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Effects of varied dietary calcium and phosphorus on renal function of male Blue Duiker antelope
Small Ruminant Research, 11 (1993) 17-32
17
© 1993 Elsevier Science Publishers B.V. All rights reserved. 0921-4488/93/$06.00
Effects of varied dietary calcium and phosphorus on renal function of male Blue Duiker antelope B.L. Roeder a, R.F. Wideman, Jr.b and G.A. V a r g a c aDepartment of Veterinary Science, Wiley Laboratory, bDepartment of Poultry Science and CDepartment of Dairy and Animal Science, The Pennsylvania State University, University Park, P4. USA (Accepted 31 August 1992 )
ABSTRACT Sixteen adult male Blue Duikers (Cephalophus monticola bicolor) were fed a basal diet with four levels of Ca and P for 4 months. Pretrial, 24-h urine clearance was studied in each Duiker while they consumed the herd diet (1.38% Ca/0.66% P) (The Pennsylvania State University (PSU) formulation ). Pretrial, 24-h urine clearance values revealed no differences (P> 0.05) among treatment groups consuming the PSU diet. However, younger males did have higher serum P and lower serum and urine Ca concentrations (P< 0.05). Posttrial renal function studies indicated age differences for [ P]elas,,a, [K]un,e, urine/plasma [K], and absolute excretion of K, which were higher in younger males, and free water clearance and tubular P excretion, which were highest in older males. Renal function was affected by the level of Ca and P in the diet. Glomerular filtration rate (GFR) and effective renal blood flow (ERBF) were lower (P < 0.05 ), and fractional excretion (FE) of electrolytes and creatinine (Cr) were higher (P<0.05) in the T 3 group (0.8% Ca/0.4% P), indicating possible onset of renal dysfunction in these Duikers. Clearance of creatinine (Ccr) was negatively correlated with [ Cr] P~a.... suggesting tubular secretion of Cr increased during decreased renal function. Endogenous Cc~ was not an accurate approximation of GFR, as determined by clearance of inulin (C~,), in Blue Duiker under varying nutritional conditions. FE of all electrolytes, as calculated by Gn, regressed against the FE calculated from C c , indicated a poor correlation except for P. This suggests that FEp as measured by Ccr may be a useful index of early renal dysfunction in Blue Duiker. It appears that Blue Duiker have the ability to maximize P retention that may be available in limited quantity in the wild. The C a / P ratio of 0.5:0.4% in the ration appears to be best suited for adult males. Key words: Duiker antelope; Renal function; Calcium; Phosphorus; Creatinine clearance
INTRODUCTION
Maintenance o f inert electrolytes and minerals is related to intake and excretion o f normal serum levels o f these substances. Homeostatic mechanisms are initiated by alterations in extracellular fluid concentrations and result in Correspondence to and present address: B.L. Roeder, Brigham Young University, Department of Animal Science, Provo, U T 84602, USA.
18
B.L. ROEDER ET AL.
increased or decreased absorption and/or excretion of minerals and electrolytes. In ruminants, homeostatic mechanisms are mediated via hormonal excretory regulation at the salivary glands and the kidneys (Horst, 1986). The ruminant kidney exhibits an efficient regulatory mechanism to help control water, Na, K, C1, Ca and P balance (Scott and Buchan, 1987; Scott, 1974; Stacy and Wilson, 1970; Stacy, 1969). Exotic ruminants indigenous to arid climates, such as dik-dik antelope (Rhynchotragus kirkii) and camels (Camelus dromedarius) appear to have superior urine concentrating abilities and renal electrolyte handling during periods of extreme ambient temperatures or dehydration (Rugangazi and Maloiy, 1987; Maloiy, 1972). Since hydration, nutritional status and disease states can affect renal electrolyte and mineral excretion patterns (Morris et al., 1984; Osbaldiston and Moore, 1971 ), assessment of these substances in urine permits an understanding of the state of homeostasis. Manipulation of electrolytes and minerals in the diet of ruminants can alter feed intake, growth rate, fecal consistency, serum composition, and urine composition (Roeder et al., 1991; Challa and Braithwaite, 1988a,b,c; Scott, 1974). In ruminants, the occurrence of struvite crystalluria (ammonium magnesium phosphate) is associated with concentrate feeding and a low Ca/ P ratio ( < 1.5 : 1) in the ration (Kallfelz et al., 1987; Jubb et al., 1985 ). Diets high in phosphate can cause a very high incidence of urolithiasis in sheep and goats due to calculi of the phosphatic (struvite) type (Kimberling and Arnold, 1985; Bushman et al., 1965a,b). In experimentally induced urolithiasis produced by dietary oxalates or calcium phosphate, calculi are initially microscopic and form in the collecting tubules (tubular microlithiasis), then encrust on the renal papilla (Jubb et al., 1985 ). Calculi are formed more frequently in animals with alkaline urine, borderline hypovitaminosis A, hypervitaminosis D, urinary stasis, and in arid climates (Powe, 1986 ). Caged Blue Duiker (Cephalophus monticola bicolor) a species of African antelope indigenous to the regions south of the Sahara desert, urinate infrequently even though water is provided ad libitum, and have highly concentrated urine (Roeder, 1990). As part of an investigation of the relationship between dietary Ca, P, and the homeostatic mechanisms controlling their extracellular fluid levels in captive Blue Duiker, evaluation of renal function was performed. This study was initiated to help identify the optimal dietary levels of these elements and the risk of urolithiasis in these animals while consuming a total mixed, pelleted diet with varied levels of Ca and P. MATERIALS AND METHODS
Sixteen clinically healthy, adult, male Blue Duikers, eight being 13 to 24 mo old and eight, 3 to 5 yr old, were obtained from the PSU Deer and Duiker
EFFECTS OF VARIED DIETARY CALCIUM AND PHOSPHORUS ON RENAL FUNCTION
19
Research Facility. Animals were housed individually in modified stainless steel cages (61 cm deep × 64 cm wide X 41 cm high ) with wire mesh flooring in a temperature (20-22 ° C ), light ( 12-h light and dark) and humidity ( 5070%) controlled environment. Animals were initially fed the PSU pelleted diet and allowed to acclimate to their new environment before initial studies were conducted to determine baseline serum and urine values. Animals were then assigned by randomized block design to one of four dietary levels of %Ca and %P of 0.5:0.4% (TI), 0.8:0.8% (T2), 0.8:0.4% (T3) , 1.25:0.4% (T4) (block = age group of Duiker) for 4 mo. Diets were complete pelleted rations with no access to hay; and dietary C a / P ratios were different ( P < 0.05 ) among treatments (T1 = 1.25: 1, T2= 1 : 1, T3=2: 1, T4=3:1 ) (Roeder et al., 1991; Roeder, 1990). While consuming the PSU diet ( 1.38% Ca/0.66% P), a pretrial, 24-h volumetric urine clearance study was performed on each Duiker. Voided urine was collected from screen covered, acid washed, stainless steel pans. To establish electrolyte and creatinine values in their blood and urine, three blood samples and two timed urine collections were obtained during a 24-h study (baseline, then a 2nd and 3rd blood sample were collected as soon as possible after the I st and 2nd urinations ). Feed and water consumption, body weight and temperature, and volumetric urine output were measured daily, and weekly blood gas analyses, venous and urine electrolytes and creatinine were determined (Roeder et al., 1991 ). A renal function study was performed after 17 wk of consuming their designated level of %Ca and P. To decrease the risk of regurgitation of ruminal contents and subsequent pulmonary aspiration associated with general anesthesia in ruminants, food was withheld for 18 h and water for 6 h prior to the procedure. To conduct kidney function studies, Duikers were anesthetized using isoflurane (Aerrane, Anaquest, Madison, WI) administered via mask for induction followed by intubation with a cuffed endotracheal tube and a light plane of anesthesia was maintained for the remainder of the experiment. The right jugular vein was cannulated cranially and caudally with sterile 20-G, 2-inch teflon intravenous (i.v.) catheters (B-D I.V. CATH non-radiopaque FEP teflon, Becton Dickinson and Co., Rutherford, NJ ) introduced with a 24-G needle. Duikers were positioned in left lateral recumbency on a towel covered, heated, circulating water pad (Aquamatic, American Medical Systems, Model K-20, Division of American Hospital Supply Corp., Cincinnati, OH ) placed on a foam cushioned surface, and the head positioned with the nostrils lower than the larynx to allow free drainage of saliva and any regurgitated fluid. Blood samples were obtained from the cranial i.v. catheters for measurement of hematocrit (PCV), osmolality, creatinine (Cr) and electrolyte (Ca, P, Na, K, C1) concentrations at times of 0, 30, and 75 min after beginning the i.v. infusion. A 2-ml bolus injection of a solution (283.5 mOsm/kg H20) containing 25 mg/ml mannitol, 1.5 m g / m l inulin, 1.5 mg/ml para-aminohip-
20
B.L. ROEDER ET AL.
puric acid (PAH) (PAH; Sigma Chemical Co., St. Louis, MO) and 25 mg/ ml dextrose was infused i.v. via infusion pump (Harvard Apparatus Co., Inc., Millis, MA) followed by an administration rate of 0.2 ml/kg B W / m i n for a 30-min equilibration period. The low dose of mannitol, an osmotic diuretic, was used to insure that adequate urine production was maintained during the clearance period. PAH was used to measure effective renal plasma flow ( C p A H = ERPF) and to assess proximal tubular secretory function. Inulin (In) was used as a filtration marker for measuring glomerular filtration rate (C~n=GFR). Dextrose 2.5% was added to the infusion solution to increase its effective osmolality to achieve isotonicity, without adding electrolytes that would influence the subsequent measurements. After 30 min of equilibration, the urinary bladder was cannulated with fenestrated PE-50 polyethylene tubing (Clay Adams, Parsippany, N J) via sterile transcutaneous cystocentesis after introduction of a sterile 16-G 1.5-inch needle into the right dorsolateral aspect of the bladder, just anterior to the pelvic canal; and all urine was collected. The PE tubing was left in situ for the 40-min clearance period, then all urine was collected in a pre-weighed inert plastic container for gravimetric determination of urine volume and flow rate. During recovery from anesthesia, animals received sterile 5% dextrose i.v. (0.2 m l / k g / m i n ) for 20 min to ensure adequate hydration and perfusion pressure were maintained after this procedure. Colorimetric assays were used to measure P, C1, and Cr as previously described (Roeder et al., 199 l; Roeder, 1990 ), inulin (Waugh, 1977 ), and PAH (Brun, 1957); flame photometry (Instrumentation Laboratory Inc., Model 443, Wilmington, MA) for Na and K (Roeder et al., 1991; Roeder, 1990); atomic absorption spectroscopy (Instrumentation Laboratory Inc., Model 55 l, Wilmington, MA) for total Ca in serum and urine. Urine pH was measured to the nearest 0.01 pH unit, using a pH meter (DigipHase pH meter, Cole Parmer, Santa Ana, CA). Urine and plasma osmolality were measured with a vapor pressure osmometer ( 5100C Vapor Pressure Osmometer, Wescor, Inc., Logan, U T ) . Urine sample volumes were converted to urine flow rates (V), and expressed as ml/kg B W / m i n for the clearance interval. Glomerular filtration rate (GFR, ml/kg B W / m i n ) was calculated as the clearance of inulin (urine inulin concentration/plasma inulin concentration × V). The osmolal clearance (Cosm, ml/kg B W / m i n ) was calculated as urine osmolality/plasma osmolality X V. Free water clearance (FWC or CH2o ) values were calculated by subtracting the osmolal clearance from V ( V - Cosm). FWC is interpreted as values greater than 0 represent the rate at which water would have to be removed from urine to maintain isotonicity with plasma (hyposmotic urine ) and values less than 0 represent the rate at which water would have to be added to urine to maintain isotonicity with plasma (hyperosmotic urine). Absolute excretion (AbsEx) rates were calculated for each sample by multi-
EFFECTSOF VARIEDDIETARYCALCIUMAND PHOSPHORUSON RENALFUNCTION
21
TABLE 1 Serum and urine electrolytes and creatinine values for male Blue Duiker consuming the PSU pelleted diet Parameter
Younger cf
Older o~
+ SE
Serum Ca ( m M / l ) Pi (mM/1) Creatinine ( mg/dl ) K (mM/l) Na ( m M / l ) CI ( m M / l )
2.6 b 3.0 a 0.64 5.5 151 118
2.9 a 2.3 b 0.58 6.3 149 118
0.06 0.18 0.03 0.30 1.43 1.79
Urine Ca (mM/1) Pi ( m M / l ) Creatinine ( m g / d l ) K (mM/1) Na (mM/1) CI ( m M / l )
0.49 b 2.71 64.1 132.4 119.5 110.0
1.30 a 3.58 96.9 115.1 128.0 105.6
0.23 1.68 15.2 19.6 16.3 11.0
a,bMeans with different superscripts differ significantly ( P < 0.05 ). n = 8 younger males ( 13 to 24 mo ) and 8 older males (3 to 5 yr).
plying the amount of each substance in urine × V and expressed as/~mol/kg BW/min. Excretion data for Ca (FEca), inorganic P (FEp), Na (FENa), K (FEK), C1 (FEcl), and Cr (FEcr) are expressed as fractional excretion rates, which were calculated for each sample by dividing the clearance of each substance (Cy=(Uy/Py)XV, where Cy=the clearance of a substance y, U = urinary concentration, P = plasma concentration, and V= urine flow rate ( m l / k g / m i n ) ) , by the GFR. Clearance values for PAH were calculated for estimations of effective renal plasma flow (ERPF, ml/kg B W / m i n ). The effective filtration fraction (EFF) was calculated as EFF = G F R / E R P F , the fraction of renal plasma flow entering the nephron by glomerular filtration. Effective renal blood flow (ERBF, ml/kg B W / m i n ) was calculated as the E R P F / ( 1 - [hematocrit/100] ). The absolute filtered water resorption rate (FWResorp) was calculated as G F R - V . The fraction of filtered water excreted (FFWExc) as urine was measured by dividing one by the concentration of urine inulin/plasma inulin = ( V/GFR ). The fraction of filtered water reabsorbed (FFWReab) from the glomerular filtrate by both kidneys was calculated as 1 minus the fraction of filtered water excreted as urine. Tubular Ca (TubCa) and P (TubP) excretion were calculated by: (their concentration in urine× V) - (their concentration in plasma × GFR). Statistical analysis was performed by least-squares analysis of variance and Duncan's multiple range test using the SAS General Linear Models procedure
22
B.L. ROEDER ET AL.
TABLE 2 Results of a 40-min clearance period in male Blue Duiker receiving an i.v. infusion of 2.5% mannitol, 0.15% PAH, 0.15% inulin, and 2.5% dextrose on baseline kidney parameters Parameter 2
kg BW PCV UpH GFR V Uosm Posm
Costa FWResorp FFWExc FFWReab ERPF ERBF EFF
Treatment group 1
2
3
4
+_SE
3.74 39.08 c 6.47 '~ 11.90 ~' 0.136 509.25 275.70 0.257 11.76 a 0.013 0.987 30.31 49.24 ~b 0.441a
4.00 46.08 a 5.52 b 7.49 ab 0.129 409.13 276.33 0.191 7.36 ab 0.019 0.981 34.69 64.06 a 0.214 b
3.79 42.70 b 6.57 a 5.58 b 0.103 527.38 279.70 0.197 5.48 b 0.021 0.979 16.53 29.32 b 0.349 ab
3.85 43,13 b 6,61 a 12.01 a 0,129 496.50 275.38 0.242 11.88 a 0.014 0.986 35.18 62.71 a 0.325 ab
0.13 0.88 0.24 1.93 0.029 81.85 4.41 0.06 1.92 0.004 0.004 6.70 10.71 0.056
a,b,¢Means with different superscripts differ significantly ( P < 0 . 0 5 ) . ~Treatment group= 0.5:0.4% ( T I ), 0.8:0.8% (T2), 0 . 8 : 0 . 4 % (T3) , 1.25:0.4% (T4).
2Abbreviations and units: kg BW, body weight in kg; PCV, erythrocytic packed cell volume or hematocrit (%); UpH, urine pH; GFR, glomerular filtration rate ( m l / k g B W / m i n ) = CIn; V, urine weight specific flow rate ( m l / k g / m i n ) ; Uosm, urine osmolality ( m O s m / k g H 2 0 ) ; Posm, plasma osmolality Uosm ( m O s m / k g H 2 0 ) ; Cosr., osmolal clearance= p---~. × V ( m l / k g B W / m i n ) ; FWResorp, filtered water clearance (Cn2o) ( m l / k g / m i n ) = V-Corm ); FFWExc, fraction of filtered water excreted ~
1
Uln//~ln
= V~
GFR; FFWReab, fraction of filtered water reabsorbed from the glomerular filtrate by both kidneys ERPF 1 - FFWExc; ERPF, CpA8 ( m l / k g B W / m i n ) ; ERBF, (ml/kg B W / m i n ) ; EFF, effective 1 - PCV/100 GFR filtration fraction = ERPF"
(SAS, 1985). The mathematical model included variations attributable to age and treatment with higher order interactions. Differences were considered significant if P values were less than 0.05. RESULTS
Pretrial, while Duikers' were consuming the PSU pelleted ration with 1.38% Ca/0.66% P (2:1 ratio), 24-h urine clearance values revealed no differences ( P > 0.05 ) among treatment groups. Younger male Duikers did have higher ( P < 0 . 0 5 ) serum P and lower ( P < 0 . 0 5 ) serum and urine Ca values than older males. Other serum and urine electrolytes and creatinine levels were not
EFFECTS OF VARIED DIETARY CALCIUM AND PHOSPHORUS ON RENAL FUNCTION
23
TABLE 3 P l a s m a a n d urine electrolyte a n d creatinine values after a 4 0 - m i n clearance period in male Blue Duiker receiving an i.v. infusion o f 2.5% m a n n i t o l , 0.15% PAH, 0.15% inulin, a n d 2.5% dextrose Parameter 2
FEca [CA]plasma [ Ca ] u,~.¢ U/P Ca AbsExCa TubCa FEP [P]Pl . . . . [P]u~i,~ U/P P AbsExP TubP FeN~ [ N a ] pl. . . . [Na]u,~ne U/P N a AbsExNa FEK [K]plasm~ [ K ] Ur~n~ U/P K AbsExK FEcl [Cl]pl . . . . [Cl]un~e U/P CI AbsExCI FEcr [Cr ]pl~m~ [Cr ]u~in~ U/P Creat CCr
T r e a t m e n t groupl
1
2
3
4
_+SE
0.0017 b 1.63 b 0.263 b 0.162 ab 0.148 - 18.98 ~b 0.012 1.69 . 1.60 0.780 0.406 - 19.77 b 0.0010 b 128.13 10.45 0.082 1.81 0.249 2.65 b 51.25 19.02 8.05 0.0023 b 110.23 20.90 0.190 3.06 1.17 b 0.475 37.90 b 82.42 b 11.99 b
0.0036 b 1.67 ab 0.306 ab 0.183 ab 0.161 - 12.49 ab 0.023 1.99 2.91 1.289 0.365 - 16.50 ~b 0.0015 ab 130.88 10.00 0.076 1.29 0.358 2.55 b 44.35 18.09 5.93
0.0067 a 1.76 ab 0.569 a 0.325 a 0.241 - 9.56 a 0.017 1.87 1.57 0.695 0.131 -8.90 a 0.0024 ~ 133.38 14.60 0.108 1.45 0.449 3.53 a 74.85 21.38 7.92 0.0051 a 108.15 26.35 0.242 2.77 7.70 a 0.250 79.05 ~ 354.08 a 35.60 ~b
0.0017 b 1.79 a 0.250 b 0.141 b 0.143 - 21.85 b 0.007 1.75 1.19 0.567 0.220 - 19.92 b 0.0013 ab 132.13 14.95 0.1 l 1 1.79 0.286 2.98 ab 76.40 26.26 10.18
0.0012 0.058 0.106 0.062 0.062 3.63 0.009 0.37 1.29 0.49 0.241 3.68 0.0005 2.31 3.90 0.028 0.66 0.087 0.21 11.46 4.58 2.42 0.0009 1.95 4.78 0.041 0.99 1.76 0.105 12.53 92.00 9.27
0 . 0 0 3 1 ab
108.08 16.38 0.152 2.19 2.72 b 0.450 36.07 b 126.80 ~b 14.90 ab
0 . 0 0 2 7 ab
112.10 24.15 0.213 3.28 3.35~b 0.325 55.37 ~b 307.08 ~b 38.36 a
abMeans with different superscripts differ significantly ( P < 0.05 ). ~Treatment g r o u p = 0 . 5 : 0 . 4 % ( T I ) , 0 . 8 : 0 . 8 % (T2), 0.8:0.4% (T3), 1.25:0.4% (T4). 2See Table 2 for a b b r e v i a t i o n s a n d units. P l a s m a a n d u r i n e values are in m M / l except creatinine ( m g / d l ) . O t h e r a b b r e v i a t i o n s in Table 3 include: FEx, fractional excretion o f s u b s t a n c e 'X',
C~=[(~ )× V]/GFR; AbsExX, absolute excretion of substance "X', [X ]u × V (llmol/kg BW /min ); T U B C a , t u b u l a r c a l c i u m excretion, ( [Ca] tmne× V) - ( [Ca]el . . . . rus excretion, ( [ P ] t m n e × V) - ( [P]et . . . . × G F R ) .
× GFR); TUBP, tubular phospho-
24
B.L. ROEDERETAL. 25 GFR=0.315*ERPF N=16
P<0.0001
R2=.93
20
0.5% ~O.'t% e Xa 2
o le~ca:o.e%P
lo! (9
Ta 3 0.1~:04%
P
Trl 4 1.25% C~:0.4% e ZX
•
0 0
I
I
i
i
i
i
10
20
30
40
50
60
70
ERPF (ml/min)
Fig. 1. Linear regression of glomerular filtration rate (GFR) against effective renal plasma flow (ERPF), by treatment. Individual Duikers within a treatment group are identified by symbols; n = 4 Duiker/trt.
70
Ccr= 4 4 . 0 3 - 5 0 . 1 8 * [Cr] plasma N=16
"~, 60
P<0.01
R2--.37 Ta 1 0.5%C~0.4% P
so
13 xrl 2 0.8% ~:0.8% P
~ 4o ~
30
~
20
Trl 3 (I.8% ~ 0 . 4 % P Trt 4 1.25% C~1:0.4% P /3.
i
i
i
i
0,2
0.4
0.6
0.8
&_ 1
Plasma Creatinine (mg/dl)
Fig. 2. Creatinine clearance (Ccr) regressed against plasma creatinine concentration by treatment. Individual Duikers within a treatment group are identified by symbol; n = 4 Duiker/trt.
affected by age (Table 1 ). Dietary %Ca intake on a bodyweight basis was approx. 0.02%=T1, 0.03%=T2 and T3, and 0 . 0 4 5 % = T 4 and significant between TI
EFFECTS OF VARIED DIETARY CALCIUM AND PHOSPHORUS ON RENAL FUNCTION CCr = 4 2 . 6 2 - 5 . 4 9 * G F R 70
N=16
+ 0.29*GFR P<.12
25
2
R2=.23
Td
o.~%c~:o.4~P Ttt 2 0.8 %Ca:0.8 %P
x-"
[]
5O
Td 3 0.8 %Ca:O.4 %P
o,) o 40
Trl 4 ~.25 %Ca:O.4 %P
E:
/ 30 C)
._~ 20 r-
[] 0
10
/
o S 0
,
i
5
10
~ 1
+
,
20
25
G F R = Inulin C l e a r a n c e ( m l / k g / m i n )
Fig. 3. Creatinine clearance (Ccr) regressed against glomerular filtration rate ( G F R ) as determined by the clearance of inulin, Individual Duikers belonging to a treatment group are identified by symbol; n = 4 Duiker/trt. The straight line within the graph is the line of equality, where points would fall if filtration = excretion. Points above this line indicate net tubular secretion and below the line indicate net tubular resorption.
Varied dietary %Ca/%P did not cause differences between age groups' serum and renal values except for [P]Plasma, [K]urine, urine K/plasma K ( U / P K), and absolute excretion of K, which were higher in younger males, and FWC and TubP excretion, which were higher in older males ( P < 0.05 ). Key differences based on diet were found for hematocrit (PCV), urine pH, [ PAH ] Urine, absolute GFR, absolute filtered water resorption rate, ERBF, EFF, [ Ca ] Urine, [Ca]Plasma, urine Ca/plasma Ca ( U / P Ca), TubCa excretion, FEca, TubP excretion, FENa, [ K ] Plasma,FEcl, [ Cr ] U~ine,urine Cr/plasma Cr, FEcr, and the creatinine clearance (Ccr) (Tables 2 and 3 ) ( P < 0.05 ). Duikers fed rations with 0.8% P (T2) had a higher ( P < 0.05 ) mean PCV, and ERBF, and lower (P<0.05) urine pH than Duikers consuming diets with 0.4% P. However, Duikers receiving the T 2 diet had decreased GFR and EFF values, tending to elevate FE values for electrolytes. Duikers consuming 0.5% Ca/0o4% P (T~) had the lowest PCV values but similar GFR, ERBF, and FE values for electrolytes to Duikers fed diets T2 and T4. The dietary Ca/P ratio of2:1 (0.8% Ca/0.04% P) in T 3 had similar urine pH values to TI and T4, and a mean PCV value similar to T4. However, the T3 diet decreased (P<0.05) GFR, ERBF, and FWResorp, and increased ( P < 0.05 ) TubCa and TubP excretion compared to the other three treatments. All FE values for electrolytes and creatinine were elevated by diet T3. Absolute excretion for Na, K, and C1 was not different ( P < 0.05 ) for Duikers fed diet T3 compared with the other diets, indicating the increase in FE values was due to the decrease in GFR rather than an absolute increase in the net loss of electrolyte ( V× [X ] U r i n e )" Urinary
26
B.L. ROEDER ET AL -
-
20
AbsExP
Trt 1 0.S%C~9.dl%P
AbsExCr
TBI2 o.e,~c ~ o ~ P T~3 0 ~ ~0.~% P
15
Te 4
1,~% c,,:o.4%P A
E E
z~ '<
<
~
5
c?
~x
D
I
l
l
l
I
l
I
I
I
I
I
2
4
6
8
10
12
14
16
18
20
22
GFR=lnulin Clearance (ml/kg/min) b
0.06
T~ I o.~'~~:o.,,~ P
FEp=. 1 7 - . 2 8 * G F R N=16
P<0.73
R2=.0~
Ta 2 0.a'~ ~ 0 . r , 6
P
0.05 Xa 3 o.e'~ ~ o . 4 % P
"6
0.04
Ta 4 1.25%Ca:0.4~P
0.03
o
0.02
[3
0.01
[] []
0 0
5
10
15
20
25
GFR (ml/kg/min) 20
Ta 1 0.5"4 C~0.4% P
FF13r=6.78-.33*GFR N=16
Tn,2 0,8%C4~'O.(P~,P
P < 0 . 0 9 R~'-.19
I::: 15 Tn3 O.e'V, C~'0.4 "/, P O Td 4
"6
1.~'W, ~ : 0 A % P
g Io
._0 5 U.
5
10
15
GFR (ml/kg/min)
2O
i _ 25
EFFECTS OF VARIED
d
0.00
DIETARY
CALCIUM
AND
PHOSPHORUS
ON RENAL
27
FEp=.005+ 1.36*FEp ( ~ ~J
N=16 P
j.J O~ 0.04 o,"6 •~
FUNCTION
J
J
•
J O5%C~0.4%P
0.03
&
~
,,2 o.a,v.c~o.a%P
0.02
Trl3 o.e~.c~o4%p
0.0t
0 __ 0
I
I
I
0.02 0.03 Fractional Excretion of P (Ccr), %
0.01
_
I 0.04
Fig. 4. Absolute excretion of phosphorus (AbsExP) and creatinine (AbsExCr) versus glomerular filtration rate (GFR) (a). Regression of the fractional excretion of phosphorus (FEp) (b) and creatinine (Cr) (c) against GFR. Regression of the FEp as performed by the clearance of inulin (C~.) against the FEp as determined by the clearance ofcreatinine (Ccr) (d). Individual Duikers within a treatment group are identified by symbol; n = 4 Duiker/trt.
creatinine concentration, Ccr , [K]Plasma, and the U / P Ca levels were also higher ( P < 0.05 ) in the Duikers fed diet T3 than in the other treatments. Plasma Ca values tended to increase as dietary Ca levels were raised. Urinary Ca excretion was highest ( P < 0.05 ) in T 3 and lowest ( P < 0.05 ) in the high Ca low P diet, T4 ( 1.25% Ca/0.4% P). Although trends were apparent for the highest plasma and urine [P] in T2, differences were nonsignificant between treatments due to the consistently elevated P values of two individuals in this group beyond those of the other T2 males. TubCa and TubP excretion were highest ( P < 0 . 0 5 ) for Duikers fed diet T 3 (0.8% Ca/0.4% P), and lowest ( P < 0 . 0 5 ) for Duikers fed the high Ca and low P diet, T4 ( 1.25% Ca/ 0.4% P). The lowest ( P < 0.05 ) TubP excretion was seen in Duikers fed diets Tl (0.5% Ca/0.4% P) and T4. There was positive correlation between GFR and ERPF (Fig. 1 ) and the slope, 31.5%, ( G F R / E R P F ) is the mean value for EFF. Although individual variation within a treatment group did cause differences, those Duikers on diets T2 or T3 with 0.8% Ca tended to have the lowest values for GFR and ERPF. Ccr was negatively correlated with Pcr concentration (Fig. 2 ). Duikers with the lowest GFR and ERPF (T2 and T 3) values tended to have the highest Ccr and low Pcr values. GFR as determined by the clearance of inulin (Cln) was poorly correlated with endogenous Ccr (Fig. 3). Most Duikers appeared to have a Ccr/Cln ratio (FEcr) greater than 1.00 indicating net tubular secretion of creatinine. Duikers receiving 0.8% Ca/0.4% P (T3) had the highest FEcr
28
B.L. R O E D E R ET AL.
(P< 0.05 ) and tended to have the lowest plasma creatinine values compared to the other three treatments. Absolute excretion of P and creatinine were relatively unaffected by GFR (Fig. 4a) and their FE values were poorly correlated with GFR as measured by C~n (Fig. 4b and c). However, when FEp, as measured by C~n, was regressed against FEp, determined by endogenous Ccr, a positive correlation was found with P < 0.001 (Fig. 4d). Similar regressions of FEca, FENa, FEK, and FEcl yielded poor nonsignificant (P> 0.05 ) correlations. DISCUSSION
This study was prompted by research of urolithiasis and renal dysfunction in domestic ruminants when consuming diets with varied Ca/P ratios beyond levels of requirements, particularly diets with a Ca/P ratio of less than 1.5: 1. Blue Duikers at PSU had been observed to have marked hyperphosphatemia, hyperkalemia, mild to moderate hypocalcemia, and reversed Ca/P ratios (particularly juveniles and young adults ) consuming the pelleted herd ration. Since these animals had been considered at risk of becoming an endangered species, all studies were conducted as survival research, experiments were designed to use primarily noninvasive procedures, and only a limited number of males were available. However, since the study was meant not only to investigate Ca and P homeostatic mechanisms but to better define requirements for these macronutrients, assessment of renal function in Blue Duikers consuming varied %Ca/% P was needed to detect impairment of kidney function that these levels may have caused. Evaluations of serum or plasma urea nitrogen, creatinine, and urine specific gravity are simple but relatively insensitive tests of renal function since normal values are compatible with up to two-thirds loss of renal parenchyma in ruminants and other mammalian species (Fetcher, 1986). The ability of the kidney to concentrate urine has been regarded as a sensitive indicator of renal function in animals when performed as urine osmolality (Bovee, 1969). The ratio of urine to plasma osmolality (u//eosm) indicates the number of times the kidney concentrates the urine over plasma; whereas, urine specific gravity provides only a crude approximation of the renal concentrating ability by measuring urine density. Performing U / P o s m in conjunction with measurements of GFR, ERPF, and FE of electrolytes and creatinine provides a more accurate assessment of renal function because changes in renal hemodynamics precede elevations in serum urea nitrogen, creatinine, and urine specific gravity (Fetcher, 1986). Calculation for FE of electrolytes using endogenous creatinine clearance (Ccr) has been advocated as useful in man and animals, including ruminants, since creatinine production within the body is relatively constant and excretion is almost entirely by glomerular filtration in most species (Neiger and
EFFECTS OF VARIED DIETARY CALCIUM AND PHOSPHORUS ON RENAL FUNCTION
29
Hagemoser, 1985; Morris et al., 1984; Kaneko, 1980; Bovee and Joyce, 1979 ). Renal FENa has been used to help differentiate renal from prerenal azotemia in humans (Espinel and Gregory, 1980; Espinel, 1976), and horses (Morris et al., 1984). When tubular dysfunction causes a reduced capacity to reabsorb filtered Na, FENa becomes elevated; alternatively, renal FENa values < 1% usually indicate prerenal azotemia with avid reabsorption of filtered Na. In horses, during feed and water deprivation, the total amount of Na excreted in urine tends to increase (Rumbaugh et al., 1982; Stacy and Wilson, 1970; Tasker, 1967a,b). Blue Duiker in this study, although fasted for 18 h prior to their renal function test still had ingesta present in their forestomachs, albeit a reduced quantity, and were adequately hydrated, as reflected by normoproteinemia and PCV values similar to unfasted Duikers in the herd. FENa (performed by CIn) did show significant differences by treatment which correlated well with levels of GFR and ERPF. The use of endogenous Ccr to perform FE calculations, however, was poorly correlated with those values obtained by the CIn except for FEp. Blue Duiker appeared to increase rate of net creatinine secretion during reduced renal function as measured by decreased GFR and ERBF thereby maintaining low levels of creatinine in plasma and enhancing urinary excretion of creatinine. Blue Duikers consuming diets with the higher levels of Ca tended to have elevated Ccr. However, Duikers receiving 0.8% Ca/0.4% P (T 3), had the lowest values for GFR, ERPF, and ERBF with the highest FE of creatinine and other electrolytes indicating this level o f % C a / % P at a 2 : 1 ratio with this diet formulation had detrimental effects on normal renal function. The FEp in the T 3 group, although not significantly different from the other treatments, was increased over the other two treatments receiving 0.4% P and approached the highest FEp values seen in animals consuming 0.8% P. Since FEp was positively correlated between its calculation by CIn and Ccr, this measurement may be a useful index of early renal dysfunction in Blue Duikers. The 24-h volumetric urine studies, although cumbersome, appear to be the most valid method of evaluating mineral metabolism via urinary clearance studies in Blue Duiker. Endogenous Ccr is affected by dietary differences in Blue Duiker similar to that which has been reported in sheep consuming diets with varied N and energy content (Faix et al., 1988 ), and camels during exposure to heat and water deprivation (Yagil and Berlyne, 1977). This, in addition to the failure of Ccr to be well correlated with Cin, suggests that endogenous Ccr is not an accurate approximation of GFR in Blue Duiker under varying C a / P conditions. EFF obtained in this study (31.5% ) was higher than what has usually been reported for most mammals, i.e., 20% or about one-fifth of the plasma entering the glomerular capillaries is filtered (Valtin, 1983 ). However, in two separate studies with cats, a species with great urine concentrating ability (even during development of azotemia compared to dogs and humans) the calcu-
30
B.L. ROEDER ET AL.
lated EFF ranged between 20-50% (Ross and Finco, 1981; Osbaldiston and Fuhrman, 1970). More studies, including the evaluation of possible anatomical differences and the extraction efficiency of PAH, need to be done to better define this difference in Blue Duiker. CONCLUSION
This study indicated that: ( 1 ) Blue Duiker fed this diet with a Ca/P ratio of 2: 1 had possible onset of renal dysfunction; (2) Blue Duiker with renal dysfunction, as measured by decreased GFR (C~n) and ERBF (CpAH), appear to increase tubular secretion of creatinine which causes plasma levels of creatinine to decrease (unlike other species with renal dysfunction where elevation of plasma creatinine concentration drives increased tubular secretion of creatinine); (3) endogenous creatinine clearance (Ccr) is not an accurate approximation of GFR in Blue Duiker under varying Ca/P conditions; (4) fractional excretion of P relative to inulin and endogenous creatinine clearances were correlated suggesting that fractional excretion of P may be a useful index for detection of early renal dysfunction in this species; and (5) filtration fractions in Blue Duikers are higher than those usually reported for other animals. These findings may indicate adaptive mechanisms for this species to survive in the wild where P intake may be limited. A higher EFF may enhance clearance of unwanted substances, and increased tubular secretion of creatinine during early renal dysfunction may prolong life. ACKNOWLEDGEMENTS
The authors thank Joyce L. Satnick, Glenda C. Loop, Donna L. Warner, and Robert C. Mothersbaugh for their technical assistance.
REFERENCES Bovee, K.C., 1969. Urine osmolarity as a definitive indicator of renal concentrating capacity. J. Am. Vet. Med. Assoc., 155: 30-35. Bovee, K.C. and Joyce, T., 1979. Clinical evaluation of glomerular function: 24 hour creatinine clearance in dogs. J. Am. Vet. Med. Assoc., 174: 488-491. Brun, C., 1957. A rapid method for the determination of para-aminohippuric in kidney function tests. J. Lab. Clin. Med., 37: 955-958. Bushman, D.H., Emerick, R.J. and Embry, L.B., 1965a. Experimentally induced ovine phosphatic urolithiasis: relationships involving dietary calcium, phosphorus and magnesium. J. Nutr., 87: 499-504. Bushman, D.H., Emerick, R.J. and Embry, L.B., 1965b. Incidence of urinary calculi in sheep as affected by various dietary phosphates. J. Anita. Sci., 24:671-675. Challa, J. and Braithwaite, G.D., 1988a. Phosphorus and calcium metabolism in growing calves with special emphasis on phosphorus homeostasis. I. Studies of the effect of changes in the
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dietary phosphorus intake on phosphorus and calcium metabolism, J. Agric. Sci., 110: 573581. Challa, J. and Braithwaite, G.D., 1988b. Phosphorus and calcium metabolism in growing calves with special emphasis on phosphorus homeostasis, 2. Studies of the effect of different levels of phosphorus, infused abomasally, on phosphorus metabolism. J. Agric. Sci., 110: 583-589. Challa, J. and Braithwaite, G.D., 1988c. Phosphorus and calcium metabolism in growing calves with special emphasis on phosphorus homeostasis. 3. Studies of the effect of continuous intravenous infusion of different levels of phosphorus in ruminating calves receiving adequate dietary phosphorus. J. Agric. Sci., 110: 591-595. Espinel, C.H., 1976. The FENa test: Use in the differential diagnosis of acute renal failure. J. Am. Med. Assoc., 236: 579-581. Espinel, C.H. and Gregory, A.W., 1980. Differential diagnosis of acute renal failure. Clin. Nephrol., 13: 73-77. Faix, S., Leng, L., Szanyiova, M. and Boda, K., 1988. Creatinine and inulin clearance at the different nitrogen and energy intake in sheep. Comp. Biochem. Physiol., 91A: 689-691. Fetcher, A., 1986. Renal disease in cattle. Part II. Clinical signs, diagnosis, and treatment. Comp. Cont. Ed. Pract. Vet., 8: $338-$345. Horst, R.L., 1986. Regulation of calcium and phosphorus homeostasis in the dairy cow. J. Dairy Sci., 69: 604-616. Jubb, K.V.F., Kennedy, P.C. and Palmer, N., 1985. Pathology of Domestic Animals. Academic Press, Inc., New York, 3rd edn., Vol. 2, pp.392-395. Kallfelz, F.A., Ahmed, A.S., Wallace, R.J., Sasangka, B.H. and Warner, R.G., 1987. D i e l a ~ magnesium and urolithiasis in growing calves. Cornell Vet., 77: 33-45. Kaneko, J.J., 1980. Clinical Biochemistry of Domestic Animals. Academic Press, New York, 3rd edn. Kimberling, C.V. and Arnold, K.S., 1985. Diseases of the urinary system of sheep and goats. Vet. Clin. North Am.: Large Anim. Pract., 5:637-640. Maloiy, G.M.O., 1972. Renal salt and water excretiion in the camel. Symp. Zool. Soc. Lond., 31 : 243-259. Morris, D.D., Divers, T.J. and Whitlock, R.H., 1984. Renal clearance and fractional excretion of electrolytes over a 24-hour period in horses. Am. J. Vet. Res., 45: 2431-2435. Neiger, R.O. and Hagemoser, W.A., 1985. Renal percent clearance ratios in cattle. Vet. Clin. Pathol., 14: 31-35. Osbaldiston, G.W. and Fuhrman, W., 1970. The clearance ofcreatinine, inulin, para-aminohippurate and phenosulphothalein in the cat. Can. J. Comp. Med., 34:138-141. Osbaldiston, G.W. and Moore, W.E., 1971. Renal function in cattle. J. Am. Vet. Med. Assoc., 159: 292-301. Powe, T.A., 1986. Diseases of the urinary system. In: Howard, J.L., ed., Current Veterinary Therapy: Food Animal Practice. W.B. Saunders Co., Philadelphia, 2nd edn., pp. 816-818. Roeder, B.L., 1990. The effect of varied dietary calcium and phosphorus on calcium and phosphorusi metabolism and acid-base balance in Blue Duiker antelope. Ph.D. Dissertation, The Pennsylvania State University, University Park, PA, USA. Roeder, B.L., Varga, G.A., and Wideman, R.F., Jr., 1991. The effect of varied dietary calcium and phosphorus on mineral metabolism and acid-base balance in Blue Duiker antelopes. Small Rumin. Res., 5: 93-107. Ross, L.A. and Finco, D.R., 1981. Relationship of selected clinical renal function tests to glomerular filtration rate and renal blood flow in cats. Am. J. Vet. Res., 42:1704-1710. Rugangazi, B.M., and Maloiy, G.M.O., 1987. Salt excretion and saline drinking in the dik-dik antelope (Rhynchotragus kirkii). Comp. Biochem. Physiol., 88A: 331-336. Rumbaugh, G.E., Carlson, G.P. and Harrold, D., 1982. Urinary production in the healthy horse and in horses deprived of feed and water. Am. J. Vet. Res., 43: 735-737.
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SAS, 1985. SAS User's Guide: Version 5 edition. SAS Institute, Inc., Cary, NC, pp.85-123. Scott, D., 1974. Changes in mineral, water and acid-base balance associated with feeding and diet. In: McDonald, I.W., and Warner, A.C.I., eds., Proceedings of the 4th International Symposium on Ruminant Physiology, Sydney, Australia, pp. 205-215. Scott, D. and Buchan, W., 1987. The effects of feeding either hay or grass diets on salivary phosphorus secretion, net intestinal phosphorus absorption and on the partition of phosphorus excretion between urine and faeces in the sheep. Q. J. Exp. Physiol., 72:331-338. Stacy, B.D., 1969. Augmented renal excretion of calcium and magnesium in sheep after feeding. Q. J. Physiol., 54: 1-10. Stacy, B.D. and Wilson, B.W., 1970. Acidosis and hypercalciuria: Renal mechanisms affecting calcium, magnesium, and sodium excretion in the sheep. J. Physiol., 210: 549-554. Tasker, J.B., 1967a. Fluid and electrolyte studies in the horse. III. Intake and output of water, sodium and potassium in normal horses. Cornell Vet., 57: 649-657. Tasker, J.B., 1967b. Fluid and electrolyte studies in the horse. IV. The effects of fasting and thirsting. Cornell Vet., 57: 658-667. Valtin, H., 1983. Renal Function: Mechanisms preserving fluid and solute balances in health. Little, Brown and Co., Toronto, 2nd edn., pp. 87-96. Waugh, W.W., 1977. Photometry of inulin and polyfructosan by use of a cysteine-tryptophan reaction. Clin. Chem., 23: 639-645. Yagil, R. and Berlyne, G.M., 1977. Renal handling of creatinine in various stages of hydration in camels. Comp. Biochem. Physiol., 56A: 15-18.
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© 1993 Elsevier Science Publishers B.V. All rights reserved. 0921-4488/93/$06.00
Effects of varied dietary calcium and phosphorus on renal function of male Blue Duiker antelope B.L. Roeder a, R.F. Wideman, Jr.b and G.A. V a r g a c aDepartment of Veterinary Science, Wiley Laboratory, bDepartment of Poultry Science and CDepartment of Dairy and Animal Science, The Pennsylvania State University, University Park, P4. USA (Accepted 31 August 1992 )
ABSTRACT Sixteen adult male Blue Duikers (Cephalophus monticola bicolor) were fed a basal diet with four levels of Ca and P for 4 months. Pretrial, 24-h urine clearance was studied in each Duiker while they consumed the herd diet (1.38% Ca/0.66% P) (The Pennsylvania State University (PSU) formulation ). Pretrial, 24-h urine clearance values revealed no differences (P> 0.05) among treatment groups consuming the PSU diet. However, younger males did have higher serum P and lower serum and urine Ca concentrations (P< 0.05). Posttrial renal function studies indicated age differences for [ P]elas,,a, [K]un,e, urine/plasma [K], and absolute excretion of K, which were higher in younger males, and free water clearance and tubular P excretion, which were highest in older males. Renal function was affected by the level of Ca and P in the diet. Glomerular filtration rate (GFR) and effective renal blood flow (ERBF) were lower (P < 0.05 ), and fractional excretion (FE) of electrolytes and creatinine (Cr) were higher (P<0.05) in the T 3 group (0.8% Ca/0.4% P), indicating possible onset of renal dysfunction in these Duikers. Clearance of creatinine (Ccr) was negatively correlated with [ Cr] P~a.... suggesting tubular secretion of Cr increased during decreased renal function. Endogenous Cc~ was not an accurate approximation of GFR, as determined by clearance of inulin (C~,), in Blue Duiker under varying nutritional conditions. FE of all electrolytes, as calculated by Gn, regressed against the FE calculated from C c , indicated a poor correlation except for P. This suggests that FEp as measured by Ccr may be a useful index of early renal dysfunction in Blue Duiker. It appears that Blue Duiker have the ability to maximize P retention that may be available in limited quantity in the wild. The C a / P ratio of 0.5:0.4% in the ration appears to be best suited for adult males. Key words: Duiker antelope; Renal function; Calcium; Phosphorus; Creatinine clearance
INTRODUCTION
Maintenance o f inert electrolytes and minerals is related to intake and excretion o f normal serum levels o f these substances. Homeostatic mechanisms are initiated by alterations in extracellular fluid concentrations and result in Correspondence to and present address: B.L. Roeder, Brigham Young University, Department of Animal Science, Provo, U T 84602, USA.
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B.L. ROEDER ET AL.
increased or decreased absorption and/or excretion of minerals and electrolytes. In ruminants, homeostatic mechanisms are mediated via hormonal excretory regulation at the salivary glands and the kidneys (Horst, 1986). The ruminant kidney exhibits an efficient regulatory mechanism to help control water, Na, K, C1, Ca and P balance (Scott and Buchan, 1987; Scott, 1974; Stacy and Wilson, 1970; Stacy, 1969). Exotic ruminants indigenous to arid climates, such as dik-dik antelope (Rhynchotragus kirkii) and camels (Camelus dromedarius) appear to have superior urine concentrating abilities and renal electrolyte handling during periods of extreme ambient temperatures or dehydration (Rugangazi and Maloiy, 1987; Maloiy, 1972). Since hydration, nutritional status and disease states can affect renal electrolyte and mineral excretion patterns (Morris et al., 1984; Osbaldiston and Moore, 1971 ), assessment of these substances in urine permits an understanding of the state of homeostasis. Manipulation of electrolytes and minerals in the diet of ruminants can alter feed intake, growth rate, fecal consistency, serum composition, and urine composition (Roeder et al., 1991; Challa and Braithwaite, 1988a,b,c; Scott, 1974). In ruminants, the occurrence of struvite crystalluria (ammonium magnesium phosphate) is associated with concentrate feeding and a low Ca/ P ratio ( < 1.5 : 1) in the ration (Kallfelz et al., 1987; Jubb et al., 1985 ). Diets high in phosphate can cause a very high incidence of urolithiasis in sheep and goats due to calculi of the phosphatic (struvite) type (Kimberling and Arnold, 1985; Bushman et al., 1965a,b). In experimentally induced urolithiasis produced by dietary oxalates or calcium phosphate, calculi are initially microscopic and form in the collecting tubules (tubular microlithiasis), then encrust on the renal papilla (Jubb et al., 1985 ). Calculi are formed more frequently in animals with alkaline urine, borderline hypovitaminosis A, hypervitaminosis D, urinary stasis, and in arid climates (Powe, 1986 ). Caged Blue Duiker (Cephalophus monticola bicolor) a species of African antelope indigenous to the regions south of the Sahara desert, urinate infrequently even though water is provided ad libitum, and have highly concentrated urine (Roeder, 1990). As part of an investigation of the relationship between dietary Ca, P, and the homeostatic mechanisms controlling their extracellular fluid levels in captive Blue Duiker, evaluation of renal function was performed. This study was initiated to help identify the optimal dietary levels of these elements and the risk of urolithiasis in these animals while consuming a total mixed, pelleted diet with varied levels of Ca and P. MATERIALS AND METHODS
Sixteen clinically healthy, adult, male Blue Duikers, eight being 13 to 24 mo old and eight, 3 to 5 yr old, were obtained from the PSU Deer and Duiker
EFFECTS OF VARIED DIETARY CALCIUM AND PHOSPHORUS ON RENAL FUNCTION
19
Research Facility. Animals were housed individually in modified stainless steel cages (61 cm deep × 64 cm wide X 41 cm high ) with wire mesh flooring in a temperature (20-22 ° C ), light ( 12-h light and dark) and humidity ( 5070%) controlled environment. Animals were initially fed the PSU pelleted diet and allowed to acclimate to their new environment before initial studies were conducted to determine baseline serum and urine values. Animals were then assigned by randomized block design to one of four dietary levels of %Ca and %P of 0.5:0.4% (TI), 0.8:0.8% (T2), 0.8:0.4% (T3) , 1.25:0.4% (T4) (block = age group of Duiker) for 4 mo. Diets were complete pelleted rations with no access to hay; and dietary C a / P ratios were different ( P < 0.05 ) among treatments (T1 = 1.25: 1, T2= 1 : 1, T3=2: 1, T4=3:1 ) (Roeder et al., 1991; Roeder, 1990). While consuming the PSU diet ( 1.38% Ca/0.66% P), a pretrial, 24-h volumetric urine clearance study was performed on each Duiker. Voided urine was collected from screen covered, acid washed, stainless steel pans. To establish electrolyte and creatinine values in their blood and urine, three blood samples and two timed urine collections were obtained during a 24-h study (baseline, then a 2nd and 3rd blood sample were collected as soon as possible after the I st and 2nd urinations ). Feed and water consumption, body weight and temperature, and volumetric urine output were measured daily, and weekly blood gas analyses, venous and urine electrolytes and creatinine were determined (Roeder et al., 1991 ). A renal function study was performed after 17 wk of consuming their designated level of %Ca and P. To decrease the risk of regurgitation of ruminal contents and subsequent pulmonary aspiration associated with general anesthesia in ruminants, food was withheld for 18 h and water for 6 h prior to the procedure. To conduct kidney function studies, Duikers were anesthetized using isoflurane (Aerrane, Anaquest, Madison, WI) administered via mask for induction followed by intubation with a cuffed endotracheal tube and a light plane of anesthesia was maintained for the remainder of the experiment. The right jugular vein was cannulated cranially and caudally with sterile 20-G, 2-inch teflon intravenous (i.v.) catheters (B-D I.V. CATH non-radiopaque FEP teflon, Becton Dickinson and Co., Rutherford, NJ ) introduced with a 24-G needle. Duikers were positioned in left lateral recumbency on a towel covered, heated, circulating water pad (Aquamatic, American Medical Systems, Model K-20, Division of American Hospital Supply Corp., Cincinnati, OH ) placed on a foam cushioned surface, and the head positioned with the nostrils lower than the larynx to allow free drainage of saliva and any regurgitated fluid. Blood samples were obtained from the cranial i.v. catheters for measurement of hematocrit (PCV), osmolality, creatinine (Cr) and electrolyte (Ca, P, Na, K, C1) concentrations at times of 0, 30, and 75 min after beginning the i.v. infusion. A 2-ml bolus injection of a solution (283.5 mOsm/kg H20) containing 25 mg/ml mannitol, 1.5 m g / m l inulin, 1.5 mg/ml para-aminohip-
20
B.L. ROEDER ET AL.
puric acid (PAH) (PAH; Sigma Chemical Co., St. Louis, MO) and 25 mg/ ml dextrose was infused i.v. via infusion pump (Harvard Apparatus Co., Inc., Millis, MA) followed by an administration rate of 0.2 ml/kg B W / m i n for a 30-min equilibration period. The low dose of mannitol, an osmotic diuretic, was used to insure that adequate urine production was maintained during the clearance period. PAH was used to measure effective renal plasma flow ( C p A H = ERPF) and to assess proximal tubular secretory function. Inulin (In) was used as a filtration marker for measuring glomerular filtration rate (C~n=GFR). Dextrose 2.5% was added to the infusion solution to increase its effective osmolality to achieve isotonicity, without adding electrolytes that would influence the subsequent measurements. After 30 min of equilibration, the urinary bladder was cannulated with fenestrated PE-50 polyethylene tubing (Clay Adams, Parsippany, N J) via sterile transcutaneous cystocentesis after introduction of a sterile 16-G 1.5-inch needle into the right dorsolateral aspect of the bladder, just anterior to the pelvic canal; and all urine was collected. The PE tubing was left in situ for the 40-min clearance period, then all urine was collected in a pre-weighed inert plastic container for gravimetric determination of urine volume and flow rate. During recovery from anesthesia, animals received sterile 5% dextrose i.v. (0.2 m l / k g / m i n ) for 20 min to ensure adequate hydration and perfusion pressure were maintained after this procedure. Colorimetric assays were used to measure P, C1, and Cr as previously described (Roeder et al., 199 l; Roeder, 1990 ), inulin (Waugh, 1977 ), and PAH (Brun, 1957); flame photometry (Instrumentation Laboratory Inc., Model 443, Wilmington, MA) for Na and K (Roeder et al., 1991; Roeder, 1990); atomic absorption spectroscopy (Instrumentation Laboratory Inc., Model 55 l, Wilmington, MA) for total Ca in serum and urine. Urine pH was measured to the nearest 0.01 pH unit, using a pH meter (DigipHase pH meter, Cole Parmer, Santa Ana, CA). Urine and plasma osmolality were measured with a vapor pressure osmometer ( 5100C Vapor Pressure Osmometer, Wescor, Inc., Logan, U T ) . Urine sample volumes were converted to urine flow rates (V), and expressed as ml/kg B W / m i n for the clearance interval. Glomerular filtration rate (GFR, ml/kg B W / m i n ) was calculated as the clearance of inulin (urine inulin concentration/plasma inulin concentration × V). The osmolal clearance (Cosm, ml/kg B W / m i n ) was calculated as urine osmolality/plasma osmolality X V. Free water clearance (FWC or CH2o ) values were calculated by subtracting the osmolal clearance from V ( V - Cosm). FWC is interpreted as values greater than 0 represent the rate at which water would have to be removed from urine to maintain isotonicity with plasma (hyposmotic urine ) and values less than 0 represent the rate at which water would have to be added to urine to maintain isotonicity with plasma (hyperosmotic urine). Absolute excretion (AbsEx) rates were calculated for each sample by multi-
EFFECTSOF VARIEDDIETARYCALCIUMAND PHOSPHORUSON RENALFUNCTION
21
TABLE 1 Serum and urine electrolytes and creatinine values for male Blue Duiker consuming the PSU pelleted diet Parameter
Younger cf
Older o~
+ SE
Serum Ca ( m M / l ) Pi (mM/1) Creatinine ( mg/dl ) K (mM/l) Na ( m M / l ) CI ( m M / l )
2.6 b 3.0 a 0.64 5.5 151 118
2.9 a 2.3 b 0.58 6.3 149 118
0.06 0.18 0.03 0.30 1.43 1.79
Urine Ca (mM/1) Pi ( m M / l ) Creatinine ( m g / d l ) K (mM/1) Na (mM/1) CI ( m M / l )
0.49 b 2.71 64.1 132.4 119.5 110.0
1.30 a 3.58 96.9 115.1 128.0 105.6
0.23 1.68 15.2 19.6 16.3 11.0
a,bMeans with different superscripts differ significantly ( P < 0.05 ). n = 8 younger males ( 13 to 24 mo ) and 8 older males (3 to 5 yr).
plying the amount of each substance in urine × V and expressed as/~mol/kg BW/min. Excretion data for Ca (FEca), inorganic P (FEp), Na (FENa), K (FEK), C1 (FEcl), and Cr (FEcr) are expressed as fractional excretion rates, which were calculated for each sample by dividing the clearance of each substance (Cy=(Uy/Py)XV, where Cy=the clearance of a substance y, U = urinary concentration, P = plasma concentration, and V= urine flow rate ( m l / k g / m i n ) ) , by the GFR. Clearance values for PAH were calculated for estimations of effective renal plasma flow (ERPF, ml/kg B W / m i n ). The effective filtration fraction (EFF) was calculated as EFF = G F R / E R P F , the fraction of renal plasma flow entering the nephron by glomerular filtration. Effective renal blood flow (ERBF, ml/kg B W / m i n ) was calculated as the E R P F / ( 1 - [hematocrit/100] ). The absolute filtered water resorption rate (FWResorp) was calculated as G F R - V . The fraction of filtered water excreted (FFWExc) as urine was measured by dividing one by the concentration of urine inulin/plasma inulin = ( V/GFR ). The fraction of filtered water reabsorbed (FFWReab) from the glomerular filtrate by both kidneys was calculated as 1 minus the fraction of filtered water excreted as urine. Tubular Ca (TubCa) and P (TubP) excretion were calculated by: (their concentration in urine× V) - (their concentration in plasma × GFR). Statistical analysis was performed by least-squares analysis of variance and Duncan's multiple range test using the SAS General Linear Models procedure
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B.L. ROEDER ET AL.
TABLE 2 Results of a 40-min clearance period in male Blue Duiker receiving an i.v. infusion of 2.5% mannitol, 0.15% PAH, 0.15% inulin, and 2.5% dextrose on baseline kidney parameters Parameter 2
kg BW PCV UpH GFR V Uosm Posm
Costa FWResorp FFWExc FFWReab ERPF ERBF EFF
Treatment group 1
2
3
4
+_SE
3.74 39.08 c 6.47 '~ 11.90 ~' 0.136 509.25 275.70 0.257 11.76 a 0.013 0.987 30.31 49.24 ~b 0.441a
4.00 46.08 a 5.52 b 7.49 ab 0.129 409.13 276.33 0.191 7.36 ab 0.019 0.981 34.69 64.06 a 0.214 b
3.79 42.70 b 6.57 a 5.58 b 0.103 527.38 279.70 0.197 5.48 b 0.021 0.979 16.53 29.32 b 0.349 ab
3.85 43,13 b 6,61 a 12.01 a 0,129 496.50 275.38 0.242 11.88 a 0.014 0.986 35.18 62.71 a 0.325 ab
0.13 0.88 0.24 1.93 0.029 81.85 4.41 0.06 1.92 0.004 0.004 6.70 10.71 0.056
a,b,¢Means with different superscripts differ significantly ( P < 0 . 0 5 ) . ~Treatment group= 0.5:0.4% ( T I ), 0.8:0.8% (T2), 0 . 8 : 0 . 4 % (T3) , 1.25:0.4% (T4).
2Abbreviations and units: kg BW, body weight in kg; PCV, erythrocytic packed cell volume or hematocrit (%); UpH, urine pH; GFR, glomerular filtration rate ( m l / k g B W / m i n ) = CIn; V, urine weight specific flow rate ( m l / k g / m i n ) ; Uosm, urine osmolality ( m O s m / k g H 2 0 ) ; Posm, plasma osmolality Uosm ( m O s m / k g H 2 0 ) ; Cosr., osmolal clearance= p---~. × V ( m l / k g B W / m i n ) ; FWResorp, filtered water clearance (Cn2o) ( m l / k g / m i n ) = V-Corm ); FFWExc, fraction of filtered water excreted ~
1
Uln//~ln
= V~
GFR; FFWReab, fraction of filtered water reabsorbed from the glomerular filtrate by both kidneys ERPF 1 - FFWExc; ERPF, CpA8 ( m l / k g B W / m i n ) ; ERBF, (ml/kg B W / m i n ) ; EFF, effective 1 - PCV/100 GFR filtration fraction = ERPF"
(SAS, 1985). The mathematical model included variations attributable to age and treatment with higher order interactions. Differences were considered significant if P values were less than 0.05. RESULTS
Pretrial, while Duikers' were consuming the PSU pelleted ration with 1.38% Ca/0.66% P (2:1 ratio), 24-h urine clearance values revealed no differences ( P > 0.05 ) among treatment groups. Younger male Duikers did have higher ( P < 0 . 0 5 ) serum P and lower ( P < 0 . 0 5 ) serum and urine Ca values than older males. Other serum and urine electrolytes and creatinine levels were not
EFFECTS OF VARIED DIETARY CALCIUM AND PHOSPHORUS ON RENAL FUNCTION
23
TABLE 3 P l a s m a a n d urine electrolyte a n d creatinine values after a 4 0 - m i n clearance period in male Blue Duiker receiving an i.v. infusion o f 2.5% m a n n i t o l , 0.15% PAH, 0.15% inulin, a n d 2.5% dextrose Parameter 2
FEca [CA]plasma [ Ca ] u,~.¢ U/P Ca AbsExCa TubCa FEP [P]Pl . . . . [P]u~i,~ U/P P AbsExP TubP FeN~ [ N a ] pl. . . . [Na]u,~ne U/P N a AbsExNa FEK [K]plasm~ [ K ] Ur~n~ U/P K AbsExK FEcl [Cl]pl . . . . [Cl]un~e U/P CI AbsExCI FEcr [Cr ]pl~m~ [Cr ]u~in~ U/P Creat CCr
T r e a t m e n t groupl
1
2
3
4
_+SE
0.0017 b 1.63 b 0.263 b 0.162 ab 0.148 - 18.98 ~b 0.012 1.69 . 1.60 0.780 0.406 - 19.77 b 0.0010 b 128.13 10.45 0.082 1.81 0.249 2.65 b 51.25 19.02 8.05 0.0023 b 110.23 20.90 0.190 3.06 1.17 b 0.475 37.90 b 82.42 b 11.99 b
0.0036 b 1.67 ab 0.306 ab 0.183 ab 0.161 - 12.49 ab 0.023 1.99 2.91 1.289 0.365 - 16.50 ~b 0.0015 ab 130.88 10.00 0.076 1.29 0.358 2.55 b 44.35 18.09 5.93
0.0067 a 1.76 ab 0.569 a 0.325 a 0.241 - 9.56 a 0.017 1.87 1.57 0.695 0.131 -8.90 a 0.0024 ~ 133.38 14.60 0.108 1.45 0.449 3.53 a 74.85 21.38 7.92 0.0051 a 108.15 26.35 0.242 2.77 7.70 a 0.250 79.05 ~ 354.08 a 35.60 ~b
0.0017 b 1.79 a 0.250 b 0.141 b 0.143 - 21.85 b 0.007 1.75 1.19 0.567 0.220 - 19.92 b 0.0013 ab 132.13 14.95 0.1 l 1 1.79 0.286 2.98 ab 76.40 26.26 10.18
0.0012 0.058 0.106 0.062 0.062 3.63 0.009 0.37 1.29 0.49 0.241 3.68 0.0005 2.31 3.90 0.028 0.66 0.087 0.21 11.46 4.58 2.42 0.0009 1.95 4.78 0.041 0.99 1.76 0.105 12.53 92.00 9.27
0 . 0 0 3 1 ab
108.08 16.38 0.152 2.19 2.72 b 0.450 36.07 b 126.80 ~b 14.90 ab
0 . 0 0 2 7 ab
112.10 24.15 0.213 3.28 3.35~b 0.325 55.37 ~b 307.08 ~b 38.36 a
abMeans with different superscripts differ significantly ( P < 0.05 ). ~Treatment g r o u p = 0 . 5 : 0 . 4 % ( T I ) , 0 . 8 : 0 . 8 % (T2), 0.8:0.4% (T3), 1.25:0.4% (T4). 2See Table 2 for a b b r e v i a t i o n s a n d units. P l a s m a a n d u r i n e values are in m M / l except creatinine ( m g / d l ) . O t h e r a b b r e v i a t i o n s in Table 3 include: FEx, fractional excretion o f s u b s t a n c e 'X',
C~=[(~ )× V]/GFR; AbsExX, absolute excretion of substance "X', [X ]u × V (llmol/kg BW /min ); T U B C a , t u b u l a r c a l c i u m excretion, ( [Ca] tmne× V) - ( [Ca]el . . . . rus excretion, ( [ P ] t m n e × V) - ( [P]et . . . . × G F R ) .
× GFR); TUBP, tubular phospho-
24
B.L. ROEDERETAL. 25 GFR=0.315*ERPF N=16
P<0.0001
R2=.93
20
0.5% ~O.'t% e Xa 2
o le~ca:o.e%P
lo! (9
Ta 3 0.1~:04%
P
Trl 4 1.25% C~:0.4% e ZX
•
0 0
I
I
i
i
i
i
10
20
30
40
50
60
70
ERPF (ml/min)
Fig. 1. Linear regression of glomerular filtration rate (GFR) against effective renal plasma flow (ERPF), by treatment. Individual Duikers within a treatment group are identified by symbols; n = 4 Duiker/trt.
70
Ccr= 4 4 . 0 3 - 5 0 . 1 8 * [Cr] plasma N=16
"~, 60
P<0.01
R2--.37 Ta 1 0.5%C~0.4% P
so
13 xrl 2 0.8% ~:0.8% P
~ 4o ~
30
~
20
Trl 3 (I.8% ~ 0 . 4 % P Trt 4 1.25% C~1:0.4% P /3.
i
i
i
i
0,2
0.4
0.6
0.8
&_ 1
Plasma Creatinine (mg/dl)
Fig. 2. Creatinine clearance (Ccr) regressed against plasma creatinine concentration by treatment. Individual Duikers within a treatment group are identified by symbol; n = 4 Duiker/trt.
affected by age (Table 1 ). Dietary %Ca intake on a bodyweight basis was approx. 0.02%=T1, 0.03%=T2 and T3, and 0 . 0 4 5 % = T 4 and significant between TI
EFFECTS OF VARIED DIETARY CALCIUM AND PHOSPHORUS ON RENAL FUNCTION CCr = 4 2 . 6 2 - 5 . 4 9 * G F R 70
N=16
+ 0.29*GFR P<.12
25
2
R2=.23
Td
o.~%c~:o.4~P Ttt 2 0.8 %Ca:0.8 %P
x-"
[]
5O
Td 3 0.8 %Ca:O.4 %P
o,) o 40
Trl 4 ~.25 %Ca:O.4 %P
E:
/ 30 C)
._~ 20 r-
[] 0
10
/
o S 0
,
i
5
10
~ 1
+
,
20
25
G F R = Inulin C l e a r a n c e ( m l / k g / m i n )
Fig. 3. Creatinine clearance (Ccr) regressed against glomerular filtration rate ( G F R ) as determined by the clearance of inulin, Individual Duikers belonging to a treatment group are identified by symbol; n = 4 Duiker/trt. The straight line within the graph is the line of equality, where points would fall if filtration = excretion. Points above this line indicate net tubular secretion and below the line indicate net tubular resorption.
Varied dietary %Ca/%P did not cause differences between age groups' serum and renal values except for [P]Plasma, [K]urine, urine K/plasma K ( U / P K), and absolute excretion of K, which were higher in younger males, and FWC and TubP excretion, which were higher in older males ( P < 0.05 ). Key differences based on diet were found for hematocrit (PCV), urine pH, [ PAH ] Urine, absolute GFR, absolute filtered water resorption rate, ERBF, EFF, [ Ca ] Urine, [Ca]Plasma, urine Ca/plasma Ca ( U / P Ca), TubCa excretion, FEca, TubP excretion, FENa, [ K ] Plasma,FEcl, [ Cr ] U~ine,urine Cr/plasma Cr, FEcr, and the creatinine clearance (Ccr) (Tables 2 and 3 ) ( P < 0.05 ). Duikers fed rations with 0.8% P (T2) had a higher ( P < 0.05 ) mean PCV, and ERBF, and lower (P<0.05) urine pH than Duikers consuming diets with 0.4% P. However, Duikers receiving the T 2 diet had decreased GFR and EFF values, tending to elevate FE values for electrolytes. Duikers consuming 0.5% Ca/0o4% P (T~) had the lowest PCV values but similar GFR, ERBF, and FE values for electrolytes to Duikers fed diets T2 and T4. The dietary Ca/P ratio of2:1 (0.8% Ca/0.04% P) in T 3 had similar urine pH values to TI and T4, and a mean PCV value similar to T4. However, the T3 diet decreased (P<0.05) GFR, ERBF, and FWResorp, and increased ( P < 0.05 ) TubCa and TubP excretion compared to the other three treatments. All FE values for electrolytes and creatinine were elevated by diet T3. Absolute excretion for Na, K, and C1 was not different ( P < 0.05 ) for Duikers fed diet T3 compared with the other diets, indicating the increase in FE values was due to the decrease in GFR rather than an absolute increase in the net loss of electrolyte ( V× [X ] U r i n e )" Urinary
26
B.L. ROEDER ET AL -
-
20
AbsExP
Trt 1 0.S%C~9.dl%P
AbsExCr
TBI2 o.e,~c ~ o ~ P T~3 0 ~ ~0.~% P
15
Te 4
1,~% c,,:o.4%P A
E E
z~ '<
<
~
5
c?
~x
D
I
l
l
l
I
l
I
I
I
I
I
2
4
6
8
10
12
14
16
18
20
22
GFR=lnulin Clearance (ml/kg/min) b
0.06
T~ I o.~'~~:o.,,~ P
FEp=. 1 7 - . 2 8 * G F R N=16
P<0.73
R2=.0~
Ta 2 0.a'~ ~ 0 . r , 6
P
0.05 Xa 3 o.e'~ ~ o . 4 % P
"6
0.04
Ta 4 1.25%Ca:0.4~P
0.03
o
0.02
[3
0.01
[] []
0 0
5
10
15
20
25
GFR (ml/kg/min) 20
Ta 1 0.5"4 C~0.4% P
FF13r=6.78-.33*GFR N=16
Tn,2 0,8%C4~'O.(P~,P
P < 0 . 0 9 R~'-.19
I::: 15 Tn3 O.e'V, C~'0.4 "/, P O Td 4
"6
1.~'W, ~ : 0 A % P
g Io
._0 5 U.
5
10
15
GFR (ml/kg/min)
2O
i _ 25
EFFECTS OF VARIED
d
0.00
DIETARY
CALCIUM
AND
PHOSPHORUS
ON RENAL
27
FEp=.005+ 1.36*FEp ( ~ ~J
N=16 P
j.J O~ 0.04 o,"6 •~
FUNCTION
J
J
•
J O5%C~0.4%P
0.03
&
~
,,2 o.a,v.c~o.a%P
0.02
Trl3 o.e~.c~o4%p
0.0t
0 __ 0
I
I
I
0.02 0.03 Fractional Excretion of P (Ccr), %
0.01
_
I 0.04
Fig. 4. Absolute excretion of phosphorus (AbsExP) and creatinine (AbsExCr) versus glomerular filtration rate (GFR) (a). Regression of the fractional excretion of phosphorus (FEp) (b) and creatinine (Cr) (c) against GFR. Regression of the FEp as performed by the clearance of inulin (C~.) against the FEp as determined by the clearance ofcreatinine (Ccr) (d). Individual Duikers within a treatment group are identified by symbol; n = 4 Duiker/trt.
creatinine concentration, Ccr , [K]Plasma, and the U / P Ca levels were also higher ( P < 0.05 ) in the Duikers fed diet T3 than in the other treatments. Plasma Ca values tended to increase as dietary Ca levels were raised. Urinary Ca excretion was highest ( P < 0.05 ) in T 3 and lowest ( P < 0.05 ) in the high Ca low P diet, T4 ( 1.25% Ca/0.4% P). Although trends were apparent for the highest plasma and urine [P] in T2, differences were nonsignificant between treatments due to the consistently elevated P values of two individuals in this group beyond those of the other T2 males. TubCa and TubP excretion were highest ( P < 0 . 0 5 ) for Duikers fed diet T 3 (0.8% Ca/0.4% P), and lowest ( P < 0 . 0 5 ) for Duikers fed the high Ca and low P diet, T4 ( 1.25% Ca/ 0.4% P). The lowest ( P < 0.05 ) TubP excretion was seen in Duikers fed diets Tl (0.5% Ca/0.4% P) and T4. There was positive correlation between GFR and ERPF (Fig. 1 ) and the slope, 31.5%, ( G F R / E R P F ) is the mean value for EFF. Although individual variation within a treatment group did cause differences, those Duikers on diets T2 or T3 with 0.8% Ca tended to have the lowest values for GFR and ERPF. Ccr was negatively correlated with Pcr concentration (Fig. 2 ). Duikers with the lowest GFR and ERPF (T2 and T 3) values tended to have the highest Ccr and low Pcr values. GFR as determined by the clearance of inulin (Cln) was poorly correlated with endogenous Ccr (Fig. 3). Most Duikers appeared to have a Ccr/Cln ratio (FEcr) greater than 1.00 indicating net tubular secretion of creatinine. Duikers receiving 0.8% Ca/0.4% P (T3) had the highest FEcr
28
B.L. R O E D E R ET AL.
(P< 0.05 ) and tended to have the lowest plasma creatinine values compared to the other three treatments. Absolute excretion of P and creatinine were relatively unaffected by GFR (Fig. 4a) and their FE values were poorly correlated with GFR as measured by C~n (Fig. 4b and c). However, when FEp, as measured by C~n, was regressed against FEp, determined by endogenous Ccr, a positive correlation was found with P < 0.001 (Fig. 4d). Similar regressions of FEca, FENa, FEK, and FEcl yielded poor nonsignificant (P> 0.05 ) correlations. DISCUSSION
This study was prompted by research of urolithiasis and renal dysfunction in domestic ruminants when consuming diets with varied Ca/P ratios beyond levels of requirements, particularly diets with a Ca/P ratio of less than 1.5: 1. Blue Duikers at PSU had been observed to have marked hyperphosphatemia, hyperkalemia, mild to moderate hypocalcemia, and reversed Ca/P ratios (particularly juveniles and young adults ) consuming the pelleted herd ration. Since these animals had been considered at risk of becoming an endangered species, all studies were conducted as survival research, experiments were designed to use primarily noninvasive procedures, and only a limited number of males were available. However, since the study was meant not only to investigate Ca and P homeostatic mechanisms but to better define requirements for these macronutrients, assessment of renal function in Blue Duikers consuming varied %Ca/% P was needed to detect impairment of kidney function that these levels may have caused. Evaluations of serum or plasma urea nitrogen, creatinine, and urine specific gravity are simple but relatively insensitive tests of renal function since normal values are compatible with up to two-thirds loss of renal parenchyma in ruminants and other mammalian species (Fetcher, 1986). The ability of the kidney to concentrate urine has been regarded as a sensitive indicator of renal function in animals when performed as urine osmolality (Bovee, 1969). The ratio of urine to plasma osmolality (u//eosm) indicates the number of times the kidney concentrates the urine over plasma; whereas, urine specific gravity provides only a crude approximation of the renal concentrating ability by measuring urine density. Performing U / P o s m in conjunction with measurements of GFR, ERPF, and FE of electrolytes and creatinine provides a more accurate assessment of renal function because changes in renal hemodynamics precede elevations in serum urea nitrogen, creatinine, and urine specific gravity (Fetcher, 1986). Calculation for FE of electrolytes using endogenous creatinine clearance (Ccr) has been advocated as useful in man and animals, including ruminants, since creatinine production within the body is relatively constant and excretion is almost entirely by glomerular filtration in most species (Neiger and
EFFECTS OF VARIED DIETARY CALCIUM AND PHOSPHORUS ON RENAL FUNCTION
29
Hagemoser, 1985; Morris et al., 1984; Kaneko, 1980; Bovee and Joyce, 1979 ). Renal FENa has been used to help differentiate renal from prerenal azotemia in humans (Espinel and Gregory, 1980; Espinel, 1976), and horses (Morris et al., 1984). When tubular dysfunction causes a reduced capacity to reabsorb filtered Na, FENa becomes elevated; alternatively, renal FENa values < 1% usually indicate prerenal azotemia with avid reabsorption of filtered Na. In horses, during feed and water deprivation, the total amount of Na excreted in urine tends to increase (Rumbaugh et al., 1982; Stacy and Wilson, 1970; Tasker, 1967a,b). Blue Duiker in this study, although fasted for 18 h prior to their renal function test still had ingesta present in their forestomachs, albeit a reduced quantity, and were adequately hydrated, as reflected by normoproteinemia and PCV values similar to unfasted Duikers in the herd. FENa (performed by CIn) did show significant differences by treatment which correlated well with levels of GFR and ERPF. The use of endogenous Ccr to perform FE calculations, however, was poorly correlated with those values obtained by the CIn except for FEp. Blue Duiker appeared to increase rate of net creatinine secretion during reduced renal function as measured by decreased GFR and ERBF thereby maintaining low levels of creatinine in plasma and enhancing urinary excretion of creatinine. Blue Duikers consuming diets with the higher levels of Ca tended to have elevated Ccr. However, Duikers receiving 0.8% Ca/0.4% P (T 3), had the lowest values for GFR, ERPF, and ERBF with the highest FE of creatinine and other electrolytes indicating this level o f % C a / % P at a 2 : 1 ratio with this diet formulation had detrimental effects on normal renal function. The FEp in the T 3 group, although not significantly different from the other treatments, was increased over the other two treatments receiving 0.4% P and approached the highest FEp values seen in animals consuming 0.8% P. Since FEp was positively correlated between its calculation by CIn and Ccr, this measurement may be a useful index of early renal dysfunction in Blue Duikers. The 24-h volumetric urine studies, although cumbersome, appear to be the most valid method of evaluating mineral metabolism via urinary clearance studies in Blue Duiker. Endogenous Ccr is affected by dietary differences in Blue Duiker similar to that which has been reported in sheep consuming diets with varied N and energy content (Faix et al., 1988 ), and camels during exposure to heat and water deprivation (Yagil and Berlyne, 1977). This, in addition to the failure of Ccr to be well correlated with Cin, suggests that endogenous Ccr is not an accurate approximation of GFR in Blue Duiker under varying C a / P conditions. EFF obtained in this study (31.5% ) was higher than what has usually been reported for most mammals, i.e., 20% or about one-fifth of the plasma entering the glomerular capillaries is filtered (Valtin, 1983 ). However, in two separate studies with cats, a species with great urine concentrating ability (even during development of azotemia compared to dogs and humans) the calcu-
30
B.L. ROEDER ET AL.
lated EFF ranged between 20-50% (Ross and Finco, 1981; Osbaldiston and Fuhrman, 1970). More studies, including the evaluation of possible anatomical differences and the extraction efficiency of PAH, need to be done to better define this difference in Blue Duiker. CONCLUSION
This study indicated that: ( 1 ) Blue Duiker fed this diet with a Ca/P ratio of 2: 1 had possible onset of renal dysfunction; (2) Blue Duiker with renal dysfunction, as measured by decreased GFR (C~n) and ERBF (CpAH), appear to increase tubular secretion of creatinine which causes plasma levels of creatinine to decrease (unlike other species with renal dysfunction where elevation of plasma creatinine concentration drives increased tubular secretion of creatinine); (3) endogenous creatinine clearance (Ccr) is not an accurate approximation of GFR in Blue Duiker under varying Ca/P conditions; (4) fractional excretion of P relative to inulin and endogenous creatinine clearances were correlated suggesting that fractional excretion of P may be a useful index for detection of early renal dysfunction in this species; and (5) filtration fractions in Blue Duikers are higher than those usually reported for other animals. These findings may indicate adaptive mechanisms for this species to survive in the wild where P intake may be limited. A higher EFF may enhance clearance of unwanted substances, and increased tubular secretion of creatinine during early renal dysfunction may prolong life. ACKNOWLEDGEMENTS
The authors thank Joyce L. Satnick, Glenda C. Loop, Donna L. Warner, and Robert C. Mothersbaugh for their technical assistance.
REFERENCES Bovee, K.C., 1969. Urine osmolarity as a definitive indicator of renal concentrating capacity. J. Am. Vet. Med. Assoc., 155: 30-35. Bovee, K.C. and Joyce, T., 1979. Clinical evaluation of glomerular function: 24 hour creatinine clearance in dogs. J. Am. Vet. Med. Assoc., 174: 488-491. Brun, C., 1957. A rapid method for the determination of para-aminohippuric in kidney function tests. J. Lab. Clin. Med., 37: 955-958. Bushman, D.H., Emerick, R.J. and Embry, L.B., 1965a. Experimentally induced ovine phosphatic urolithiasis: relationships involving dietary calcium, phosphorus and magnesium. J. Nutr., 87: 499-504. Bushman, D.H., Emerick, R.J. and Embry, L.B., 1965b. Incidence of urinary calculi in sheep as affected by various dietary phosphates. J. Anita. Sci., 24:671-675. Challa, J. and Braithwaite, G.D., 1988a. Phosphorus and calcium metabolism in growing calves with special emphasis on phosphorus homeostasis. I. Studies of the effect of changes in the
EFFECTS OF VARIED DIETARY CALCIUM AND PHOSPHORUS ON RENAL FUNCTION
31
dietary phosphorus intake on phosphorus and calcium metabolism, J. Agric. Sci., 110: 573581. Challa, J. and Braithwaite, G.D., 1988b. Phosphorus and calcium metabolism in growing calves with special emphasis on phosphorus homeostasis, 2. Studies of the effect of different levels of phosphorus, infused abomasally, on phosphorus metabolism. J. Agric. Sci., 110: 583-589. Challa, J. and Braithwaite, G.D., 1988c. Phosphorus and calcium metabolism in growing calves with special emphasis on phosphorus homeostasis. 3. Studies of the effect of continuous intravenous infusion of different levels of phosphorus in ruminating calves receiving adequate dietary phosphorus. J. Agric. Sci., 110: 591-595. Espinel, C.H., 1976. The FENa test: Use in the differential diagnosis of acute renal failure. J. Am. Med. Assoc., 236: 579-581. Espinel, C.H. and Gregory, A.W., 1980. Differential diagnosis of acute renal failure. Clin. Nephrol., 13: 73-77. Faix, S., Leng, L., Szanyiova, M. and Boda, K., 1988. Creatinine and inulin clearance at the different nitrogen and energy intake in sheep. Comp. Biochem. Physiol., 91A: 689-691. Fetcher, A., 1986. Renal disease in cattle. Part II. Clinical signs, diagnosis, and treatment. Comp. Cont. Ed. Pract. Vet., 8: $338-$345. Horst, R.L., 1986. Regulation of calcium and phosphorus homeostasis in the dairy cow. J. Dairy Sci., 69: 604-616. Jubb, K.V.F., Kennedy, P.C. and Palmer, N., 1985. Pathology of Domestic Animals. Academic Press, Inc., New York, 3rd edn., Vol. 2, pp.392-395. Kallfelz, F.A., Ahmed, A.S., Wallace, R.J., Sasangka, B.H. and Warner, R.G., 1987. D i e l a ~ magnesium and urolithiasis in growing calves. Cornell Vet., 77: 33-45. Kaneko, J.J., 1980. Clinical Biochemistry of Domestic Animals. Academic Press, New York, 3rd edn. Kimberling, C.V. and Arnold, K.S., 1985. Diseases of the urinary system of sheep and goats. Vet. Clin. North Am.: Large Anim. Pract., 5:637-640. Maloiy, G.M.O., 1972. Renal salt and water excretiion in the camel. Symp. Zool. Soc. Lond., 31 : 243-259. Morris, D.D., Divers, T.J. and Whitlock, R.H., 1984. Renal clearance and fractional excretion of electrolytes over a 24-hour period in horses. Am. J. Vet. Res., 45: 2431-2435. Neiger, R.O. and Hagemoser, W.A., 1985. Renal percent clearance ratios in cattle. Vet. Clin. Pathol., 14: 31-35. Osbaldiston, G.W. and Fuhrman, W., 1970. The clearance ofcreatinine, inulin, para-aminohippurate and phenosulphothalein in the cat. Can. J. Comp. Med., 34:138-141. Osbaldiston, G.W. and Moore, W.E., 1971. Renal function in cattle. J. Am. Vet. Med. Assoc., 159: 292-301. Powe, T.A., 1986. Diseases of the urinary system. In: Howard, J.L., ed., Current Veterinary Therapy: Food Animal Practice. W.B. Saunders Co., Philadelphia, 2nd edn., pp. 816-818. Roeder, B.L., 1990. The effect of varied dietary calcium and phosphorus on calcium and phosphorusi metabolism and acid-base balance in Blue Duiker antelope. Ph.D. Dissertation, The Pennsylvania State University, University Park, PA, USA. Roeder, B.L., Varga, G.A., and Wideman, R.F., Jr., 1991. The effect of varied dietary calcium and phosphorus on mineral metabolism and acid-base balance in Blue Duiker antelopes. Small Rumin. Res., 5: 93-107. Ross, L.A. and Finco, D.R., 1981. Relationship of selected clinical renal function tests to glomerular filtration rate and renal blood flow in cats. Am. J. Vet. Res., 42:1704-1710. Rugangazi, B.M., and Maloiy, G.M.O., 1987. Salt excretion and saline drinking in the dik-dik antelope (Rhynchotragus kirkii). Comp. Biochem. Physiol., 88A: 331-336. Rumbaugh, G.E., Carlson, G.P. and Harrold, D., 1982. Urinary production in the healthy horse and in horses deprived of feed and water. Am. J. Vet. Res., 43: 735-737.
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