as shown in Fig. 3, thefrequency of micturitions of both the males and females increase systematically. Thenumber of micturitions of the males increases at a higher rate until the male micturitionfrequency is signi®cantly higher that of the females. As indicated by Fig. 3 this occursduring the early morning hours between 02:00 and 08:00.

Volume per Micturition

Figure 4 shows the pattern of diurnal values of the volume voided per micturition,corresponding to the frequency illustrated above in Fig. 3. As shown in Fig. 4 while thevolume voided by females is signi®cantly higher than males during some of the periodsevaluated, this difference is not as prominent as frequency. Instead, the diurnal pattern ofvolume/micturition mirrors urine production more closely.

Fig. 1. Typical micturition patterns of a male and a female rat housed in metabolic cages as detected by adigital balance. The cumulative weight of urine is shown to increase after each micturition in stepwiseincrements. As indicated the pattern of micturition in the females consists of relatively constant volumesduring the 24-hour period of observation while the males void more frequently smaller amounts of urineduring the early hours of the morning.

290 Schmidt et al.

DISCUSSION

The present study demonstrated that frequency and associated volume voidedduring each micturition are in¯uenced by gender as well as the time of day or night atwhich observations are made. In addition it is shown that there is a gender difference inthe amount of water consumed which in turn may be responsible for modulating thefrequency/volume characteristics. Overall the evidence suggests that female ratsdemonstrate the highest diurnal variability in both intake and output. When normalizedfor animal weight the reported gender differences in water consumption are in con¯ictwith the observations of Chun et al. [1990] in terms of the parameters of waterconsumption and urine output but in agreement with those reported by Longhurst et al.[1992a]. The discrepancy in these observations may be explained by the fact that Chunet al. [1990] did not normalize their data with reference to animals' body weight.

It was speculated that hormonal variations may be a possible reason for theobserved higher water consumption by the females. This possibility has been examinedby Longhurst et al. [1992b], who evaluated the in¯uence of ovariectomy and estradiolreplacement on bladder function, demonstrating that ovariectomy had no effects onmicturition characteristics. However, estradiol treatment of ovariectomized rats didcause signi®cant increases in water consumption and urine excretion, and in mean andmaximal micturition volumes compared to both ovariectomized and sham-operatedrats. Although estradiol reversed the effects of ovariectomy on bladder function,indirect effects were evident which were probably the result of estradiol-induced

Fig. 2. Comparison of the diurnal rhythm of urine production between males and females over a 24-hourtime period; data corrected for body weight. As shown, throughout the period of monitoring the urineproduction by the females is signi®cantly higher than that by males.

Gender and Micturition 291

polyuria. Subsequent substantiation of the importance of the hormonal effect in maleswas demonstrated by Eika et al. [1994] who evaluated the modulation of bladderfunction in castrated and shamoperated males and females. These studies showed thatdifferences in water consumption and urine excretion between male and female were inagreement with our data in the present study when corrected for body weight. Inaddition the data of Eika et al. [1994] suggest that gender differences in waterconsumption and urine production were not found in diabetic animals. A possibleinterpretation of this observation may be that the higher rates of water consumption andurine production by the female can be considered the normal state, and it is only in casesof pharmacological intervention, such as streptozotocin in this case, that deviationsfrom the physiological state may occur. The results of the present study show that duringperiods with a high urine production rate it is shown that both genders void at largervolumes than during periods of low urine production rate. This suggests that the storage

Fig. 3. Gender differences in the variation of the diurnal pattern of micturition frequency as averaged overthree intervals of time. In male and female animals, during the daytime hours, the number of micturitions isapproximately equal. With the onset of the night, divergence of observed frequencies is evident betweengenders with the male voiding more frequently than the female. The number of micturitions is signi®cantlyhigher in the males during the early hours of the morning.

292 Schmidt et al.

function of the bladder could be in¯uenced by urine production rate. These observationsare consistent with the concept that urine production in¯uences bladder function whichhas been introduced by Tammela et al. [1994] and Levin et al. [1995] in studies thatevaluated the temporal changes in bladder function during Furosemide treatment. Thesestudies have shown that modest increases in bladder mass and increases in contractilefunction are a normal and bene®cial physiologic response to diuresis and demonstratethat functional bladder capacity increases immediately upon the initiation of diuresiswith sucrose or Furosemide, as does the frequency of micturition. In view of theseobservations the importance of water intake and consequently urine production, on thefunction of the bladder has been extended by Ohnishi et al. [1999] who demonstratedexplicitly that diuresis causes an increase in contractile strength consequentlyprotecting the bladder from the contractile dysfunction associated with partialobstruction. In addition to the in¯uence of diuresis noted above, it is appropriate toconsider that there are gender differences in the central and peripheral distribution ofreceptors [Latifpour et al., 1990; Vaughan and Satchel, 1995; Morita et al., 1998] thatmay affect the contractile function of the bladder. In addition to variations in a and breceptors contractility may be in¯uenced by estrogen and NO shown to reduce bladderblood ¯ow [Pontari and Ruggieri, 1999] and whole body production of NO is greater in

Fig. 4. Gender differences in the volume voided/micturition. While the voided volumes are approximatelyequal during daytime, the females void with larger volumes during the night.

Gender and Micturition 293

healthy women than in men [Forte et al., 1998]. Indeed the age-related effects of 17b-estradiol supplementation on micturition of ovariectomized rats show that estrogenimpacts on bladder function in an age-dependent way, depending at which point in timeit is administered, and the different age-related responses to exogenous estrogenindicate that endogenous estrogen plays a major role in the development of normalbladder function [Diep and Constantinou, 1999].

Finally the results obtained from the present study suggest that, in experimentalprotocols designed to be used for the pharmacological evaluation of new agents thataffect bladder function, it may be essential to consider the contribution of gender anddiurnal variation as contributing factors affecting the outcome. Indeed considerationshould be given to the possibility that speci®c gender and diurnal in¯uence of the drugmay directly in¯uence urine production and thereby not only affect the demands placedon the storage function of the bladder but also in¯uence the symptom scores used toevaluate the ef®cacy of the applied pharmacological treatment. In view of theseobservations it is important to consider whether the observed diurnal and gendervariability in water intake and frequency/volume is present in humans.

ACKNOWLEDGMENTS

This study was supported by a grand from the Enuresis Foundation to FS and by aParalyzed Veterans Summer Fellowship to SK. Research work was carried out at theSpinal Injury Center, Of®ce of Research and Development, Rehabilitation R and DService, Department of Veteran Affairs.

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