Kidney International, Vol. 37 (1990), pp. 36—43

Reaction of MDCK cells to crystals of monosodium urate

monohydrate and uric acid BRYAN T. EMMERSON, MICHAEL CROSS, JULIE M. OSBORNE, and Ro A. AXELSEN Department of Medicine, University of Queensland, and Department of Anatomical Pathology, Princess Alexandra Hospital, Brisbane, Queensland, Australia

Reaction of MDCK cells to crystals of monosodium urate monohydrate and uric acid. Madin-Darby canine kidney (MDCK) cells exhibit many

urate or uric acid [6], the former being principally interstitial and the latter principally intra-luminal. Although the processes of the characteristics of cells of the cortical collecting tubule. Since hypotheses concerning the development of gouty and uric acid involved in their formation may be independent and different nephropathy involve a reaction between such cells and crystals, either of monosodium urate monohydrate (MSUM) or uric acid, the reaction between MDCK cells in culture and the above crystals was studied, both morphologically and functionally. In monolayer cultures, reaction sites developed within four hours of exposure to urate crystals. These increased in number for up to 72 hours and subsided gradually after removal of the crystals. At these reaction sites, crystals were observed to have passed beneath the cell surface and could be demonstrated both

[7], many of the pathological studies suggest that the two

processes may not be entirely distinct, and it has been postulated that some intra-luminal crystals might erode the tubular basement membrane of the renal tubules and pass into the renal interstitium [8], inducing in the process an inflammatory reaction. Although kidney disease has been questioned as a sequel of

within intra-cellular lysosomes as well as within the inter-cellular gouty arthritis at a time when allopurinol is used extensively [9], spaces. When the MDCK cells were maintained as single cells in suspension, phagocytosis of crystals by the majority of the cells could earlier studies indicated that the most consistent abnormality in be observed, but the response was much more rapid than in monolayers. During the cell/crystal reaction, significant amounts of lysosomal enzymes and prostaglandin E2 were released and, to a less significant degree, cytosolic enzymes, presumably due to cell lysis. This enzyme release did not occur in MDCK cells grown in protein-free medium, and protein coating of the crystals was necessary for reactivity with cells. In

the kidneys of patients with chronic gouty arthritis was the presence of interstitial urate deposits [5]. In view of the role of

the kidney in urine concentration, it is not surprising that

adverse effects in the form of urate/uric acid crystal deposition might develop in the kidney and lead to a tissue response similar this regard, coating with IgG or lysozyme was more effective than to that in other organs. albumin. The reaction with uric acid crystals revealed a reactivity Experimental studies in rats fed uric acid together with the which was lesser in degree but qualitatively similar to that of urate uricase inhibitor, oxonate [10, 11], showed the development of crystals. These studies support the hypothesis that one of the earliest significant tubular damage affecting particularly the epithelial lesions in uric acid or gouty nephropathy occurs between intra-luminal crystals and the epithelial cells of the distal portion of the nephron, and cells of the collecting tubules. Inflammatory cells were present that some of the subsequent renal damage is secondary to this reaction. near tubules or in the interstitium related to damaged tubules. Thus, there is much to suggest that at least one of the mecha-

nisms of renal damage from urate and uric acid involves a Evidence from both genetic over-producers of urate [I] and

patients with acute over-production of urate following the chemotherapy of malignancy [2] indicates that excessive renal excretion of urate can lead to renal damage [3]. Acute renal

reaction between a crystal, either of urate or uric acid, and the tubular lining cell, and that this is most likely to occur in the

distal portion of the nephron. Such pathology has been ob-

served in the uric acid nephropathy of leukemia [12]. Because insufficiency in such a patient can often be reversed by an of this, we undertook a study of the reaction between urate and intense alkaline diuresis, and has been attributed to tubular uric acid crystals and cultured renal tubular cells to examine the earliest stages of this reaction. Since they have been reported to obstruction by intra-luminal deposits of uric acid crystals [2, 4]. possess many of the characteristics of collecting duct cells In hyperuricemic and gouty subjects, deposits of monosodium [13—15], we chose to study the Madin-Darby canine kidney urate monohydrate (MSUM or urate) may lead to renal micro(MDCK) cell line which can be maintained in continuous cell tophi, which may develop insidiously within the renal medulla culture. and be associated with more chronic and less reversible renal insufficiency [5]. X-ray crystallography has confirmed the naMethods ture of the crystals involved in these processes as being either Cell cultures

MDCK cells were obtained from Commonwealth Serum Laboratories (Melbourne, Australia) and were maintained in

Received for publication February 24, 1989 and in revised form August 18, 1989 Accepted for publication September 1, 1989

Medium 199 (Flow Laboratories, Helsinki, Finland) with 20 mti

HEPES (Sigma Chemical Co., St. Louis, Missouri, USA) and either 5% or 10% fetal calf serum (FCS) in 75 cm2 flasks. Cells

© 1990 by the International Society of Nephrology 36

Emmerson et a!: Reaction of MDCK cells to crystals of MSUM and uric acid

37

were subcultured using 0.075% trypsin in 0.6 mi EDTA. Cells solution by lowering the pH rapidly with acetic acid. This in suspension culture were maintained as single cells after resulted in rod-shaped crystals, most of which were small but a trypsinization of a monolayer culture by constant movement of few of which were larger and needed to be crushed prior to the culture medium with a magnetic stirrer for up to four hours. study. Diamond crystals used as a control for the MSUM Culture medium with a reduced pH (6.5 to 6.7) and reduced crystals were Micron SND 4 to 8 m, purchased from de sodium concentration was prepared by replacing much of the Beer's, Melbourne, Australia. These crystals were heat sterilsodium with choline chloride. This medium was based on ized before they were introduced to cultures. No contamination Commonwealth Serum Laboratories' Medium 199 in which of these diamond crystals with silicon dioxide could be detected NaCl and NaHCO3 were replaced by choline chloride to yield (...-

wE 5

are homogeneous insofar as they both result in urate overproduction. The first of these is the deficiency of hypoxanthine guanine phosphoribosyl transferase (HGPRT) [1] and the sec0 0 200 400 600 800 1000 ond is the urate overproduction due to hemopoietic malignancy, especially after treatment with cytotoxic agents [2]. Both are Crystal concentration, pg/m/ associated with excessive urinary excretion of urate, often prior Fig. 7. Release of the lysosomal enzymes $-glucuronidase (E) and to the development of elevation of the serum urate concentracx-galactosidase (0) from MDCK cells in monolayer culture exposed to tion. This urate overproduction is often associated with the increasing concentrations of MSUM (urate) crystals for 72 hours. development of renal damage, usually referred to as a uric acid nephropathy, and it has been postulated that uric acid crystals formed within the lumen are important in the pathogenesis of * 12

this type of renal disease. In addition, it is postulated that tubular epithelial cells react with the crystals as an initiating

disease mechanism. The present study supports this hypothesis in that it demonstrates a definite response by cells with many characteristics of renal tubular epithelial cells to have contact

10

with crystals of urate and uric acid. In this reaction, crystals

:t%8

aEE 6

pass initially into the epithelial cells, as well as into the inter-cellular spaces, and from there to a site beneath the epithelium. The release of lysosomal enzymes indicates in-

**

wE

** 2

0 LDH -glucuronidase -gaIactosdase Fig. 8. Release of the enzymes 13-glucuronidase, a-galactosidase and lactate dehydrogenase after exposure of MDCK cells in monolayer

volvement of lysosomes in the reaction and the lesser extent of the release of cytsolic enzymes indicates disruption of some of these cells. However, most cells in the reaction sites excluded trypan blue, even at 72 hours, and by this criterion, were still viable. These studies imply that crystals can pass through the epithelium by several mechanisms and may induce subsequent pathological changes. Similar structures to those reported here were observed in pig kidneys exposed to intra-luminal xanthine

and oxipurinol crystals [8]. A variety of cytokines may be released in the process, which may be followed in vivo by the

culture to MSUM (urate) and diamond crystals (500 jg/ml) for 72 arrival of neutrophils and macrophages, which together result in hours. Each study was done in triplicate (* P = 0.05, ** P < 0.01 in an inflammatory process and the possibility of pathological comparison with control cells. Symbols are: () control; (ff111) MSUM; changes. In this regard, the similarity of the MDCK cell line to () diamond.

Discussion

cells of the collecting duct epithelium has been well documented [15], and suggests that the response of these cells to crystals might resemble that of epithelial cells of the distal nephron in vivo. The culture system under study could there-

Findings in the kidneys of patients with gout have shown a fore serve as a model to assess the importance of the interaction wide variety of renal lesions, but this is probably not surprising of tubular cells with crystals in isolation from other physiologwhen it is realized that urate metabolism in gouty subjects is ical and cellular interference, such as tubular obstruction and very heterogeneous [24]. In addition, the kidneys of gouty leucocytic invasion. Because of the lower intra-luminal pH, most of the crystals to subjects are often subjected to damage from conditions other than MSUM or uric acid crystals such as hypertension, hyper- which renal epithelial collecting duct cells might be exposed in lipidemia, degenerative vascular disease and urinary infection, vivo would be expected to be uric acid rather than urate.

each of which may contribute to renal pathology. This has However, it should be noted that needle-shaped crystals of resulted in much conflicting evidence concerning the nature and MSUM (urate) have been found in the urine of patients with pathogenesis of gouty nephropathy. However, renal disease is a gout [25], so that the distinction may not be as important as frequent finding in two different, but allied, conditions which might be suggested theoretically. However, because of the

42

Emmerson et al: Reaction of MDCK cells to crystals of MSUM and uric acid

** *

*

I

Fig. 9. Enzyme release by MDCK cells in suspension after 4 hours exposure to MSUM (urate) crystals (500 j.Lg/ml). Cells were cultured and assayed in serum-free and protein-free medium. The MSUM crystals were either uncoated or coated with albumin (BSA), lysozyme or gamma globulin (* P < P < 0.001 when 0.05, ** P < 0.01, compared with control). Symbols are: (J)

75 50

as 0 /3-glucuronidase

LDH

a-galactosidase

Table 2. Enzyme release from MDCK cells in suspension on exposure to increasing concentrations of crystals of uric acid for 4 hours Initial uric acid

Final uric acid

. % Cells concen- concenf3-glucuronidase a-galactosidase takin tration tration Final release release up j.gIml (pmol/min/106 cells) pH crystals

0 125

0 55

6.40

0

6.35 6.31

8.7

11.8 14.0 14.8 16.6 15.6 17.5

3.5 4.1

control; (•) MSUM; () BSA; () lysozyme; () gamma globulin.

coating on the reactivity between cells and crystals. In this regard, neutrophil leucocytes are particularly active [261. How-

ever, fibroblasts [27, 28] and macrophages are known to be involved actively in crystal cell reactions. In most of these studies, it has been shown that crystal coating with IgG greatly enhances the cell reactivity and increases the release of lysosomal enzymes [291. This has been attributed to the orientation

of the adsorbed IgO being such that the Fc portion of the molecule was exposed and presumably able to bind to Fe receptors in the cell surface [30]. Direct studies on crystals

within the phagolysosomes of neutrophils have confirmed IgG as the major coating in vivo, although other proteins such as C3 and fibrinogen were present in small amounts [311. The findings 285 30.8 4.8 1000 767 6.21 62.4 5.2 in the present study of renal epithelial cell reactivity are similar to those reported for other more highly reactive cells such as neutrophils, in that phagocytosis is followed by the release of difficulty of maintaining cell cultures in a medium of low pH lysosomal enzymes [32]. The renal celllcrystal response in the (similar to urine) to which uric acid crystals could be added present study also appeared consistent with a crystal membrane without dissolving, the major aspects of the present study were interaction initially, followed by the formation of a phagosome undertaken with urate crystals and the cell crystal reaction and a phagolysosome, which subsequently released lysosomal documented first in these cells. Later, with a progressive enzymes. Crystals coated with IgG again showed greater reacmodification of the pH and sodium concentration in the medium tivity. The amount of lysosomal enzyme released in 72 hours to prevent the formation of MSUM crystals, it was possible to (approximately 2.5% of total intra-cellular enzyme) suggests study the reaction between uric acid crystals and the MDCK that only a small proportion of the cells were involved in the cell cells. Thus, because of their different solubilities at different pH crystal reaction. This compared with 15 to 20% of neutrophils values, uric acid and urate crystals required different optimal involved in reactions with comparable concentrations of culture and study conditions. However, their reactions were MSUM crystals [29]. Together with the slower time of the similar in both the morphological and functional (enzyme re- reaction, this suggests that the degree of reactivity of the lease) studies, and it appeared that the reactions with the two MDCK cells with crystals, while definite, was still of a lesser order than that of the highly reactive neutrophil, although the types of crystal were qualitatively similar. Crystal associated disease may affect many organs, and factors governing reactivity with crystals remained basically the special study has been undertaken of the influence of protein same. 250 375 500

144 179

6.22 6.22

12.5 16.9

4.6 5.1

Emnerson et a!: Reaction of MDCK cells to crystals of MSUM and uric acid

Reactions between crystals and other cells result in the

43

9. BECK LH: Requiem for gouty nephropathy. Kidney mt 30:280—287,

1986 release of a range of inflammatory mediators and/or cytokines which can cause symptoms and sometimes the development of 10. WAISMAN J, MWASI LM, BLUESTONE R, KLINENBERG JR: Acute hyperuncemic nephropathy in rats: An electron microscopic study. tissue damage [33]. Neutrophil leucocytes have again been the Am J Pathol 8 1:367—378, 1975 model cell and, in their reaction with urate crystals, have been 11. BLUESTONE R, BRADY S. WAISMAN J, KLINENBERG JR: Experimental hyperuricemic nephropathy: A model for human urate shown to release oxygen-derived free radicals, chemotactic deposition disease. Arthr Rheum (suppl) 18:823—834, 1975 factors such as leukotriene B4 (LTB4) and the prostaglandins, 12. KANWAR YS, MANALIGOD JR: Leukemic urate nephropathy. Arch particularly PGE2. Crystallmacrophage reactions can cause Pathol 99:467—472, 1975 formation of all of the above mediators as well as interleukin- 1 13. LEIGHTON J, ESTES LW, MANSUKHANI S, BRADA Z: A cell line (IL-i). Crystal/synovial fibroblast reactions may additionally derived from normal dog kidney (MDCK) exhibiting qualities of

promote formation of collagenase and neutral proteases. A variety of other clotting factors and complement components

have also been reported and the reaction is potently chemotactic. In the present study of crystal reactions with MDCK cells, no leukotriene B4 was produced. However, significant amounts of prostaglandin E2 were formed, and this may be an important finding in the pathogenesis of the renal damage by virtue of the local vasodilation and edema which could result. In vivo, this

papillary adenocarcinoma and of renal tubular epithelium. Cancer 26:1022—1028, 1970

14. RINDLER MJ, CHUMAN LM, SHAFFER L, SAIER M: Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK). J Cell Biol 81:635—648, 1979 15, VALENTICH JD: Morphological similarities between the dog kidney cell line MDCK and the mammalian cortical collecting tubule. Ann NYAcad Sci 372:384—404, 1981 16. TAUB M, CHUMAN L, SAIER MH JR, SATO G: Growth of Madin-

could promote the arrival of neutrophils and macrophages

Darby canine kidney epithelial cell (MDCK) line in hormone supplemented, serum free media. Proc Natl Acad Sci USA 76:

which may then react with the urate crystals which have passed through the epithelium.

17. SIMPsoN RU, GOODFRIEND TL: Angiotensin and prostaglandin

epithelium, after which crystals may infiltrate the interstitium to

metric DNA determination in biological material using 33258 Ho-

3338—3342, 1979

interactions in cultured kidney tubules. J Lab Clin Med 103:

These experiments on MDCK cells suggest that cultured 255—271, 1984 tubular cells of the distal nephron have a specific response to 18. FAIRES iS, MCCARTY DJ: Acute arthritis in man and dog after intrasynovial injection of sodium urate crystals. Lancet 2:682—685, urate and uric acid crystals that is similar to that observed by 1962 kidney tubule cells in vivo. The reaction of MDCK cells, if 19. HARRIS RI, STONE PCW, STUART i: An improved chromogenic extended to kidney tubules, suggests that crystals can cause substrate endotoxin assay for clinical use. J Clin Pathol 36: epithelial damage directly, independently of any invasion of 1145—1147, 1983 leucocytes, and can permit the transfer of crystals across the 20. CESARONE CF, BOLOGNESI C, SANTI L: Improved microfluonechst. Anal Biochem 100:188-197, 1979 produce an inflammatory reaction. Some other facets of the 21. SPILBERG A, GALLACHER A, MENDELL B: Studies on crystalepithelial cell/crystal reaction yet to be explained include induced chemotactic factor II: Role of phagocytosis. J Lab Clin whether they produce other factors such as chemotactic factors Med 85:631—636, 1975

and cytokines involved in inflammation. Understanding the basic 22. BRUNK UT, ERIcssoN JLE: Cytochemical evidence for the leakage of acid phosphatase through ultrastructurally intact lysosomal pathological responses should assist in their ultimate prevention. Acknowledgments We are grateful for support for various aspects of this study from the National Health & Medical Research Council of Australia, the Mayne

Bequest Fund of the University of Queensland and the Australian Kidney Foundation.

membranes. Histochem J 4:479—491, 1972 23. LEIGHTON i, ZBYNEK B, ESTES LW, JUSTIL G: Secretory activity and oncogenicity of a cell line (MDCK) derived from canine kidney. Science 163:472—473, 1969 24. WYNGAARDEN JB, KELLEY WN: Gout and Hyperuricemia. New York, Grune & Stratton, 1976, pp. 133—175

25. EMMER5ON BT: Heredity in primary gout. Australas Ann Med 9:168—175, 1960

Reprint requests to Professor B.T. Emmerson, University of Queensland, Department of Medicine, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia 4102.

26. K0zIN F, MCCARTY Di: Protein adsorption to monosodium urate, calcium pyrophosphate dihydrate and silica crystals. Art hr Rheum

References

27. HA5SELBACHER F: Stimulation of synovial fibroblasts by calcium

1. EMMERSON BT, GORDON RB, JOHNSON LA: Urate kinetics in HGPRT deficiency: Their significance in the understanding of gout. QJMed45:49—61, 1976 2. BARRY KG, HUNTER RH, DAVIS TE, CROSBY WH: Acute uric acid nephropathy: AMA. Arch intern Med 111:453—459, 1963 3. EMMERsON BT, Row PG: An evaluation of the pathogenesis of the gouty kidney. Kidney mt 8:65—71, 1975 4. EMMERSON BT, THOMPSON L: The spectrum of hypoxanthineguanine phosphoribosyl-transferase deficiency. Q J Med 42:423— 440, 1973

5. TALBOTT JH, TERPLAN KL: The kidney in gout. Medicine 39: 405—462, 1960

6. SEEGMILLER JE, FRAZIER PD: Biochemical considerations of the renal damage of gout. Ann Rheum Dis 25:668—672, 1966 7. BLUEsTONE R, WAI5MAN J, KLINENBERG JR: The gouty kidney. SeminArthrRheum 7:97—113, 1977 8. FAREBROTHER DA, HATFIELD P, SIMMONDS HA, CAMERON JS,

JONES AS, CADENHEAD A: Experimental crystal nephropathy (one year study in the pig). Cliii Nephrol 4:243—250, 1975

19:433—438, 1976

oxalate and monosodium urate monohydrate. J Lab Clin Med 100:977—985, 1982

28. WIGLEY FM, FINE IT, NEWCOMBE DS: The role of the human synovial fibroblast in monosodium urate crystal induced synovitis. JRheumato! 10:602—611, 1983 29. KOzIN F, GINSBERG MH, SKOSEY iL: Polymorphonuclear leuko-

cyte responses to monosodium urate crystals: Modification by adsorbed serum proteins. J Rheumatol 6:519—526, 1979 30. KozIN F, MCCARTY Di: Molecular orientation of immunoglobulin G adsorbed to microcrystalline monosodium urate monohydrate. J Lab Clin Med 95:49—58, 1980 31. CHERIAN PV, SCHUMACHER HR: Immunochemical and ultrastructural characteristics of serum proteins associated with monosodium urate crystals (MSU) in synovial fluid cells from patients with gout. Ultrastruct Pathol 10:209—219, 1986 32. SHIRAHAMA T, COHEN AS: Ultrastructural evidence for leakage of lysosomal contents after phagocytosis of monosodium urate crystals. Am J Pathol 76:501—512, 1974 33. WOOLF AD, DIEPPE PA: Mediators of crystal-induced inflammation in the joint. Br Med Bull 43:429—444, 1987