The OS/+ mouse: a genetic animal model of reduced renal mass

The OS/+ mouse: a genetic animal model of reduced renal mass RUDOLFS K. ZALUPS Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia 31207 Zalups, Rudolfs K. The OS/+ mouse: a genetic animal model of reduced renal mass. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F53-F60, 1993.-The ROP [Ra/+ (ragged), OS/+ (oligosyndactyly), and Pt/+ (pintail)] mouse possessing the gene for oligosyndactylism (OS) was evaluated as a potential genetic animal model of reduced renal mass. Young male ROP mice that were heterozygotes with respect to the OS gene (OS/+) and their normal homozygote litter mates (+/+) were used in the present study. Approximately 50% fewer nephrons were present in the OS/+ mice than in the +/+ mice. Hypertrophic changes were detected in the epithelial cells in proximal convoluted and proximal straight tubules as well as in cortical collecting ducts in the kidneys of the OS/+ mice. Glomerular hypertrophy was also noted in the kidneys of these mice. After unilateral nephrectomy, further hypertrophic changes occurred in both OS/+ and +/+ mice, but the magnitude of compensatory growth was greater in the +/+ mice. From a functional standpoint, the OS/+ mice appeared to be capable of maintaining normal fluid and electrolyte homeostasis, even after unilateral nephrectomy. The findings from the present study indicate that the ROP OS/+ mouse can be a useful animal model to study the effects of reduced renal mass. oligosyndactylism; ROP mouse; compensatory renal growth A SELECTIVE BREEDING and irradiation experiment with mice at Oak Ridge National Laboratory in 1953 induced the formation of a mutant gene that causes the expression of a musculoskeletal abnormality called oligosyndactylism (4). Oligosyndactylism (OS) in mice is a condition in which fewer digits form on the feet (generally all four), mainly as a result of the fusion of the phalanges, metacarpals, and/or metatarsals in the second and third digits. The presence of oligosyndactylism can be diagnosed in utero as early as the 11th day of development, where there is a reduction of the preaxial margin of the foot plates (5). The OS gene is semidominant and is expressed in heterozygotes (OS/+). In homozygotes, the OS gene is lethal. In the late 1960s it was observed that some stocks of mice possessing the OS gene had nephrogenic concentrating defects (2). A couple of groups of investigators examined these mice as models of diabetes insipidus (9, 10). As part of their studies, they made some very interesting and important observations concerning the morphology of the kidneys in OS/+ mice. They found that the kidneys of the OS/+ mice were significantly smaller than the kidneys of +/+ littermates. This was due to the fact that the kidneys of the OS/+ mice contained only one-quarter to one-third the number of nephrons present in the kidneys of the +/+ mice, presumably due to some alteration in nephrogenesis. The investigators also noted hypertrophy in glomeruli and in tubular segments in the kidneys of the OS/+ mice. The hypertrophic changes observed in various segments of the nephron in the OS/+ mice could very well reflect adaptive changes that are similar to those that occur as a result of compensatory renal growth after a significant reduction of renal mass. Thus the OS/+ mouse may serve as a useful genetic mouse model to study both structural and functional changes associated with reduced renal mass. Unfortunately, very little, if any, further study of the OS/+ mouse as a model of reduced renal mass has been carried out since the two studies in the late 1960s. Therefore, it was the aim of the present study to further examine some structural and functional characteristics of the kidneys of OS/+ mice to determine the usefulness of the OS/+ mouse as a model of reduced renal mass. As part of this aim, structural and functional characteristics of the kidneys of OS/+ mice and +/+ mice were compared with each other and with those of OS/+ and +/+ mice that had a kidney removed surgically. The data from these experiments provide information on how closely the functional and structural changes in the OS/+ mice parallel the structural and functional changes associated with compensatory renal growth in +/+ mice that have undergone unilateral nephrectomy. In addition, the findings from the unilaterally nephrectomized OS/+ mice provide information on the capacity of the remaining renal tissue in the OS/+ mouse to adapt to a further deficit in the number of functioning nephrons. MATERIALS Animals AND METHODS Male heterozygous ROP [ Ra/+ (ragged), OS/+ (oligosyndactyly), and Pt/+ (pintail)] mice possessing the gene for the expression of oligosyndactylism were bred from offspring of a pair of breeder mice obtained from the Jackson Laboratory. The pair consisted of a male heterozygote mouse (OS/+) and a normal female littermate not possessing the gene for oligosyndactylism (+/+). All breeding of ROP OS/+ and +/+ mice was carried out in the animal care facility at Mercer University School of Medicine. During the process of breeding, the animals were provided a special laboratory food designed for the breeding mice. In the present study, 12 male ROP OS/+ and 12 male ROP +/+ mice were used. To study the process of compensatory renal growth in the male OS/+ and +/+ mice, unilateral nephrectomy was performed on six OS/+ and six +/+ mice. After surgery, the animals were allowed to recover for 12 days to allow for compensatory renal growth to occur. During the period of recovery, all the animals in the study were housed individually in plastic cages and were allowed water and a standard commercial laboratory diet (containing 20% protein) for mice ad libiturn. Surgical Procedure Before surgery, the OS/+ and +/+ mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). Once anesthesia was achieved, a small flank incision, penetrating the skin and abdominal muscles, was made on the right side of the body using a scalpel with a no. 11 blade. The right kidney was carefully removed from its retroperitoneal position and was exteriorized from the body using blunt dissection. A sterile 4.0 silk suture was tied around the right renal artery and vein and right ureter. Once the ligature was tightly secured, the kidney was excised distal to the ligature using a 0363~6127/93 $2.00 Copyright 0 1993 The American Physiological Society F53

l F54 THE OS/+ MOUSE AS A MODEL OF REDUCED RENAL MASS small set of surgical scissors. After the removal of the right kidney, the abdominal muscles were sewn together using sterile 4.0 silk suture, and the skin was approximated using sterile g-mm wound clips. Balance Studies At the end of the 12th day after surgery, the 24 animals in the study were placed in plastic metabolic cages. After a couple of days of acclimation to the cages, the animals were fasted for 24 h. During the subsequent 24 h, the animals were provided food ad libitum and urine was collected. At the conclusion of the 24-h collection period, the amount of urine excreted by the mice was determined gravimetrically and then the urine was stored for later analysis. In addition, all the animals were weighed and then anesthetized with an overdose of pentobarbital sodium (100 mg/kg). Once an animal was anesthetized, a O.&ml sample of blood was obtained from the inferior vena cava and the kidney was removed, trimmed of fat, and weighed quickly. The left kidney was cut in half along the transverse plain. A 2-mm midtransverse slice was obtained and placed in fixative for histological and morphometric analysis. The blood was spun down in a microcentrifuge, and the plasma fraction was removed and stored for future analysis. Analysis of Blood and Urine The concentration of creatinine in samples of plasma and urine from all the animals was determined spectrophotometritally using the Lancer Creatinine II Rapid Stat Diagnostic Kit (St. Louis, MO). Blood urea nitrogen (BUN) was determined spectrophotometrically using the Urea Nitrogen Calorimetric Endpoint Kit by Sigma (St. Louis, MO). The concentrations of sodium and potassium in samples of plasma and urine were determined by standard methods for flame spectrophotometry using a Bacharach flame photometer (Pittsburgh, PA). Processing of Tissue for Histological Analysis The midtransverse section of the left kidney from each mouse was fixed for 48 h at 4 C in a fixative containing 4% formaldehyde (vol/vol) and 1% glutaraldehyde (vol/vol) buffered with 11.6 g/l NaH,PO, and 2.7 g/l NaOH. The ph of the fixative was adjusted to 7.35, and the total osmolality of the fixative was -1,120 mosmol/kgh,o. After fixation, the samples of kidney were dehydrated in an ascending graded series of ethanols, cleared in xylene, and then embedded in paraffin wax. Sections (3-5 pm) were obtained from the blocks of embedded tissue using standard microtomy and were stained with hematoxylin and eosin. Morphometric Analysis All sections were first analyzed qualitatively using a Nikon Optiphot light microscope. Then the number of glomeruli present on each midtransverse section of kidney was determined. Subsequently, the following morphometric determinations were made using a computerized morphometric system: measurement of the area of each midtransverse section of kidney; measurement of the diameter of 35-50 proximal convoluted tubules in the cortex and 35-50 proximal straight tubules at the corticomedullary junction; measurement of the cross-sectional area of all the glomeruli on a section; and measurement of the approximated area of 35-50 epithelial cells in proximal convoluted tubules in the cortex, in proximal straight tubules at the corticomedullary junction, and in cortical collecting ducts. The cells that were evaluated in the cortical collecting ducts were presumed to be principal cells. The morphometric system used consisted of a Nikon Optiphot light microscope with a camera lucida drawing tube attachment, a 12-in. X 12-in. digitizing tablet, a computer with an Intel 386 microprocessor, and software that allows for morpho- metric measurements. The diameter of the tubules or the area of the glomeruli, tubular epithelial cells, or midtransverse sections was determined first by tracing the diameter of the tubules or the perimeter of the glomeruli, cells, or midtransverse sections on white paper using the camera lucida drawing tube. The midtransverse sections were viewed at a magnification of ~25, the glomeruli were viewed at x200, and the tubules and epithelial cells were viewed at a magnification of x400. Some estimations were made with respect to defining the lateral margins of the epithelial cells. The lengths of the traced diameters and areas of the traced images were determined later by digitizing the images using the digitizing tablet. After careful calibration of magnification using a stage micrometer, the actual diameters and areas were determined by computer analysis. The computer analysis provided a mean for each type of measurement for each animal. To minimize introduction of bias to the analysis, all the sections of kidney were coded before analysis and were decoded only after all the sections were analyzed. Calculations Glomerular filtration rate (GFR) was estimated by the clearance of creatinine using the following equation GFR=;xV Cr where [U,,] is the concentration of creatinine in milligrams per deciliter in the urine, [P,,] is the concentration of creatinine in milligrams per deciliter in the plasma, and V is the rate of urine flow in milliliters per minute. The absolute excretion of either sodium or potassium was calculated by the equation ux+ x v24 where UX+ is the concentration of either sodium or potassium in milliequivalents per liter and V,, is the amount of urine excreted in 24 h. Finally, the fractional excretion (FE) of either sodium or potassium was determined using the equation FE,+ ux+ =-A- p X+ where P xt is the concentration of either sodium or potassium in the plasma. Both glomerular filtration rate and the absolute excretion of either sodium or potassium were factored by renal mass in grams. Statistical Analysis Values in this manuscript are expressed as means & SE. Differences between means for any parameter measured in the four groups of mice were evaluated using the two-way analysis of variance, where one factor was genotype and the other factor was surgery. When F values from an analysis were found to be statistically significant at the 0.05 level of significance, Tukey s multiple comparison t test was used to determine which means were significantly different from one another. The level of significance used in this study was chosen a priori. RESULTS Body Weight and Renal Mass The weights of the animals and their kidneys at the time they were killed is presented in Table 1. There was no significant difference in body weight between the four groups of mice. However, there were some differences with respect to renal mass. The OS/+ mice had -38% less renal mass than the +/+ mice. This was true even when renal mass was factored by body weight (Table 1). As a result of unilateral nephrectomy, compensatory renal growth occurred in both +/+ and OS/+ mice during u,, p Cr

THE OS/+ MOUSE AS A MODEL OF REDUCED RENAL MASS F55 Table 1. Body and kidney weight data from normal and uninephrectomized male ROP OS/+ and +/+ mice Body Left Kidney Weight, g Weight, g Midtransverse Cross-Sectional Area of I,eft Kidney, mm2 Left Kidney/ Body Weight, 5%, Total Renal Mass, g Total Renal Mass/ Body Weight, 5% +/+ 20.7+1.3 0.133t0.007 15.54kO.71 0.64+0.01 0.263t0.014 1.28t0.03 os/+ 20.8kl.O 0.083t0.006* 11.60t0.58* 0.40~0.01* 0.163&0.009* 0.79t0.02* NPX +/+ 21.1t0.8 0.199t0.009t 22.19+_0.53f- 0.94+_0.02t 0.199t0.009f- 0.94+0.02t NPX OS/+ 18.2kl.O 0.102tO.O03*t 11.84t0.44* 0.56k0.02*+ 0.102+0.003*t 0.56&0.02*-t Values are means t SE; n = 6 for all groups. OS/+, mice possessing the gene for the expression of oligosyndactylism; +/+, normal littermates of OS/+ mice; NPX, uninephrectomized. * Significantly different (P < 0.05) from the mean for the corresponding group of +/+ mice. t Significantly different (P < 0.05) from the mean for the corresponding group of mice that were not uninephrectomized. the period between surgery and death, although the magnitude of compensatory renal growth appeared to be greater in the uninephrectomized (NPX) +/+ mice. The weight of the remaining left kidney in the +/+ mice increased by -50% during the period allotted for compensatory renal growth, whereas the weight of the remaining left kidney in the NPX OS/+ mice increased by only -23%. These data indicate that the capacity for the hypertrophic response induced by unilateral nephrectomy was significantly lower in the OS/+ mice than in the +/+ mice. Histological and Morphometric Data Area of midtransverse sections of left kidney. The area of the midtransverse sections of the left kidney was found to correlate well with the mass of the left kidney (Table 1). Differences in the mass of the left kidney between the four groups of mice used in this study were reflected by similar differences in the midtransverse cross-sectional area of the left kidney. Glomerular data. Histological analysis revealed that the midtransverse sections of the left kidney from the OS/+ mice contained approximately one-half the number of glomeruli that were present in the midtransverse sections of the left kidney from the +/+ mice (Fig. 1). Assuming that the two-dimensional density of glomeruli in the midtransverse sections reflects the approximate volumet- Fig. 1. Number of glomeruli per midtransverse section of left kidney from normal and uninephrectomized (NPX) male ROP OS/+ and +/+ mice. Values are means t SE. * Significantly different (P < 0.05) from mean for corresponding group of +/+ mice. ric density of glomeruli per cubic millimeter of tissue throughout the kidneys of the OS/+ and +/+ mice, it appears that the kidneys of OS/+ mice contain one-half the number of glomeruli present in the kidneys of +/+ mice. The number of glomeruli in the midtransverse sections of the left kidney from both the OS/+ and +/+ mice was not altered as a result of nephrectomy (Fig. l), indicating that no new glomeruli were formed as a result of a unilateral reduction of renal mass. At a qualitative level, the size of the glomeruli (particularly those in the inner cortex) appeared to be substantially greater in the sections of the left kidney from the OS/+ mice than in the sections of the left kidney from the +/+ mice (Fig. 2). Morphometric analysis confirmed the qualitative observations by revealing that the average area of the glomeruli in the sections of the left kidney from the OS/+ mice was -46% greater than that of the glomeruli in the sections of the left kidney from the +/+ mice (Fig. 3). Moreover, the average area of the inner cortical glomeruli was -34% greater in the sections of kidney from the OS/+ mice than in the sections of kidney from the +/+ mice (Fig. 4). There was a slight, but significant, increase in the size of the glomeruli in the left kidney of the OS/+ mice after unilateral nephrectomy. This was reflected by the area of the glomeruli in the midtransverse sections of the left kidney (Fig. 3), particularly those in the inner cortex (Fig. 4). No evidence of statistically significant glomerular hypertrophy after unilateral nephrectomy was found in the sections of kidney from the +/+ mice (Figs. 3 and 4). Tubular data. Under the light microscope, segments of the nephron appeared to be larger in the kidneys of the OS/+ mice than in the kidneys of the +/+ mice. The most striking differences in size were observed in segments of the proximal tubule, particularly in the pars recta (Fig. 5). Segments of the nephron in the kidneys of the NPX OS/+ and NPX +/+ mice also appeared hypertrophied compared with the size of the corresponding segments of the nephron in the normal +/+ mice. Tubular hypertrophy after unilateral nephrectomy appeared to be greater in the NPX OS/+ mice than in the NPX +/+ mice. Tubular hypertrophy observed during routine histological examination was confirmed with computerized morphometry. The diameters of the proximal convoluted and proximal straight tubules in the kidneys of both OS/+ and NPX OS/+ mice were significantly greater than those in the kidneys of corresponding +/+ and NPX +/+ mice (Fig. 6). Some of the increase in tubular diameter could be

F56 THE OS/+ MOUSE AS A MODEL OF REDUCED RENAL MASS.- 3 9 t E 0 8. OS./+ NPX +/+ NPX OS/+ Fig. 3. Cross-sectional area of glomeruli in midtransverse sections of left kidney from normal and uninephrectomized (NPX) male ROP OS/+ and +/+ mice. Values are means + SE. * Significantly different (P < 0.05) from mean for corresponding group of +/+ mice. + Significantly different (P < 0.05) from mean for OS/+ mice. Fig. 2. Photomicrographs of an inner cortical glomerulus in a midtransverse section of left kidney from a male ROP OS/+ mouse (A) and an inner cortical glomerulus in a midtransverse section of left kidney from a male ROP +/+ mouse (B). Note that glomerulus in A is substantially greater than glomerulus in B. Both micrographs were printed at same magnification. accounted for by hypertrophy of the tubular epithelial cells. Morphometric analysis revealed that the epithelial cells in the proximal convoluted and proximal straight tubules were larger in the kidneys of the OS/+ mice than in kidneys of the +/+ mice (Fig. 7). Moreover, the epithelial cells in these segments of the nephron in the remaining left kidney of both the NPX OS/+ and NPX +/+ mice were also hypertrophied. The magnitude of hypertrophy was greater than that found in the kidneys of the OS/+ mice (Fig. 7). Some cellular hypertrophy was also confirmed in a tubular segment beyond the proximal tubule in the OS/+, NPX OS/+, and NPX +/+ mice. Marked hypertrophy i 0 1 +I+ os/+ Fig. 4. Cross-sectional area of inner cortical glomeruli in midtransverse sections of left kidney from normal and uninephrectomized (NPX) male ROP OS/+ and +/+ mice. Values are means -+ SE. * Significantly different (P < 0.05) from mean for corresponding group of +/+ mice. + Significantly different (P < 0.05) from mean for OS/+ mice. occurred in a cell type present in the cortical collecting duct. It was assumed that this cell type was the principal cell, since hypertrophic changes have been shown to occur in principal cells in the cortical collecting duct after renal mass has been reduced significantly (11, 12). The size of the hypertrophied cells in the renal cortical collecting duct was greater in the OS/+ and NPX OS/+ mice, with the greatest degree of hypertrophy in the NPX OS/+ mice (Fig. 8). Functional Data The rate of urine flow factored by renal mass during the 24-h period allowed for the collection of urine was greater in the OS/+ mice than in the +/+ mice (Fig. 9). In addition, unilateral nephrectomy caused the rate of urine flow NPX OS - +

THE OS/+ MOUSE AS A MODEL OF REDUCED RENAL MASS F57 Fig. 5. Photomicrographs of proximal straight tubules at corticomedullary junction in a midtransverse section of left kidney from a male ROP OS/+ mouse (A) and proximal straight tubules at corticomedullary junction in a midtransverse section of left kidney from a male ROP OS/+ mouse (B). Note that size of proximal tubules in A are substantially greater than those in B. Both micrographs were printed at same magnification. to double in both the OS/+ and +/+ mice. Glomerular filtration rate (factored by renal mass), which was used as a index of renal function, was unaffected in the OS/+ mice compared with that in +/+ mice (Fig. lo), despite the fact that the OS/+ mice possessed -50% fewer nephrons (Fig. 2). Unilateral nephrectomy also did not affect glomerular filtration rate in the +/+ mice. It did, however, cause glomerular filtration rate to increase significantly per gram kidney in the OS/+ mice. As a result of this adaptive change, the concentration of creatinine in plasma did not change in NPX OS/+ animals (Table 2). In fact, the concentration of creatinine in plasma was similar in all four groups of mice. BUN was measured in the four groups of mice in the present study in the hope that this measurement could be used as a confirmatory index of renal function. Interest- ingly, BUN did not correlate with or correspond with the concentration of creatinine in plasma or the patterns of glomerular filtration rate found in the animals. In both the OS/+ and NPX OS/+ mice, BUN was nearly twice that found in the corresponding +/+ and NPX +/+ mice (Fig. 11). In addition, unilateral nephrectomy did not affect BUN in either the OS/+ or +/+ mice. Thus BUN does not appear to accurately reflect renal function in OS/+ mice. All four groups of mice appeared to maintain normal sodium and potassium homeostasis, since there was no change in either plasma sodium or potassium in any of the mice (Table 2). The absolute excretion of sodium and potassium (factored by renal mass) was similar in the OS/+ and +/+ mice (Table 2). However, the absolute excretion of sodium and potassium was significantly

F58 THE OS/+ MOUSE AS A MODEL OF REDUCED RENAL MASS PCT I CCD Cells I.. + * T m ' G;// /" / Fig. 6. Diameters of proximal convoluted tubules (PCT) and proximal straight tubules (PST) in midtransverse sections of left kidney from normal and uninephrectomized (NPX) male ROP OS/+ and +/+ mice. Values are means & SE. * Significantly different (P < 0.05) from corresponding mean for corresponding group of +/+ mice. + Significantly different (P < 0.05) from corresponding mean for +/+ mice. cd&- z!g40- a- 220. 200-180- 160-120- PCT Cells + ;,(, P / ' liilzl =; :,/Jp PST Cells c <diyhloo 2% at 125 75 50 25 0 T \,,,,,,,- I i - +I+ * T P r + T Fig. 8. Area of epithelial cells (presumed to be principal cells) in cortical collecting ducts (CCD) in midtransverse sections of left kidney from normal and uninephrectomized (NPX) male ROP OS/+ and +/+ mice. Values are means k SE. * Significantly different (P < 0.05) from corresponding mean for corresponding group of +/+ (either normal or NPX) mice. + Significantly different (P < 0.05) from corresponding mean for corresponding group normal (+/+ or OS/+) mice. + * 1 100-80/ 207f 0 * Fig. 7. Area of epithelial cells in proximal convoluted tubules (PCT) and proximal straight tubules (PST) in midtransverse sections of left kidney from normal and uninephrectomized (NPX) male ROP OS/+ and +/+ mice. Values are means + SE. * Significantly different (P < 0.05) from corresponding mean for +/+ mice. + Significantly different (P < 0.05) from corresponding mean for corresponding group normal (+/+ or OS/+) mice. elevated in the NPX OS/+ and NPX +/+ mice. Consequently, the fractional excretion of sodium and potassium was also elevated tremendously in the NPX OS/+ and NPX +/+ mice (Table 2). DISCUSSION The total renal mass in the ROP OS/+ mice used in the present study was -60% of that in the corresponding +/+ litter mates of comparable weight, even though the kidneys in the OS/+ mice contained -50% fewer nephrons than the kidneys in the +/+ mice. Morphometric analysis confirmed cellular hypertrophy in both proximal convoluted and proximal straight tubules and in a cell type in the cortical collecting duct that was presumed to be the principal cell. Not only were some tubular segments hypertrophic, but a number of glomeruli, particularly those in the inner cortex, were also enlarged in the kidneys of the OS/+ mice. These findings indicate that some level of compensatory hypertrophy takes place dur- NPX +/+ NPX OS/+ Fig. 9. Rate of urine flow or production, averaged over a 24-h period, in normal and uninephrectomized (NPX) male ROP OS/+ and +/+ mice. Values are means t SE. * Significantly different (P < 0.05) from mean for corresponding group of +/+ (either normal or NPX) mice. + Significantly different (P < 0.05) from mean for corresponding group normal (+/+ or OS/+) mice. ing some stage of nephrogenesis and/or renal development in the OS/+ mice. Similar hypertrophic changes have also been observed in the kidneys of different strains of OS/+ mice that were bred during the 1960s (9, 10). In these strains, however, the OS/+ mice contained far fewer number of nephrons (only ZO-30%) relative to the number of nephrons in corresponding +/+ littermates than the ROP OS/+ mice used in the present study. This difference in the number of nephrons per kidney between the strains of OS/+ mice appears to indicate that the OS gene and/or some modifying gene(s) has been altered significantly over time as a result of selective breeding. Compensatory hypertrophic changes also occurred in the kidneys of both the OS/+ and +/+ mice after unilateral nephrectomv. The evidence for this comes from the

increases in weight of the remnant kidney and the morphometric data indicating marked cellular hypertrophy in both the convoluted and straight portions of the proximal tubule as well as in the cortical collecting duct. Based on the quantitative data, the magnitude of compensatory c9 0.25 THE OS/+ MOUSE AS A MODEL OF REDUCED RENAL MASS F59 Fig. 10. Glomerular filtration rate (estimated by renal clearance of creatinine), averaged over a 24-h period, in normal and uninephrectomized (NPX) male ROP OS/+ and +/+ mice. Values are means t SE. * Significantly different (P < 0.05) from mean for group of NPX +/+ mice. 60 n * Fig. 11. Concentration of blood urea nitrogen (BUN) in normal and uninephrectomized (NPX) male ROP OS/+ and +/+ mice. Values are means t SE. * Significantly different (P < 0.05) from mean for corresponding group of +/+ (either normal or NPX) mice. Table 2. Urinary excretion of sodium and potassium in normal and uninephrectomized male ROP OS/+ and +/+ mice renal growth after unilateral nephrectomy appears to be greater in +/+ mice than in OS/+ mice. This is probably due to the fact that the epithelial cells in proximal and distal segments of the nephron in the kidneys of the OS/+ mice have already undergone some compensatory changes as a result of a 50% deficit in the number of functioning nephrons, which resulted during nephrogenesis. The hypertrophy of some of the renal tubular epithelial cells in the OS/+ mice after unilateral nephrectomy indicates that the kidneys in the OS/+ mice possess a significant compensatory reserve for loss of nephrons. From a functional standpoint, no significant differences in plasma creatinine, sodium, and potassium, in glomerular filtration rate and in the absolute and fractional excretion of sodium and potassium were detected between the normal OS/+ and +/+ mice used in the present study. These data indicate that, for the most part, the ROP OS/+ mouse is fully capable of maintaining normal fluid and electrolyte balance. In a previous study, it was determined that BUN was significantly greater in OS/+ mice than in corresponding +/+ mice (9). It was suspected that the increased level of urea nitrogen in the blood was due to decreased renal clearance of this plasma solute. In the present study, BUN was also found to be significantly greater in OS/+ mice than in +/+ mice. However, the increased level of BUN was not associated with a change in the concentration of creatinine in plasma. This leads one to believe that the increased concentration of urea nitrogen in blood may be related to some metabolic abnormality associated with the generation and/or metabolism of urea nitrogen, rather than being due to a decrease in the renal function. Further evidence for this notion comes from the fact that BUN did not change in either the OS/+ or +/+ mice after unilateral nephrectomy. One would have expected BUN to increase to a greater extent in the OS/+ mice after unilateral nephrectomy if the increase in BUN in the normal OS/+ mice was in fact due to decreased renal function associated with reduced renal mass. Other investigators (29, 10) have observed that OS/+ mice produce more urine than corresponding +/+ mice. One particular strain of OS/+ mice, designated as DI OS/+ (Z), produced large quantities of dilute urine and demonstrated signs of diabetes insipidus (9). The rate of urine excreted by the ROP OS/+ mice used in the present study was greater than twice that in corresponding +/+ mice. However, there was no evidence of massive volume loss in the ROP OS/+ mice that could be associated with [ Creatinine] in Plasma, mddl [Na+] in Plasma, [K+] in Plasma, medl n-w/l Absolute Excretion of Na+, meq. 24 h- -g- Absolute Excretion of K+, meq. 24 h-l-g- Fractional Excretion of Na+, % Fractional Excretion of K+, % +/+ os/+ NPX +/+ NPX OS/+ 0.43t0.06 148tl 4.ltO.l 1.66t0.33 0.06kO.O 1 1.81k0.25 2.45t0.56 0.48t0.03 147tl 4.7kO.4 2.92t0.27 0.15_to.o1 2.24t0.2 1 3.67k0.54 0.61zkO.15 144tl 4.5&O. 1 5.06&0.53* 2.30&0.20* 4.76&1.28* 71.2t19.3* 0.41t0.02 145tl 4.3t0.3 8.87t0.95* 4.14&0.40* 3.78&0.36* 53.8k6.13 Values are means t SE; n = 6 for all groups. OS/+, mice possessing the gene for the expression oligosyndactylism; +/+, normal litter mates of OS/+ mice; NPX, uninephrectomized. * Significantly different (P < 0.05) from the mean for the corresponding group of mice that were not uninephrectomized.

F60 THE OS/+ MOUSE AS A MODEL OF REDUCED RENAL MASS a form of diabetes insipidus. This indicates that the diabetes insipidus component in the expression of oligosyndactyly has been altered after selective breeding. It is possible that the mechanism for the increased rate of excretion of urine in the OS/+ mouse is related to some compensatory changes in glomerular and tubular fluid dynamics associated with the 50% deficit in the number of functioning nephrons. Evidence for this hypothesis comes from NPX OS/+ and NPX +/+ mice, in which there is an associated increase in the rate of urine excreted after a unilateral reduction of renal mass. When unilateral nephrectomy was performed on either the OS/+ or +/+ mice, the concentration of creatinine in plasma did not increase significantly. This was particularly unexpected in the OS/+ mice, inasmuch as these animals contained only -25% of the number of renal glomeruli that were present in the normal +/+ mice. For the concentration of creatinine not to have increased in the NPX OS/+ mice, there needed to be a substantial reserve capacity for filtration in the remaining glomeruli. Glomerular filtration rate per gram kidney (as estimated by creatinine clearance) doubled in the OS/+ mice after unilateral nephrectomy, which indicates that there was indeed a significant reserve capacity for filtration. The mechanism for the increased capacity for the glomeruli in the OS/+ mice to filter plasma at a greater rate may be related to the fact that on average, the renal glomeruli in the OS/+ mice were significantly greater in size than those in the +/+ mice. It would seem logical that with larger glomeruli there would be greater endothelial surface area to allow for filtration. Increases in glomerular volume have also been observed in other strains of OS/+ mice (lo), as well as in rats, rabbits, and dogs that have had their renal mass reduced (8). It is not clear whether the increase in the volume of the renal glomeruli in the OS/+ mice occurs during early phases of nephrogenesis, or is a functional adaptive change that occurs during early neonatal development, or is a combination of both. There is evidence from the present study, however, that further glomerular enlarge- ment can occur in the OS/+ mouse after unilateral nephrectomy. This would support the hypothesis that increases in glomerular volume in the OS/+ mouse occur as a result of the altered physiological demands induced by reducing renal mass. All the animals in the present study appeared to maintain normal sodium and potassium balance. Part of the maintenance of potassium balance in the NPX OS/+ and NPX +/+ mice was apparently due to increased frac- tional excretion of potassium. Some of the increased fractional excretion of potassium may have been due to increased secretion of potassium in the cortical collecting duct, which is known to occur after a significant reduction in renal mass (1, 3, 7). Moreover, the increased size in the presumed principal cells of the cortical collecting ducts in the NPX OS/+ and NPX +/+ mice is also consistent with findings in rats that show an association between hypertrophy of principal cells in cortical collecting ducts and increased fractional excretion of potassium after renal mass is reduced significantly (11, 12). In summary, the data from the present study strongly indicate that the ROP OS/+ mouse is a useful genetic animal model to study the effects of reduced renal mass. An additional benefit of this animal model is that the OS/+ mouse can be identified without surgery as early as the 11th day of development in utero. The OS/+ mouse may also be a very useful animal model in which nephrogenesis can be studied. Because the OS/+ mouse develops fewer nephrons than the normal mouse (+/+), there must be some important clues in this mouse that would aid us in understanding some of the mechanisms involved in the regulation and control of nephrogenesis. As a final note, it should be pointed out that the OS gene is located on the proximal portion of chromosome 8 in the mouse (6), which is homologous to the portion on chromosome 16 in humans that contains the gene for adult polycystic kidney disease (PKDl). Thus the OS/+ mouse may also be useful in study of the genetics of some renal diseases. The author acknowledges the technical assistance rendered by Steven Deal. This project was supported in part by National Institutes of Health Grant ES-05157. Received 27 May 1992; accepted in final form 24 August 1992. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Bank, N., and H. S. Aynedjian. A micropuncture study of potassium excretion by the remnant kidney. J. CZin. Inuest. 52: 1480-1490, 1973. 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