SECTION XI – URINARY LITHIASIS AND ENDOUROLOGY

Chapter 42 – Urinary Lithiasis: Etiology, Epidemiology, and Pathogenesis MARGARET S. PEARLE, MD, PhD   YAIR LOTAN, MD    EPIDEMIOLOGY OF RENAL CALCULI

   PHYSICOCHEMISTRY

   MINERAL METABOLISM

  PATHOGENESIS OF UPPER URINARY TRACT CALCULI

Although stone disease is one of the most common afflictions of modern society, it has been described since antiquity. With Westernization of global culture, however, the site of stone formation has migrated from the lower to the upper urinary tract and the disease once limited to men is increasingly gender blind. Revolutionary advances in the minimally invasive and noninvasive management of stone disease over the past 2 decades have greatly facilitated the ease with which stones are removed. However, surgical treatments, although they remove the offending stone, do little to alter the course of the disease. Indeed the overall estimated annual expenditure for individuals with claims corresponding to a diagnosis of nephrolithiasis was nearly $2.1 billion in 2000, reflecting a 50% increase since 1994 ( Pearle et al, 2005 ). Given the frequency with which stones recur, the development of a medical prophylactic program to prevent stone recurrences is desirable. To this end, a thorough understanding of the etiology, epidemiology, and pathogenesis of urinary tract stone disease is necessary.

EPIDEMIOLOGY OF RENAL CALCULI

The lifetime prevalence of kidney stone disease is estimated at 1% to 15%, with the probability of having a stone varying according to age, gender, race, and geographic location. In the United States, the prevalence of stone disease has been estimated at 10% to 15% ( Norlin et al, 1976 ; Sierakowski et al, 1978 ; Johnson et al, 1979 ). Using data derived from the United States National Health and Nutrition Examination Survey dataset (NHANES II and III), Stamatelou and colleagues (2003) established a 5.2% prevalence of kidney stone disease from 1988 to 1994, which represents a 37% increase from 1976 to 1980 for which a 3.8% prevalence rate was determined. The finding of an increase in the prevalence of stone disease has been observed by others ( Norlin et al, 1976 ; Yoshida and Okada, 1990 ; Serio et al, 1999 ; Trinchieri et al, 2000 ).

Gender

Stone disease typically affects adult men more commonly than adult women. By a variety of indicators, including inpatient admissions, outpatient office visits, and emergency department visits, men are affected two to three times more frequently than women ( Hiatt et al, 1982 ; Soucie et al, 1994 ; Pearle et al, 2005 ). However, there is some evidence that the difference in incidence between men and women is narrowing. Using the National Inpatient Sample dataset representing hospital discharges, Scales and colleagues found that while overall population-adjusted discharges for a diagnosis of renal or ureteral calculus increased by only 1.6% from 1997 to 2002, discharges for women increased by 17%, whereas discharges for men decreased by 8.1% ( Scales et al, 2005 ). This trend reflects a change in the ratio of male-to-female discharges from 1.7 in 1997 to 1.3 in 2002. Indeed, in 2002, hospital discharges were the same for men and women in this dataset. Whether trends

in hospital discharges accurately reflect trends in the overall prevalence of the disease, however, is not clear. Stamatelou and associates (2003) also noted a slight decrease in the male-to-female ratio of stone disease, from 1.75 (between 1976 and 1980) to 1.54 (between 1988 and 1994) using the NHANES dataset.

Race/Ethnicity

Racial/ethnic differences in the incidence of stone disease have been observed. Among U.S. men, Soucie and colleagues (1994) found the highest prevalence of stone disease in whites, followed by Hispanics, Asians, and African Americans, who had prevalences of 70%, 63%, and 44% of whites, respectively. Among U.S. women, the prevalence was highest among whites but lowest among Asian women (about half that of whites). Others found an even higher differential (threefold to fourfold) between whites and African Americans ( Sarmina et al, 1987 ). Interestingly, despite differences in prevalence of stone disease according to ethnicity, Maloney and colleagues (2005) observed a remarkably similar incidence of metabolic abnormalities between white and nonwhite stone formers from the same geographic region, although the distribution of abnormalities differed, suggesting that dietary and other environmental factors may outweigh the contribution of ethnicity in determining stone risk.

The gender distribution of stone disease varies according to race. Sarmina and colleagues (1987) noted a male-to-female ratio among whites of 2.3 and among African Americans of 0.65. Michaels and coworkers (1994) also noted a reversal of the male predisposition to stone disease in Hispanics and African Americans, reporting a male-to-female ratio of 1.8 among Asians, 1.6 among whites, 0.7 among Hispanics, and 0.5 among African Americans, among a group of patients undergoing extracorporal shock wave lithotripsy. Soucie and associates (1994) observed a similar trend in the male-to-female ratio of the lifetime incidence of stone disease of 3.4 among Asians, 2.6 among whites, 2.1 among Hispanics, and 1.8 among African Americans, although the actual ratios differed in the two studies. Dall'era and colleagues (2005) reviewed emergency department records to identify patients presenting with symptomatic renal or ureteral calculi and found a male-to-female ratio of 1.17 among Hispanic patients compared with 2.05 for white patients.

Age

Stone occurrence is relatively uncommon before age 20 but peaks in incidence

in the fourth to sixth decades of life ( Marshall et al, 1975 ; Johnson et al, 1979 ; Hiatt et al, 1982 ). It has been observed that women show a bimodal distribution of stone disease, demonstrating a second peak in incidence in the sixth decade of life, corresponding to the onset of menopause ( Marshall et al, 1975 ; Johnson et al, 1979 ). This finding, and the lower incidence of stone disease in women compared with men, has been attributed to the protective effect of estrogen against stone formation in premenopausal women, owing to enhanced renal calcium absorption and reduced bone resorption ( McKane et al, 1995 ; Nordin et al, 1999 ). Indeed, Heller and colleagues (2002) identified lower urinary saturation of calcium oxalate and brushite in women compared with men. Moreover, urinary calcium was lower in women than in men until age 50, after which it reached equivalence in the two groups. Estrogen-treated postmenopausal women had lower urinary calcium and saturation of calcium oxalate than untreated women.

Alternatively, Fan and associates (1999) found that androgens increased and estrogens decreased urinary and serum oxalate in an experimental rat model, perhaps accounting for the reduced risk of stone formation in women. However, Van Aswegen and coworkers (1989) found lower levels of urinary testosterone in stone formers compared with non—stone-forming control subjects, further confusing the issue.

Geography

The geographic distribution of stone disease tends to roughly follow environmental risk factors; a higher prevalence of stone disease is found in hot, arid, or dry climates such as the mountains, desert, or tropical areas. However, genetic factors and dietary influences may outweigh the effects of geography. Finlayson (1974) reviewed several worldwide geographic surveys and found that areas of high stone prevalence included the United States, British Isles, Scandinavian and Mediterranean countries, northern India and Pakistan, northern Australia, Central Europe, portions of the Malay peninsula, and China. Within the United States, Mandel and Mandel (1989a, 1989b) [235] [236] identified the highest rates of hospital discharge for patients with calcium oxalate stones in the Southeast and for uric acid stones in the East, among the veteran patient population. Soucie and associates (1994) found increasing age-adjusted prevalence rates in both men and women going from north to south and west to east, with the highest prevalence observed in the Southeast ( Fig. 42-1 ). After controlling for other risk factors, the authors determined that ambient temperature and sunlight were independently associated with stone prevalence ( Soucie et al, 1996 ).

Climate

Seasonal variation in stone disease is likely related to temperature by way of fluid losses through perspiration and perhaps by sunlight-induced increases in vitamin D. Prince and Scardino (1960) noted the highest incidence of stone disease in the summer months, July through September, with the peak occurring within 1 to 2 months of maximal mean temperatures ( Prince et al, 1956 ). Likewise, Bateson (1973) reported a peak incidence of stone disease between December and March in Australia, corresponding to the summer season.

The study of military personnel translocated to desert locations has provided a unique opportunity to study the effect of climate on a defined population. Pierce and Bloom (1945) reported that American soldiers in an undisclosed desert location had an increase in symptomatic episodes of renal colic during the summer season. Another study of military personnel who developed symptomatic stones after arrival in Kuwait and Iraq disclosed a mean time interval to stone formation of 93 days ( Evans et al, 2005 ). Finally, Parry and Lister (1975) measured urinary calcium and magnesium levels in soldiers before and 10 days after transfer to the Persian Gulf and noted increased urinary calcium levels from baseline in those soldiers transferred during the summer months but not among those transferred during the “cold season,” which was attributed to sunlight-induced increased production of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). Thus it is likely that climate and geography influence the prevalence of stone disease indirectly, through effects on temperature and possibly sunlight.

Occupation

Heat exposure and dehydration constitute occupational risk factors for stone disease as well. Cooks and engineering room personnel, both of whom are exposed to high temperatures, were found to have the highest rates of stone formation among personnel of the Royal Navy ( Blacklock, 1969 ). Likewise, Atan and colleagues (2005) found a significantly higher incidence of stones among steel workers exposed to high temperatures (8%) compared with those working in normal temperatures (0.9%). Metabolic evaluation of these two groups of workers showed a higher incidence of low urine volume and hypocitraturia among the workers in the hot area. Borghi and colleagues (1993) also noted differences in the incidence of stone disease and urinary stone risk factors among workers at a glass plant who were or were not chronically exposed to high temperatures causing massive perspiration. Those exposed to high temperatures exhibited lower urine volumes and pH, higher uric acid levels, and higher urine specific gravity, leading to higher urinary saturation of uric acid. Accordingly, those workers who formed stones had a remarkably high incidence of uric acid stones (38%).

Individuals with sedentary occupations, such as those in managerial or professional positions, have been found to carry an increased risk of stone formation for unclear reasons ( Blacklock, 1969 ). This finding is consistent with the work of Robertson and associates (1980b) , who reported an increased risk of stone disease in affluent individuals, countries, and societies, which may be reflective of a more indulgent diet and lifestyle.

Body Mass Index and Weight

The association of body size and incidence of stone disease has been investigated. In two large prospective cohort studies of men and women, the prevalence and incident risk of stone disease were directly correlated with weight and body mass index in both sexes, although the magnitude of the association was greater in women than men ( Curhan et al, 1998 ; Taylor et al, 2005 ). Although these investigators identified a reduced risk of incident stone formation with high intake of fluid (men and women) and low protein intake (men) (Curhan et al, 1993, 1997 [76] [78]), they found that obesity and weight gain were independent risk factors for incident stone formation and could not be accounted for by diet alone ( Taylor et al, 2005 ). Recent evidence linking obesity and insulin resistance with low urine pH and uric acid stones (Maalouf et al, 2004a, 2004b [230] [231]) as well as an association between hyperinsulinemia and hypercalciuria ( Kerstetter et al, 1991 ; Shimamoto et al, 1995 ; Nowicki et al, 1998 ) could account for an increased risk of uric acid and/or calcium stones in obese patients.

Water

The beneficial effect of a high fluid intake on stone prevention has long been recognized. In two large observational studies, fluid intake was found to be inversely related to the risk of incident kidney stone formation (Curhan et al, 1993, 1997 [76] [78]). Furthermore, in a prospective, randomized trial assessing the effect of fluid intake on stone recurrence among first-time idiopathic calcium stone formers, urine volume was significantly higher in the group assigned to a high fluid intake compared with the control group receiving no recommendations, and, accordingly, stone recurrence rates were significantly lower (12% vs. 27%, respectively) ( Borghi et al, 1996 ).

Geographic differences in the incidence of stone disease have been ascribed in some cases to differences in the mineral and electrolyte content of water in different areas. Although several investigators reported a lower incidence of stone disease in geographic regions with a “hard” water supply compared with a “soft” water supply, where water “hardness” is determined by content of calcium carbonate ( Churchill et al, 1978 ; Sierakowski et al, 1979 ), others found no difference. Schwartz and coworkers (2002) found no association between water hardness and incidence of stone episodes, although they did observe a correlation between water hardness and urinary magnesium, calcium, and citrate levels.

PHYSICOCHEMISTRY

The physical process of stone formation is a complex cascade of events that occurs as the glomerular filtrate traverses the nephron. It begins with urine that becomes supersaturated with respect to stone-forming salts, such that dissolved ions or molecules precipitate out of solution and form crystals or nuclei. Once formed, crystals may flow out with the urine or become retained in the kidney at anchoring sites that promote growth and aggregation, ultimately leading to stone formation. The discussion that follows describes the process of stone formation from a physicochemical standpoint.

State of Saturation

A solution containing ions or molecules of a sparingly soluble salt is described by the concentration product, which is a mathematical expression of the product of the concentrations of the pure chemical components (ions or molecules) of the salt. For example, the concentration product (CP) expression for sodium chloride is CP = [Na+][Cl-]. A pure aqueous solution of a salt is considered saturated when it reaches the point at which no further added salt crystals will dissolve. The concentration product at the point of saturation is called the thermodynamic solubility product, Ksp, which is the point at which the dissolved and crystalline components are in equilibrium for a specific set of conditions. At this point, addition of further crystals to the saturated solution will cause the crystals to precipitate unless the conditions of the solution, such as pH or temperature, are changed.

In urine, despite concentration products of stone-forming salt components, such as calcium oxalate, that exceed the solubility product, crystallization does not necessarily occur because of the presence of inhibitors and other molecules that allow higher concentrations of calcium oxalate to be held in solution before precipitation or crystallization occurs. In this state of saturation, urine is considered to be metastable with respect to the salt. As concentrations of the salt increase further, the point at which it can no longer be held in solution is reached and crystals form. The concentration product at this point is called the formation product, Kf.

The solubility product and the formation product differentiate the three major states of saturation in urine: undersaturated, metastable, and unstable ( Fig. 42-2 ). Below the solubility product, crystals will not form under any circumstances and dissolution of crystals

is theoretically possible. At concentrations above the formation product, the solution is unstable and crystals will form. In the metastable range, between the solubility product and the formation product and in which the concentration products of most common stone components reside, spontaneous nucleation or precipitation does not occur despite urine that is supersaturated. It is in this area that modulation of factors controlling stone formation can take place and therapeutic intervention is directed.

In the metastable range of concentration products, although crystal growth can occur on existing crystals, de novo formation of crystals cannot occur in the length of time it normally takes for the filtered urine to reach the bladder. However, crystal formation can occur in this range under certain circumstances. First, in parts of the nephron local concentration products may exceed the formation product for long enough time periods to allow nucleation to occur. Second, local areas of obstruction or stasis in the upper urinary tract may prolong urinary transit time and allow crystal formation to occur in the metastable urine. Finally, microscopic impurities or other constituents in the urine can facilitate the nucleation process by adsorption of the crystal components in a geometric way that resembles the native crystal. The energy required for this “heterogeneous nucleation” process is much less than that required for “homogeneous nucleation.”

To estimate the state of saturation for any given crystal system, such as calcium oxalate or calcium phosphate, Pak and Chu (1973) developed a mathematical formula, the activity product ratio, that takes into account urine pH and the ionic activities of all major ion species directly involved in the stone-forming process or those that affect the overall ionic strength of the urine. Finlayson subsequently developed a computer program to measure the state of saturation, EQUIL 2, which is commonly used today ( Werness et al, 1985 ). The relative saturation ratio or concentration product ratio is defined as the ratio of the concentration product of the urine to the solubility product of the specified stone-forming salt. A reduction in the numerator will lead to undersaturation of the urine with respect to the stone-forming salt and consequently reduce the likelihood of precipitation. This can be accomplished by reducing the urinary concentrations of the stone components (e.g., calcium or oxalate), by reducing the filtered load, or by increasing urinary reabsorption. In addition, complexation with substances such as citrate, reduce available free ionic calcium and decrease the relative saturation ratio. On the other hand, manipulation of factors such as pH can significantly impact the concentration of ions such as phosphate, the generation of which is highly pH dependent. Manipulation of pH has little effect on oxalate concentration, however, because oxalic acid is a strong acid (pK = 4) and pH changes within the physiologic range will have little effect on oxalate concentration.

Historically, urinary oxalate has been considered a more important contributor to calcium oxalate stone formation than urinary calcium ( Nordin et al, 1972 ; Robertson and Peacock, 1980 ). This assumption is based on the findings of Nordin and colleagues (1972) , who noted that a rise in urinary calcium concentration had less of an effect than a rise in oxalate concentration in increasing urinary saturation of calcium oxalate. They further showed that at high urinary calcium concentrations the saturation of calcium oxalate reached a plateau that did not exceed the theoretic formation product of calcium oxalate, whereas high oxalate concentrations did, thereby increasing the risk of calcium oxalate crystal formation. Pak and colleagues (2004) , however, challenged the notion that urinary oxalate exerts a greater pathogenetic role than calcium in calcium oxalate stone formation. They demonstrated that the choice of stability constant used for calculating relative saturation ratio determines the

relative effects of urinary calcium and oxalate concentration. Using the commonly accepted stability constant of 2.746 × 103 (used in the EQUIL 2 program), the effect of urinary calcium and oxalate proved to be equivalent. Thus, they concluded that urinary calcium and oxalate are both important and equal contributors to calcium oxalate stone formation. As such, reduction in both calcium and oxalate will be effective in reducing the relative saturation ratio, and intervention to prevent stone formation can be directed at either.

Nucleation and Crystal Growth, Aggregation, and Retention

In normal human urine, the concentration of calcium oxalate is four times higher than its solubility in water. Urinary factors favoring stone formation, including low volume and citrate and increased calcium, oxalate, phosphate and uric acid, all increase calcium oxalate supersaturation. Once the concentration product of calcium oxalate exceeds the solubility product, crystallization can potentially occur. However, in the presence of urinary inhibitors and other substances, calcium oxalate precipitation occurs only when supersaturation exceeds the solubility by 7 to 11 times.

Homogeneous nucleation is the process by which nuclei form in pure solution. Nuclei are the earliest crystal structure that will not dissolve. Small nuclei are unstable; below a critical size threshold, dissolution of the crystal is favored over crystal growth. If the driving force (supersaturation level) and the stability of the nuclei are adequate and the lag time to nucleation is sufficiently short compared with the transit time of urine through the nephron, the nuclei will persist. Inhibitors, like citrate, destablilize nuclei, whereas promoters stabilize nuclei by providing a surface with a binding site that accommodates the crystal structure of the nucleus. In urine, crystal nuclei usually form through heterogeneous nucleation by adsorption onto existing surfaces of epithelial cells ( Umekawa et al, 2001 ), cell debris ( Fasano et al, 2001 ), or other crystals ( Kok, 1997 ).

Within the time frame of transit of urine through the nephron, estimated at 5 to 7 minutes, crystals cannot grow to reach a size sufficient to occlude the tubular lumen. However, if enough nuclei form and grow, aggregation of the crystals will form larger particles within minutes that can occlude the tubular lumen. Inhibitors can prevent the process of crystal growth or aggregation. Magnesium and citrate inhibit crystal aggregation. Nephrocalcin, an acidic glycoprotein made in the kidney, inhibits calcium oxalate nucleation, growth, and aggregation ( Nakagawa et al, 1987 ; Asplin et al, 1991 ). Tamm-Horsfall mucoprotein, the most abundant protein in urine, inhibits aggregation ( Hess et al, 1991 ), and uropontin inhibits crystal growth ( Shiraga et al, 1992 ). Bikunin, the light chain of inter-alpha inhibitor, has been shown to be an efficient inhibitor of crystal nucleation and aggregation.

Opposing views regarding the formation and growth of crystal particles have led to controversy over the concept of free crystal particle growth versus fixed particle growth. Although it was initially concluded that free particle stone formation was impossible within the normal transit time through the nephron ( Finlayson et al, 1978b ), later recalculation using current nephron dimensions, supersaturation, and crystal growth rates determined that crystalline particles can be formed that are large enough to be retained during normal transit time through the kidney ( Kok and Khan, 1994 ).

Fixed particle growth theory presupposes an anchoring site to which crystals bind, thereby prolonging the time the crystals are exposed to supersaturated urine and facilitating crystal growth and aggregation. A number of mechanisms have been proposed to account for crystal

fixation. One favored theory proposes that oxalate-induced injury to renal tubular epithelial cells promotes adherence of calcium oxalate crystals ( Miller et al, 2000 ). In animal models of stone formation in which administration of high oxalate loads leads to calcium oxalate crystal formation, elevated urinary levels of enzyme markers of cell injury, including N-acetyl-β-glucosidase and alkaline phosphatase, imply damage to renal tubular epithelial cells ( Khan et al, 1992 ; Thamilselvan and Khan, 1998 ). Although the exact mechanism of oxalate-induced cell injury is not known, several studies have suggested that it is mediated by free radical formation ( Thamilselvan and Khan, 1998 ; Thamilselvan et al, 1999 ). Not only are high concentrations of oxalate toxic to renal tubular cells, but calcium oxalate crystals themselves have also been shown to promote damage to cells (Khan et al, 1993, 1999 [191] [192]; Thamilselvan and Khan, 1998 ; Thamilselvan et al, 1999 ). Despite these findings in animal models and in-vitro systems, evidence for oxalate-induced tubular damage in humans has been lacking. Indeed, Holmes and Assimos (2004) observed no increase in markers of oxidative stress or renal cell injury in normal individuals after ingestion of a large oxalate load, although the response in stone formers has not yet been determined.

How oxalate-induced renal tubular cell damage potentially promotes crystal retention is not known. Randall (1937) first observed areas of damage associated with subepithelial plaques on the renal papillae. Later, structural analyses in hyperoxaluric rats demonstrated crystals attached to the injured epithelium lining the collecting ducts ( Khan, 1991 ). In-vitro studies confirmed increased binding of calcium oxalate crystals to injured renal epithelial cells in culture ( Verkoelen et al, 1998 ). Whether the renal tubular cells or the interstitium constitutes the primary site of stone formation is unclear. Evidence of endocytosis of calcium oxalate crystals into renal tubular cells has been demonstrated in patients with disorders of oxalate metabolism ( Saxon et al, 1974 ; Mandell et al, 1980 ; Lieske et al, 1992 ). Intracellular incorporation of these crystals could potentially lead to cell death and deposition of crystals in the interstitium, or transport of the crystals from the luminal to the basement membrane side could promote cell damage

and subsequent erosion through to the papillary surface. Knoll and colleagues (2004) demonstrated in cell culture that oxalate-induced damage was more pronounced in renal nontubular compared with tubular cell lines and, further, that renal epithelial cells were more vulnerable to the toxic effects of oxalate on their basolateral side compared with their apical (luminal) side, implicating the interstitium as a possible site of primary stone formation.

In light of these recent findings, a number of investigators have revisited the role of Randall's plaques in the pathogenesis of stone formation. Low and Stoller (1997) mapped the papillae of patients undergoing endoscopic stone removal, as well as control subjects undergoing endoscopy for unrelated reasons, and found that papillary plaques occurred in 74% of stone formers compared with only 43% of control subjects. Stoller and colleagues (2004) hypothesized that the inciting event in the pathogenesis of stones may be vascular injury to the vasa recta near the renal papilla. Repair of damaged vessel walls could involve an atherosclerotic-like reaction that results in calcification of the endothelial wall, followed by erosion into the papillary interstitium and then into the collecting ducts, where it could serve as a nidus for stone formation.

Evan and coworkers presented an alternative view of the pathogenesis of stone formation based on extensive analysis of papillary plaques derived from biopsies obtained during percutaneous nephrolithotomy in idiopathic calcium oxalate stone formers. They localized the origin of the plaque to the basement membrane of the thin limbs of the loops of

Henle and demonstrated that the plaque subsequently extends through the medullary interstitium to a subepithelial location ( Fig. 42-3 ) ( Evan et al, 2003 ). Once these plaques, which are invariably composed of calcium apatite, erode through the urothelium, they constitute a stable, anchored surface on which calcium oxalate crystals can nucleate and grow as attached stones. They further noted that among idiopathic calcium oxalate stone formers, the volume of papillary surface covered by plaque correlated negatively with urine volume and positively with hypercalciuria (Kuo et al, 2003a, 2003b [212] [213]) and the number of stones formed ( Kim et al, 2005a ), providing further corroborating clinical evidence for the sequence of events they proposed.

In contrast, patients with enteric hyperoxaluria due to intestinal bypass for obesity demonstrated no plaque but did show apatite crystal deposits plugging the terminal collecting duct lumen and associated epithelial cell damage with inflammation and fibrosis of the interstitium ( Fig. 42-4 ) ( Evan et al, 2003 ). Patients who form brushite stones were found to have intermediate pathology between the calcium oxalate stone formers and the intestinal bypass patients, demonstrating interstitial apatite plaque and apatite plugging of the terminal collecting ducts, along with associated collecting duct injury and interstitial fibrosis ( Fig. 42-5 ) ( Evan et al, 2005 ). The pathogenesis of brushite stones was postulated to occur by way of crystallization of apatite in the collecting duct, followed by collecting duct injury and cell death leading to enlarged collecting ducts, interstitial inflammation as a result of the injured cells, and, finally, progressive involvement of adjacent renal tissue.

The finding of distinct morphologic subtypes characterizing particular patient phenotypes is consistent with their divergent underlying pathophysiologic abnormalities and supports the role of both urine chemistry/state of saturation as well as local factors in the pathogenesis of stone formation.

Inhibitors and Promoters of Crystal Formation

At the concentrations that most stone-forming salt components are present in urine, including calcium, oxalate, and phosphate, urine is supersaturated, thereby favoring crystal formation. However, the presence of molecules that raise the supersaturation needed to initiate crystal nucleation or reduce the rate of crystal growth or aggregation prevents stone formation from occurring on a routine basis. Although inhibitors have been identified for calcium oxalate and calcium phosphate, no specific inhibitors are known that affect uric acid crystallization.

Whole urine, when added to a solution of calcium phosphate, raises the supersaturation level required to initiate calcium phosphate crystallization (formation product) ( Fleisch et al, 1962 ). Inorganic pyrophosphate was found to be responsible for 25% to 50% of the inhibitory activity of whole urine against calcium phosphate crystallization. By using different methodology, however, citrate, magnesium, and pyrophosphate together were noted

to account for approximately 20% of the inhibitory activity of whole urine, with citrate comprising the most important factor of the three ( Bisaz et al, 1978 ).

Citrate acts as an inhibitor of calcium oxalate and calcium phosphate stone formation by a variety of actions. First, it complexes with calcium, reducing the availability of ionic calcium to interact with oxalate or phosphate ( Meyer et al, 1975 ; Pak et al, 1982 ). Second, it directly inhibits the spontaneous precipitation of calcium oxalate ( Nicar et al, 1987 ) and prevents the agglomeration of calcium oxalate crystals ( Kok et al, 1986 ). Although it has limited inhibitory effect on calcium oxalate crystal growth, it has more potent activity in reducing calcium phosphate growth ( Meyer et al, 1975 ). Lastly, citrate prevents heterogeneous nucleation of calcium oxalate by monosodium urate ( Pak and Peterson, 1986 ).

The inhibitory activity of magnesium is derived from its complexation with oxalate, which reduces ionic oxalate concentration and calcium oxalate supersaturation ( Meyer et al, 1975 ). In addition, magnesium reduces the rate of calcium oxalate crystal growth in vitro ( Desmars et al, 1973 ).

Polyanions, including glycosaminoglycans, acid mucopolysaccharides, and RNA have been shown to inhibit crystal nucleation and growth. Among the glycosaminoglycans, heparin sulfate interacts most strongly with calcium oxalate monohydrate crystals ( Yamaguchi et al, 1993 ).

Two urinary glycoproteins, nephrocalcin and Tamm-Horsfall glycoprotein, are potent inhibitors of calcium oxalate monohydrate crystal aggregation ( Nakagawa et al, 1987 ). Nephrocalcin is an acidic glycoprotein containing predominantly acidic amino acids that is synthesized in the proximal renal tubules and

the thick ascending limb. In simple solution, nephrocalcin strongly inhibits the growth of calcium oxalate monohydrate crystals ( Nakagawa et al, 1987 ). Nephrocalcin has been identified in four isoforms; non—stone formers excrete greater quantities of two isoforms associated with the most inhibitory activity, whereas stone formers excrete urine enriched for the two isoforms lacking inhibitory activity ( Nakagawa, 1997 ). The isoforms with inhibitory activity were found to contain γ-carboxyglutamic acid residues that were lacking in the isoforms isolated from stone formers.

Tamm-Horsfall protein is expressed by renal epithelial cells in the thick ascending limb and the distal convoluted tubule as a membrane-anchored protein that is released into the urine after cleavage of the anchoring site by phospholipases or proteases. Tamm-Horsfall is the most abundant protein found in the urine and a potent inhibitor of calcium oxalate monohydrate crystal aggregation, but not growth. The role of Tamm-Horsfall as an inhibitor, promoter, or bystander is controversial and may depend on the state of the molecule itself, which can self-aggregate under certain conditions. A recent study using a Tamm-Horsfall (THP) knockout mouse model demonstrated spontaneous formation of calcium oxalate crystals in the kidneys of mice fed ethylene glycol and vitamin D, suggesting a protective role of Tamm-Horsfall protein against crystallization of calcium salts ( Mo et al, 2004 ).

Osteopontin, or uropontin, is an acidic phosphorylated glycoprotein expressed in bone matrix and renal epithelial cells of the ascending limb of the loop of Henle and the distal tubule.

Osteopontin has been shown to inhibit nucleation, growth, and aggregation of calcium oxalate crystals as well as to reduce binding of crystals to renal epithelial cells in vitro ( Asplin et al, 1998 ; Wesson et al, 1998 ). In an osteopontin knockout mouse model, intratubular calcium oxalate crystals can be induced in mice exposed to high levels of oxalate by ethylene glycol feeding ( Wesson et al, 2003 ). Interestingly, in a THP knockout mouse model, mice

fed ethylene glycol and vitamin D exhibited a dramatic increase in osteopontin levels over baseline but still formed calcium oxalate crystals ( Mo et al, 2004 ). The authors concluded that osteopontin may constitute an inducible inhibitor of calcium oxalate crystallization that works in conjunction with constitutively expressed Tamm-Horsfall protein to prevent crystallization.

Lastly, inter-α-trypsin is a glycoprotein synthesized in the liver that is composed of three polypeptides (two heavy chains and one light chain), of which bikunin comprises the light chain. Bikunin is a strong inhibitor of calcium oxalate crystallization, aggregation, and growth in vitro ( Hochstrasser et al, 1984 ; Atmani et al, 1999 ), and its expression has been shown to be upregulated in a rat model when exposed to oxalate.

Matrix

Renal calculi consist of both crystalline and noncrystalline components. The noncrystalline component is termed matrix, which typically accounts for about 2.5% of the weight of the stone ( Boyce and Garvey, 1956 ). In some cases, matrix comprises the majority of the stone (up to 65%), usually in association with chronic urinary tract infection ( Boyce and Garvey, 1956 ; Allen and Spence, 1966 ). The exact composition of matrix is difficult to ascertain because only 25% of it is soluble ( Ryall, 1993 ); however, chemical analysis reveals a heterogeneous mixture consisting of 65% protein, 9% non-amino sugars, 5% glucosamine, 10% bound water, and 12% organic ash ( Boyce, 1968 ). Among the proteins incorporated into the matrix substance are Tamm-Horsfall protein, nephrocalcin, a γ-carboxyglutamic acid—rich protein, renal lithostathine, albumin, glycosaminoglycans, free carbohydrates, and a mucoprotein called matrix substance A ( Hess et al, 1996 ). Boyce and associates (1962) found that substance A is immunologically unique and present in the matrix component of all stone formers. Moore and Gowland (1975) determined that substance A is composed of three or four distinct antigens unique to stones that were detected in the urine of 85% of stone formers but in no normal individuals. The exact role of matrix in stone formation, whether as a promoter, an inhibitor, or a passive bystander has yet to be elucidated.

MINERAL METABOLISM Calcium

Thirty to 40 percent of dietary calcium is absorbed from the intestine, with most being absorbed in the small intestine and only approximately 10% absorbed in the colon ( Bronner et al, 1999 ). By a process of intestinal adaptation, absorption of calcium varies with calcium intake. At times of low calcium intake, fractional calcium absorption is enhanced; during high calcium intake, fractional calcium absorption is reduced. With a calcium-rich diet, a nonsaturable, paracellular pathway for calcium absorption predominates. A saturable, vitamin D—dependent transcellular pathway constitutes the major pathway for intestinal calcium absorption when calcium intake is limited; this pathway is downregulated by a diet replete in

calcium ( Buckley et al, 1980 ; Bronner et al, 1986 ). Because of the saturable component of calcium transport, a larger portion of calcium is absorbed when it is divided into several doses taken hours apart compared with a large single dose ( Phang et al, 1968 ). A small amount of calcium is secreted into the lumen of the intestine, thereby reducing net calcium absorption, such that, overall, 100 to 300 mg of a total average calcium intake of 600 to 1200 mg daily will be absorbed.

Calcium is absorbed in the ionic state, and incomplete calcium absorption is due in part to formation of soluble calcium complexes in the intestinal lumen. As such, substances that complex calcium such as phosphate, citrate, oxalate, sulfate, and fatty acids reduce the availability of ionic calcium for absorption ( Allen, 1982 ). Calcium readily complexes with phosphate in the intestinal lumen, but because calcium phosphate formation is dependent on pH (pK = 6.1), high luminal pH favors calcium phosphate complexation, thereby reducing calcium availability. On the other hand, calcium oxalate complex formation displays less pH dependence and complex formation is less reversible. Consequently, an oxalate-rich diet reduces calcium absorption. Transcellular calcium absorption is mediated by 1,25(OH)2D3 (calcitriol), which is reported to enhance calcium permeability at the brush border of the intestinal epithelial cells ( Fontaine et al, 1981 ).

The active form of vitamin D, 1,25(OH)2D3, is the most potent stimulator of intestinal calcium absorption. After conversion of 7-dehydrocholesterol in the skin to previtamin D3

promoted by sunlight, previtamin D3 is hydroxylated in the liver to 25-hydroxyvitamin D3, which is further hydroxylated in the proximal renal tubule to 1,25(OH)2D3. The conversion of 25-hydroxyvitamin D3 to 1,25(OH)2D3 is stimulated by parathyroid hormone (PTH) and by hypophosphatemia. A decrease in serum calcium increases secretion of PTH, which in turn directly stimulates the enzyme 1α-hydroxylase, which is located in the mitochondria of the proximal renal tubule. After transport via the bloodstream to the intestine, 1,25(OH)2D3, binds to the vitamin D receptor in the brush border membrane epithelial cells to enhance calcium absorption.

Calcitriol also acts on the bone and kidney in addition to its action in increasing calcium absorption from the intestine. In the bone, 1,25(OH)2D3, along with PTH, promotes the recruitment and differentiation of osteoclasts that subsequently mobilize calcium from the bone. Consequently, the filtered load of calcium and phosphate increases. However, PTH increases renal calcium reabsorption and enhances phosphate excretion, leading to a net increase in serum calcium, which ultimately suppresses further PTH secretion and synthesis of 1,25(OH)2D3.

PTH is critical in maintaining normal concentration of calcium in the extracellular fluid. PTH is an 84 amino acid protein that is the cleavage product of the precursor protein preproPTH. Only mature PTH is secreted from the parathyroid gland, and the most potent stimulus for its secretion is a decrease in serum calcium ( Sherwood et al, 1968 ). PTH stimulates mobilization of calcium from bone through the action of osteoclasts, further raising serum calcium and phosphorus. The action of PTH is mediated through changes in cyclic adenosine monophosphate (AMP) and phospholipase C ( Dunlay et al, 1990 ; Muff et al, 1992 ). At the kidney, PTH enhances renal calcium reabsorption and reduces renal tubular reabsorption of phosphate. It also stimulates synthesis of 1,25(OH)2D3, which leads to enhanced intestinal calcium and phosphate absorption. PTH has no direct effect on intestinal calcium absorption.

Phosphorus

Like calcium, inorganic phosphate absorption is dependent on both saturable, transcellular and nonsaturable, paracellular transport. At low phosphorus concentrations (1 to 3 mmol/L), saturable, absorptive transport occurs. At higher phosphorus levels, absorption increases without saturation ( Walton et al, 1979 ). Approximately 60% of the phosphate in the diet is absorbed by the intestine. Active absorption of phosphate from the intestine involves a 1,25(OH)2D3-regulated, sodium-dependent transport process ( Danisi et al, 1980 ; Lee et al, 1986 ). Phosphate absorption is highly pH dependent; low luminal pH reduces phosphate transport, whereas high luminal pH enhances phosphate transport.

Approximately 65% of absorbed phosphate is excreted by the kidney and the remainder by the intestine. In normal healthy adults, 80% to 90% of the filtered load of phosphate is reabsorbed by the renal tubules and 10% to 20% is excreted in the urine. Regulation of renal phosphate handling is primarily by way of PTH, which inhibits renal tubular reabsorption of filtered phosphate.

Magnesium

Magnesium is absorbed from the intestine by passive dif-fusion or active transport, although passive diffusion accounts for most of the net magnesium absorption. Magnesium is absorbed in both the large and small intestine, with the majority absorbed from the distal small intestine. Hormonal regulation of magnesium is primarily through vitamin D.

Oxalate

Oxalate metabolism differs markedly from calcium metabolism. Whereas 30% to 40% of ingested calcium is absorbed from the intestine, only 6% to 14% of ingested oxalate is absorbed ( Holmes et al, 1995 ; Hesse et al, 1999 ). Oxalate absorption occurs throughout the intestinal tract, with about half occurring in the small intestine and half in the colon ( Holmes et al, 1995 ). Although oxalate absorption is difficult to measure directly, it has been estimated by urinary oxalate excretion. A recent study suggested that hyperoxaluric stone formers absorb more oxalate in response to an oral oxalate load than stone formers with normal oxalate excretion ( Krishnamurthy et al, 2003 ). Likewise, in patients with small bowel disease or intestinal resection and an intact colon, oxalate absorption is markedly increased ( Barilla et al, 1978a ). Historically, the contribution of dietary oxalate to urinary oxalate was presumed to be no greater than 20%. However, Holmes and colleagues (2001) demonstrated that the relationship between ingested oxalate and urinary oxalate is curvilinear, owing to higher absorption of oxalate at low intake compared with high intake.

A number of factors can influence oxalate absorp-tion, including the presence of oxalate-binding cations such as calcium or magnesium and oxalate-degrading bacteria. Co-ingestion of calcium and oxalate-containing foods leads to formation of a calcium oxalate complex, which limits the availability of free oxalate ion for absorption ( Liebman and Chai, 1997 ; Hess et al, 1998 ). Oxalate-degrading bacteria, notably Oxalobacter formigenes, utilize oxalate as an energy source and consequently reduce intestinal oxalate absorption. Recent studies have demonstrated that stone formers have reduced levels or absent colonization with Oxalobacter compared with non—stone-forming control subjects, and those individuals lacking the bacteria have higher urinary oxalate levels ( Sidhu et al, 1999 ; Mikami et al,

2003 ; Troxel et al, 2003 ). The contribution of Oxalobacter to the overall risk of stone formation is not fully understood at this time.

Absorbed oxalate is nearly completely excreted in the urine ( Hodgkinson et al, 1974 ; Prenan et al, 1982 ). However, up to 80% of urinary oxalate is derived from endogenous production in the liver (40% from ascorbic acid, 40% from glycine) and 20% or more is derived from dietary sources. It is estimated that greater than 98% of oxalate is filterable, but oxalate reabsorption is negligible. However, there is evidence from a number of animal models of a secretory pathway for oxalate that likely resides in the proximal renal tubules ( Cattell et al, 1962 ; Williams et al, 1971 ; Knight et al, 1981 ; Tremaine et al, 1985 ).

PATHOGENESIS OF UPPER URINARY TRACT CALCULI Classification of Nephrolithiasis

The most common component of urinary calculi is calcium, which is a major constituent in nearly 75% of stones. Calcium oxalate makes up about 60% of all stones; mixed calcium oxalate and hydroxyapatite, 20%; and brushite stones, 2%. Both uric acid and struvite (magnesium ammonium phosphate) stones occur approximately 10% of the time, whereas cystine stones are rare (1%) ( Table 42-1 ) ( Wilson, 1989 ). Stones associated with medications and their by-products such as triamterene, adenosine, silica, indinavir, and ephedrine are very uncommon and usually preventable.

Most classification systems for nephrolithiasis differentiate stones on the basis of the underlying metabolic or environmental abnormalities with which they are associated ( Table 42-2 ). A number of pathophysiologic derangements contribute to calcium stone formation either alone or in combination, including hypercalciuria ( Zerwekh et al, 1988 ), hypocitraturia ( Pak and Fuller, 1986 ; Pak, 1987 ; Coe et al, 1992 ; Bushinsky, 1998 ), hyperuricosuria, and hyperoxaluria. Uric acid, cystine, and struvite stones form in relatively unique settings: uric acid stones form only in an acid urine ( Halabe et al, 1994 ; Asplin, 1996 ), cystine stones are the result of impaired renal reabsorption of cystine ( Marshall et al, 1976 ; Stephens, 1989 ; Joly et al, 1999 ; Nakagawa et al, 2000 ), and infection stones occur in an alkaline urine produced by urease-producing bacteria ( Marshall et al, 1976 ). For some stones, such as cystine, knowledge of the chemical composition of the stone may provide sufficient information to initiate appropriate therapy. However, due to the multiple causes associated with calcium-based stones, an understanding of the underlying metabolic disorders and environmental factors that predispose to stone formation is required to implement a rational treatment plan ( Wall et al, 1988 ; Wall and Tiselius, 1990 ). Recent investigation into the molecular and genetic causes of stone formation may ultimately translate into newer treatment strategies ( Frick et al, 2003 ; Langman, 2004 ).