Confirming the promise to prevent physiological disorders of organs: urolithiasis in laying hens 1

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1 C 2015 Poultry Science Association Inc. Confirming the promise to prevent physiological disorders of organs: urolithiasis in laying hens 1 Robert F. Wideman, Jr. 2,3 Center of Excellence for Poultry Science, University of Arkansas, Fayetteville Primary Audience: Nutritionists, Pathologists SUMMARY Several problems caused by organ system disorders, such as urolithiasis (kidney stone formation) in laying hens, occur sporadically in poultry flocks and are likely to remain unresolved unless the scientific method is rigorously applied to develop and validate a research model for inducing similar pathology in experimental flocks. Successful research models confirm key triggering mechanisms and stressors, reveal innate organ system limitations or susceptibilities, and provide a reliable test bed for evaluating practical prophylactic and therapeutic strategies. Researchers have a professional responsibility to confirm their research model s limitations and relevance before making recommendations to poultry producers. Our experiments confirmed that feeding diets containing high Ca with low available phosphorus (HCLP) to experimental groups of immature pullets consistently triggered the pathognomonic symptoms associated with field outbreaks of urolithiasis. The HCLP research model also demonstrated that affected hens would survive and remain in production when their dietary cation:anion balance was adjusted to acidify the urine and thereby solubilize the kidney stones. Urinary acidification was rapidly adopted as the method of choice for treating commercial flocks experiencing outbreaks of urolithiasis. Key words: scientific method, experimental model, urolithiasis, acidifiers, calcium, kidney damage, methionine, infectious bronchitis virus 2016 J. Appl. Poult. Res. 25: DESCRIPTION OF THE PROBLEM Problems attributable to organ system limitations or damage arise regularly in poultry production. Examples include sudden death syndrome (SDS, heart attacks, flipover), pulmonary arterial hypertension syndrome (PAH or 1 Presented as part of the Informal Nutrition Symposium Response Measurements and Decisions at the Poultry Science Association s annual meeting in Louisville, Kentucky, on July 27, Corresponding author: rwideman@uark.edu 3 The research on live animals described in this review was conducted in accordance with guidelines approved by the Institutional Animal Care and Use Committee. PHS, ascites syndrome), proventriculitis, lameness (numerous musculoskeletal pathologies), and diuresis or visceral gout (kidney damage or failure). Producers initially address these issues by asking technical professionals for advice based on prior experience. Existing products may be evaluated under poorly controlled conditions. Descriptive reports and case histories may be published, but the initial outbreaks typically do not motivate sustained research. If production losses continue, then university scientists with specific expertise may be asked to help. This is exactly what occurred on my first morning as a new assistant professor specializing in kidney anatomy and physiology in the

2 WIDEMAN: KIDNEY STONES IN HENS 293 Poultry Science Department at Penn State University. Two poultry veterinarians, Dr. Dwight Schwartz (Penn State University) and Dr. Edward T. Mallinson (University of Maryland) appeared at my office door and announced that we were leaving immediately to visit flocks of laying hens in Pennsylvania and Maryland. Hens in full production were dying from urolithiasis, which is an acquired degenerative kidney disease characterized by focal mineralization of the kidneys and progressive obstruction of the ureters by uroliths (kidney stones). The causes were unclear, no effective treatments were available, and flock mortalities ranged from 2 to 50%. A variety of competing hypotheses had been proposed but none had been rigorously tested. The industry obviously needed help, but addressing this problem was daunting and our resources were limited. Furthermore, outbreaks of urolithiasis were sporadic, making it impractical to use commercial flocks to evaluate the pathogenesis or develop treatments. Commercial outbreaks would provide an opportunity to observe the problem, define key characteristics, and obtain anecdotal evidence, but we clearly needed to develop a research model that would consistently replicate most if not all of the pathognomonic symptoms of urolithiasis. Successful research models confirm key triggering mechanisms and stressors, reveal innate organ system limitations or susceptibilities, and provide a reliable test bed for evaluating practical prophylactic and therapeutic strategies. Our goals were prioritized as follows: define the essential characteristics of urolithiasis outbreaks in commercial flocks; develop and validate a research model for triggering similar pathology in experimental flocks; and use the model to evaluate potential treatment strategies. Researchers have a professional responsibility to confirm their research model s limitations and relevance before making recommendations to poultry producers. APPLYING THE SCIENTIFIC METHOD The scientific method includes the following steps: (1) Observe the problem and accumulate descriptive information; (2) Review the existing literature; (3) Form an initial hypothesis; (4) Design and conduct experiments to test the hypothesis; (5) Evaluate the results to determine whether or not the hypothesis has been supported; and (6) Revise the hypothesis as necessary and conduct additional experiments accordingly. With regard to the evolution of a working hypothesis, Occam s razor provides valuable guidance: First evaluate the least complex hypothesis with the fewest assumptions, and thereafter increase the level of complexity only as necessitated by compelling additional evidence. The scientific method was rigorously applied during our investigation of the etiology and pathogenesis of urolithiasis. Observing the Problem and Accumulating Descriptive Information The initial series of farm visits and necropsy sessions organized by Dwight Schwartz and Edward T. Mallinson, combined with additional farm visits arranged by Mr. Herbert C. Jordan and Dr. Owen D. Keene (Penn State Poultry Extension) led to a clear overview of the pathological progression leading to terminal urolithiasis. In clinically healthy hens each kidney has 3 divisions that are uniform in color and fill the space available to them in the bony synsacrum (pelvis). The 3 divisions are drained by a single ureter. Normally the left and right kidneys are bilaterally symmetrical in shape and weight (Figure 1A). During urolithiasis outbreaks, discrete focal areas of mineralization develop within one or more of the kidney divisions. Affected divisions undergo atrophy, often with no apparent alteration in the macroscopic appearance of the remaining divisions. Small uroliths may form in a primary ureteral branch draining the affected division, or larger uroliths may occlude an entire ureter. Tissue upstream from the occlusion atrophies, whereas kidney divisions that remain un-occluded undergo compensatory hypertrophy (Figure 1B). Accordingly, the left and right kidneys in affected hens lack symmetry in both shape and weight. For example, dividing the weight of the heavier kidney by the weight of the lighter kidney yields a heavy/light kidney weight ratio that rarely exceeds 1.10 in clinically healthy hens, but routinely is much higher in hens with urolithiasis [1, 2]. These pathological findings indicate that outbreaks in

3 294 JAPR: Review Article * * * (A) (B) (C) (D) Figure 1. (A) The kidneys of clinically healthy hens normally are bilaterally symmetrical in shape and weight. (B) A large stone or urolith blocks an entire ureter (arrows), causing the obstructed kidney tissue to atrophy (asterisks). The unobstructed kidney remains macroscopically normal in appearance. (C)Affected hens survive and continue to lay eggs as long as one third of the normal renal mass remaines functional, in spite of bilateral ureteral obstruction by occlusive uroliths (arrows). (D) Urolith blockage of the caudal-most segments of both ureters (arrows) rapidly leads to terminal renal insufficiency, visceral gout (visceral urate deposition), and death.

4 WIDEMAN: KIDNEY STONES IN HENS 295 the United States closely resembled outbreaks reported in Great Britain [3,4]. We selected hens from commercial flocks with existing uroliths and missing kidney divisions, identified by digital palpation via the cloaca, and housed them in isolation units for further observation and kidney function studies. Kidney function (urine flow rates, glomerular filtration rates, renal plasma flow rates, tubular transport of minerals and electrolytes) fell within normal ranges when adjusted for differences in functional renal mass. Histopathological evaluations provided further evidence that urolithiasis is a progressive disease associated with a gradual, discrete loss of entire nephrons due to occlusion of the collecting ducts and ureteral branches. Our observations indicated that affected hens could survive and continue to lay eggs as long as at least one third of the normal renal mass remained functional (Figure 1C), after which further obstruction of the ureter causes renal insufficiency and the precipitation of retained nitrogenous wastes (uric acid and urate salts) on the internal (visceral) organs (Figure 1D). This condition is called visceral gout or visceral urate deposition, and is a typical observation in birds that die from urolithiasis [1, 2]. Reviewing the Existing Literature During the 1960s through the early 1980s, urolithiasis outbreaks occurred fairly frequently in Great Britain [3 5], but not in the United States or Canada [2, 6]. Water deprivation was suspected of contributing to outbreaks of urolithiasis in two flocks in Canada [6]. Blaxland et al. [3] suggested that either excessive dietary calcium levels or nephropathogenic infectious bronchitis virus (IBV) might be involved. The nephrotropism of some IBV strains in young birds was well established [4, 7]. Urolithiasis is not consistently caused by high Ca diets [4, 8, 9], but Shane et al. [10, 11] triggered urolithiasis-like symptoms and visceral gout by feeding immature pullets diets containing high Ca (HC) in combination with low levels of available phosphorus (ap) high Ca with lowavailable phosphorus (HCLP). These observations resonated with anecdotal evidence from our visits to affected flocks. Several outbreaks of urolithiasis in Pennsylvania and Maryland had occurred after pullets had been fed a HC pre-laying ration, or had exhibited adverse reactions to a new IBV vaccination protocol or different IBV vaccine strain. Problems during pullet growout clearly were implicated during a major urolithiasis outbreak in the United States, with pullets from one source developing much higher instances of urolithiasis than pullets from a second source, despite subsequently having been housed, managed, and fed together in the same cage layer facility after 20 wk of age [2]. Renal interstitial inflammation and nephritis typical of nephrotropic IBV were common observations in affected hens, but attempts to isolate IBV were not successful and the results of serological profiles were ambiguous [2]. The most extreme urolithiasis outbreak in the eastern United States (56% total flock mortality) occurred in laying hens that had been fed layer ration continuously beginning at 7 wk of age [12]. Forming an Initial Hypothesis Our working hypothesis is diagrammed in Figure 2, which is based on the observations reported by Shane et al. [10, 11] as well as our basic knowledge of the role of vitamin D 3 and parathyroid hormone (PTH) in co-regulating the concentrations of Ca and P in blood [13, 14]. This hypothesis is based on the practice of feeding immature pullets a HC ( 2% Ca) and low ap (LP: 0.4% ap) pre-laying ration intended to support bone mineralization in preparation for the onset of egg production. Laying hens typically were fed a HC ( 3.5% Ca) LP ( 0.3% ap) layer diet to support optimal eggshell mineralization while minimizing bone mineral loss and osteoporosis. These HCLP diets worked quite well for their intended purposes. Low ap in the diet reduces blood P concentrations, which in turn directly stimulates 25-hydroxyvitamin D- 1α-hydroxylase to increase the synthesis of the active vitamin D 3 metabolite, 1,25(OH) 2 vitamin D 3 (1,25[OH] 2 D 3 ). The active D 3 metabolite stimulates intestinal P absorption for the purpose of restoring normal blood P concentrations, but 1,25[OH] 2 D 3 also unavoidably stimulates enhanced intestinal Ca absorption even when existing blood Ca concentrations are normal. The flood of excess Ca into the blood directly supports eggshell mineralization and

5 296 JAPR: Review Article HCLP diet Blood P Bone Ca & P Mineral Improved Bone Mineral Blood Ca Ca P 1,25(OH) 2 D 3 Parathyroid Hormone Intestine P Ca + Blood P Blood Ca Kidneys Reduced Ca Reabsorption IBV (tubular debris) Improved Eggshell Quality Shell Gland Shell Ca Urine Ca Increased risk of Urolithiasis (Ca~Na ~urate) ACIDIFY! (Cl, PO 4, SO 4 ) Figure 2. Diets containing high Ca (HC; 3.5% Ca) and low phosphorus (LP; 0.3% P) support optimal eggshell mineralization while minimizing bone mineral loss and osteoporosis. The low P in the diet reduces blood P levels, which in turn directly stimulates 25-hydroxyvitamin D-1α-hydroxylase to increase the synthesis of the active vitamin D 3 metabolite, 1,25(OH) 2 vitamin D 3 (1,25[OH] 2 D 3 ). This active metabolite stimulates intestinal P absorption for the purpose of restoring normal blood P concentrations, but 1,25[OH] 2 D 3 also unavoidably stimulates enhanced intestinal Ca absorption even when existing blood Ca concentrations are normal. The flood of excess Ca into the blood directly supports eggshell mineralization and prevents osteoporosis by supporting bone mineral accretion rather than dissolution. However, elevated blood Ca levels also suppress the secretion of parathyroid hormone, thereby inhibiting renal tubular Ca reabsorption and allowing high levels of Ca to be excreted in the urine. prevents osteoporosis by supporting bone mineral accretion rather than dissolution. However, elevated blood Ca levels also inhibit the secretion of parathyroid hormone by the parathyroid glands [10]. A key role of parathyroid hormone is to prevent hypocalcemia (low blood Ca) by mobilizing bone mineral and minimizing Ca excretion in the urine. When elevated blood Ca concentrations suppress parathyroid hormone secretion, or when the parathyroid glands are experimentally removed, then Ca concentrations in the urine increase dramatically [14]. These hypercalcemic and hypercalciuric responses to HCLP diets substantially enhance the risk of nephron calcification and kidney stone formation. After the kidneys initially are damaged by Ca-induced mineralization or IBV, cellular debris obstructs the collecting ducts and ureteral branches, causing pressure-induced degeneration and then necrotic atrophy of the tissue upstream from the blockage. Various combinations of localized atrophy and compensatory hypertrophy account for the asymmetry in kidney shapes and weights in affected birds. The necrotic (obstructed) tissue serves as a focal area for additional mineralization. Tissue debris and Caurates solidify into mineral deposits in the collecting ducts and ureteral branches, thereby initiating the process of urolith accretion [2, 15, 16]. Field studies had shown that non-laying (cull) hens are highly likely to develop urolithiasis in commercial flocks, presumably because non-layers consuming a HC layer ration excrete the extra Ca in their urine rather than using it to form eggshells [16]. Accordingly, it was our hypothesis that immature pullets would develop urolithiasis when they were fed HCLP rations, and the incidence of urolithiasis would increase further if the HCLP diets were accompanied by exposure to nephrotropic strains of IBV that enhance the cumulative damage to kidney tissues. Conducting Experiments to Test the Hypothesis When research resources are limited and a variety of alternatives must be evaluated, an efficient approach is to conduct pilot studies designed to impose the most likely challenges upon which the hypothesis is predicated. Pilot studies are intended to contribute to the

6 WIDEMAN: KIDNEY STONES IN HENS 297 development of a research model that, under well controlled and adequately replicated experimental conditions, will consistently recreate the pathognomonic symptoms of the field problem. Our initial pilot study was conducted by undergraduate students with the specific objective of developing a protocol for triggering urolithiasis in experimental flocks [12]. SCWL chicks were fed a NC (1% Ca) and NP (0.6% ap) diet throughout, or a HCLP diet (3.25% Ca, 0.4% ap) beginning at 4 wk of age. The HCLP group, but not the NCLP group, also received an aerosol inoculation with the nephrotropic Gray strain IBV at 10 wk of age. This pilot study did not allow differentiation between the roles of the HCLP diet and IBV, but 32.5% of the birds in the HCLP+IBV group did develop characteristic urolithiasis lesions, whereas no lesions developed in the NCNP group by wk 18 when the experiment was terminated. Urolithiasis triggered considerable kidney asymmetry in the HCLP+IBV group, whereas the kidneys from pullets in the NCNP group were highly symmetrical in shape and weight. Extensive nephron degeneration accompanied by compensatory hypertrophy of the remaining kidney tissue was demonstrated in the HCLP+IBV group when compared with NCLP group [12]. A follow-up study was conducted to independently evaluate the impact of the HCLP diet without purposefully exposing the birds to IBV [17]. Beginning at 7 wk of age, SCWL pullets were fed diets containing NC (1% Ca) or HC (3.5% Ca), and NP (0.6% ap) or LP (0.4% ap). Half of the pullets were necropsied at 18 wk of age, and the remaining birds were transferred to laying cages where all were fed the same commercial layer ration until they were necropsied at wk 51. As shown in Table 1, feeding the HCLP diet during pullet growout successfully triggered urolithiasis in both pullets and laying hens. It also was of interest that the HCNP diet did not cause kidney damage in 18-week-old pullets but did subsequently trigger urolithiasis when the hens reached maturity. Obviously it would be exceptionally difficult to deduce the epidemiology of a urolithiasis outbreak in 40-week-old hens if the initiating source of the problem occurred much earlier during pullet growout. Kidney function studies demonstrated that feeding immature pullets the HCLP diet flooded their urine with Ca, reduced urinary P excretion, and significantly increased the urine ph (Table 2) [17]. Urine with reduced acidity and that contains high concentrations of Ca provides an ideal medium for the precipitation of Ca-containing kidney stones. As described by Mongin [18], HCLP diets shift the dietary fixed cation:anion balance, determined as [(Na + + K + + Ca ++ + Mg ++ ) (Cl + PO 4 = + SO 4 = )], toward a less acidic range due to the net increase in fixed cations (Ca ++ ) combined with the net reduction in fixed anions (PO 4 = ). Table 1. Incidence of urolithiasis (%; n affected/n evaluated) in pullets or hens raised from 7 to 18 wk of age on diets containing normal or high calcium (NC = 1% Ca; HC = 3.5% Ca), and normal or low available phosphorus (NP = 0.6% ap; LP = 0.4% ap). Diet during pullet growout NCNP, % HCNP, % NCLP, % HCLP, % Pullets, wk 18 0 a (0/95) 1 a (1/88) 0 a (0/92) 14 b (13/93) Hens, wk 51 0 a (0/111) 12 b (13/108) 2 a (2/107) 14 b (15/111) a,b Values with different superscripts differed significantly at P < Table 2. Kidney function in 18-week-old pullets fed diets containing normal or high calcium (NC = 1% Ca; HC = 3.5% Ca), and normal or low available phosphorus (NP = 0.6% ap; LP = 0.4% ap). Diet during pullet growout Variable NCNP HCNP NCLP HCLP Glomerular filtration rate (ml/kgbw/min) 1.42 a,b 1.34 b 1.56 a 1.25 b Fractional Ca excretion 0.04 b 0.10 b 0.08 b 0.98 a Fractional Pi excretion 0.70 a 0.35 b 0.37 b 0.07 c Urine ph 5.52 b 5.40 b 5.46 b 5.85 a a c Values with different superscripts within a variable differed significantly at P < 0.05.

7 298 JAPR: Review Article Table 3. Incidence of urolithiasis (%; n affected/n evaluated) in pullets or hens raised from 7 to 18 wk of age on diets containing normal or high calcium (NC = 1% Ca; HC = 3.25% Ca), and normal or low available phosphorus (NP = 0.6% ap; LP = 0.4% ap), and that were dehydrated or received water ad libitum during pullet growout or during the laying phase. Diet during pullet growout Treatment group NCNP, % HCNP, % NCLP, % HCLP, % Pullets, hydrated 0 b (0/49) 2.1 a,b (1/48) 0 b (0/49) 12.5 a (6/48) Pullets, dehydrated 0 b (0/46) 0 b (0/40) 0 b (0/43) 15.5 a (7/45) Hens, hydrated 0 b (0/68) 11.4 a (8/70) 1.4 b (1/71) 16.4 a (12/73) Hens, dehydrated 0 b (0/43) 13.2 a (5/38) 2.8 a,b (1/36) 7.9 a (3/38) a,b Values with different superscripts within a treatment group differed significantly at P < Subsequent independent studies confirmed the effects of dietary Ca and ap on urinary Ca excretion, and demonstrated that low ap diets stimulate the synthesis of 1,25[OH] 2 D 3, which in turn increases intestinal Ca absorption and floods the blood and urine with Ca [19 23]. The role of dehydration in triggering urolithiasis also was evaluated. Achieving low manure moisture is essential in cage layer facilities, and managers use specific strategies to reduce water consumption and manure moisture. One report implicated water deprivation as a factor that contributed to outbreaks of urolithiasis in 2 flocks of laying hens [6]. To test this possibility, SCWL pullets were divided into 4 groups at 7 wk of age and were fed NCNP, HCNP, NCLP, and HCLP diets. Beginning at 12 wk of age, water was withheld from 25 pullets from each diet treatment on 5 consecutive d per wk for 5 consecutive wk, whereas the remaining pullets had ad libitum access to water. At 18 wk of age all dehydrated pullets were necropsied, as were representative pullets from each diet treatment that had ad libitum access to water. The remaining pullets were moved into individual layer cages and were fed a commercial layer ration. At 37 wk of age, one third of the hens were subjected to a dehydration regimen for 5 wk, consisting of no water for 3 consecutive d followed by ad libitum access to water for the next 2 d. All surviving hens were necropsied at wk 51. Dehydration reduced body weights and kidney weights in the pullets, but the incidence of urolithiasis was not enhanced in either pullets or hens that had been subjected to repeated episodes of severe dehydration (Table 3). Demonstrating an independent role of IBV in urolithiasis outbreaks was problematic. Nephrotropic strains of IBV had been isolated from the kidneys of affected hens during urolithiasis outbreaks in the Midwestern and southeastern United States, but experimental birds inoculated with these strains failed to develop characteristic urolithiasis pathology [15, 24, 25]. Indeed, exposing pullets to IBV without first feeding them a HC diet did not lead to an amplified incidence of urolithiasis [16, 19, 20]. However, IBV inoculations consistently elicited higher urolithiasis incidences when pullets were fed HCLP diets prior to the inoculation. In a breakthrough experiment, pullets were fed NCNP or HCLP diets beginning at 6 wk of age, and either remained unexposed to IBV or were exposed to the nephrotropic Gray strain IBV at 14 wk of age. All surviving birds were necropsied at 21 wk of age to assess the incidence of urolithiasis and gross kidney damage. No uroliths or kidney asymmetry were detected in pullets fed the NCNP diet regardless of IBV exposure. The HCLP diet did trigger a significant incidence of urolithiasis and gross kidney damage, and both of these responses were further amplified by subsequent exposure to nephrotropic IBV (Table 4). Renal function studies did not reveal specific IBV-induced alterations in urinary Ca or P excretion, in urine ph, or in the ability of the kidneys to acidify the urine. Instead, exposure to nephrotropic IBV caused widespread destruction of kidney tubules as reflected by reductions in the total number of nephrons per kidney and associated reductions in the glomerular filtration rate [19, 20]. The available evidence suggests that IBV usually does not trigger urolithiasis during pullet growout because pullet diets normally contain approximately 1% Ca, and consequently their urine normally is not flooded with Ca. IBV can serve as an effective amplifier of urolithiasis when high Ca pre-laying diets predispose

8 WIDEMAN: KIDNEY STONES IN HENS 299 Table 4. Incidence of urolithiasis and macroscopic kidney damage (%; n affected/ n evaluated) in 21-week-old pullets reared from 6 wk of age on diets containing normal or high calcium (NC = 1% Ca; HC = 3.5% Ca), and that either remained unexposed to infectious bronchitis virus (N) or were exposed at 14 wk of age to nephropathogenic Gray strain infectious bronchitis virus (IBV). Diet and IBV Treatments Pathology NCN, % NC+IBV, % HCN, % HC+IBV, % Urolithiasis 0 c (0/103) 0 c (0/66) 12.5 b (12/96) 26.2 a (22/84) Macroscopic damage 0 c (0/104) 0 c (0/66) 19.8 b (19/96) 33.3 a (28/84) a c Values with different superscripts within a pathology category differed significantly at P < pullets toward hypercalciuria (increased urinary Ca excretion), or when pullets that are not yet in full production are first moved to laying cages and fed a high Ca diet. Moving pullets to laying cages and the subsequent onset of egg production are stressful events that can suppress the immune system and permit IBV that had been sequestered within renal proximal tubule cells to emerge and reinitiate viremia. Urolithiasis typically develops in commercial flocks shortly after the hens first come into lay, at a time when stressinduced viremia may coincide with providing HCLP diets to pullets that are just beginning to sporadically produce eggs. These studies suggest that kidney tissue damaged by IBV releases necrotic debris into the tubule lumen, plugging the collecting ducts and ureteral branches and initiating Ca-urate precipitation that initiates an ongoing process of urolith accretion [16, 19]. Evaluating Whether the Hypothesis Has Been Supported The experiments summarized above strongly support the hypothesis diagrammed in Figure 2. Feeding HC diets to experimental groups of immature pullets, particularly HCLP diets, consistently triggered the pathognomonic symptoms associated with field outbreaks of urolithiasis. Nephrotropic IBV amplified the incidence of Ca-induced urolithiasis, but only when pullets were fed HC diets prior to or contemporaneously with IBV inoculation (e.g., after the birds already have been flooded with Ca). Nutrition interactions between dietary Ca and ap levels, and Nutrition x Disease interactions between dietary Ca and IBV, are clear, and the requisite sequence of events necessary for this interaction to occur helps to explain the difficulties encountered in attempts to use field isolates of IBV as the sole trigger for urolithiasis in experimental flocks. Including IBV inoculations in an experimental model adds substantially to the complexity and expense, because inoculated pullets must be maintained under biosecurity constraints in isolation facilities. Accordingly, we subsequently emphasized the HCLP challenge as a triggering mechanism in our experimental model. DEVELOPING A PRACTICAL SOLUTION Composition of Uroliths from a Variety of Sources Detailed micro-anatomical and renal function experiments repeatedly indicated that the physiological impact of urolithiasis arises predominately from progressive reductions in functional renal mass attributable to sequential occlusion of the ureters and their branches by uroliths, rather than from constitutively or pathologically inappropriate renal tubular transport processes. This was a consistent observation regardless of whether hens with urolithiasis were obtained during spontaneous outbreaks in commercial flocks [1], or from experimental flocks in which urolithiasis was triggered by HCLP diets alone [17] or in combination with nephrotropic IBV [16, 19, 20]. In the absence of a primary defect in renal function, hens in affected flocks were considered highly likely to survive and remain in egg production if new uroliths could be prevented from forming and existing uroliths could be dissolved. In order to modify the urine composition to prevent urolith accretion, it first was necessary to determine the chemical composition of the urolith mineral. Uroliths collected from outbreaks in commercial flocks, and from experimental flocks in which urolithiasis was induced by IBV and/or feeding immature pullets

9 300 JAPR: Review Article HCLP diets, were tested by x-ray diffractometry, infrared spectrophotometry, and emission spectrography. In virtually all cases the uroliths had a unique calcium-sodium-urate composition, with occasional minor or major substitutions of ammonium hydrogen urate. These uroliths were found to be formed by gradual accretion of homogeneous mineral [26]. Qualitative analyses of uroliths from previous outbreaks in the United States and Great Britain also demonstrated the presence of calcium-sodium-urate including some ammonium salts of uric acid [2, 4, 27]. It was inferred from the overwhelming prevalence of the unique calcium-sodium-urate mineral that a similar process of urolith formation likely occurs worldwide, regardless of the etiology of the urolithiasis outbreak. Indeed, it appeared highly likely that treatments that prevent ureteral obstruction when urolithiasis is induced experimentally with HCLP diets also would be effective when applied to commercial outbreaks. Based on the calcium-sodium-urate urolith mineral composition, it was predicted that increasing the hydrogen ion concentration of the urine (e.g., urinary acidification) should help dissolve pre-formed uroliths and prevent the accretion of new uroliths [26]. Dietary Supplementation with Urinary Acidifiers Effectively Treats Urolithiasis Urine with reduced acidity and containing high concentrations of Ca is an ideal medium for the formation of calcium-sodium-urate uroliths. Several nutritional factors that tend to increase the incidence of urolithiasis may do so by affecting the fixed cation:anion balance in such a way that urine acidity is reduced. According to the cation:anion balance equation (see above) [18], HCLP diets reduce the urine acidity because HC increases the fixed cations while LP reduces the fixed anions. When the chloride (Cl ) content of a layer ration is reduced to achieve lower manure moisture, urine acidity also is reduced due to the net reduction in fixed anion. In some cases, layer diets are formulated specifically to be alkaline as a means of improving eggshell quality. For example, sodium bicarbonate may be added rather that sodium chloride, thereby reducing the Cl content of the diet and reducing the urine acidity. To assess the possible role of dietary Cl as an effective acidifier, experiments were conducted to evaluate the efficacy of supplementing HCLP diets with ammonium chloride (NH 4 Cl). In the initial experiment 60% of the pullets fed the HCLP diet from 4 through 16 wk of age developed macroscopic kidney lesions and urolithiasis, whereas only 30% of the pullets fed the HCLP diet containing 0.5% NH 4 Cl developed macroscopic kidney lesions and urolithiasis [16]. Subsequently it was demonstrated that feeding HCLP diets with 1% NH 4 Cl during pullet growout and during the 32- to 52-week laying period successfully acidified the urine, reduced the incidence of urolithiasis and reduced kidney asymmetry ratios when compared with pullets and hens that had been fed a normal HCLP diet or a HCLP diet alkalinized by adding 1% sodium bicarbonate. However, and as fully anticipated, hens fed the HCLP diet containing 1% NH 4 Cl had significantly higher water consumption and manure moisture when compared with hens fed the normal HCLP diet [28]. Increased water consumption and manure moisture create serious problems in cage layer facilities, and we were concerned that hens with limited functional renal mass and partial ureteral obstruction might not tolerate the dual challenges of enhanced acid loads (the kidneys are responsible for excreting metabolic acid) plus higher rates of urine flow. On the other hand, it also was possible that high urine flow rates helped to prevent urinary tract obstruction by briskly flushing cellular debris from the collecting ducts and ureters [16, 28]. To acidify the urine but avoid increasing manure moisture, we evaluated the efficacy of supplementing HCLP diets with sulfur that, per the Mongin [18] equation (see above), would be excreted as the fixed anion sulfate (SO = 4 ) plus hydrogen ions, thus essentially adding sulfuric acid (H 2 SO = 4 ) to the urine. In mammals, increased protein intake is associated with enhanced urinary acidification, an effect partially attributable to increased urinary excretion of H 2 SO = 4 generated when excess sulfur-containing amino acids (cysteine and methionine) are metabolized. However, increasing the levels of protein in diets fed to hens with urolithiasis would be counterproductive because excess protein also increases the nitrogenous waste load (e.g., uric acid) and

10 WIDEMAN: KIDNEY STONES IN HENS 301 increases water consumption and urine flow [29]. Instead we evaluated adding excess methionine to HCLP diets, either in the form of methionine per se, or in the form of methionine hydroxy analog (MHA, D,L-2-hydroxy-4- [methylthio]butanoic acid). From 5 to 17 wk of age, pullets were fed NCNP or HCLP diets with or without supplemental MHA (0, 0.3, or 0.6%). Relative to pullets fed the NCNP diet, pullets fed the HCLP diet without MHA excreted less acidic urine, had high urinary Ca concentrations, and developed kidney asymmetry and urolithiasis. Supplementing the HCLP diet with 0.6% MHA significantly acidified the urine without increasing water consumption or urine flow, and significantly reduced kidney asymmetry and the incidence of urolithiasis when compared with the un-supplemented HCLP diet [29]. Further studies demonstrated that either DL-methionine or equimolar levels of MHA could be added to HCLP diets to attenuate Ca-induced kidney damage and urolith formation during pullet growout. At biologically effective levels (0.3 or 0.6% DL-methionine, 0.34 or 0.68% MHA), neither of these methionine supplements adversely affected subsequent production parameters including hen-day egg production, egg mass, eggshell mass, percentage eggshell, or bone mineralization [30]. Laying hens fed layer rations containing methionine supplements at levels of up to 1% for DL-methionine or 1.13% for MHA did not exhibit feed avoidance, gross or histopathological renal or hepatic changes, changes in egg production or eggshell quality, deterioration of femur quality, or increased mortality. These observations confirmed that an adequate margin of safety existed for producers who chose to treat urolithiasis outbreaks by supplementing their layer rations with DL-methionine or MHA within the recommended ranges of 0.6 and 0.68%, respectively [31, 32]. Additional studies demonstrated that adding 0.53% ammonium sulfate [(NH 4 ) 2 SO 4 ] to HCLP diets was significantly more effective and less expensive than adding 0.6% DL-methionine for reducing gross kidney damage and the incidence of urolithiasis in 12- and 18-week-old pullets. Both supplements tended to acidify the urine when compared with the un-supplemented HCLP diet, but neither DL-methionine nor ammonium sulfate caused systemic metabolic acidosis, nor did they consistently affect water consumption, urine flow rates, or manure moisture [33, 34]. Dietary Acidification: The Industry Standard for Treating Urolithiasis Dietary supplementation with 0.5% ammonium sulfate became the treatment of choice for dissolving existing uroliths and preventing new uroliths from forming in flocks experiencing an outbreak of urolithiasis. Acidifiers must be used moderately and continuously in affected flocks. The kidneys are responsible for excreting excess acid. If the diet is supplemented with too much acidifier (>1%), then hens with moderate to severe kidney damage will be unable to rapidly excrete the acid load, and a substantial spike in mortality may be noted for several wk immediately following introduction of the acidified diet. Even when reasonable acidifier levels are used (e.g. 0.5% ammonium sulfate), a brief peak of mortality may occur as hens having the most marginal renal capacity succumb to the challenge. The efficacy of using high acidifier levels to cull hens with damaged kidneys from a flock prior to molting has not been evaluated experimentally. In any case, flocks that have experienced significant urolithiasis mortality are not promising prospects for molting, because substantial numbers of apparently healthy hens may harbor subclinical damage that will progressively worsen during the stress of re-stimulation for egg production and re-introduction of the HC layer ration. Once treatment with dietary acidifiers begins and urolithiasis mortality has been eliminated from a flock, the treatment must nevertheless continue for the remainder of the production cycle. Otherwise, uroliths will begin to form once again in hens with damaged kidneys, and within 2 wk after acidifier withdrawal substantial urolithiasis mortality should be expected to resume. Genetic Susceptibility to Urolithiasis During the late 1980s and early 1990s the majority of urolithiasis outbreaks occurred primarily in flocks of one particular SCWL strain. Experiments verified that SCWL strain A was much more susceptible to urolithiasis induced by HCLP diets than another popular SCWL

11 302 JAPR: Review Article Table 5. Incidence (%) of urolithiasis and gross kidney damage at 18 wk of age in pullets from strains A or B raised from 5 wk of age on diets containing normal or high calcium (NC = 1% Ca; HC = 3.5% Ca; both containing 0.4% ap), with(as) or without 0.5% supplemental ammonium sulfate. Diet during pullet growout Pathology NC NC+AS HC HC+AS Strain A urolithiasis 0 b 0 b 38 a 0 b Strain B urolithiasis 0 b 0 b 0 b 0 b Strain A macroscopic damage 0 b 0 b 63 a 0 b Strain B macroscopic damage 0 b 0 b 50 a 13 a,b a,b Values with different superscripts within a pathology category differed significantly at P < strain B of that era (Table 5) [33]. Interestingly, healthy pullets of these strains that had not been challenged with IBV or high dietary Ca, nevertheless were found to have measurable strain-specific differences in the microscopic anatomy of their kidneys. The kidneys of strain A had a superior capacity to conserve water by concentrating the urine, based on having higher proportions of nephrons with long loops of Henle to serve as countercurrent multipliers. Pullets of strain B had a lower proportion of nephrons with loops of Henle, and consequently these pullets consumed and excreted slightly more water [35 37]. We hypothesized that strain A had been genetically selected for reduced water consumption and manure moisture, both of which were desirable traits for commercial laying hens. However, the accompanying changes in kidney function and structure apparently unintentionally rendered this line more susceptible to urolithiasis [33, 37]. Perhaps a low urine flow rate and high concentrations of solutes in the urine help to accelerate the precipitation and accretion of urolith mineral. Alternatively, unpublished pilot studies indicated that during the pathogenesis of calcium-induced urolithiasis, the earliest site of mineralization within the kidneys occurred within the thick ascending limb of the loop of Henle [35]. Perhaps kidneys having higher proportions of nephrons with loops of Henle provide proportionally more opportunity for the site-specific initiation of tissue mineralization. The biological basis for strain-related differences in urolithiasis susceptibility remains to be confirmed, but poultry managers should be aware that nutrition, vaccination, and management strategies may require adjustment if a transition from one strain to another is accompanied by an outbreak of urolithiasis. COLLATERAL BENEFITS During the initial necropsies conducted on hens with urolithiasis the possibility existed that the unilateral or bilateral absence of entire cranial and/or medial kidney divisions might indicate a congenital or developmental abnormality [2, 38]. However, missing kidney divisions were exceptionally rare in hens from healthy flocks but were common in flocks affected by urolithiasis. Furthermore, in affected flocks the missing kidney divisions almost always were associated with uroliths in the ipsilateral ureter, indicating focal ureteral blockage was followed by progressive degeneration of the tissue upstream from the obstruction [1, 2]. To confirm the latter hypothesis, techniques were developed in SCWL chicks to surgically clamp one or both ureters on the caudal surface of the medial kidney division [39], or to clamp or surgically remove a one mm segment of one ureter at the level of the ischiadic artery [40, 41]. Several wk after ureteral obstruction or transection the kidney divisions upstream from the site of blockage or injury had degenerated and virtually disappeared, and compensatory hypertrophy by the unobstructed kidney divisions restored essentially normal functional renal mass and normal kidney function [39 41]. These responses reproduced our observations in hens with urolithiasis, and provided convincing evidence that the degenerative pathology associated with urolithiasis was primarily, if not wholly, attributable to ureteral obstruction. Enabling affected hens to remain productive clearly required preventing progressive ureteral obstruction by new or pre-existing uroliths. An unanticipated consequence of these experiments arose from our observations that the major renal blood vessels remained intact after the cranial and medial divisions had degenerated. In normal avian

12 WIDEMAN: KIDNEY STONES IN HENS 303 kidneys the renal arteries, renal veins, and renal portal veins are surrounded by kidney tissue and are inaccessible to experimental manipulation. By clamping or transecting one ureter to cause ipsilateral degeneration of the cranial and medial divisions, the arterial, renal venous, and renal portal vasculature of the caudal renal division became accessible for experimental manipulation [39 41]. A serendipitous consequence of our urolithiasis research was the development and validation of a new model that for the first time allowed us to directly evaluate the roles of compensatory hypertrophy and arterial and renal portal hemodynamics on avian kidney function [40 45]. CONCLUSIONS AND APPLICATIONS 1. The scientific method was used to develop and validate a research model to induce the pathognomonic symptoms associated with field outbreaks of urolithiasis. 2. The model allowed us to confirm that HCLP diets and IBV are key triggers for urolithiasis outbreaks. A true nutrition x disease x genetic interaction was demonstrated. 3. Detailed micro-anatomical and renal function experiments repeatedly indicated that the physiological impact of urolithiasis arises predominately from progressive reductions in functional renal mass attributable to sequential occlusion of the ureters and their branches by uroliths, rather than from constitutively or pathologically inappropriate renal tubular transport processes. 4. Our model allowed us to confirm key triggering mechanisms and stressors, reveal innate organ system limitations or susceptibilities, and provide a reliable test bed for evaluating practical prophylactic and therapeutic treatments. 5. The cation:anion balance of pullet and layer diets can be adjusted to acidify the urine and prevent urolith mineral accretion. Urinary acidification has been widely adopted as the method of choice for treating commercial flocks experiencing urolithiasis outbreaks. REFERENCES AND NOTES 1. Wideman, R. F., E. T. Mallinson, and H. Rothenbacher Kidney function of pullets and laying hens during outbreaks of urolithiasis. Poult. Sci. 62: Mallinson, E. T., H. Rothenbacher, R. F. Wideman, D. B. Snyder, E. Russek, A. I. Zuckerman, and J. P. Davidson Epizootiology, pathology, and microbiology of an outbreak of urolithiasis in chickens. Avian Dis. 28: Blaxland, J. D., E. D. Borland, W. G. Siller, and L. Martindale An investigation of urolithiasis in two flocks of laying fowls. Avian Pathol. 9: Siller, W. G Renal pathology of the fowl. Avian Pathol. 10: Randall, C. J., T. B. Blandford, E. D. Borland, N. H. Brooksbank, and S. A. Hall A survey of mortality in 51 caged laying flocks. Avian Pathol. 6: Julian, R Water deprivation as a cause of renal disease in chickens. Avian Pathol. 11: Siller, W. G., and R. B. Cumming The histopathology of an interstitial nephritis in the fowl produced experimentally with infectious bronchitis virus. J. Pathol. 114: Page, R. K., O. J. Fletcher, and P. Bush Calcium toxicosis in broiler chicks. Avian Dis. 24: Chandra, M., B. Singh, G. L. Soni, and S. P. Ahuja Renal and biochemical changes produced in broilers by high-protein high-calcium, urea-containing, and vitamin A-deficient diets. Avian Dis. 28: Shane, S. M., R. J. Young, and L. Krook Metabolic disturbances in replacement pullets produced by high calcium diets. Proc. Cornell Nutr. Conf Shane, S. M., R. J. Young, and L. Krook Renal and parathyroid changes produced by high calcium intake in growing pullets. Avian Dis. 13: Niznik, R. A., R. F. Wideman, B. S. Cowen, and R. E. Kissell Induction of urolithiasis in Single Comb White Leghorn pullets: effect on glomerular number. Poult. Sci. 64: Norman, A. W The vitamin D endocrine system. Physiologist 28: Wideman, R. F., Renal regulation of avian calcium and phosphorus metabolism. J. Nutr. 117: Cowen, B. S., R. F. Wideman, M. O. Braune, and R. L. Owen An infectious bronchitis virus isolated from chickens experiencing a urolithiasis outbreak. I. In vivo characterization studies. Avian Dis. 31: Wideman, R. F., and B. S. Cowen Effect of dietary acidification on kidney damage induced in immature chickens by excess calcium and infectious bronchitis virus. Poult. Sci. 66: Wideman, R. F., J. A. Closser, W. B. Roush, and B. S. Cowen Urolithiasis in pullets and laying hens: Role of dietary calcium and phosphorus. Poult. Sci. 64: Mongin, P., Role of acid-base balance in the physiology of egg shell formation. World s Poult. Sci. J. 24: Glahn, R. P., R. F. Wideman, and B. S. Cowen Effect of Gray strain infectious bronchitis virus and high dietary calcium on renal function of SCWL pullets at 6, 10 and 18 weeks of age. Poult. Sci. 67: Glahn, R. P., R. F. Wideman, and B. S. Cowen Order of exposure to high dietary calcium and Gray strain

13 304 JAPR: Review Article infectious bronchitis virus (IBV) alters renal function and the incidence of urolithiasis. Poult. Sci. 68: Rao, K. S., and D. A. Roland Influence of dietary calcium and phosphorus on urinary calcium in commercial leghorn hens. Poult. Sci. 69: Rao, S. K., D. A. Roland, and J. L. Orban Influence of dietary cholecalciferol, calcium, and phosphorus on urinary calcium in commercial laying hens. Poult. Sci. 70: Frost, T. J., D. A. Roland, and D. N. Marple The effect of various dietary phosphorus levels on the circadian patterns of 1,25-dihydroxycholecalciferol, total calcium, ionized calcium, and phosphorus in laying hens. Poult. Sci. 70: Cowen, B. S., R. F. Wideman, H. Rothenbacher, and M. O. Braune An outbreak of urolithiasis on a large commercial egg farm. Avian Dis. 31: Brown, T. P., J. R. Glisson, G. Rosales, P. Villegas, and R. B. Davis Studies of avian urolithiasis associated with an infectious bronchitis virus. Avian Dis. 31: Oldroyd, N. G., and R. F. Wideman Characterization and composition of uroliths from domestic fowl. Poult. Sci. 65: Manning, R. A., and B. J. Blaney Identification of uroliths by infrared spectroscopy. Austr. Vet. J. 63: Glahn, R. P., R. F. Wideman, and B. S. Cowen Effect of dietary acidification and alkalinization on urolith formation and renal function in SCWL laying hens. Poult. Sci. 67: Wideman, R. F., W. B. Roush, J. L. Satnick, R. P. Glahn, and N. O. Oldroyd Methionine hydroxy analog (free acid) reduces avian kidney damage and urolithiasis initiated by excess dietary calcium. J. Nutr. 119: Wideman, R. F., B. C. Ford, R. M. Leach, D. F. Wise, and W. W. Robey Liquid methionine hydroxy analog (free acid) and D-L methionine attenuate calcium-induced kidney damage in domestic fowl. Poult. Sci. 72: Dibner, J. J., M. L. Kitchell, W. W. Robey, A. G. Yersin, P. A. Dunn, and R. F. Wideman Liver damage and supplemental methionine sources in the diets of laying hens. J. Appl. Poult. Res. 3: Wideman, R. F., B. C. Ford, J. J. Dibner, W. W. Robey, and A. G. Yersin Responses of laying hens to diets containing up to 2% DL-methionine or equimolar (2.25%) 2-hydroxy-4(methylthio)butanoic acid. Poult. Sci. 73: Lent, A., and R. F. Wideman Susceptibility of two commercial Single Comb White Leghorn strains to calcium-induced urolithiasis: efficacy of dietary supplementation with D-L methionine and ammonium sulfate. Br. Poult. Sci. 34: Lent, A. J., and R. F. Wideman Hypercalciuric response to dietary supplementation with dl-methionine and ammonium sulfate. Poult. Sci. 73: Wideman, R. F Kidney Anatomy and Physiology. Pages in CRC Critical Rev. in Poult. Biol. Volume 1, Dietert, R. R., ed. CRC Press. 36. Wideman, R. F Maturation of glomerular size distribution profiles in domestic fowl (Gallus domesticus). J. Morphol. 201: Wideman, R. F., and A. C Nissley Kidney structure and responses of two commercial Single Comb White Leghorn strains to saline in the drinking water. Br. Poult. Sci. 33: Tudor, D. C Congenital defects in poultry. Worlds Poult. Sci. J. 35: Wideman Jr., R. F., and G. Laverty Kidney function in domestic fowl with chronic occlusion of the ureter and caudal renal vein. Poult. Sci. 65: Wideman Jr., R. F., and C. M. Gregg Model for evaluating avian renal hemodynamics and glomerular filtration rate autoregulation. Am. J. Physiol. 254: R925 R Gregg, C. M., and R. F. Wideman Morphological and functional comparisons of normal and hypertrophied kidneys of adult domestic fowl (Gallus gallus).am.j.physiol. 258:F403 F Wideman, R. F Autoregulation of renal plasma flow: contribution of the renal portal system. J. Comp. Physiol. B 160: Wideman Jr., R. F., R. P. Glahn, W. G. Bottje, and K. R. Holmes Use of a thermal pulse decay system to assess avian renal blood flow during reduced renal arterial perfusion pressure. Am. J. Physiol. 262: R90 R Wideman Jr., R. F., H. Nishimura, W. G. Bottje, and R. P. Glahn Reduced renal arterial perfusion pressure stimulates renin release from domestic fowl kidneys. Gen. Comp. Endocrinol. 89: Glahn, R. P., W. G. Bottje, P. Maynard, and R. F. Wideman Response of the avian kidney to acute changes in arterial perfusion pressure and portal blood supply. Am. J. Physiol. 264:R428 R434.