recent advances in the pathophysiology of nephrolithiasis

Recent advances in the pathophysiology ofnephrolithiasisKhashayar Sakhaee1

1Department of Internal Medicine, Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of TexasSouthwestern Medical Center at Dallas, Dallas, Texas, USA

Over the past 10 years, major progress has been made in the

pathogenesis of uric acid and calcium stones. These advances

have led to our further understanding of a pathogenetic link

between uric acid nephrolithiasis and the metabolic

syndrome, the role of Oxalobacter formigenes in calcium

oxalate stone formation, oxalate transport in Slc26a6-null

mice, the potential pathogenetic role of Randall’s plaque as a

precursor for calcium oxalate nephrolithiasis, and the role of

renal tubular crystal retention. With these advances, we may

target the development of novel drugs including (1) insulin

sensitizers; (2) probiotic therapy with O. formigenes,

recombinant enzymes, or engineered bacteria; (3) treatments

that involve the upregulation of intestinal luminal oxalate

secretion by increasing anion transporter activity (Slc26a6),

luminally active nonabsorbed agents, or oxalate binders; and

(4) drugs that prevent the formation of Randall’s plaque

and/or renal tubular crystal adhesions.

Kidney International (2009) 75, 585–595; doi:10.1038/ki.2008.626;

published online 10 December 2008

KEYWORDS: calcium oxalate; kidney stone; metabolic syndrome;

nephrolithiasis; uric acid

Calcium oxalate is the most prevalent type of kidney stonedisease in the United States and has been shown to occur in70–80% of the kidney stone population.1 The prevalence ofrecurrent calcium oxalate stones has progressively increasedin untreated subjects, approaching a 50% recurrence rateover 10 years.2 The lifetime risk for kidney stone diseasecurrently exceeds 6–12% in the general population.3,4 In thefinal quarter of the twenty-first century, the prevalence ofkidney stone disease increased in both gender and ethnicity.4

Although kidney stone nephrolithiasis is perceived as anacute illness, there has been growing evidence that nephro-lithiasis is a systemic disorder that leads to end-stage renaldisease.5–7 It is also associated with an increased risk ofhypertension,8–12 coronary artery disease,13,14 the metabolicsyndrome (MS),15–20 and diabetes mellitus.19–24 Nephro-lithiasis without medical treatment is a recurrent illness witha prevalence of 50% over 10 years.2 Nephrolithiasis hasremained a prominent issue that imposes a significantburden on human health and is a considerable financialexpenditure for the nation. In 2005, based on inpatient andoutpatient claims, this condition was estimated to cost over$2.1 billion.25 A novel strategy for the development of newdrugs has been hampered largely by the complexity of thisdisease’s pathogenetic mechanism and its moleculargenetic basis. Our further understanding of theseunderlying pathophysiologic mechanisms will be the keystep in developing more effective preventive and therapeuticmeasures.

ETIOLOGIC MECHANISMS OF URIC ACID STONE FORMATION

Three major factors for the development of uric acid (UA)stones are low urine volume, acidic urine pH, andhyperuricosuria. However, abnormally acidic urine is theprincipal determinate in UA crystallization. The etiologicmechanisms for UA stone formation are diverse, and includecongenital, acquired, and idiopathic causes.26 The mostprevalent cause of UA nephrolithiasis is idiopathic. In itsinitial description, the term ‘gouty diathesis’ was coined.27

The clinical and biochemical presentation of idiopathic UAnephrolithiasis (IUAN) cannot be attributed to an inbornerror of metabolism 26,28,29 or secondary causes such aschronic diarrhea,30 strenuous physical exercise,31 and a highpurine diet.32

http://www.kidney-international.org r e v i e w

& 2009 International Society of Nephrology

Received 15 August 2008; revised 14 October 2008; accepted 21

October 2008; published online 10 December 2008

Correspondence: Khashayar Sakhaee, Department of Internal Medicine,

Charles and Jane Pak Center for Mineral Metabolism and Clinical Research,

University of Texas Southwestern Medical Center at Dallas, 3523 Harry Hines

Blvd, Dallas, Texas 75390-8885, USA.

E-mail: [email protected]

Kidney International (2009) 75, 585–595 585

http://dx.doi.org/10.1038/ki.2008.626

http://www.kidney-international.org

mailto:[email protected]

PHYSICOCHEMICAL CHARACTERISTICS OF URIC ACID

In humans and higher primates, UA is an end product ofpurine metabolism. Owing to their lack of the hepaticenzyme, uricase, which converts UA to soluble allantoin, theirserum and urinary levels of UA are considerably higher thanin other mammals.33 Normally, urinary UA solubility islimited to 96 mg/l. In humans with a urinary UA excretion of600 mg/day, this should generally exceed the limit ofsolubility and susceptibility to precipitation.34 Moreover,urine pH is another important factor in UA solubility. UA is aweak organic acid with an ionization constant (pKa) of5.5.35,36 Therefore, at a urine pH less than 5.5, the urinaryenvironment becomes supersaturated with sparingly soluble,undissociated UA that precipitates to form UA stones21,37,38

(Figure 1).

EPIDEMIOLOGY OF URIC ACID NEPHROLITHIASIS AND THEMETABOLIC SYNDROME

The MS is an aggregate of features that increase the risk oftype 2 diabetes mellitus (T2DM) and atheroscleroticcardiovascular disease.15–17 In a retrospective analysis a stoneregistry in Dallas initially showed a high prevalence offeatures of the MS in IUAN patients, leading to thedetermination that patients with IUAN share characteristicssimilar to those of the MS. Numerous epidemiologic studieshave shown that obesity, weight gain, and T2DM areassociated with an increased risk of nephrolithiasis.39,40

Despite the large sample size, stone composition was notreported among these studies. This center first reported thehigh prevalence of UA stones as the main stone constitutefound in T2DM. In addition, recent retrospective and cross-sectional studies have noted an increased prevalence of UAstones among obese and T2DM patients.23,41–44 However,T2DM and a greater body mass index were shown to beindependent risk factors for nephrolithiasis.44

PATHOPHYSIOLOGY OF LOW URINE pH IN IDIOPATHIC URICACID NEPHROLITHIASIS

The metabolic defect suspected for low urinary pH in UAstone formation was described almost four decades ago.45

Defective ammoniagenesis or excretion was attributed as apossible pathogenetic mechanism. Initial studies showingabnormalities in glutamine metabolism, which resulted in theimpaired conversion of glutamine to a-ketoglutarate andconsequently resulted in reduced renal ammonium (NH4

þ )excretion, were not supported by further investigation.46–49

Mechanistic studies, however, have shown that the two majorfactors responsible for abnormally low urine pH are acombination of defective NH4

þ excretion and increased netacid excretion (NAE).

Defective ammonium excretion

Under normal circumstances, a tight acid–base balance ismaintained with a high capacity buffer, ammonia, (pKa 9.2),which effectively buffers most protons while the remainingprotons are buffered by titratable acids. This process works tosustain a normal urinary pH. In contrast, the defective NH4

þ

excretion in IUAN requires the urine to be buffered mainly bytitratable acids to maintain this equilibrium, thus promotingan acidic urinary pH and providing an environment highlyconducive for UA precipitation (Figure 2).

Increased acid production alone may not be sufficient incausing abnormally acidic urine, as the excreted acid isneutralized by urinary buffers. Evidence of defective NH4

þ

excretion was provided in IUAN patients under a fixed,metabolic diet.21,23 Therefore, an unduly acidic urine pH inthe IUAN population is not related to environmental factorsbut it is, in part, related to the higher body weight in thesesubjects.50 The defective NH4

þ production in these subjectswas further explored by the administration of an acute acidload, which amplified the ammoniagenic effect21 (Figure 3).Similar findings were also demonstrated in IUAN patients ona random diet.22 Furthermore, it has been shown that innormal persons, urinary pH and NH4

þ /NAE ratio fallswith increasing features of the MS, indicating that renal

H+ + Urate Uric acid

pKa = 5.5

pH < 5.5

Undissociated uric acid

Uric acid or uricacid/CaOx stones

Figure 1 | Physicochemical scheme for the development ofuric acid stones.

Renal tubule lumen

NH3

NH4+

A–

HA

H+ H+

NH3

NH4+

Normal UA nephrolithiasis

A–

HA

Figure 2 | Mechanisms of urinary acidification.

586 Kidney International (2009) 75, 585–595

r e v i e w K Sakhaee: Pathophysiology of nephrolithiasis

ammoniagenesis and low urine pH may be features of thegeneral MS and not IUAN specific.51

Several studies have provided evidence supporting arelationship between UA nephrolithiasis, obesity, and insulinresistance.21,23,41–44 The mechanistic connection betweenperipheral insulin resistance, urinary pH, and NH4

þ , wasfirst demonstrated using the hyperinsulinemic-euglycemicclamp technique in patients with IUAN.24 These studiessupport the potential role of insulin resistance in an impairedurinary NH4

þ excretion and low urinary pH. Insulinreceptors are expressed in various portions of the ne-phron.52,53 Furthermore, in vitro studies have shown thatinsulin has a stimulatory function in renal ammoniagen-esis.54,55 In addition, NH4

þ secretion is regulated by thesodium–hydrogen exchanger NHE3.56 As NHE3 has a keyfunction in the transport or trapping of NH4

þ in the renaltubular lumen,56 insulin resistance may potentially lead todefective renal NH4

þ excretion. One other plausible mechan-ism may be substrate competition by substituting circulatingfree fatty acid for glutamine, which is increased in the MS,thereby reducing the proximal renal tubular cell utilization ofglutamine and renal ammoniagenesis.57

Increased net acid excretion

An elevated NAE may occur due to increased endogenousacid production or because of dietary influences such as lowdietary alkali or the increased consumption of acid-richfoods.36 Metabolic studies comparing subjects on fixed, low-acid ash diets showed a higher NAE in IUAN patientscompared to control subjects, suggesting that endogenousacid production may increase in IUAN 34 (Figure 4). Inaddition, the urinary NAE for any given urinary sulfate

(a surrogate marker of acid intake) tended to be higher inpatients with T2DM.22 These studies also implied that thepathophysiologic mechanism accounting for increased NAEis related to obesity/insulin resistance. Supporting thiscorrelation, additional studies have shown increased organicacid excretion with higher body weight and higher bodysurface area.58,59 The nature of these putative organic anionsand their link to obesity and/or UA stones has not been fullystudied.

POTENTIAL ROLE OF RENAL LIPOTOXICITY

Under standard metabolic conditions, when caloric intakeand caloric utilization are well balanced, triglyceridesaccumulate in adipocytes.60,61 A disequilibrium in this tightbalance leads to the accumulation of fat to non-adipocytetissues.61 This process of fat redistribution, termed lipotoxi-city, typically affects tissues such as cardiac myocardial cells,pancreatic b-cells, skeletal muscle cells, and parenchymalliver cells.61–66

Cellular injury is primarily due to the accumulation ofnonesterified fatty acids and their toxic metabolites includingfatty acyl CoA, diacylglycerol, and ceramide.60,67,68 It hasbeen shown that fat redistribution is accompanied withimpaired insulin sensitivity,63 cardiac dysfunction,65 andsteatohepatitis.62,69 There is an emerging interest in therole of renal lipotoxicity in the pathogenesis of renaldisease.67,70,71 A few studies have revealed a mechanistic linkbetween obesity, obesity-initiated MS, and chronic kidneydisease.70,71 Additional studies have shown a possible role ofsterol-regulating element-binding proteins in renal fataccumulation and injury.72–74 At the present time, there isinsufficient data available to suggest whether renal lipotoxi-city influences endogenous acid production and reducesrenal ammoniagenesis, consequently leading to abnormallyacidic urine.

From the above information, one may propose a three-hitmechanism for the development of low urinary pH and thepropensity for UA stone formation. The first mechanism isrelated to excessive dietary acid intake and/or increasedendogenous acid production. However, this alone may not besufficient in lowering urinary pH. Therefore, the secondmechanism is associated with defective NH4

þ excretion.Together, these two defects lower urinary pH adequatelyenough to convert urate salt into undissociated UA. This isnecessary but not sufficient for the formation of UA stones.Finally, the possible absence of inhibitors or presence ofpromoters of UA precipitation is operative in triggering UAstone formation.

CALCIUM OXALATE NEPHROLITHIASIS

Although it affects both genders, calcium oxalate nephro-lithiasis generally tends to occur in more men than women.In the calcium oxalate stone former, urinary oxalate andurinary calcium are equally conducive in raising urinarycalcium oxalate supersaturation.75 Hyperoxaluria is encoun-tered in 8–50% of kidney stone formers.76–78 The main

NH

3/C

r �m

ol/m

g (p

re a

nd p

ost)

NH

4+/C

r �m

ol/m

g (p

re a

nd p

ost)

Urin

ary

pH (

pre

and

post

)

0.001

0.002

0.003

0.004

0.005

10

20

30

40

NH3/Cr �mol/mg (pre and post)

NH4+/Cr �mol/mg (pre and post)

Normal subjects

Urinary pH (pre and post)

6.5

6.0

5.5

5.0

Uric acidstone formers

NH3 + H+ NH4+

Figure 3 | Acute acid loading. Previously published in Sakhaeeet al.21


K Sakhaee: Pathophysiology of nephrolithiasis r e v i e w

etiologic causes of hyperoxaluria can be classified into threegroups: (1) increased oxalate production as a result of aninborn error in metabolism of the oxalate synthetic pathway,(2) increased substrate provision from dietary oxalate-richfoods or other oxalate precursors, and (3) increased intestinaloxalate absorption.1 With the study of Oxalobacter formigenes(OF)79,80 and the role of putative anion transporter Slc26a681

as potential tools in the treatment of primary hyperoxaluria,our knowledge of the pathophysiologic mechanisms ofoxalate metabolism has advanced significantly over thepast decade.82

PHYSICOCHEMICAL PROPERTIES OF OXALATE

The human serum oxalate concentration ranges between 1and 5 mM, however, due to water reabsorption in thekidney, its concentration is 100 times higher in the urine.1,83

At a physiologic pH, oxalate will form an insoluble salt withcalcium. As the solubility of calcium oxalate in anaqueous solution is limited to approximately 5 mg/l at apH of 7.0, assuming that normal urine volume rangesbetween 1 and 2 l/day and normal urinary oxalate excretion isless than 40 mg/day, normal urine is often supersaturatedwith calcium oxalate. However, under normal conditions,the blood is undersaturated with respect to calcium oxalate.As seen in patients with primary hyperoxaluria and renalinsufficiency, when the serum oxalate concentrationincreases to above 30 mM, the blood becomes supersaturatedwith calcium oxalate.84 In the plasma, oxalate is notsignificantly bound to protein and is freely filtered by thekidneys. A recent study reported that urinary calcium is asimportant as urinary oxalate in raising calcium oxalatesupersaturation.75

OXALATE HOMEOSTASISHepatic production

In mammals, oxalate is an end product of hepaticmetabolism.79 The major precursor for hepatic oxalateproduction is glyoxalate metabolism within hepatic peroxi-somes. This metabolic conversion is mediated by enzymealanine–glyoxalate aminotransferase. Under normal circum-stances, the metabolism of glyoxalate to glycolate and glycinedetermines the conversion of glyoxalate to oxalate. Glyoxalateis also metabolized to glycolate by enzyme D-glyceratedehydrogenase, which has both glyoxalate/hydroxypyruvatereductase activity.85 An inborn error in metabolism withalanine–glyoxalate aminotransferase and glyoxalate/hydroxy-pyruvate reductase deficiency leads to oxalate overproduc-tion, which results in type 1 and type 2 primaryhyperoxaluria.79,81,85 Several other metabolic precursors ofoxalate metabolism, including the breakdown of ascorbicacid, fructose, xylose, and hydroxyproline, have also beenincriminated. However, their influences on oxalate produc-tion, under normal physiologic circumstances, have not beenfully accepted.86–88

Intestinal absorption

Dietary oxalate intake is important in urinary oxalateexcretion. The estimated intake of oxalate ranges between50 and 1000 mg/day.77,78,89 Oxalate-rich foods primarilyinclude seeds, such as chocolate that is derived from tropicalcacao tree, and leafy vegetation, including spinach, rhubarb,and tea. Approaching approximately 45%, the contributionof dietary oxalate to urinary oxalate excretion has been shownto be much higher than previously described.90 In addition,with intestinal oxalate absorption ranging between 10 and

mE

q/da

y

Normal subjects

Net acidexcretion32 mEq

NH4+

NH4+

NH4+

TA Net acid excretion61 mEq

HCO3–

Cit

Type 2 diabetics

TA

Cit

HCO3– HCO3

–

UA Stone formers

Net acid excretion61 mEq

TA

Cit

*P<0.001 vs NV

*

*

*

–20

–10

0

10

20

30

40

50

60

70

80

90

Figure 4 | Inpatient net acid excretion. Net acid excretion¼NH4þþTA – (HCO3

�þCit).



72%, this relationship between oxalate absorption anddietary oxalate intake has not been shown to be linear.90

In humans, the exact intestinal segment participating inoxalate absorption has not been determined. Indirectevidence suggests that oxalate absorption occurs throughouta large segment of the small intestine. This has been proposedas the main percentage of absorption occurs during the first4–8 h after the ingestion of oxalate-rich foods.91–93 Thisinference has been made based on the reported 5-h intestinaltransit time from the stomach to the colon. However, it hasalso been suggested that the colon may also participate inoxalate absorption, but to a lesser extent.93 In addition, theparacellular intestinal oxalate flux has been suggested tooccur in the early segment of the small intestine largely due tothe negative intestinal luminal potential and higher luminaloxalate concentration compared to the blood.94

Role of the putative anion exchange transporter Slc26a6

Recently, the putative anion exchange transporter Slc26a6 hasbeen shown to be involved in intestinal oxalate transport.82

The Slc26a6 is expressed in the apical portion of varioussegments of the small intestine such as the duodenum,jejunum, and ileum. It can also be found in the largeintestine, but to a smaller percentage.95 In vitro studies usingthe Ussing chamber technique demonstrated defective netoxalate secretion in mice with a targeted inactivation of theSlc26a6.96 Moreover, in vivo studies in the Slc26a6-null miceon a controlled oxalate diet reported high urinary oxalateexcretion, increased plasma oxalate concentration, anddecreased fecal oxalate excretion.96 The differences in urinaryoxalate excretion, plasma oxalate concentration, and fecaloxalate excretion were abolished following a 7-day equilibra-tion on an oxalate-free diet. These findings suggest that thereduction of net oxalate secretion in Slc26a6-null miceincreases net oxalate absorption, raising plasma oxalateconcentrations and consequently raising urinary oxalateexcretion. This study concluded that the Slc26a6 anionexchanger has a key function in urinary oxalate excretion.96

These results were also associated with bladder stones andYasue-positive crystals in the kidney. Staining of the kidneyspecimen with Yasue stain demonstrated evidence ofbirefringent crystal deposits in the luminal cortical collectingducts and, to a minimal extent, in the inner medullarycollecting ducts (IMCD). Calcium oxalate stones were foundin the renal pelvis and bladder. The renal tubular epithelialcells were surrounded by lymphocytic infiltration anddistorted morphology. However, unlike with the kidneys inidiopathic calcium oxalate stone formers, no abnormality wasfound in the medullary interstitial space.

Role of O. formigenes

Among many other bacteria including Eubacteriumlentum, Enterococcus faecalis, Lactobacillus, Streptococcusthermophilus, and Bifidobacterium infantis, OF have beenreported to degrade oxalate.94 OF was first isolated inruminates 97 and has since been found in many animal

species as well as in humans.98 However, OF is not found ininfancy. The bowel becomes colonized with this bacterium atapproximately 6–8 years of age. It decreases in later years andmay only be found in the feces of 60–80% of the adultpopulation.99

OF is a Gram-negative obligate anaerobe microorganismthat primary utilizes oxalate as a source of energy for cellularbiosynthesis.100 Through this electrogenic process, oxalateenters the oxalobacter through an oxalate–formate antiporter.It then utilizes its own enzymes, formyl CoA transferase andoxalyl-CoA decarboxylase, to convert oxalate into formateand CO2.101 In this process, one proton is utilized and createsa chemical gradiant due to the cell alkalinity. The electro-chemical gradients created by these processes facilitate protonentry and ATP synthesis101 (Figure 5).

The clinical importance of OF colonization is primarilysuggested for patients with recurrent calcium oxalate nephro-lithiasis,102–104 in patients with enteric hyperoxaluria,105,106 andin those with cystic fibrosis.107 Studies in patients withurolithiasis and cystic fibrosis have shown that the prolongeduse of antibiotics may abrogate the bowel colonization of OFand may irreversibly destroy these bacteria. Very recently, acase–control study of 274 patients with recurrent calciumoxalate stones and 259 normal subjects matched for age andgender displayed that the prevalence of OF was significantlylower in the stone formers. In this study, 17% of stoneformers were positive for OF vs 38% of normal subjects. Thisrelationship persisted with age, gender, race, ethnic back-ground, region, and antibiotic use108 (Figure 6).

The colonization of OF may be regulated by dietaryoxalate intake. This has been shown in animal modelswhere a significant decrease in urinary oxalate resulted fromthe administration or in the upregulation of OF coloniza-

Formyl-CoA transferase

Oxalyl-CoA decarboxylase

+ + + + + + + +– – – – – – – –

O– O–

O–

O

O

O O

O O

O

H

H+

H

Oxalate2– Formate1–

F0 F1

3H+

ADP

CO2

ATP

1

SCoASCoA

Formyl-CoA

Oxalyl-CoA

2

Figure 5 | Oxalate catabolism and energy conservation inOxalobacter formigenes.



tion.102,109 It has been recently shown in rodents,ex vivo using the Ussing chamber method, that the role ofOF in oxalate metabolism is not solely dependent on itscapacity to degrade intestinal luminal oxalate or to lowermucosal to serosal oxalate flux, but also on its capacity tostimulate the net intestinal oxalate secretion.110 Given thisexperimental design, increased net oxalate secretion cannotbe explained by transepithelial oxalate gradients. One mayspeculate that OF interacts with mucosal epithelial cells,enhancing luminal oxalate secretion.

The result of these animal experiments has been recentlyconveyed into human diseases.80 One such study conductedin patients with type 1 primary hyperoxaluria, in subjectswith normal renal function, and in patients with chronicrenal insufficiency, reported the reduction of urinary oxalatethat ensued following the oral administration of OF.80 Themajor drawbacks of the use of OF are (1) the lack of large,long-term, controlled studies in calcium oxalate kidney stoneformers and in subjects with enteric hyperoxaluria, such aspatients with cystic fibrosis or those following a gastro-intestinal bypass procedure; (2) the variable response to OFadministration; and (C) OF’s short life span on the completeutilization of its primary nutrient source, oxalate. Futurelong-term studies and the development of target drugs toeither upregulate the intestinal secretion of oxalate bystimulating Slc26a6 provide the enzyme products of OF toallow for its persistent oxalate-degrading capacity, provideengineered bacteria that are not entirely dependent onoxalate as a substrate for nutrients, or contain a luminallyactive agent that binds intestinal luminal oxalate content arenecessary in overcoming these deficiencies.

Renal excretion

The kidney has an important function in oxalate excretion.With impaired kidney function, plasma oxalate concentra-tions progressively rise and result in kidney damage.Eventually, with further impairment, there is a robust spike

in plasma oxalate concentration that exceeds its saturation inthe blood and thereby increases the risk of systemic tissueoxalate deposition. It has been recently demonstrated thatSlc26a6 is also expressed in the apical portion of the proximalrenal tubule111 and influences the activity of various apicalanion exchangers.112 In Slc26a6-null mice, it has been shownthat Cl–oxalate exchange activity is completely inhibited, andthe activity of Cl�/OH� and Cl�/HCO3 is significantlydiminished. However, the significance of this putative aniontransporter in calcium oxalate stone formation has not beenfully elucidated.

RANDALL’S PLAQUE IN THE PATHOGENESIS OF CALCIUMOXALATE STONES

Several mechanisms have been proposed for the formation ofcalcium stones. First, it has been suggested that the increasedsupersaturation of stone-forming salts are responsible for theprocess of homogenous nucleation in the lumen of thenephron. This process, followed by crystal growth, ultimatelyresults in an obstruction in the distal nephron. Second, it hasbeen suggested that crystal forms in the renal tubular lumenadhere to the luminal renal tubular cells. This adhesion theninduces renal cell injury resulting in the formation of a fixednuclei that interacts with the supersaturated urinaryenvironment and results in crystal growth. These processesboth lead to nephron obstruction and consequently result inintratubular calcification.113 However, the theory of fixed andfree crystal growth attachments in the nephron has not beenfully described as a mechanism of kidney stone formation. Asoccurs in intestinal bypass and cystine patients, if anintraluminal crystal plug attachment occurs at the openingof the Bellini duct, it is possible that this mineral plug canprotrude to the minor calyx, resulting in stone growth.

Dr Alexander Randall was the first to argue thatintraluminal plugging is an infrequent occurrence in kidneystone formers.114 Conversely, he suggested that interstitialcalcium phosphate deposits are initial niduses that anchorurinary crystals beneath the normal uroepithelial cells of therenal papilla. The erosion of the overlying uroepitheliumexposes these deposits, referred to as plaques, to thesupersaturated urine that then propagate calcium oxalatestones. He found these lesions to be interstitial as opposed tointraluminal, and without any inflammatory reactions. Healso showed these deposits to be mainly found beneath thetubular basement membrane and in the interstitial collagen.Randall’s hypothesis was primarily disputed since it wascarried out in cadaveric kidney specimens and not in atargeted kidney stone-forming population.114 His majordiscovery, however, was a small stone propagated in therenal pelvis that was attached to a calcium plaque found inthe papillae of the kidney.

Characteristics of the interstitial plaques

Randall’s initial observations were recently followed withthe development of modern techniques for determiningmineral composition. These techniques have been used to

Positive(17%)

Oxalobacter formigenes status

Negative(83%)

Positive(38%)Negative

(62%)

Recurrent CaOxstone formers

(n = 247)

Non-stone formers(n = 259)

Figure 6 | Oxalobacter formigenes in stool among patientswith recurrent calcium oxalate kidney stones and non-stoneformers. Previously published as a modification of informationobtained from Kaufman et al.108



characterize the nature of crystals attached to these plaquesand to develop novel techniques to visualize Randall’s plaquein vivo in patients with nephrolithiasis.115,116 An analysis ofover 5000 stones showed the main mineral composition ofinterstitial plaque to be mainly carbapatite. However,amorphous carbonated calcium phosphate, dosium hydrogenurate, and UA were found to a smaller extent.117 Anotherstudy utilizing m-CT determined that apatite crystalsurrounded by calcium oxalate was the main mineralcomposition of Randall’s plaque.118

It was first shown that Randall’s plaques occur morefrequently in patients with kidney stones as compared tonon-stone formers undergoing an endoscopic evaluation.119

Furthermore, a relationship was found between metabolicabnormalities in patients with calcium stones and thenumber of plaques.120 The result of this study was reachedusing digital video and endoscopic techniques to estimateaccurately the extent of Randall’s plaque in both calciumstone-forming and non-stone-forming subjects.121 In thisstudy, the main biochemical profiles correlating with theformation of interstitial plaque were urinary volume, urinarypH, and urinary calcium excretion. Higher urinary calciumand lower urinary volume showed an increased coverage ofthe renal papilla with plaque. This study supports amechanistic relationship between water reabsorption in therenal medulla and papilla with plaque formation. In addition,a separate retrospective study, using nephroscopic papillarymapping with representative still images and Moving PicturesExpert Group movies in 13 calcium oxalate kidney stoneformers, determined the percent of plaque coverage to bedirectly correlated with the number of kidney stonesformed.122

LOCALIZATION OF RANDALL’S PLAQUE

The basement membrane of thin descending loops of Henleis the principal site of Randall’s plaque localization.115 Thethin descending limb basement membrane is made up ofcollagen and mucopolysaccharides, which attract calcium andphosphate ions.123 Once attracted to this protein matrix, thecrystallization processes begins. In the interaction following,calcium phosphate crystals grow and propagate to thesurrounding collagen and mucopolysaccharide-rich renalinterstitium.124 This complex then makes its way throughthe urothelium and serves as a nidus for calcium oxalatedeposition, ultimately resulting in calcium oxalate kidneystone formation. Randall’s plaque has only been localized inthe basement membranes and in the interstitium. It has neverbeen found in the tubular lumen within epithelial cells orvessels. Within the basement membrane, this plaque consistsof coated particles of overlying regions of crystalline materialand organic matrix116 (Figure 7).

MECHANISM OF PLAQUE FORMATION

The mechanism of interstitial plaque formation has not beenfully elucidated. Our limitations in this area are based on thelack of availability of an animal model that mimics this

human disease. A few clinical studies have suggested acorrelation between urine volume, urinary calcium, andseverity of stone disease with the fraction of papillaryinterstitium covered by Randall’s plaque.119–122 Althoughthis link is not causal, however, it indicates some correlationbetween plaque formation and kidney stone disease inidiopathic hypercalciuric patients. It is plausible to proposethat plaque formation in the thin descending limb of Henleoccurs because of an increase in interstitial calcium andphosphate concentration as well as an increase in renalpapillary osmolality as a result of water reabsorption in thisnephron segmet.125 Moreover, whether increasing interstitialfluid pH affects the abundance of plaque formation has beensuggested but has never been fully explored.116

ABSENCE OF RANDALL’S PLAQUEFollowing gastric bypass surgery

Hyperoxaluria and calcium oxalate stones are a commonoccurrence in patients following intestinal bypass surgery dueto morbid obesity.116,126 In these subjects, there is no plaqueobserved in the renal papilla. However, crystal aggregates arefound in the IMCD. Moreover, in contrast to conditions inidiopathic calcium oxalate stone formers, there is evidence ofrenal IMCD cell injury, interstitial fibrosis, and inflammationadjacent to the crystal aggregates. The IMCD crystalaggregates are usually composed of apatite crystals. Thedeposition of these apatite crystals occurs despite an acidicurinary environment, implying that tubular pH may bedifferent from the final urinary pH.116,126

Figure 7 | Sites and characteristics of crystal deposition. Atransmission electron micrograph showing a crystalline structurecomposed of concentric layers of crystalline material (light) andmatrix protein (dark). Previously published in Evan et al.127



Brushite stone formers

In brushite stone formers, similar to calcium oxalate stoneformers following gastric bypass surgery, there is evidence ofcell injury and interstitial fibrosis in the IMCD adjacent toapatite crystal deposits. Although brushite stone formers,much like idiopathic calcium oxalate stone formers, haveplaque in the renal papilla, the stones have not been shown toattach to the plaque.127 This may be, in part, due to clinicaland technical difficulties as the high burden of brushitestones may affect the structural integrity of the renal papillae,making it difficult to detect smaller stones that may beattached to the plaque. In addition, the extent of Randall’splaque is minimal in brushite stone formers so attachedstones are not commonly anticipated. One importantpredisposition to distortion of structural integrity of therenal papillae in these subjects may be acquired and is relatedto the number of shockwave lithotripsy in this popula-tion.127,128

THE ROLE OF RENAL TUBULAR CRYSTAL RETENTION

Although the crystallization process is necessary, it alone isnot sufficient for the formation of kidney stones. Threedecades ago, it was initially proposed that the accumulationof crystals in the renal calices are involved in the pathogenesisof nephrolithiasis.129 It was further hypothesized that tubularnephrocalcinosis is preceded by renal stone formation. Thisscheme does not refute Randall’s theory that interstitialnephrocalcinosis and plaque formation are precursors for thedevelopment of kidney stones.114 However, it has becomeincreasingly recognized that both mechanisms may besignificant in the formation of kidney stones.130,131 Thefurther elaboration of these two pathogenic pathways isimportant as stone formation may occur in the absence ofplaque in the kidney.132 Furthermore, experimental evidencehas suggested that crystal binding to the surface of theregenerating/redifferentiating renal tubular cell is regulatedby the expression of a number of luminal membranemolecules, including hyaluronic acid, osteopontin, theirtransmembrane receptor protein CD44, and p38 mitogen-activated protein kinase.130,133–137 In addition, several othermolecules expressed in the renal tubular apical membranesuch as Annexin-II138 and an acidic fragment of nucleolin-related protein139 have been proposed as active bindingprotein regions for calcium oxalate crystals. The clinicalimplications of this experimental evidence are progressivelyemerging in the field.

In addition, the increased incidence of tubular nephro-calcinosis in preterm infants may possibly occur fromexposure of differentiating renal tubular epithelial cellsfollowing crystalluria caused by furosemide treatment.140,141

Moreover, tubular nephrocalcinosis has been seen in a largenumber of renal allografts, suggesting that ischemic injuryresulting in increased expression of hyaluronic acid andosteopontin precedes crystal retention.142,143 From the abovediscussion, one can conclude that under normal conditions,crystals do not adhere to renal tubular epithelial cells and are

readily excreted in the urine. However, with antecedent renaltubular epithelial damage and during the process of renaltubular repair,144–146 specific crystal-binding proteins areexpressed at the apical surface of the renal epithelial cell,predisposing crystal adhesion and possibly stone formation.Whether this process has a pathogenic function in manyclinical conditions associated with tubular nephrocalcinosisand nephrolithiasis deserves intense future investigation.130

CONCLUSION

Kidney stone disease remains a major public health burden.Its pathophysiologic mechanisms are complex, mainlybecause it is a polygenic disorder, and it involves an intricateinteraction between the gut, kidney, and bone. In addition,an exact animal model to recapitulate the human disease hasnot yet been defined. Despite these limitations, ourcomprehension of UA stone formation’s link to insulinresistance and renal lipotoxicity, the underlying mechanismsof intestinal oxalate transport, the role of renal papillaryplaque in idiopathic calcium oxalate stone formation andrenal tubular crystal bindings, has advanced significantly overthe past decade. These elucidations can potentially lead us tothe development of novel drugs targeting basic metabolicabnormalities that abrogate stone formation.

DISCLOSUREThe author declared no competing interest.

ACKNOWLEDGMENTSThis work was supported by the National Institutes of Health grants(P01-DK20543 and M01-RR00633). We acknowledge HadleyArmstrong for her primary role in the preparation and editorial reviewof this paper.

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