Influence of Dietary Protein on Renal Function in Dogs
KEHHETH C. BOVÉE2
School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104
ABSTRACT Two previously published studies in dogs with reduced renal function are reviewed. In the first study, renal function and biochemical responses to di etary changes were studied in four dogs with stable chronic renal failure. The objective was to determine if dogs with moderate stable failure adjust to diets with varied protein and electrolyte content. These dogs were found to have the capacity to adapt to a wide range of dietary protein and electrolyte intake. The only excep tion was found in dogs fed a reduced-protein diet, which failed to appropriately adjust renal tubular excretion of sodium and phosphate. The only advantage of reduced dietary protein in this study was a reduction in blood urea nitrogen (BUN). Disadvantages of reduced-protein diets were reduced glomerular filtration rate (GFR) and renal plasma flow. In the second study, the hypothesis that large amounts of dietary protein sustain renal hyperfunction and produce progressive glomerulosclerosis in dogs as previously reported in rats was tested.
Results failed to find a pattern of deterioration of renal function over 4 y. Light microscopic changes and elec tron microscopy also failed to find glomerular injury similar to that reported in rodents. These results do not support the hypothesis that feeding a high protein diet had a significant adverse effect on renal function or morphology. J. Nutr. 121:8128-8139, 1991.
INDEXING KEY WORDS:
- symposium - dogs - renal function - dietary protein
There has been considerable confusion in the vet erinary literature about dietary protein and renal function in dogs. Confusion and controversy has arisen from both nutritionists and clinicians due to three major problems: 1) imprecise terminology about renal failure, 2) confusion between progression of renal dis ease and treatment of renal failure and 3) dietary effects in rodents that have been unfortunately transposed to other species. The purpose of this paper is to attempt to clarify these points and review two studies from our laboratory. Discussion will focus on adaptation of renal function to loss of renal mass and progression of renal failure. There are no reliable studies on the treatment of naturally occurring renal failure in ani mals similar to those described in humans. Completion of such a study would be extremely difficult and ex pensive and would face even more severe limitations than those encountered by the current multicenter study in human medicine. Therefore, present data are generated from experimental models that have proven useful to elucidate the mechanisms of uremia in dogs and rats (1).
Terminology. These terms are generally used in nephrology to describe the various stages of reduced kidney function: reduced renal mass, permanent loss of a major portion of renal tissue, usually quantified by reduction in glomerular filtration rate (GFR); renal insufficiency, reduction of renal function in the ab sence of clinical signs with mild azotemia or no ab normalities in standard clinical chemistry measures (reduction in function may range up to 60% loss of GFR); progression of renal failure, gradual reduction in renal function as measured by an objective method such as GFR (rate or absence of progression may vary due to etiology of the disease and between individual animals); renal failure, overt clinical signs of failure with metabolic abnormalities of uremia, which may be stable or progressive; end stage failure, severe met abolic abnormalities that can no longer be managed.
Progression of renal failure. Renal insufficiency may progress to failure through mechanisms that are independent of the initiating insult. Renal diseases that progress to failure do so when glomerular filtration rate has been reduced by some substantial level. The trigger point of progression and subsequent rates of progression are primarily independent of the initial disease process.
The measurement of progression of renal failure re quires specific definition. The use of clinical symp toms, signs, reduced urinary concentration, elevated blood urea nitrogen (BUN), elevated plasma creatinine, hyperphosphatemia, acidosis, proteinuria, the recip rocal of serum creatinine versus time, and clearance of creatinine have all been shown to be misleading and inadequate as a precise measure of progression (2, 3).
The most reliable method is the measure of GFR by using inulin or labeled iothalamate.
Progression to failure may be estimated on the basis of morphologic measures and/or functional measures, i.e., GFR. The agreement of morphologic and func tional measures is extremely complex and appears to vary with the form of disease. A synthesis or compar ison of functional and morphologic measures may be helpful in some cases. However, results may or may not agree. Although histologie or electron microscopic alterations may tell us something about the pathophysiology, their relationship to progression of failure may be difficult to quantify. It is commonly difficult to quantify histologie lesions because they are not uniformly distributed, they are confused by compen satory hypertrophy and hyperplasia, and in some forms of disease fibrosis may obscure histologie architecture.
Several factors have been suggested as possible trig gers to the progression of failure, including reduced renal mass, increased energy intake, increased dietary protein intake, hyperfiltration of remnant nephrons, increased dietary phosphate intake, high blood pres sure and metabolic generation of oxalate, or a com bination of these (4, 5). Most studies have used rodent models, the results of which may not be applicable to the dog. Our current knowledge using the dog is lim ited to models of reduced renal mass, dietary protein intake and phosphate intake.
Species Differences. Rodents have been widely used to study possible causative factors of progression of renal failure. Some strains of rats have a high in cidence of spontaneous glomerular and tubular lesions associated with age (6-11). The progression and se verity of these lesions can be enhanced by increased dietary protein (12-14), sodium (7)and phosphate (15). Surgical reduction in renal mass hastens these changes as does diabetes (16-18). Although these observations are limited to rodents, they have influenced many in vestigators by establishing the possibility of a dietaryprotein- induced nephropathy in other species. A hy pothesis has been advanced that ingestion of excessive dietary protein leads to sustained glomerular hyperfiltration and progressive glomerular sclerosis (19).
There is little data on the effects of dietary protein on renal function or morphology in nonrodent species.
There is evidence that high protein diets enhance renal function in dogs, including GFR and renal blood flow, on an acute and chronic basis (20-22). An increase in renal hemodynamics is seen in many species, including humans after consuming a meat meal (23). This has recently been referred to as the renal reserve or ability of the kidney to vasodilate and may be used as a prog nostic indicator of the severity of reduced function.
Presumably, when GFR is dramatically reduced, the renal reserve will be limited or lost. Many factors have been shown to cause increased GFR and renal blood flow, including dietary protein, oral intake of amino acids, intravenous infusion of amino acids, diabetes mellitus, glucagon and other hormones. It is presently unclear whether this renal vasodilation that can be stimulated by dietary protein intake is the same phe nomenon known as hyperfiltration, which may be pathologic in rodent remnant nephrons. There is clear evidence in some rodent strains that reduced dietary protein will diminish the progression of renal failure, presumably due to lowering of GFR in remnant neph rons.
REDUCED RENAL HEMODYNAMICS IN UREMIC DOGS FED REDUCED PROTEIN DIETS
The purpose of this study was to determine the renal and metabolic effects of feeding dissimilar commercial diets with different protein contents to dogs with chronic renal failure (24). Specifically, can dogs with moderate stable renal failure adjust to diets with varied protein and electrolyte content? Furthermore, is the renal reserve or hemodynamic response to varied pro tein intake present in the face of moderate failure?
Methods. Four adult mixed-breed dogs, ages 2-4 y, (7-18 kg) with 7/8 reduction in renal mass were studied 60 d after the reduction of renal mass to assure a steady state of renal function. The study followed a Latin square design using the four dogs, four diets and four periods, each of 14 d. Moderate renal failure was defined by a mean BUN of 23.9 mmol/L and mean serum creatinine of 194.4 mmol/L. GFR was ~20% of normal and clinical evidence of renal failure in cluded persistent polyuria, nonresponsive anemia and moderate weight loss.
Dogs were fed four commercial diets with protein content varying from 53 to 7.8% (dry matter basis) (Table 1). The electrolyte content of the diets varied considerably in regard to calcium, phosphorus and so dium; some markedly divergent from the National Research Council (NRC, 1974) recommendations for healthy dogs. Diets were fed on an energy basis de termined to maintain body weight. The quantity of food consumed was measured daily.
Dogs received water ad libitum. Body weight was recorded three times weekly.
Renal function was measured on the 11th and 13th day of each 14-d period. Renal clearance measurements were performed in quadruplicate at each measurement; the mean of eight measurements performed on 2 d were taken as the value of renal function of each dietary regimen. Renal function studies were per formed in unanesthetized dogs after 18 h of fasting. Dogs were trained to stand quietly in slings for 2-3 h during the clearance measurements with inulin (GFR)and paraminohippurate (renal plasma flow). The de tails of these methods and other clinical chemistries were previously described from our laboratory (24).
Table 1 - Composition of diets
| Diet | |||||
|---|---|---|---|---|---|
| Nutrient | A1 | B2 | C3 | D4 | NRC5 |
| Protein | 537 | 258 | 177 | 78 | 220 |
| Fat | 343 | 114 | 212 | 132 | 50 |
| Carbohydrate | 30 | 511 | 544 | 690 | - |
| Fiber | 16 | 34 | 16 | 50 | - |
| Ash | 74 | 83 | 51 | 50 | - |
| Calcium |
10.7 | 16.7 | 8.2 | 3.9 | 11 |
| Phosphorus |
10.3 | 10.3 | 10.1 | 1.8 | 9 |
| Potassiun |
9.9 | 7.8 | 4.4 | 4.3 | 6 |
| Sodium |
9.5 | 5.8 | 2.5 | 11.7 | 4.4 |
| Magnesium |
0.4 | 1.8 | 1.1 | 0.3 | 0.4 |
| Cross energy |
|
|
|
|
|
| Joules/g |
26.9 | 19.6 | 22.1 | 19.1 | - |
| Kcal/g |
6.45 | 4.69 | 5.30 | 4.59 | - |
| Dry matter, % |
24.2 | 93.6 | 31.6 | 28.1 | - |
1Diet of water, beef byproducts, beef, animal fat.
2Diet of corn, soybean meal, cereals, fat.
3Diet of water, corn, chicken, brewers rice.
4Diet of water, corn starch, animal fat, brewers rice.
5Nutrient requirements for "adequate nutrition" of healthy dogs
(NRC, 1974); modified from table in réf2.4.
The renal clearance of electrolytes and nitrogenous substances was also measured. Results of sodium and phosphate were expressed as percent reabsorption of filtered load and the absolute excretion rate. Results of potassium and uric acid were expressed as the ratio of urinary excretion divided by filtered load because these solutes have bidirectional transport. Urinary ex cretion of urea, uric acid and creatinine were also ex pressed in absolute terms.
The data were submitted to a one-way analysis of variance with repeated measurements. This parti tioned variance between diets, dogs, duplicate mea surements and residual error.
Results. The clinical appearance of the dogs was the same during and after each dietary change. Body weight decreased a mean of 6.0 ±2.1% SEM (range 0-9%) during dietary change. A pattern of weight change related to a specific diet was not seen.
The most dramatic difference in renal function among the four diets was reduced renal hemodynamics when diets with reduced protein were fed (Table 2).
GFR and renal plasma flow were significantly higher when diets containing 54 and 26% protein were fed than when reduced-protein diets were fed. There were no differences in these two measures between these latter two diets. Although the two lower protein diets were not different from each other, GFR was 21% lower than when diets A and Bwere fed. Renal plasma flow, measured by clearance of para-aminohippurate (PAH), was 28% higher in dogs fed diets A and Bthan in dogs fed diets C and D.
Table 2 - Glomerular filtration rate and effective renal plasma flow in dogs fed diets with 54,26,18 and 8% protein1
| |
Diet2 | |||
|---|---|---|---|---|
| |
A | B | C | D |
| % Dietary protein |
54 | 26 | 18 | 8 |
| |
|
ml/min-m2 |
|
|
| CIN | 19.5±2.9a | 18.5±1.2a | 14.8±2.7b | 15.5±2.9b |
| CPAH | 71.7±14.8a | 77.7±5.4a | 55.2±10.3b | 52.8±10.3b |
1Values are means ±SE; n = 8 measures for each of 4 dogs with each diet. Data were submitted to one-way analysis of variance with repeated measurements. This partitioned variance between diets, dogs, duplicate measures and residual error. Modified from table in ref.24. CIN, clearance of inulin; CPAH,clearance of para-aminohip purate.
2Means not sharing a common superscript letter are significantly different at P < 0.05.
The fractional reabsorption of sodium was signifi cantly different among the four diets due to varied intake (Table 3). The lowest fractional reabsorption was found when dogs were fed Diet D, the diet with the highest sodium content. When sodium handling was expressed as mmol/min, there were significant dif ferences (P < 0.05). Sodium excretion was the same when diets B and C were fed but was different from the effects of Diet A and D (P < 0.01). The effect of Diet A was different than the effect of Diet D (P < 0.05). Urinary sodium excretion, expressed as frac tional reabsorption or absolute excretion, was gener ally proportional to dietary sodium intake. Diet C was the exception; the lowest sodium intake was not as sociated with the lowest excretion.
The fractional reabsorption of phosphate was not significantly different among the four diets. When urinary phosphate excretion was expressed as mg/min, however, dogs fed diets A and Bhad a threefold greater phosphate excretion compared with the other two diets (P < 0.05). Diet D, with a reduced phosphate content, produced a higher fractional reabsorption and lower phosphate excretion. Diet C, with a standard phosphate intake, produced the highest fractional phosphate reabsorption and lowest excretion. Renal handling of potassium, expressed as filtered load divided by excretion rate, was not significantly different among diets (Table 3). Urinary excretion of potassium, expressed as mmol/min, was significantly higher in Diet A compared with the other three diets. The serum concentration of sodium, phosphate and potassium remained in the normal range and was not significantly affected by diet.
The renal clearance of urea was not significantly affected by diet (24). The urinary excretion of urea was significantly higher when diets with higher protein content were fed. Urinary excretion of uric acid was significantly higher (P < 0.005) when diets containing higher protein were fed. When uric acid excretion was expressed as filtered load divided by urinary excretion, a significant difference was not found. Urinary excre tion of creatinine was significantly higher when diets containing higher protein were fed, which would cor relate with increased GFR.
A significant difference in BUN value was found related to diet. BUN values were highest when Diet A was fed and were lowest when Diet D was fed. Serum creatinine concentration was not significantly affected by diet. These findings emphasize the effect of dietary protein on BUN independent of renal func tion. All other serum chemistries, electrolytes and en zymes remained in the normal range and were not sig nificantly effected by diet.
Protein scores of the diets were calculated as the ratio of essential amino acid in greatest deficit in the test protein compared with the corresponding value in egg protein (25).This test is based on the assumption that a deficit of any essential amino acid limits the value of the whole protein. Methionine was limiting in Diet Band C, isoleucine in Diet D and tryptophane in Diet A. Essential amino acid indices were calculated as the geometric means of ratios of the essential amino acids to the corresponding value in egg protein. On these two bases, protein score and essential amino acid index, protein quality was best in Diet D, moderate in diets A and C and poorest in Diet B.
Table 3 - Duly dietary intake and renal handling of sodium, phosphate and pot anniurn1
| Diet | |||||
|---|---|---|---|---|---|
| A | B | C | D | P Value | |
| Dietary Na intake, mg/Kg BW |
105 | 122 | 35 | 164 | |
| Na, % reab. |
94.1±1.6 | 96.0±0.55 | 93.3±1.7 | 90.6±1.8 | <0.01 |
| Una, mmol/min |
0.107 | 0.085 | 0.087 | 0.147 | <0.05 |
| Dietary phosphate intake, % reab. |
113 | 217 | 144 | 25 | |
| phosphate, % reab. | 68.9±10.2 | 66.5±11.7 | 82.2±7.3 | 80.2±8.4 | NS |
| Uphos, mmol/min | 0.23±0.07 | 0.25±0.05 | 0.08±0.3 | 0.09±0.03 | <0.05 |
| Dietary K intake, mg/Kg BW | 109 | 128 | 62 | 60 | |
| K, filtered load excretion |
0.63±0.13 | 0.49±0.05 | 0.56±0.18 | 0.52±0.08 | NS |
| UK, mmol/min |
0.035±0.005 | 0.028±0.006 | 0.023±0.005 | 0.023±0.003 | <0.001 |
1Values are means ±SE;modified from table in ré2f.4. UN., Uph04and UKrepresent urinary excretion of sodium, phosphate and potassium, respectively.
Daily intakes of amino acids are listed in Table 4. Apart from methionine and tryptophan, intakes of es sential amino acids were lower in diets C and D than A and B. Comparing averages of the other eight es sential amino acids, Diet C intakes were 64 ±7% of Diet B, and Diet D intakes were 33 ±6% of Diet B. The most critical amino acid was arginine, Diet C pro viding 54% and Diet D only 25% of the level found in Diet B, the standard protein diet. Correspondingly, Diet C provided 31% and Diet D provided only 14% of the arginine intake of Diet A.
After termination of dietary change, dogs were fed Diet B for 2 wk and found to be clinically unchanged from the predietary period. Complete blood count was similar to earlier measurements, indicating that the severity of anemia was unchanged. Plasma chemical values and GFR, measured by clearance of creatinine, were unchanged. Discussion. In the review of these results, it is clear that there was no clinical or biochemical evidence of extraneous factors that may have affected renal function in these dogs. Anorexia, vomiting or other gastrointestinal signs commonly associated with ure mia were not present. Food intake was stable with re gard to all diets and body weight remained relatively constant.
Table 4 - Daily intakes of amino acida per metabolic body size1
| |
Diet | |||
|---|---|---|---|---|
| |
A | B | C | D |
| |
mg/Kg 0.75 | |||
| Methionine | 222 | 68 | 71 | 46 |
| Isoleucine | 407 | 236 | 152 | 84 |
| Tryptophan | 74 | 46 | 52 | 23 |
| Phenylalanine | 445 | 301 | 195 | 93 |
| Valine | 568 | 333 | 209 | 110 |
| Leucine | 865 | 570 | 357 | 152 |
| Threonine | 457 | 253 | 167 | 93 |
| Lysine | 815 | 341 | 233 | 150 |
| Arginine | 753 | 428 | 233 | 108 |
| Histidine | 296 | 166 | 114 | 46 |
1Modified from table in ref.24.
The increased GFR and renal plasma flow due to protein intake in these dogs was similar to previous reports in normal dogs (20-22), normal humans (23) and rats (16). Increased GFR was reported with rats with reduced renal mass when fed increased protein (13, 14). The infusion of amino acids acutely increases GFR and renal plasma flow in normal dogs (21, 26).
The mechanisms whereby increased dietary protein causes vasodilation and increased renal function is not understood. It is interesting to note that, in this study, the phenomenon is not an acute postprandial episode but is maintained even after fasting. A more dramatic effect might be seen in the immediate postprandial period, which was not studied in these dogs.
Although the macronutrient content of the diets varied, there is no evidence that this altered renal function given the short interval of feeding each diet. The fractional reabsorption of sodium and quantity of urinary sodium closely approximated the dietary intake in the case of three diets. The significant dif ferences in sodium handling, expressed as fractional reabsorption or absolute urinary excretion, may be ex plained on the basis of dietary intake. The diet con taining the highest sodium content would be expected to produce the greatest urinary loss and lowest frac tional reabsorption. These physiological adaptations maintain sodium homeostasis in the presence of highly varied sodium intake. Diet C, which contained lower sodium, did not follow this pattern and produced en hanced urinary sodium loss. The reason for this finding is unclear.
Fractional phosphate reabsorption is expected to be reduced (90% is normal) in dogs with reduced renal mass. Reduction was present when all four diets were fed but was also influenced by phosphate intake. Al though slightly increased fractional reabsorption of phosphate would be expected in Diet D, which con tained the lowest phosphate level, one would expect a lower fractional reabsorption when Diet C was fed with a normal phosphate level. Again, dogs responded appropriately to varying phosphate levels with the ex ception of Diet C, where enhanced phosphate reabsorption was found and could exacerbate hyperphosphatemia, hyperparathyroidism and soft-tissue calci fication. This unexpected finding in Diet C is unclear. It may be speculated that there is a relationship be tween the low sodium diet and normal phosphate level in this diet that may influence the tubular reabsorption of these solutes.
The renal handling of potassium was adjusted to enhance excretion despite varied intakes of potassium in the four diets. An avid tubular excretory mechanism provides homeostasis of this electrolyte.
The mean urinary excretion of urea was signifi cantly related to dietary protein intake (r = 0.96, P < 0.05) (data not shown). When urea was expressed as a clearance value taking into account the GFR, there were no significant differences between diets. It had previously been claimed (27) that diets containing ex cessive sodium will aid in the urinary excretion of metabolic wastes, which is not supported by these findings. The urinary excretion of uric acid was not related to dietary protein content. Urinary excretion rate of creatinine approximated dietary protein intake (r = 0.99).
In conclusion, renal hemodynamics were signifi cantly reduced when diets with <20% protein were fed. BUN was elevated when diets with higher protein were fed despite increased renal hemodynamics with these diets emphasizing the dissociation between BUN and renal function. The clinical appearance of the dogs and serum chemistries were unchanged with the four diets, with the exception of BUN. The urinary excre tion of nitrogenous products, urea, creatinine and uric acid was not related to dietary sodium or renal he modynamics, but was closely related to dietary intake (24). The renal tubular handling of electrolytes was appropriately adjusted, with the exception of the moderately reduced protein diet (Diet C), where in appropriate handling of sodium and phosphate were present.
These results indicate that dogs with moderate sta ble renal failure have the capacity to adapt to a wide range of dietary protein and solute alterations. The only advantage of reduced dietary protein in this study was a reduction in BUN. Possible disadvantages of re duced protein diets are reduced GFR and renal plasma flow. Whether these differences are important in the maintenance of renal function or survival in dogs with renal failure for prolonged periods is unknown.
LONG-TERM RESPONSES TO VARIED PROTEIN DIETS IN DOGS WITH 75% NEPHRECTOMY
The objective of this study was to determine the effect of dietary protein on renal function and struc ture by use of two experimental models for periods up to 4 y. Renal function was measured before and after the induction of pyelonephritis and/or the sur gical ligation of ~75% of renal mass. This reduction of renal mass was expected to condense the time frame of renal aging and uncover possible progression to failure.
Methods. Experiments were performed using 35 healthy female Beagle dogs from 6 to 12 mo of age at the onset. The method of surgical ligation to reduce renal mass was previously reported (28). Pyelonephri tis was produced by using a known pathogenic strain of Escherichia coli cultured from dogs as previously described (28). The majority of dogs had sterile urine 4 mo after the initiation of infection. Renal mass was then further reduced by surgical ligation.
Dogs were fed exclusively one of three diets 30 d before and after the reduction of renal mass (Table 5).
These commercial diets contained protein of 56, 27 and 19%. All three diets were studied in the surgical ligation model, whereas only diets with 56 and 27% protein were fed to dogs with pyelonephritis. Daily protein intake with these three diets was 13.2, 6.9 or 3.2 g/kg body weight. While there was some variation in the electrolyte content of the diets, they were all considered complete for maintenance of adult dogs.
Phosphate content of the three diets was identical.
Blood pressure was measured in each dog during and after reduction in renal mass and was found to be normal throughout the study. Routine laboratory measurements and renal function measurements in cluded a complete blood count, total plasma protein, the albumin:globulin ratio, plasma creatinine, BUN, 24-h creatinine clearance, 24-h urinary protein excre tion, routine urinalysis and urine culture. Renal clear ances of creatinine, inulin and paraminohippuric acid and the fractional reabsorption of sodium reabsorption were measured at various stages of the reduction of renal mass and at 13, 22, 30, 36, 42, 45 and 48 mo after the beginning of the study in the surgical ligation model. Dogs in the pyelonephritis group were killed at 42 mo and those in the ablation group were killed at 48 mo.
Dogs were killed at the end of the study for com plete necropsy. Dogs that died spontaneously before the end of the study underwent necropsy and histo logie evaluation. Four pathologists interpreted dupli cate slides of kidneys without knowledge of dietary group. Histologie changes were scored on a predeter mined scale of 1-4 (minimal to severe). Two of the pathologists also evaluated electron micrographs of kidney samples and graded glomerular lesions, in cluding foot process effacement, mesangial prolifera tion, membrane irregularities, presence of inflamma tory cells and platelet aggregates and the presence or absence of dense deposits. These gradings were also done without knowledge of diet group. T tests were performed on scores of morphologic data by using the mean square of error from the analysis of variance.
Table 5 - Composition of dry mutter of diets1
| |
Diet | ||
|---|---|---|---|
| Nutrient | A2 | B3 | C4 |
| |
|
mg/Kg dry matter | |
| Protein | 560 | 270 | 190 |
| Fat | 240 | 80 | 220 |
| Carbohydrate | 50 | 520 | 520 |
| Fiber | 20 | 40 | 20 |
| Ash | 130 | 90 | 50 |
| Calcium |
10.7 | 16.7 | 8.2 |
| Phosphorus |
10.3 | 10.3 | 10.1 |
| Potassium |
9.9 | 7.8 | 4.4 |
| Sodium |
9.5 | 5.8 | 2.5 |
| Magnesium |
0.4 | 1.8 | 1.1 |
1Modified from table in ref. 28.
2Diet of water, beef byproducts, beef, animal fat.
3Diet of corn, soybean meal, cereals, animal fat.
4Diet of water, corn, chicken, brewers rice.
The source of variation consisted of percentage of protein in diet, dogs in each dietary group, and the interaction between pathologist and diet group.
Statistical analysis of functional data was performed by using analysis of variance, which partitioned total variance in the data into partial variances attributable to diet, time of observation, diet times time interaction and residual variation within dogs. One dog was ex cluded from the analysis because of death before the 13th month. The Hotelling-Lawley trace statistic was used to test the multivariate null hypothesis of no dif ference among the three diets over all the dependent variables. In the case of a significant effect attributable to diet, diets were compared pairwise to identify sig nificant differences for all variables.
Results. Dogs remained healthy, ate normally, and were not azotemic throughout the study. Twelve of the 21 dogs in the surgical ligation group survived the 4-y period. There were no apparent relationships be tween deaths, or death attributable to renal failure and diet (28). The survival rate in the pyelonephritis group was identical to the surgical ligation model.
Analysis of variance of variables in the pyelone phritis model through 42 mo indicated no significant diet effect. Thus, individual variable comparisons were not statistically analyzed for this model. There were no significant interactions of diet and time in this model. The mean values of major variables, measured at all study periods, indicate no significant difference.
Dogs fed Diet Bhad higher GFR measured by clearance of inulin and creatinine, and higher clearance of PAH compared with dogs fed Diet A. Most important is the absence of reduced renal function over time.
In the surgical ligation model, renal plasma flow and filtration rate were affected by diet before reduc tion in renal mass. The baseline measurements were higher in dogs fed diets A and B after 30 d compared with Diet C (Figs. 1 and 2). This reduction in renal function with the lowest-protein diet is similar to the finding of our previous report using uremie dogs. After 75% nephrectomy, analysis of variance indicated sig nificant differences attributable to dietary protein in BUN, 24-h creatinine clearance, clearance of inulin and creatinine and clearance of PAH (Table 6). There were no significant differences in 24-h urinary protein excretion or plasma creatinine related to diet. The clearance of inulin was significantly higher in dogs fed Diet B (27%) compared with Diet C (19%) (P < 0.05). Clearance of PAH was significantly higher in dogs fed Diet B compared with Diet C (P < 0.05). Diet A dogs had significantly higher 24-h clearance of creatinine, serum albumin and BUN compared with Diet C. There were significant time effects and interaction of these variables over the 4-y study period. There were no interactions between diet and time for the most sen sitive indicators of renal function, i.e., GFR and plasma flow.
The mean values of major variables measured at all study periods for the three diets are shown in Table 7. Diet B was associated with the highest clearance of PAH. Twenty-four-hour creatinine clearance was highest in dogs fed Diet A. Diet C was associated with the lowest clearance of PAH and lowest GFR using all methods, lowest BUN, and lowest serum albumin (data not shown).
When all measurements of GFR and renal plasma flow are plotted against time, it is obvious that dogs fed the lowest-protein diet, Diet C (19%), had the lowest values at all study periods (Figs. 1 and 2). Again, there was no reduction of renal function in any of the diets related to time after surgical ligation.
No significant differences were found in complete blood counts, plasma chemistries, electrolytes or en zymes, except for higher serum cholesterol in dogs fed Diet A. Routine urinalyses were not remarkable and no changes attributable to diet were found. The body weights of dogs were maintained constant. Energy intakes, expressed in terms of body weight0 75,metabolic body size, were 24 and 10% greater in dogs fed Diet A and B, respectively, than in those fed Diet C. Protein intakes were 295 and 120% greater in dogs fed Diet A and B, respectively, than in those fed Diet C.
Table 6 - Analysis of variance of results through 48 mo from dogs with 75 % nephrectomy due to surgical ligation1,2
| |
Significance of effect attributable to | |||
|---|---|---|---|---|
| Variable3 | Diet |
Time | Interaction |
R2 |
| BUN | P<0.001 | P<0.001 | P<0.001 | .777 |
| Plasma creatinine |
NS | P<0.001 | P/,0.001 | .699 |
| 24- hr CCR |
p<0.003 | P<0.001 | P<0.05 | .820 |
| 24 - hr urinary protein |
NS | P<0.001 | NS | .560 |
| CIN | P<0.012 | P<0.001 | NS | .733 |
| CCR | P<0.003 | P<0.001 | NS | .728 |
| CPAH | P<0.001 | P<0.001 | NS | 697 |
1 Multivariate test diet effect over all dependent variables: Hotelling- Lawley Trace = 28.307, P < 0.002. Modified from table in ref. 28.
2 R2 is the proportion of variation that is explained by the sta
tistical model for each variable. It is a measure of the fit of the model.
3 CCR,clearance of creatinine; CIN, clearance of inulin, CPAH/ clearance of para- aminohippurate; NS, not significant.
Histopathologic examination of kidneys revealed a difference in response to the method of reduction of renal mass. In the pyelonephritis model, numerous foci of cortical destruction with glomerular dropout were present. The remaining glomeruli in these foci showed glomerulosclerosis. Examination of this population of glomeruli sectioned at 1.0 /*m showed the glomeru losclerosis to be focal and segmental. Interstitial fibrosis in the cortex was present with lymphoid and plasma cell infiltration. Medullary fibrosis was present and variable, accompanied by lymphoid and plasma cell infiltration. Around the renal pelvis, and partic ularly in the parietal surface, a lymphoid and plasma cell infiltrate was present with a tendency to form lymphoid follicles. These changes were associated with the mechanical trauma to induce pyelonephritis as the nontraumatized areas of the kidney appeared normal.
In the surgical ligation model, a portion of the kid ney with vascular ligation showed extensive fibrosis of the cortex and medulla. The remaining renal paren chyma showed variable degrees of glomerulosclerosis, shrinkage of glomerular tufts with distortion of Bow man space. A focal interstitial fibrosis was seen in the cortex and medulla. The overall incidence of glomer ular lesions was graded minimal (Tables 8and 9).Dogs fed Diets B and C had glomerular lesions in 10-20% of glomeruli. Small focal infiltrates of lymphocytes and plasma cells were present in the interstitium. There were scattered and focal areas of minimal interstitial fibrosis. Some tubules were dilated and filled with amorphous eosinophilic debris.
Table 7 - Results of renal function tests in dogs with 75% nephrectomy due to surgical ligation1
Variable |
Dieta A (n= 49) |
Dieta B (n= 28) |
Dieta C (n= 34) |
|---|---|---|---|
| BUN, mmol/L Plasma creatinine |
8.1±0.4 | 5.5±0.2 | 4.1±0.1 |
| mmol/L 24 - h CCR |
79.5±2.6 | 70.7±35.3 | 79.5±2.6 |
| ml/(min-m2) urinary protein, |
38.8±1.8 | 33.1±4.0 | 30.0±0.4 |
| g/d | 0.25±0.6 | 0.02±0.00 | 0.17±0.06 |
| CIN ml/(min-m2) |
30.0±1.5 | 30.8±1.8 | 23.1±1.1 |
| CCR ml/(min-m2) |
30.6±1.4 | 31.7±1.6 | 23.1±1.2 |
| CPAH ml/(min-m2) |
104±4.3 | 119±6.8 | 79.4.2 |
1Values are means ±SEM;n = number of measurements of vari ables over the 4-y study period. The number of dogs fed diets A, B and C at the outset were 10, 5 and 6, respectively. Modified from table in ref. 28.
Two of four dogs fed Diet A had lesions similar in incidence and severity to those described for the other two diets. The other two dogs had moderate sclerotic lesions in ~40% of the glomeruli. Most of the effected glomeruli had minimal lesions, which consisted of fo cal segmental sclerosis and tuft adhesions. Tubulointerstitial lesions in these two dogs were similar in in cidence and severity to the other dogs in their diet group and to those in the other diet groups. There was no correlation of the incidence or severity of tubulointerstitial lesions with a dietary effect in any group.
The incidence and severity of glomerular lesions evaluated by light microscopy increased as the level of dietary protein increased (Tables 8 and 9) (29).
However, none of the dogs had a correlation of the degree of glomerular pathology to renal functional impairment as assessed by GFR, renal plasma flow, proteinuria and clinical pathology measurements. This lack of correlation emphasizes the minimal effect of these lesions on renal function.
In contrast to the light microscopic findings, no sta tistically significant difference in glomerular pathology could be found between diet groups with electron mi croscopy (Table 10).A minimal amount of foot process effacement occurred in all dogs in all dietary groups. This lesion was present in a scattered focal distribution and appeared as areas of fusion of three to five foot processes. Proliferation of mesangial cells and matrix was seen in all dietary groups. Most dogs had focal areas of irregularity in the glomerular basement mem brane. These irregularities included splitting and fold ing of the membrane with humps and lytic areas within the glomerular basement membrane. The incidence and severity of these lesions was not correlated with dietary protein. Lesions of this type are common in dogs of this age and breed.
Other glomerular lesions, such as the presence of dense deposits, tuft adhesions and the presence of in flammatory cells occurred in all dietary groups and was not correlated with dietary protein. Likewise, there was no correlation of the occurrence of ultrastructural glomerular lesions with any impairment of renal function. There appeared to be little effect of dietary protein on glomeruli when evaluated with the electron microscope.
Discussion. These data from dogs do not agree with previous data from rats. Dogs with reduced renal mass in this study had higher renal function when fed 56 or 27% protein compared with dogs fed 19% pro tein. The higher GFR and renal plasma flow were pres ent in dogs fed higher protein diets before reduction of renal mass and persisted throughout 4 y without a pattern of deterioration.
Table 8 - Incidence and severity of light microscopic glomerular pathology in 11 dogs with reduced renal mass fed diets with either 56,27 or 19%protein content1
| Diet used/animal |
% Protein |
Pathologist 1 |
Pathologist 2 |
Pathologist 3 | Pathologist 4 |
Group mean |
|---|---|---|---|---|---|---|
| A/1 | 56 | 4 | 3.5 | 3.5 | 3.5 | 3.625 |
| A/2 | 56 | 3.5 | 3.5 | 2 | 2.5 | 2.075 |
| A/3 | 56 | 2.5 | NE | 1 | 1 | 1.500 |
| A/4 | 56 | 2 | 2 | 1 | 1 | 1.500 |
| B/5 | 27 | 2 | 1 | 1.5 | 1 | 1.375 |
| B/6 | 27 | 2 | 2 | 1 | 0 | 1.250 |
| B/7 | 27 | 1 | 2 | 1 | 1.5 | 1.275 |
| C/8 | 19 | 0 | 1.5 | 1 | 0 | 0.650 |
| C/9 | 19 | 1 | 2 | 1 | 0 | 1.000 |
| C/10 | 19 | 2 | 2 | 0 | 0 | 1.000 |
| C/11 | 19 | 2 | 2 | 2 | 1.5 | 1.875 |
1Grades of severity of pathology: 0, no changes; 1, minimal; 2, moderate; 3, marked; 4, severe; NE, not examined. One dog in the 56% protein group (diet A) died of postsurgical sepsis in the 46th mo of the study. Microscopy of kidneys from this dog is not included in this analysis. Modified from table in ref. 29
While there was some variation in the amount of urinary protein excreted by these dogs and there was increased protein excretion per surviving nephron, there was no significant dietary effect on protein ex cretion in the urine. Dogs fed 56% protein excreted an average of 0.25 g/d; dogs fed 19% protein excreted 0.17 g/d; and those fed 27% protein excreted 0.02 g/ d. The values found when 27% protein was fed are within the normal range, whereas the high and low protein diets were associated with slight proteinuria. As stated previously, there was not correlation of light and microscopic lesions with urinary protein values or other functional tests.
Much of the previous work that supports a delete rious role of dietary protein in progressive glomerular disease was done in rats. Several investigators have established that high protein feeding accelerates the development of chronic nephrosis and proteinuria.
Saxton and Kimball (12) found that rats fed a diet of 30-40% protein had over three times the incidence of chronic nephrosis compared with rats fed a 10% diet. They also found a higher incidence of chronic ne phrosis in rats fed a 33% casein-lactalbumin protein diet than rats fed a 41% dry liver protein diet, sug gesting the type of protein may also play a role.
In other rodent studies, renal mass was reduced or experimental disease was induced to compromise renal function, and then dietary manipulation was per formed. Hostetter et al. (30) studied renal function and appearance of glomerular lesions in Munich-Wistar rats 7 d after 90% renal ablation. They found higher single-nephron GFR in rats fed a normal protein diet (24%) compared with those fed a low protein diet (6%) or sham-operated controls. Minimal glomerular le sions were seen in rats fed a normal dietary protein.
Glomerular lesions of these types in rats fed low pro tein diets or sham-operated controls were much less severe. No sclerotic lesions were seen in any rats, pos sibly due to the short duration of the study. Glomer ular lesions were reported by Shimamura and Morrison (16) in Wistar rats fed a normal protein diet for 10- 50 wk after 85% renal ablation. Glomerular hyalinization began by 25 wk and was extensive by 50 wk in remnant kidneys. Foot process effacement, mesangial proliferation and mesangial capillary occlusion were seen by electron microscopy.
Table 9 - Statistical analysis of glomerular pathology gradings by light microscopy1
| Diet used/ % protein | Pathologist 1 | Pathologist 2 | Pathologist 3 | Pathologist 4 | Mean±SEM |
|---|---|---|---|---|---|
| A/56% | 3.00±0.91 | 3.00±0.87 | 1.88±1.18 | 2.00±1.22 | 2.43±1.10 |
| B/27% | 1.67±0.58 | 1.67±0.58 | 1.17±0.29 | 0.83±0.76 | 1.33±0.62 |
| C/19% | 1.25±0.96 | 1.88±0.25 | 1.00±0.81 | 0.38±0.75 | 1.13±0.87 |
1Grading system same as in Table 8. Significance of effects of dietary protein: 56 vs. 19%, P <0.01; 56 vs. 27%, P < 0.05; 27 vs. 19%,not significant; there is a significant (P < 0.01) linear dose-response trend for increased incidence and severity of glomerular pathology withincreasing levels of dietary protein. Modified from table in ref. 29
Although these studies in rodents clearly establish functional and structural abnormalities related to di etary protein, similar changes were not found in dogs in the present study. We were unable to establish sig nificant effects of high protein feeding on the incidence of glomerular lesions. We were also unable to establish a protective effect of reduced protein feeding on the incidence of glomerular pathology. Glomerular lesions such as protein résorptiondroplets, attenuation of visceral epithelial cell cytoplasm and mesangial pro liferation, shown in rats to be associated with ablationinduced hyperfiltration, were not seen in the present canine study. Such lesions were not significant in dogs fed 27 or 56% protein, even though elevated GFR, and presumably hyperfiltration, were present. Rats with reduced renal mass fed high protein diets became proteinuric and azotemic (17), whereas dogs in this pres ent study did not.
Both light and electron microscopic evaluation of glomerular pathology were used in the present study and there were significant differences between the re sults obtained with each technique. There was a trend toward increasing incidence and severity of glomerular lesions with increasing levels of dietary protein, as as sessed by light microscopy. However, this trend was not seen with electron microscopy. Electron micro scopic samples taken at random allow evaluation of fewer glomeruli but with greater precision for diag nosis of subtle lesions, which may be indicative of hyperfiltration/ hyperfunction lesions. We believe the electron microscope sampling procedure may be less biased than light microscopy, where the observation of prominently stained sclerotic glomeruli may influ ence grading of lesions in all glomeruli.
Table 10 - Incidence and severity of u/fcrastrue tura/ glomerular pa thology1
| |
|
Foot process Fusion |
Mesangial proliferation |
Basement membrane irregularities |
Mesangial froth |
Dense deposits |
Tuft adhesions |
||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Animal |
% Protein |
A |
B |
A |
B |
A |
B |
A |
B |
A |
B |
A |
B |
| 1 | 56 | 2.0 | 3.0 | 3.0 | 3.0 | 2.5 | 2.0 | 3.0 | 3.0 | 2.0 | 2.0 | 2.5 | 2.0 |
| 2 | 56 | 3.0 | 3.0 | 3.0 | 4.0 | 3.5 | 2.5 | 3.0 | 2.5 | 3.5 | 4.0 | 1.0 | 1.0 |
| 3 | 56 | 1.0 | 1.0 | 1.5 | 1.5 | 3.0 | 1.0 | 3.0 | 1.0 | 2.0 | 1.0 | 0 | 0 |
| 4 | 56 | 1.0 | 1.5 | 2.5 | 2.5 | 3.0 | 1.5 | 1.0 | 2.0 | 1.0 | 1.5 | 0 | 0.5 |
| 5 | 27 | 1.0 | 1.5 | 2.5 | 1.0 | 3.5 | 2.5 | 2.0 | 1.0 | 2.0 | 1.0 | |
0 |
| 6 | 27 | 2.5 | 1.5 | 1.0 | 2.0 | 3.5 | 2.5 | 3.0 | 1.0 | 2.0 | 1.0 | 0 | 0 |
| 7 | 27 | 2.0 | 2.0 | 2.0 | 1.0 | 2.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 0 | 0 |
| 8 | 19 | 1.0 | 1.5 | 1.0 | 2.0 | 2.5 | 2.5 | 2.0 | 3.0 | 1.0 | 2.0 | 0 | 1.5 |
| 9 | 19 | 1.5 | 1.1 | 2.0 | 2.5 | 1.5 | 1.0 | 2.0 | 3.0 | 1.5 | 2.0 | 0 | 1.0 |
| 10 | 19 | 2.5 | 2.0 | 3.0 | 3.0 | 1.5 | 2.0 | 2.5 | 4.0 | 1.0 | 2.0 | 2.5 | 3.0 |
| 11 | 19 | 1.5 | 1.0 | 2.5 | 0.5 | 3.5 | 2.5 | 2.5 | 3.0 | 0 | 1.0 | 1.5 | 3.0 |
1Grading system same as Table 9. A and B, scores of two pathologists. Modified from table in ref. 29.
These data using dogs with 75% nephrectomy fed varied protein diets indicate that dogs do not respond to high dietary protein the same as rats. There may be several reasons for these differences. Dogs and rats have a different spectrum of age-related glomerular diseases. Middle-aged and old albino rats of several different strains (Wistar, Sprague-Dawley) have a high incidence of spontaneous primary glomerulosclerosis (6-11), but dogs do not (31-35). Dogs are much more commonly affected by membranous and membranoproliferative glomerulonephritis, amyloidosis and tubulointerstitial disease when older (31-35). Thus, spontaneous glomerulosclerosis may develop in rats fed high levels of dietary protein, which is a lesion that does not seem to occur frequently in dogs.
The influence of systemic hypertension on arterial sclerosis or glomerulosclerotic changes in dogs is un known. The lack of hypertension in the present dog study appears to be another species difference com pared with rats. Hypertension is not found in dogs with 75% nephrectomy unless salt loading is used (36).
One would not expect hypertension in the present study because all diets contained <1% sodium. It is interesting to note that Diet A contained the highest protein and highest sodium content, yet hypertension or deterioration of renal hemodynamics was not found. In the hypertensive rat, or those with reduced renal mass, it has been shown that intrarenal hyper tension is more critical than systemic blood pressure to cause glomerular morphologic changes (30, 37).
The dietary content of linoleic acid has been shown to influence the progression of glomerulosclerosis in rats with 75% nephrectomy (38). The high dietary in take of linoleic acid, 27 versus 2% of energy in a nor mal diet, prevented glomerulosclerosis, presumably by increased prostaglandin E2 (PGE2) generation in the renal cortex, which led to vasodilation or altered co agulation status. In the present study, the diets con tained varied fat content, but, in all cases, a low linoleic acid content. Diet B, with the lowest fat content, had the highest linoleic acid, 0.58%. Therefore, this low linoleic acid content would be unlikely to have an in fluence on the progression of renal disease in these dogs.
While these results indicate that the dog is different from the rat in response to dietary protein, several important questions are raised that cannot be answered with this study. Progressive deterioration of function was not reached in these dogs. What portion of renal mass removal is critical in dogs? It may be that a greater portion of the renal mass must be removed in dogs compared with rats to trigger progressive deteriora tion, or a longer time may be required to trigger de terioration. Whole kidney functional loss may not have been apparent with the present measurements until more advanced damage has occurred. What is the nature of hyperfiltration in the remnant dog kid ney? The present study could not assess the influence of dietary protein on the single-nephron hyperfiltra tion response. It is possible that single-nephron hyperperfusion resulting from high protein intake could mask the effects of glomerular damage. To investigate these possibilities, further studies to measure intrarenal hypertension and glomerular hyperfiltration are needed in the dog model.
Thus, dogs fed a high protein diet for 4 y after 75% nephrectomy show no deterioration of GFR or clear ance of PAH. It was assumed that glomerular hyper filtration occurred in these dogs when renal mass was reduced. These results of increased GFR agree with previous reports from dog studies (1). These results suggest that the deleterious effects of high protein feeding seen in rats do not occur in dogs with com parable reduction in renal mass.
ACKNOWLEDGMENTS
The author thanks the many collaborators who have assisted with these studies, including D. S. Kronfeld, M. Goldschmidt, G. S. Hill, J. E. Tomaszewski, J. L. Robertson and C. Ramberg.
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