, Volume 43, Supplement 1, pp 77–92 | Cite as

Biomimetic Randall’s plaque as an in vitro model system for studying the role of acidic biopolymers in idiopathic stone formation

  • Archana Chidambaram
  • Douglas Rodriguez
  • Saeed Khan
  • Laurie Gower
Invited Review


Randall’s plaque (RP) deposits seem to be consistent among the most common type of kidney stone formers, idiopathic calcium oxalate stone formers. This group forms calcium oxalate renal stones without any systemic symptoms, which contributes to the difficulty of understanding and treating this painful and recurring disease. Thus, the development of an in vitro model system to study idiopathic nephrolithiasis, beginning with RP pathogenesis, can help in identifying how plaques and subsequently stones form. One main theory of RP formation is that calcium phosphate deposits initially form in the basement membrane of the thin loops of Henle, which then fuse and spread into the interstitial tissue, and ultimately make their way across the urothelium, where upon exposure to the urine, the mineralized tissue serves as a nidus for overgrowth with calcium oxalate into a stone. Our group has found that many of the unusual morphologies found in RP and stones, such as concentrically laminated spherulites and mineralized collagenous tissue, can be reproduced in vitro using a polymer-induced liquid precursor (PILP) process, in which acidic polypeptides induce a liquid phase amorphous precursor to the mineral, yielding non-equilibrium crystal morphologies. Given that there are many acidic proteins and polysaccharides present in the renal tissue and urine, we have put forth the hypothesis that the PILP system may be involved in urolithiasis. Therefore, our goal is to develop an in vitro model system of these two stages of composite stone formation to study the role that various acidic macromolecules may play. In our initial experiments presented here, the development of “biomimetic” RP was investigated, which will then serve as a nidus for calcium oxalate overgrowth studies. To mimic the tissue environment, MatriStem® (ACell, Inc.), a decellularized porcine urinary bladder matrix was used, because it has both an intact epithelial basement membrane surface and a tunica propria layer, thus providing the two types of matrix constituents found associated with mineral in the early stages of RP formation. We found that when using the PILP process to mineralize this tissue matrix, the two sides led to dramatically different mineral textures, and they bore a striking resemblance to native RP, which was not seen in the tissue mineralized via the classical crystal nucleation and growth process. The interstitium side predominantly consisted of collagen-associated mineral, while the luminal side had much less mineral, which appeared to be tiny spherules embedded within the basement membrane. Although these studies are only preliminary, they support our hypothesis that kidney stones may involve non-classical crystallization pathways induced by the large variety of macromolecular species in the urinary environment. We believe that mineralization of native tissue scaffolds is useful for developing a model system of stone formation, with the ultimate goal of developing strategies to avoid RP and its detrimental consequences in stone formation, or developing therapeutic treatments to prevent or cure the disease. Supported by NIDDK grant RO1DK092311.


Randall’s plaque Nephrolithiasis Biomineralization mechanisms Acidic proteins Osteopontin Calcium phosphate PILP 



Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH) under Award Number R01DK092311. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors gratefully acknowledge Drs. Sharon W. Matthews and Dr. Jill W. Verlander of COM electron microscopy Core for assisting with TEM sectioning, as well as ACell, Inc. for kindly providing the Matristem® samples. We would also like to thank the Major Analytical Instrumentation Center for use of their SEM and TEM.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Randall A (1940) Papillary pathology as precursor of primary renal calculus. J Urol 44:580–589Google Scholar
  2. 2.
    Randall A (1937) The origin and growth of renal calculi. Ann Surg 105:1009–1027PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Coe F et al (2010) Three pathways for human kidney stone formation. Urol Res 38(3):147–160PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Evan A (2010) Physiopathology and etiology of stone formation in the kidney and the urinary tract. Pediatr Nephrol 25(5):831–841PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Al-Atar U et al (2010) Mechanism of calcium oxalate monohydrate kidney stones formation: layered spherulitic growth. Chem Mater 22(4):1318–1329CrossRefGoogle Scholar
  6. 6.
    Evan A et al (2006) Randall’s plaque: pathogenesis and role in calcium oxalate nephrolithiasis. Kidney Int 69(8):1313–1318PubMedGoogle Scholar
  7. 7.
    Evan AP (2007) Histopathology predicts the mechanism of stone formation. AIP Conf Proc 900(1):15–25CrossRefGoogle Scholar
  8. 8.
    Evan AP et al (2007) Mechanism of formation of human calcium oxalate renal stones on Randall’s plaque. Anat Rec: Adv Integr Anat Evol Biol 290(10):1315–1323CrossRefGoogle Scholar
  9. 9.
    Evan AP et al (2005) Apatite plaque particles in inner medulla of kidneys of calcium oxalate stone formers: osteopontin localization. Kidney Int 68(1):145–154PubMedCrossRefGoogle Scholar
  10. 10.
    Evan AP et al (2003) Randall’s plaque of patients with nephrolithiasis begins in basement membranes of thin loops of Henle. J Clin Investig 111(5):607–616PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Low RK, Stoller ML (1997) Endoscopic mapping of renal papillae for Randall’s plaques in patients with urinary stone disease. J Urol 158(6):2062–2064PubMedCrossRefGoogle Scholar
  12. 12.
    Bagga HS et al (2013) New insights into the pathogenesis of renal calculi. Urol Clin North Am 40(1):1–12PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Grases F et al (2013) Renal papillary calcification and the development of calcium oxalate monohydrate papillary renal calculi: a case series study. BMC Urol 13:14PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Khan SR et al (2012) Association of Randall plaque with collagen fibers and membrane vesicles. J Urol 187(3):1094–1100PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Matlaga BR et al (2006) Endoscopic evidence of calculus attachment to Randall’s plaque. J Urol 175(5):1720–1724PubMedCrossRefGoogle Scholar
  16. 16.
    Miller NL et al (2009) A formal test of the hypothesis that idiopathic calcium oxalate stones grow on Randall’s plaque. BJU Int 103(7):966–971PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Sepe V et al (2006) Henle loop basement membrane as initial site for Randall plaque formation. Am J Kidney Dis 48(5):706–711PubMedCrossRefGoogle Scholar
  18. 18.
    Sayer JA, Carr G, Simmons NL (2004) Nephrocalcinosis: molecular insights into calcium precipitation within the kidney. Clin Sci 106(6):549–561PubMedCrossRefGoogle Scholar
  19. 19.
    Vervaet BA et al (2009) Nephrocalcinosis: new insights into mechanisms and consequences. Nephrol Dial Transplant 24(7):2030–2035PubMedCrossRefGoogle Scholar
  20. 20.
    Ghadially FN (2001) As you like it, Part 3: a critique and historical review of calcification as seen with the electron microscope. Ultrastruct Pathol 25(3):243–267PubMedCrossRefGoogle Scholar
  21. 21.
    Stoller ML et al (1996) High resolution radiography of cadaveric kidneys: unraveling the mystery of Randall’s plaque formation. J Urol 156(4):1263–1266PubMedCrossRefGoogle Scholar
  22. 22.
    Stoller ML et al (2004) The primary stone event: a new hypothesis involving a vascular etiology. J Urol 171(5):1920–1924PubMedCrossRefGoogle Scholar
  23. 23.
    Khan SR (1997) Calcium phosphate/calcium oxalate crystal association in urinary stones: implications for heterogeneous nucleation of calcium oxalate. J Urol 157(1):376–383PubMedCrossRefGoogle Scholar
  24. 24.
    Tiselius HG (2011) A hypothesis of calcium stone formation: an interpretation of stone research during the past decades. Urol Res 39(4):231–243PubMedCrossRefGoogle Scholar
  25. 25.
    Bazin D, Daudon M (2012) Pathological calcifications and selected examples at the medicine-solid-state physics interface. J Phys D Appl Phys 45(38):383001CrossRefGoogle Scholar
  26. 26.
    Amos F et al (2009) Mechanism of formation of concentrically laminated spherules: implication to Randall’s plaque and stone formation. Urol Res 37(1):11–17PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Khan S (2006) Renal tubular damage/dysfunction: key to the formation of kidney stones. Urol Res 34(2):86–91PubMedCrossRefGoogle Scholar
  28. 28.
    Olszta MJ et al (2007) Bone structure and formation: a new perspective. Mater Sci Eng R Rep 58(3–5):77–116CrossRefGoogle Scholar
  29. 29.
    Ohman S, Larsson L (1992) Evidence for Randall’s plaques to be the origin of primary renal stones. Med Hypotheses 39(4):360–363PubMedCrossRefGoogle Scholar
  30. 30.
    Khan SR, Finlayson B, Hackett R (1984) Renal papillary changes in patient with calcium oxalate lithiasis. Urology 23(2):194–199PubMedCrossRefGoogle Scholar
  31. 31.
    Tiselius HG et al (2009) Studies on the role of calcium phosphate in the process of calcium oxalate crystal formation. Urol Res 37(4):181–192PubMedCrossRefGoogle Scholar
  32. 32.
    Khan SR, Canales BK (2011) Ultrastructural investigation of crystal deposits in Npt2a knockout mice: are they similar to human Randall’s plaques? J Urol 186(3):1107–1113PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Nancollas G, Henneman Z (2010) Calcium oxalate: calcium phosphate transformations. Urol Res 38(4):277–280PubMedCrossRefGoogle Scholar
  34. 34.
    Hug S et al (2012) Mechanism of inhibition of calcium oxalate crystal growth by an osteopontin phosphopeptide. Soft Matter 8(4):1226–1233CrossRefGoogle Scholar
  35. 35.
    Saw NK, Rao PN, Kavanagh JP (2008) A nidus, crystalluria and aggregation: key ingredients for stone enlargement. Urol Res 36(1):11–15PubMedCrossRefGoogle Scholar
  36. 36.
    Thurgood LA et al (2010) Comparison of the specific incorporation of intracrystalline proteins into urinary calcium oxalate monohydrate and dihydrate crystals. J Proteome Res 9(9):4745–4757PubMedCrossRefGoogle Scholar
  37. 37.
    Achilles W (1997) In vitro crystallisation systems for the study of urinary stone formation. World J Urol 15(4):244–251PubMedCrossRefGoogle Scholar
  38. 38.
    Christmas KG et al (2002) Aggregation and dispersion characteristics of calcium oxalate monohydrate: effect of urinary species. J Colloid Interface Sci 256(1):168–174PubMedCrossRefGoogle Scholar
  39. 39.
    Hirose M et al (2012) Role of osteopontin in early phase of renal crystal formation: immunohistochemical and microstructural comparisons with osteopontin knock-out mice. Urol Res 40(2):121–129PubMedCrossRefGoogle Scholar
  40. 40.
    Kolbach AM et al (2012) Relative deficiency of acidic isoforms of osteopontin from stone former urine. Urol Res 40(5):447–454PubMedCrossRefGoogle Scholar
  41. 41.
    Okada A et al (2008) Morphological conversion of calcium oxalate crystals into stones is regulated by osteopontin in mouse kidney. J Bone Miner Res 23(10):1629–1637PubMedCrossRefGoogle Scholar
  42. 42.
    Okada A et al (2010) Renal macrophage migration and crystal phagocytosis via inflammatory-related gene expression during kidney stone formation and elimination in mice: detection by association analysis of stone-related gene expression and microstructural observation. J Bone Miner Res 25(12):2701–2711PubMedCrossRefGoogle Scholar
  43. 43.
    Lan M et al (2007) Renal calcinosis and stone formation in mice lacking osteopontin, Tamm-Horsfall protein, or both. Am J Physiol Renal Physiol 293(6):F1935–F1943CrossRefGoogle Scholar
  44. 44.
    Liu Y et al (2010) Progressive renal papillary calcification and ureteral stone formation in mice deficient for Tamm-Horsfall protein. Am J Physiol Renal Physiol 299(3):F469–F478PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Grohe B et al (2009) Crystallization of calcium oxalates is controlled by molecular hydrophilicity and specific polyanion-crystal interactions. Langmuir 25(19):11635–11646PubMedCrossRefGoogle Scholar
  46. 46.
    Kleinman JG et al (1995) Expression of osteopontin, a urinary inhibitor of stone mineral crystal growth, in rat kidney. Kidney Int 47(6):1585–1596PubMedCrossRefGoogle Scholar
  47. 47.
    Khan SR, Kok DJ (2004) Modulators of urinary stone formation. Front Biosci 9:1450–1482PubMedCrossRefGoogle Scholar
  48. 48.
    Kim IW Biomimetic and bioinspired crystallization with macromolecular additivesGoogle Scholar
  49. 49.
    Marangella M et al (1985) Urine saturation with calcium salts in normal subjects and idiopathic calcium stone-formers estimated by an improved computer model system. Urol Res 13(4):189–193PubMedCrossRefGoogle Scholar
  50. 50.
    Wesson JA et al (2003) Osteopontin is a critical inhibitor of calcium oxalate crystal formation and retention in renal tubules. J Am Soc Nephrol 14(1):139–147PubMedCrossRefGoogle Scholar
  51. 51.
    Xie A-J et al (2009) Formation of calcium oxalate concentric precipitate rings in two-dimensional agar gel systems containing Ca2+ –RE3+(RE=Er, Gd and La)–C2O4 2−. Colloids Surfaces A: Physicochem Eng Aspects 332(2):192–199CrossRefGoogle Scholar
  52. 52.
    Khan SR, Finlayson B, Hackett RL (1982) Experimental calcium oxalate nephrolithiasis in the rat. Role of the renal papilla. Am J Pathol 107(1):59PubMedCentralPubMedGoogle Scholar
  53. 53.
    Gnessin E, Lingeman JE, Evan AP (2010) Pathogenesis of renal calculi. Turkish J Urol 36(2):190–199CrossRefGoogle Scholar
  54. 54.
    Khan SR (2012) Reactive oxygen species as the molecular modulators of calcium oxalate kidney stone formation: evidence from clinical and experimental investigations. J Urol 189(3):803–811PubMedCrossRefGoogle Scholar
  55. 55.
    Gower LB, Amos FF, Khan SR (2010) Mineralogical signatures of stone formation mechanisms. Urol Res 38(4):281–292PubMedCrossRefGoogle Scholar
  56. 56.
    Grover PK, Kim DS, Ryall RL (2002) The effect of seed crystals of hydroxyapatite and brushite on the crystallization of calcium oxalate in undiluted human urine in vitro: implications for urinary stone pathogenesis. Mol Med 8(4):200–209PubMedCentralPubMedGoogle Scholar
  57. 57.
    Amos FF et al. (2007) Relevance of a polymer-induced liquid-precursor (PILP) mineralization process to normal and pathological biomineralization. In: biomineralization—medical aspects of solubility. Wiley, New York. pp. 125–217Google Scholar
  58. 58.
    Olszta MJ, Douglas EP, Gower LB (2003) Scanning electron microscopic analysis of the mineralization of type I collagen via a polymer-induced liquid-precursor (PILP) process. Calcif Tissue Int 72(5):583–591PubMedCrossRefGoogle Scholar
  59. 59.
    Gower LB, Odom DJ (2000) Deposition of calcium carbonate films by a polymer-induced liquid-precursor (PILP) process. J Cryst Growth 210(4):719–734CrossRefGoogle Scholar
  60. 60.
    Jee S–S, Thula TT, Gower LB (2010) Development of bone-like composites via the polymer-induced liquid-precursor (PILP) process. Part 1: influence of polymer molecular weight. Acta Biomater 6(9):3676–3686PubMedCrossRefGoogle Scholar
  61. 61.
    Gower LB (2008) Biomimetic model systems for investigating the amorphous precursor pathway and its role in biomineralization. Chem Rev 108(11):4551–4627PubMedCrossRefGoogle Scholar
  62. 62.
    Ryall R (2008) The future of stone research: rummagings in the attic, Randall’s plaque, nanobacteria, and lessons from phylogeny. Urol Res 36(2):77–97PubMedCrossRefGoogle Scholar
  63. 63.
    Golub E (2011) Biomineralization and matrix vesicles in biology and pathology. Semin Immunopathol 33(5):409–417PubMedCentralPubMedCrossRefGoogle Scholar
  64. 64.
    Nudelman F et al (2010) The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nat Mater 9(12):1004–1009PubMedCentralPubMedCrossRefGoogle Scholar
  65. 65.
    Bradt J-H et al (1999) Biomimetic mineralization of collagen by combined fibril assembly and calcium phosphate formation. Chem Mater 11(10):2694–2701CrossRefGoogle Scholar
  66. 66.
    Rodriguez DE et al (2014) Multifunctional role of osteopontin in directing intrafibrillar mineralization of collagen and activation of osteoclasts. Acta Biomater 10(1):494–507PubMedCrossRefGoogle Scholar
  67. 67.
    Thula TT et al (2010) Mimicking the nanostructure of bone: comparison of polymeric process-directing agents. Polymers 3(1):10–35CrossRefGoogle Scholar
  68. 68.
    Kim YK et al (2010) Mineralisation of reconstituted collagen using polyvinylphosphonic acid/polyacrylic acid templating matrix protein analogues in the presence of calcium, phosphate and hydroxyl ions. Biomaterials 31(25):6618–6627PubMedCentralPubMedCrossRefGoogle Scholar
  69. 69.
    Baumann JM, Affolter B, Casella R (2011) Aggregation of freshly precipitated calcium oxalate crystals in urine of calcium stone patients and controls. Urol Res 39(6):421–427PubMedCrossRefGoogle Scholar
  70. 70.
    Silverman L, Boskey AL (2004) Diffusion systems for evaluation of biomineralization. Calcif Tissue Int 75:494–501PubMedCentralPubMedCrossRefGoogle Scholar
  71. 71.
    Viswanathan P et al (2011) Calcium oxalate monohydrate aggregation induced by aggregation of desialylated Tamm-Horsfall protein. Urol Res 39(4):269–282PubMedCentralPubMedCrossRefGoogle Scholar
  72. 72.
    Gericke A et al (2005) Importance of phosphorylation for osteopontin regulation of biomineralization. Calcif Tissue Int 77(1):45–54PubMedCentralPubMedCrossRefGoogle Scholar
  73. 73.
    Hunter GK et al (1985) Inhibition of hydroxyapatite formation in collagen gels by chondroitin sulphate. Biochem J 228(2):463–469PubMedCentralPubMedGoogle Scholar
  74. 74.
    Hunter GK, Goldberg HA (1993) Nucleation of hydroxyapatite by bone sialoprotein. Proc Natl Acad Sci 90(18):8562–8565PubMedCentralPubMedCrossRefGoogle Scholar
  75. 75.
    Boskey AL et al (2012) Post-translational modification of osteopontin: effects on in vitro hydroxyapatite formation and growth. Biochem Biophys Res Commun 419(2):333–338PubMedCentralPubMedCrossRefGoogle Scholar
  76. 76.
    Badylak SF, Freytes DO, Gilbert TW (2009) Extracellular matrix as a biological scaffold material: structure and function. Acta Biomater 5(1):1–13PubMedCrossRefGoogle Scholar
  77. 77.
    Freytes DO et al (2008) Hydrated versus lyophilized forms of porcine extracellular matrix derived from the urinary bladder. J Biomed Mater Res A 87A(4):862–872CrossRefGoogle Scholar
  78. 78.
    Azuma N et al (2006) A rapid method for purifying osteopontin from bovine milk and interaction between osteopontin and other milk proteins. Int Dairy J 16(4):370–378CrossRefGoogle Scholar
  79. 79.
    Sørensen E, Petersen T (1993) Purification and characterization of three proteins isolated from the proteose peptone fraction of bovine milk. J Dairy Res 60:189–197PubMedCrossRefGoogle Scholar
  80. 80.
    Thula TT et al (2011) In vitro mineralization of dense collagen substrates: a biomimetic approach toward the development of bone-graft materials. Acta Biomater 7(8):3158–3169PubMedCentralPubMedCrossRefGoogle Scholar
  81. 81.
    Bewernitz MA et al (2012) A metastable liquid precursor phase of calcium carbonate and its interactions with polyaspartate. Faraday Discuss 159:291–312CrossRefGoogle Scholar
  82. 82.
    Kwak S-Y et al (2009) Role of 20-kDa amelogenin (P148) phosphorylation in calcium phosphate formation in vitro. J Biol Chem 284(28):18972–18979PubMedCentralPubMedCrossRefGoogle Scholar
  83. 83.
    Deshpande AS et al (2011) Primary structure and phosphorylation of Dentin Matrix Protein 1 (DMP1) and Dentin Phosphophoryn (DPP) uniquely determine their role in biomineralization. Biomacromolecules 12(8):2933–2945PubMedCentralPubMedCrossRefGoogle Scholar
  84. 84.
    LeBleu VS, MacDonald B, Kalluri R (2007) Structure and function of basement membranes. Exp Biol Med 232(9):1121–1129CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Archana Chidambaram
    • 1
  • Douglas Rodriguez
    • 1
  • Saeed Khan
    • 2
  • Laurie Gower
    • 1
  1. 1.Department of Materials Science and EngineeringUniversity of FloridaGainesvilleUSA
  2. 2.Department of Pathology, College of MedicineUniversity of FloridaGainesvilleUSA

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