Review Article

Reactive Oxygen Species as the Molecular Modulators of Calcium

Oxalate Kidney Stone Formation: Evidence from Clinical

and Experimental Investigations

Saeed R. Khan*From the Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida

Purpose: Idiopathic calcium oxalate kidney stones form while attached to Ran-dall plaques, the subepithelial deposits on renal papillary surfaces. Plaque for-mation and growth mechanisms are poorly understood. Plaque formation else-where in the body is triggered by reactive oxygen species and oxidative stress.This review explores possible reactive oxygen species involvement in plaqueformation and calcium oxalate nephrolithiasis.Materials and Methods: A search of various databases for the last 8 yearsidentified literature on reactive oxygen species involvement in calcium oxalatenephrolithiasis. The literature was reviewed and results are discussed.Results: Under normal conditions reactive oxygen species production is con-trolled, increasing as needed and regulating crystallization modulator produc-tion. Reactive oxygen species overproduction or decreased antioxidants lead tooxidative stress, inflammation and injury, and are involved in stone comorbidity.All major chronic inflammation markers are detectable in stone patient urine.Patients also have increased urinary excretion of the I�I and the thrombinprotein families. Results of a recent study of 17,695 participants in NHANES III(National Health and Nutrition Examination Survey) showed significantly lowerantioxidants, carotene and �-cryptoxanthin in those with a kidney stone history.Animal model and tissue culture studies revealed that high oxalate, calciumoxalate and calcium phosphate crystals provoked renal cell reactive oxygenspecies mediated inflammatory responses. Calcium oxalate crystals induce reninup-regulation and angiotensin II generation. Nonphagocytic NADPH oxidaseleads to reactive oxygen species production mediated by protein kinase C. TheP-38 MAPK/JNK transduction pathway is turned on. Transcriptional and growthfactors, and generated secondary mediators become involved. Chemoattractantand osteopontin production is increased and macrophages infiltrate the renalinterstitium around the crystal. Phagocytic NADPH oxidase is probably acti-vated, producing additional reactive oxygen species. Localized inflammation,extracellular matrix and fibrosis develop. Crystallization modulators have asignificant role in inflammation and tissue repair.Conclusions: Based on available data, Randall plaque formation is similar to ex-tracellular matrix mineralization at many body sites. Renal interstitial collagenbecomes mineralized, assisting plaque growth through the interstitium until themineralizing front reaches papillary surface epithelium. Plaque exposure to pelvicurine may also be a result of reactive oxygen species triggered epithelial sloughing.

Key Words: kidney; nephrolithiasis; calcium oxalate; reactive oxygen species;calcification, physiologic

Abbreviation

and Acronyms

AP-1 � activator protein 1

CaP � biological calciumphosphate

I�I � inter-�-inhibitor

LDH � lactate dehydrogenase

MCP-1 � monocytechemoattractant protein-1

NADPH � nicotinamide adeninedinucleotide phosphate

NAG � N-acetyl-�-glucosaminidase

NF-�B � nuclear factor �-light-chain-enhancer of activated Bcells

OPN � osteopontin

OS � oxidative stress

Ox � oxalate

phox � phagocytic oxidase

PKC � protein kinase

ROS � reactive oxygen species

RP � Randall plaque

THP � Tamm-Horsfall protein

Supported by National Institutes of HealthGrant RO1-DK078602 and the University of Flor-ida Center for the Study of Lithiasis.

* Correspondence: Department of Pathology,Immunology and Laboratory Medicine, Universityof Florida College of Medicine, Box 100275,Gainesville, Florida 32610 (telephone: 352-392-3574; e-mail: khan@pathology.ufl.edu).

0022-5347/13/1893-0803/0 http://dx.doi.org/10.1016/j.juro.2012.05.078THE JOURNAL OF UROLOGY® Vol. 189, 803-811, March 2013© 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC. Printed in U.S.A.

www.jurology.com 803

REACTIVE OXYGEN SPECIES AS MODULATORS OF KIDNEY STONE FORMATION804

IT is estimated that symptomatic stone disease de-velops in 5% to 15% of the American population byage 70 years. The worldwide prevalence of urolithi-asis in men by age 70 years varies from approxi-mately 4% in England to 20% in Saudi Arabia. Dataindicate that between 1976 to 1980 and 1988 to 1994the incidence of kidney stones increased by 37% ineach gender in the United States. Geographically,kidney stones are more common in places with highhumidity and temperature. With the expected ele-vated temperature worldwide an increase of 1.6 to2.2 million lifetime cases of kidney stones by 2050has been predicted, particularly in the southeasternregions of the United States.1

Urolithiasis is a chronic disease with a 60%chance of recurrence within 10 years. Reports indi-cate that the incidence in children has increased.Unfortunately, they have more difficulty passingcalculi and often require invasive surgery. In addi-tion, the increased CaP, particularly brushite in re-current stones, leads to increased stone hardness,making them more difficult to comminute by shockwave lithotripsy. A cycle of tissue damage and re-current stone formation is thought to be the result oflithotripsy.2

Despite increased understanding of crystalliza-tion and stone formation,3 and effective currenttherapies advances in medical management havebeen modest because of the slow progress in deter-mining stone formation pathophysiology. CaOx stoneformation is associated with a number of metabolicdisorders that cause urine to become sufficientlysupersaturated for CaOx/CaP crystals to form. Re-nal epithelial cells become exposed to slightly higherlevels of Ox, CaP and/or CaOx crystals during stoneformation. Based on experimental investigations,we hypothesized that stone formation depends onthe type of renal cellular response to the changingurinary environment with significant ROS involve-ment.4 This article provides a brief overview of ex-perimental and clinical data supporting the role ofROS and inflammation in CaOx stone formation.

REACTIVE OXYGEN SPECIES

Free radicals, atoms or molecules with unpairedelectrons and their metabolites are collectivelycalled ROS. Under normal conditions ROS are gen-erated by tightly controlled enzymes. They serve asmediators in various regulatory processes and sig-naling pathways, including the proliferation, activa-tion or inactivation of regulatory biomolecules andregulation of transcriptional activity. Major cellularROS include superoxide anion (O2

–●), nitric oxideradical (NO●), hydroxyl radical (OH●) and hydrogenperoxide (H2O2), which are generated by several

pathways. O2

–● anions are produced by NADPH ox-

idase, xanthine oxidase, lipooxygenase, cyclooxygen-ase and heme oxygenase, and they are a byproductof the mitochondrial respiratory chain.4 Lipid radi-cals can also produce O2

–●. NO● radicals are pro-duced by endothelial nitric oxide synthase mediatedoxidation of L-arginine. Endothelial nitric oxide syn-thase can also produce O2

–●. The reaction betweensuperoxide and nitric oxide can produce the highlyreactive peroxynitrite (ONOO–). Signaling mole-cules regulated by ROS include but are not limitedto protein tyrosine kinases and phosphatases, ser-ine/theorine kinases and phosphatases, RAS, vari-ous phospholipases and many calcium signals. ROSalso regulate such genes as c-fos, c-myc and c-jun,and transcription factor AP-1 and NF-�B. In addi-tion, ROS also participate in the initiation and im-plementation of apoptosis.

Since ROS have many significant regulatoryroles, they normally occur at steady state levels, aregenerated as needed, and are then cleared by theactivity of various antioxidants and scavengers.They are short lived with tightly controlled actionsand localized to specific sites where needed. They donot travel too far because of their potential to doharm.

ROS can also produce chemical modifications ofand damage to proteins, lipids, carbohydrates andnucleotides, and they modulate renal and cardiovas-cular systems through redox dependent signalingpathways. Uncontrolled generation of reactive oxy-gen and/or a decrease in endogenous antioxidantcapacity creates OS, which may lead to inflamma-tion and injury.5 Most cells respond to OS by boost-ing levels of intracellular antioxidants, such as glu-tathione. Pathological changes may result from thedamaging effects of ROS and from ROS mediatedchanges in gene expression and signal transduction.Because most ROS are short lived and do not travela long distance, the presence of OS is generallyrecognized by the abundance of byproducts of ROSinteraction with cell constituents. Malondialdehyde,isoprostanes and oxidized lipids are among the mostcommon byproducts of ROS induced OS.

CLINICAL DATA

Analysis of stone patient urine suggests that ROSinduced renal cellular injury and inflammation arelikely involved in the pathogenesis of idiopathicstone disease. Higher than normal levels of renalenzymes, �-glutamyl transpeptidase, angiotensin 1converting enzyme, �-galactosidase and NAG, indic-ative of tubular injury, were found in the urine ofidiopathic CaOx stone formers.6 Results of recentstudies indicate that urine from CaOx stone patientshad significantly increased thiobarbituric acid-reac-

tive substances, in addition to renal enzymes, indi-

REACTIVE OXYGEN SPECIES AS MODULATORS OF KIDNEY STONE FORMATION 805

cating that renal injury was most likely caused byROS production. Urinary 8-hydroxydeoxyguanos-ine, a marker of DNA oxidative damage, was in-creased in stone patients and correlated positivelywith tubular damage, as assessed by urinary NAGexcretion.7 Recently, a group reported urinary excre-tion of the anti-inflammatory proteins calgranulin,�-defensin and myeloperoxidase8 by stone patientsand the presence of these proteins in the inner coreof CaOx stones, indicating a putative role in neph-rolithiasis.

Other studies of recurrent idiopathic calciumstone formers suggest an association between stoneformation and decreased antioxidant levels.9 Inves-tigation revealed that antioxidant deficit is commonand unrelated to the presence or absence of stones.The investigators concluded that stone formationstarted with oxidatively damaged cells. In anotherrecent study serum levels of antioxidants were ex-amined in adult participants of 1988 to 1994NHANES III.10 Mean levels of antioxidants, �-car-otene, �-carotene and �-cryptoxanthin were signifi-cantly lower in participants with a self-reported his-tory of kidney stones, while those with higher serum�-carotene and �-cryptoxanthin showed a tendencytoward a decreasing prevalence of kidney stones.

Stones, such as cystine, brushite and CaOx cal-culi in primary hyperoxaluria, calculi that developafter bariatric surgery and CaP stones in those withprimary hyperparathyroidism, are attached to tubu-lar crystal deposits in the ducts of Bellini.3 Crystaldeposition is associated with renal cell injury, cellloss, inflammation and fibrosis.3,11 Most idiopathicstones form while connected to RPs, which start asdeposits of poorly crystalline biological apatite inthe basement membrane of the loops of Henle,3,12

collecting ducts13 or vasa recta.14 Although Ran-dall considered plaque a product of a natural re-pair process for some form of tubule damage,15

recent reports indicate absent tissue injury andinflammation in association with RP in idiopathicstone formers.3,12 Collagen is a part of interstitialCaP deposits and it is mineralized during plaqueformation (fig. 1).16

OS is also a common feature of many stonecomorbidities, such as hypertension, diabetes mel-litus, atherosclerosis and myocardial infarction.17

Interestingly, adherence to a DASH (Dietary Ap-proaches to Stop Hypertension) diet, which reducesthe risk of stroke and cardiovascular disease, alsoreduces the risk of stone formation up to 45%.18 TheDASH diet decreases blood pressure by increasingantioxidant capacity and lowering OS. Anotherstudy of 57,326 hyperlipidemic active duty and re-tired members of the United States Armed Forcesand their dependents showed a 50% decreased risk

of stone formation in patients who used statins com-

pared to those who did not. Statins not only lowercholesterol but also decrease the expression and ac-tivity of NADPH oxidase and inhibit ROS produc-tion.

TISSUE CULTURE

Tissue culture studies, in which renal epithelial orother cells are exposed to Ox and/or CaOx or CaPcrystals, have provided new insights into the patho-genesis of nephrolithiasis. The cell response is timeand concentration dependent, and cell specific. Crys-tals bind rapidly to the surface of epithelial cells andare internalized. Finger-like cell surface projectionsor microvilli appear to probe the crystals, followedby crystal endocytosis into the cells (fig. 2).

The response of renal epithelial cells to CaOxmonohydrate crystals is characterized by increasedexpression of specific encoding genes, transcrip-tional activators, extracellular matrix regulators,growth factors, and production of proinflammatoryand anti-inflammatory molecules, such as OPN,MCP-1, prostaglandin E2, bikunin and componentsof I�I, �-1 microglobulin, CD-44, calgranulin, hepa-ran sulfate, osteonectin, fibronectin and matrix glaprotein.11,19,20 Although many of these moleculesare integral to inflammation and fibrosis, they alsomodulate biomineralization (see Appendix).21

Renal cells exposed to CaOx crystals secrete su-peroxide in real time, as measured by an electro-chemical superoxide biosensor.22 Mitochondria4,23

and NADPH oxidase11,19,24 are involved in ROS pro-duction. Ox or crystal induced cellular responses canbe inhibited by antioxidants as well as by NADPHoxidase inhibitors. Cellular injury could be amelio-rated by antioxidants and free radical scaven-gers.4,19,25 Catalase decreased LDH release, and hy-

Figure 1. Transmission electron microscopy. A, spherulitic CaPdeposits in kidneys of idiopathic stone former in interstitiumand around necrosing tubules (arrows). Scale bar indicates 10�m. B, higher magnification reveals CaP (arrowhead) and colla-gen fibers (arrows). Bar indicates 1 �m.

drogen peroxide and 8-isoprostane production by

REACTIVE OXYGEN SPECIES AS MODULATORS OF KIDNEY STONE FORMATION806

LLC-PK1 and MDCK cells exposed to brushite crys-tals. Similarly, free radical scavengers, catalase andsuperoxide dismutase provided protection from Oxinduced injury to the 2 cell types and reducedMCP-1 mRNA as well as protein in Ox, CaOx, CaPand uric acid treated NRK52E renal epithelial cells.The NADPH oxidase inhibitor diphenyleneiodiumalso decreased Ox, CaOx, brushite and uric acidinduced up-regulation of OPN and MCP-1 in renalepithelial cells.19 MCP-1 mRNA expression and pro-tein production were increased after exposure of thehuman renal tubular cell line HK-2 to Ox or CaOxcrystals.26 Pretreatment with free radical scaven-gers, catalase and superoxide dismutase signifi-cantly decreased the expression and production ofMCP-1.

NADPH oxidase is a major source of ROS in thekidneys, particularly in the presence of angiotensinII. Exposure of the human renal epithelial derivedcell line HK-2 to Ox and CaOx crystals resulted in

Figure 2. Scanning electron microscopy shows NRK52E cells onexposure to 133 �g/cm2 hydroxyapatite. A, normal cells. B,higher magnification shows surface covered with short mi-crovilli. C, crystals are evenly dispersed on cell surface. D, at 3hours of treatment crystals (x) appear to be in contact with thinprojections from cell surface. E, cell appears to take in crystals.Arrows indicate cell leading edge. F, epithelial cell appears tohave phagocytosed crystals. Arrows indicate cell leading edge.

increased LDH release, 8-isoprotane production, in-

creased NADPH oxidase activity and superoxideproduction.24 Exposure to CaOx crystals resulted insignificantly higher activity than Ox exposure alone.Exposure to Ox and CaOx crystals affected the ex-pression of various NADPH oxidase subunits withconsistent increases in the expression of membranebound p22phox and cytosolic p47phox. Strong, signifi-cant correlations were seen between NADPH oxi-dase activity, and p22phox and p47phox expression,superoxide production and LDH release when cellswere exposed to CaOx crystals. Expression of nei-ther p22phox nor p47phox correlated significantly withincreased NADPH oxidase activity after Ox expo-sure. Ox induced injury is mediated by Rac-1, andPKC-� and � dependent activation of NADPH oxi-dase.27,28

Mitochondria are generally the most commonsource of superoxide and H2O2 in most cells andtissues. Selective probes, substrates and inhibitorsshow that mitochondria are a major site of CaOxcrystal induced superoxide production and glutathi-one depletion in LLC-PK1 and MDCK cells. Expo-sure of LLC-PK1 cells to Ox significantly increasedcellular ceramides, which could be blocked by N-ace-tylcysteine pretreatment. Furthermore, Ox expo-sure can decrease mitochondrial membrane potential inMDCK cells. Isolated mitochondria responded to Oxexposure by the accumulation of ROS, lipid perox-ides and oxidized thiol proteins. Administration ofexogenous citrate to LLC-PK1 and MDCK cellsbolstered antioxidant defenses and decreased cel-lular injury caused by exposure to increased Oxand CaOx crystals.29 Citrate in culture mediumwas associated with a significant increase in glu-tathione, and decreased production of H2O2 and8-isoprostane, an end product of lipid breakdown.Cell viability significantly improved, as demon-strated by decreased LDH release and increasedtrypan blue exclusion.

ANIMAL MODEL

A number of animal models have been developed toinvestigate the pathogenesis of different types ofkidney stones.30 CaOx stone disease is establishedby inducing hyperoxaluria by administering Ox orprecursors such as glyoxylate, ethylene glycol andhydroxyl-L-proline, which lead to intratubular CaOxdeposition (fig. 3). Experimental induction of hyper-calciuria by dietary or genetic manipulation leads toCaP crystal deposition in the tubular lumina and/orpelvic space. Thus, neither hyperoxaluria nor hyper-calciuria produces Ca deposits similar to idiopathicstones. However, animal model studies provide in-formation on the renal cellular response to hyper-calciuria, hyperoxaluria, and CaOx and CaP crys-

tals. Hyperoxaluria and CaOx crystal deposition

REACTIVE OXYGEN SPECIES AS MODULATORS OF KIDNEY STONE FORMATION 807

triggers various morphological and pathophysiolog-ical changes in the kidneys and alters urinary com-position.30,31 These changes indicate an inflamma-tory response by renal cells to high Ox and CaOxcrystals. Renal expression of OPN,20 I�I, �-1 micro-globulin,32 calgranulin, heparan sulfate and matrixgla protein33 is increased (see Appendix and fig. 4).The expression of NF-�B, kidney injury molecule,proliferating cell nuclear antigen, CD44 and e-cad-herin was also increased in the renal tubules ofhyperoxaluric rats (fig. 4), particularly in associa-tion with CaOx crystal deposits.31 Urinary excretionof many of these molecules is increased.11 As indi-cated by ED4 positive cells, monocytes and macro-phages migrate to crystal deposition sites (fig. 4).

Animal model studies also provide evidence thatrenal responses to Ox and crystals are mediated byROS. Lipid peroxides increased in renal tissue andurine in rats with hyperoxaluria and CaOx nephro-lithiasis.34 Vitamin E treatment improved tissuelevels of antioxidant enzymes, decreased peroxida-tive tissue injury and totally eliminated CaOx crys-tal deposition in the kidneys. CaOx crystal deposi-tion in the kidneys was associated with a reductionin total renal cellular glutathione and an increase in

Figure 3. Light microscopy. A, rat kidney with significant CaOxcrystal deposition as seen under polarized light. H & E, reducedfrom �1.2. B, in kidney section crystals appear as dark deposits.Pizzalato stain, reduced from �10. C, kidney of hyperoxaluric rattreated with apocynin demonstrates highly significant decreasein CaOx crystal deposition. Reduced from �10.

lipid peroxides. Rats that received the angiotensin

converting enzyme inhibitor losartan, which is knownto decrease OS, showed a significant increase inglutathione and a decrease in thiobarbituric acid-reactive substances in the kidneys. Catalase and Mnsuperoxide dismutase activities increased in the kid-neys, while � and �-glutathione-S-transferase in-creased in the urine of hyperoxaluric rats.35

ROS are at least in part produced with the in-volvement of NADPH oxidase through activation ofthe renin-angiotensin system. Decreasing angioten-sin production by inhibiting angiotensin convertingenzyme as well as blocking angiotensin receptor re-

Figure 4. Immunohistochemical staining of kidneys of hyperoxaluricrats. A, ED-1 positive cells were present in renal interstitium next tobirefringent CaOx crystal containing tubules, indicating infiltration ofmonocytes and macrophages (inflammatory response). Reducedfrom �20. B, tubular epithelial cells stained positive for CD-44,which is receptor for hyaluronic acid and other ligands, such asOPN and collagens. Reduced from �20. C, renal papillary sur-face epithelium in renal fornix, particularly outer surface, showsheavy staining for OPN, modulator of biomineralization andinflammation. Reduced from �40. D, tubular epithelial cellsdemonstrate positive staining for proliferating cell nuclear anti-gen, indicating cellular regeneration and proliferation. Reducedfrom �20. E, increased staining for OPN in renal cortical tubules.Reduced from �20. F, epithelial cells lining cortical renal tubulesstained positive for E-cadherin, which has important role in celladhesion. Reduced from �20. G, tubular epithelial cells showedincreased staining for NF-�B, mediator of inflammation andinjury. H, crystal deposition was associated with increased ex-pression of kidney injury molecule in tubular cells and their

luminal surfaces. Reduced from �20.

REACTIVE OXYGEN SPECIES AS MODULATORS OF KIDNEY STONE FORMATION808

duces crystal deposition and ameliorates the associ-ated inflammatory response. CaOx crystal deposi-tion in rat kidneys activated the renin-angiotensinsystem, increased renin expression in the kidneysand serum, and blocked angiotensin receptor de-creased OPN expression and CaOx crystal deposi-tion in hyperoxaluric rats.20

NADPH oxidase inhibition by apocynin treatmentreduced ROS production and CaOx crystal renaldeposition in hyperoxaluric rats.31 Atorvastatin,which decreases the expression of Nox-1 and p22phox

subunits of NADPH oxidase, also inhibited crystaldeposition in rats with experimentally induced hy-peroxaluria.36

DISCUSSION

Experimental studies suggest that exposure to highOx, and/or CaOx or CaP crystals results in increasedgene expression and production of molecules in-volved in tissue remodeling, inflammation andbiomineralization (fig. 5). Stone formation is influ-enced by a number of crystallization modulators/inhibitors, which affect crystal nucleation, growth,aggregation and retention, and can be modified byROS. The loss of inhibitory activity of THP in pa-tients with hyperoxaluria was suggested to be me-diated primarily by oxidative damage.37 Almost allmacromolecules regarded as crystallization inhibi-tors/modulators are known participants in inflam-matory processes and are generated through redoxdependent signaling pathways (see Appendix).

The up-regulated macromolecules have signifi-cant roles in the inflammatory process. Heparansulfate regulates extracellular matrix production.Bikunin, a constituent of I�I, is a proteinase inhib-itor associated with inflammation that stabilizesthe extracellular matrix. Acute inflammatory con-ditions are known to up-regulate or down-regulatetranscription of I�I genes. THP, seen in the renalinterstitium in several forms of tubulointerstitial dis-ease, produces interstitial inflammation and scarring.Interestingly, inactivating the THP gene in mouseembryonic stem cells resulted in spontaneous forma-tion of CaOx crystals in adult kidneys.38 THP canactivate alternate pathways, interact with neutro-phils and bind to certain cytokines. Prothrombin isthe precursor of thrombin, and fragments 1 and 2.Thrombin is involved in platelet aggregation andblood coagulation, and it has a major role in therecruitment and activation of infiltrating immunecells.

Inflammation is above all a protective response.In the case of pathological mineralization (stone for-mation), one would expect the body to produce min-eralization inhibitors/modulators. For example, OPN,

which is involved in inflammation and fibrosis,39 is a

known mineralization inhibitor as well as a che-moattractant.11,21 Its presence would attract mono-cytes/macrophages to the mineral formation, first toinhibit crystallization and in the case of mineralformation attract inflammatory cells to then elimi-nate the crystals.11 Thus, being part of the inflam-matory cascade and being a mineralization inhibitorare not mutually exclusive.

Most idiopathic stones form while connected toRP, which starts as deposits of poorly crystallinebiological apatite in the basement membrane of theloops of Henle, collecting ducts13 or vasa recta.14 Itwas proposed that the deposits, consisting of aggre-gated CaP spherules, then move through the intersti-tium toward the renal papillary epithelium, wherethey eventually ulcerate to the surface. A number ofquestions remain unanswered. Does urinary super-saturation affect CaP crystallization in the renalinterstitium without intraluminal crystallization?How and why do the crystals deposit in the base-ment membrane? How do the crystals move throughthe interstitium and then ulcerate to the papillarysurface? Crystals generally stay where they areformed except when they deposit unattached in thetubular lumina. We noted that collagen is a part ofinterstitial CaP deposits (fig. 1).16 It is mineralizedduring plaque formation and may have a significantrole in plaque growth and development. Collagen isdeposited during fibrosis.40 It is an excellent nucle-ator of CaP and has a critical role in the biominer-alization processes of the body.41

Interestingly, all RPs are not associated withstones and the kidneys of nonstone formers alsoshow RP.42 It was suggested that RP is formed with-out causing renal injury and inflammation. SinceRPs are CaP deposits at abnormal sites, RP shouldbe recognized as a foreign body and an inflammatoryresponse should be expected unless it is activelysuppressed. Alternatively, the inflammatory re-sponse may be localized, short lived and quicklyresolved, leaving only telltale signs, or perhapsplaque is a relic of an earlier injury, as suggested byRandall.15

Our examinations of RP have revealed injuredtubules. The presence of molecules such as OPN,12

heavy chain of I�I,12 collagen42–44 and zinc45 in in-terstitial plaques strongly suggests that inflamma-tion may be an early local participant.11 Biopsies aretaken at stone removal many months after its for-mation and only a small amount of tissue from alimited number of patients has been investigated todate. Inflammation is a complex biological responseto irritants, in this case high Ox, and/or CaOx andCaP crystals. OPN and I�I may be protective medi-ators that are most likely produced to inhibit crys-tallization. In normal human kidneys OPN is local-

ized primarily to the distal nephron and strongly

tripho

REACTIVE OXYGEN SPECIES AS MODULATORS OF KIDNEY STONE FORMATION 809

expressed in the thick ascending limbs of the loops ofHenle and papillary surface epithelium. OPN ex-pression is increased during inflammation and in-terstitial fibrosis.46

A number of pathways of RP formation have beenproposed14,47,48 but to our knowledge none havebeen experimentally tested to date. As mentioned,idiopathic stones start as deposits of biological apa-

P47phox

COX 2

Ox/CaO

Renal angiotensin II

ATR

PLA2

PKC

PTK R

LA

P38 MAPK/JNK

PGE2

Inflamma�on

Macrophage Infiltra�on

Phagocy�c NADPH oxidase

Collagen Mi

Growth of Ran

Figure 5. Pathways involved in NADPH oxidase regulation and Rmechanical stress associated with crystal deposition in kidneyscytic NADPH oxidase is activated and ROS are produced, metranslocation of Rac1 and p47phox to membrane. P-38 mitogen acpathway is turned on. Various transcriptional and growth factobecome involved. Secondary mediators are generated, suchProduction of chemoattractants, such as MCP-1 and crystallizastitium around crystal deposits. Activation of phagocytic NADPHcollagen deposition and mineralization develop, leading to grangiotensin II type 1 receptor. COX 2, cyclooxygenase 2. LPC, lyA2. PTK, protein tyrosine kinase. Rac GTP, �-related guanosine

tite or RP in the renal papillary interstitium, at the

junction of the loops of Henle, and the collectingducts and vasa recta. Changes in local availability ofCa, phosphate and citrate as well as pH may have acritical role in crystal formation. After CaP crystalsare deposited, nearby cells respond to the foreignbody by generating ROS, producing chemoattrac-tants such as MCP-1 and OPN. Monocytes/macro-phages infiltrate the interstitium. Crystals are

Non-phagocy�c NADPH oxidase

ROS

B

-1

AP-1

TGF-ß

calized Inters��al Fibrosis

P67phoxp40phox

Gp91phox Nox4 Nox1

Rac GTP

P22phox

P47phox

iza�on

Plaque

oduction. Renal cell exposure to high Ox, CaOx/CaP crystals ando renin up-regulation and angiotensin II generation. Nonphago-by PKC. Activation involves phosphorylation of p47phox, andprotein kinase/jun N-terminal kinase (MAPK/JNK) transduction

luding NF-�B, AP-1 and transforming growth factor-� (TGF-�),prostanes, cytoplasmic phospholipase A2 and prostaglandin.odulator OPN, is increased. Macrophages infiltrate renal inter-se results in additional ROS production. Inflammation, fibrosis,of interstitial CaP deposits or RP. AA, arachidonic acid. ATR,sphatidylcholine. PGE2, prostaglandin E2. PLA2, phospholipasesphate.

X/CaP

ac

PC A

NF-κ

OPNMCP

Lo

neral

dall’s

OS prleads tdiatedtivatedrs, incas isotion m

oxidaowthsopho

phagocytized and eliminated or fenced by a wall of

REACTIVE OXYGEN SPECIES AS MODULATORS OF KIDNEY STONE FORMATION810

macromolecules adsorbed to crystal surfaces, ren-dering them harmless.49 This is likely the case withinterstitial plaques, which are common in the kid-neys of stone formers and nonstone formers. How-ever, if the conditions that promoted crystal forma-tion persist and crystals continue to form, localizedinjury and inflammation develop, followed by fibro-sis and collagen deposition around the CaP crystaldeposits, initiating the second phase of stone patho-genesis. Collagen is mineralized, leading to plaquegrowth, which eventually reaches the papillary epi-thelium, ulcerates to the surface and develops into astone nidus. After it is exposed to pelvic urine, theplaque is overgrown by CaOx crystals, leading to theformation of an idiopathic CaOx kidney stone at-tached to the subepithelial RP.11

CONCLUSIONS

Experimental and clinical studies provide evidencefor ROS production and OS development in the kid-neys of stone forming patients. Initially ROS areinvolved in the production of crystallization inhibi-

tors, preventing stone formation. However, de-

REFERENCES

2010; 38: 239. 1161.

creased antioxidant capacity or persistently super-saturated urine, leading to increased crystallization,may result in OS and stone disease. Pretreatmentwith antioxidants and inhibitors of ROS generatingenzymes substantially decreases renal CaOx crystaldeposition, which is a surrogate marker of nephro-lithiasis in animal models. OS is also suggested to bea mediator of other chronic renal diseases that si-multaneously occur with kidney stones. OS genera-tion in one renal disease may predispose the kidneysto another disease. Surgical treatment, particularlyshock wave lithotripsy, also generates ROS, whichmay in part be responsible for stone recurrence. Acombination of antioxidants and free radical scaven-gers may provide renal protection against shockwave induced renal injury50 as well as stone recur-rence.

We hypothesize that localized OS induced inflam-mation resulting from interstitial CaP deposition inthe renal papillae promotes fibrosis and collagenformation. Collagen mineralization promotes CaPpropagation and RP growth. Antioxidants and anti-inflammatory therapy may impede RP growth and

inhibit stone recurrence.

APPENDIXCrystallization Modulating Macromolecules in Renal Epithelial Cells in response to Ox or CaOx crystal exposure

Nephrolithiasis Role Inflammation � Repair Role Ox � CaOx Crystal Response

THP Crystal aggregation inhibitor Renoprotective, elicits immune response Up-regulation, down-regulation, no changeOPN Free OPN inhibits crystal nucleation, growth,

aggregation � attachment, � immobilizedOPN promotes crystal attachment

Ca binding, renoprotective, tissue repair �inflammation, monocyte/macrophagechemoattractant

Up-regulation

Prothrombin fragment-1 Crystal growth � aggregation inhibitor Ca binding, coagulation Up-regulation , no changeBikunin � I�I family Crystal nucleation, growth, aggregation �

attachment inhibitorMetastasis, tissue repair � remodeling Up-regulation

�-1 Microglobulin Crystallization inhibitor Immunosuppressive, mitogenic Up-regulationHyaluronic acid Major stone matrix constituent Important extracellular matrix constituentCD-44 Crystal attachment promoter Tissue repair � remodeling Up-regulationCalgranulin Crystal growth � aggregation inhibitor Ca binding, tissue remodeling �

inflammationUp-regulation

Heparan sulfate Crystal aggregation � attachment inhibitor Tissue remodeling Up-regulationOsteonectin Ca binding, tissue remodeling Up-regulationFibronectin Crystal aggregation, attachment �

endocytosis inhibitorMorphogenesis, wound healing �metastasis

Up-regulation

Matrix gla protein Crystal deposition inhibitor Biomineralization inhibitor Up-regulation

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