Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation

Kidney International, Vol. 66 (2004), pp. 1159–1166

Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation LAN MO, HONG-YING HUANG, XIN-HUA ZHU, ELLEN SHAPIRO, DAVID L. HASTY, and XUE-RU WU Department of Urology, Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York; Department of Microbiology, Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York; Veterans Affairs Medical Center, New York, New York; Department of Anatomy and Neurobiology, University of Tennessee, Memphis, Tennessee; and Veterans Affairs Medical Center, Memphis, Tennessee

Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation. Background. The tubular fluid of the mammalian kidney is often supersaturated with mineral salts, but crystallization rarely occurs under normal conditions. The unique ability of the kidney to avoid harmful crystal formation has long been attributed to the inhibitory activity of the urinary macromolecules, although few in vivo studies have been carried out to examine this hypothesis. Here we examined the role of Tamm-Horsfall protein (THP), the principal urinary protein, in urinary defense against renal calcium crystal formation, using a THP knockout model that we recently developed. Methods. Wild-type and THP knockout mice were examined for the spontaneous formation of renal calcium crystals using von Kossa staining. The susceptibility of these mice to experimentally induced renal crystal formation was evaluated by administering mice with ethylene glycol, a precursor of oxalate, and vitamin D 3 , which increases calcium absorption. Renal calcium crystals were visualized by von Kossa stain, dark field microscopy with polarized light and scanning electron microscopy. Results. Inactivating the THP gene in mouse embryonic stem cells results in spontaneous formation of calcium crystals in adult kidneys. Excessive intake of calcium and oxalate, precursors of the most common type of human renal stones, dramatically increases both the frequency and the severity of renal calcium crystal formation in THP-deficient, but not in wild-type mice. Under high calcium/oxalate conditions, the absence of THP triggers a marked, adaptive induction in renal epithelial cells of osteopontin (OPN), a potent inhibitor of bone mineralization and vascular calcification. Thus, OPN may serve as an inducible inhibitor of calcium crystallization, whereas THP can serve as a constitutive and apparently more effective inhibitor. Conclusion. These results provide the first in vivo evidence that THP is a critical urinary defense factor and suggest that its deficiency could be an important contributing factor in human nephrolithiasis, a condition afflicting tens of millions of people in the world annually. Key words: Tamm-Horsfall protein, uromodulin, knockout, calcium oxalate stones, nephrolithiasis, osteopontin, macromolecules. Received for publication February 24, 2004 and in revised form March 18, 2004 Accepted for publication April 7, 2004
C

2004 by the International Society of Nephrology

Renal stone disease or nephrolithiasis is an exceedingly common clinical condition in the industrialized world. Up to 15% of white males and 6% of all females will have at least one renal stone during their lifetime, and half of these individuals will experience recurrence [1, 2]. In the United States, the disease afflicts more than 1 million people annually and accounts for nearly 10 of every 1000 hospital admissions. The incidence of this disease appears to be on the rise during recent decades, although the exact cause for this increase is unknown. Among the various types of renal stones, those composed of calcium oxalate are by far the most prevalent, accounting for 75% of all stones [3]. Renal stone formation is believed to be contingent upon two critical factors. The first is the state of supersaturation in which there is an overabundance of stoneforming ions, exceeding the metastable limit [1, 2, 4]. If unopposed, supersaturation can lead to crystallization, triggering a series of pathophysiologic events that include nucleation, crystal aggregation, growth, and attachment to epithelia [5, 6]. The second critical factor is the presence of urinary macromolecules that can exert inhibitory effects on various phases of renal crystal formation [7, 8]. It has been suggested that the level and functional status of specific urinary macromolecules is an important contributing factor in individual susceptibility to renal stones [4, 7]. Two notable candidates for inhibition of crystal formation are osteopontin (OPN) and Tamm-Horsfall protein (THP), also known as uromodulin. OPN is an acidic phosphorylated glycoprotein initially isolated from bone matrix. It is believed to play a role in modulating mineralization of normal bone by enhancing osteoclast activity and halting hydroxyapatite crystal growth [9]. Targeted deletion of the murine OPN gene did not result in a mineralization defect, but it enhanced vascular calcification in mice also deficient for the extracellular matrix Gla protein, suggesting that these two proteins can act in concert to inhibit ectopic calcification [10]. In addition to being expressed in bone, OPN is also 1159

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expressed by normal renal epithelial cells, particularly cells of the ascending limb of Henle’s loop and the distal convoluted tubules, and it is secreted into the urine at about 4 mg per day [11, 12]. Evidence supporting a role for OPN as an inhibitor of renal stone formation includes the observations that: OPN is a major component of human renal stones [13]; purified OPN inhibits the nucleation, growth, and aggregation of calcium oxalate crystals in vitro and reduces the binding of the crystals to renal epithelial cells in vitro [8]; OPN is up-regulated in renal tubules upon overexposure to calcium oxalate in experimental models [14]; and OPN knockout mice develop experimentally induced renal calcium oxalate crystals after exposure to high levels of oxalate, but not under normal conditions [15, 16]. THP is expressed by the same kidney epithelial cells that express OPN, but unlike OPN, THP is kidneyspecific [17, 18]. THP is initially synthesized as a glycosylphosphatidylinositol (GPI)–anchored membrane protein and released into the urine upon the cleavage of the anchorage by phospholipases or proteases [19–21]. Released THP is the most abundant protein found in urine of all placental animals, amounting to 100 mg daily in humans. Because of its urinary abundance and its consistent presence in human renal stones, it has been proposed that THP may play a role in regulating stone formation. However, in vivo evidence is lacking and controversy persists as to whether THP is a promoter, inhibitor, or innocent bystander during stone formation [1, 22, 23]. In this study, we investigated the in vivo role of THP as a host defense factor against renal crystal formation using THP knockout mice that we recently generated [24]. METHODS THP knockout and wild-type mice Inactivation of murine THP gene was achieved by the deletion of a THP gene fragment containing the proximal promoter as well as exons 1 to 4, through homologous recombination in embryonic stem cells [24]. Mice homozygous for the mutated THP allele lacked renal expression of THP mRNA by both Northern blotting and reverse transcription-polymerase chain reaction (RTPCR) analyses, and did not synthesize any THP protein as demonstrated by Western blotting of the total kidney and urinary proteins and by immunohistochemical staining. Both THP knockout and wild-type control mice were bred continuously in a 129/SvEv genetic background. Animals in the sixth and seventh generations were used in the current study. The genotype of the experimental animals was confirmed by Southern blotting of EcoRI-digested tail DNA using a targeting event–specific probe. All animal studies were conducted in accordance with National Institutes of Health (NIH) guidelines for the use and care of laboratory animals and under an active protocol ap-

proved by the investigators’ institute’s animal care and use committee. Detection of renal calcium crystals Spontaneous formation of renal calcium crystals in THP knockout mice (2 to 4 months old) was assessed by von Kossa histochemical staining, which specifically reacts with calcium deposits in the tissue [25]. Briefly, 4 l thick sagittal sections of paraffin-embedded mouse kidneys were deparaffinized, hydrated, and exposed to 2% silver nitrate under bright light for 60 minutes. After washing in 5% sodium thiosulfate for 5 minutes, the sections were counterstained with neutral red and examined under a light microscope. Alternatively, the paraffin sections were stained with hemotoxylin and eosin, followed by microscopic visualization of the birefringent crystals under dark field illumination with polarized light (Axioplan II microscope) (Zeiss, Oberkochen, Germany). Experimental induction of renal calcium crystal formation Adult male THP knockout and wild-type mice (both 2 to 4 months of age) were administered 1% ethylene glycol (Sigma Chemical Co., St. Louis, MO, USA) and 4 IU/mL Vitamin D 3 (Twin Laboratories, Inc., Hauppange, NY, USA) in the drinking water continuously for 1 month. Treated mice were then sacrificed and their kidneys processed for either histochemical or biochemical analyses. Scanning electron microscopy and x-ray elemental analysis of renal crystals Five 10 lm thick paraffin sections of kidney were floated on a warm water bath and mounted on carbon planchets. Sections were deparaffinized while they were on the planchet. Uncoated specimens were analyzed by scanning electron microscopy with a Philips XL30 ESEM microscope (FEI Philips, Boston, MA, USA) in the backscatter electron mode. Dense materials were easily detected in the backscatter electron (BSE) mode because they were much brighter than surrounding tissue. Elemental spectra of crystals were determined by energy dispersive x-ray analysis (Edax Corp., Mahwah, NJ, USA). RNA and protein analyses For the assessment of renal expression of OPN and THP under various normal and experimental conditions, total RNA was isolated using an RNA extraction kit (Promega, Madison, WI, USA). Fifteen micrograms of the total RNA were resolved by formaldehyde-agarose gel, transferred onto a nylon membrane, and hybridized with cDNA probes specific for mouse OPN and mouse THP cDNA probe. After autoradiography, the nylon

Mo et al: Tamm-Horsfall protein in renal calcium crystal formation

membrane was heat-stripped and rehybridized with a bactin probe. For the assessment of protein expression, fresh mouse kidney tissues were directly homogenized in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) loading buffer. Solubilized total kidney proteins were electrophoresed, transferred onto an immobilonpolyvinylidine difluoride (PVDF) membrane and reacted with an anti-OPN antibody (Assay Designs, Inc., Ann Arbor, MI, USA). The reaction was visualized by an enhanced chemiluminescence (ECL) detection system (Amersham, Piscataway, NJ, USA). Immunoreaction to mitogen-activated protein kinase (MAPK) (New England Biolabs, Inc., Beverly, MA, USA) was used as a loading control.

Immunohistochemistry Freshly dissected kidney tissues were fixed in 10% buffered formalin and processed routinely for paraffin embedding. Four micron tissue sections were microwaved in a citrate buffer (pH 6.0) for 15 minutes for antigen retrieval and were then stained with the anti-OPN antibodies.

Statistical analysis Differences in spontaneous and experimentally induced renal crystal formation were statistically analyzed using the chi-square (v 2 ) test. P values < 0.05 were considered significant.

RESULTS Spontaneous formation of renal calcium crystals in THP knockout mice Our laboratory recently generated transgenic mice lacking the THP gene. The mice were completely lacking the THP message and THP protein [24]. The availability of these transgenic mice provided the first opportunity for us to examine the hypothesis that THP plays a role in inhibiting renal calcium crystal formation in vivo. When we stained kidney sections of the THP knockouts using von Kossa stain, which specifically reacts with calcium deposits, we observed dark, dense intratubular crystal aggregates (Fig. 1B to D). These crystals were located primarily in the collecting ducts in deep medulla and renal papillae, with some of the crystals clearly attached to the luminal surfaces of the tubular epithelial cells (Fig. 1B, inset, and D). Four out of 25 THP knockout mice examined, but none out of 25 wild-type littermates, harbored significant numbers of renal crystals (P < 0.05) (Table 1) strongly suggesting that renal calcium crystal formation was a direct result of THP deficiency.

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THP deficiency dramatically increased susceptibility of mice to experimentally induced renal crystal formation To test whether the absence of THP also reduces the ability of the urinary system to respond to mineral overload, we fed THP knockout and wild-type littermates in the drinking water with ethylene glycol, an oxalate precursor, and vitamin D 3 , which enhances calcium absorption and produces a hypercalciuric condition. Wild-type mice remained completely crystal-free under high calcium/high oxalate conditions (Fig. 2A and E) (Table 1), but THP knockout mice developed large numbers of renal crystals that were both reactive with silver nitrate of the von Kossa solution (Fig. 2B to D) and were distinctively light-reflective upon dark field microscopy with polarized light illumination (Fig. 2F). The outer medulla of the kidney, a region in which ascending limbs of Henle’s loop are concentrated and where THP is normally expressed (Fig. 2B, boxed area), harbored the greatest number of crystals. In addition, crystals were found in the collecting ducts of the deep medulla and renal papillae. Intratubular crystals were also seen in tissue sections analyzed by scanning electron microscopy in the backscatter electron mode (Fig. 3A and B). Elemental analysis determined by energy dispersive x-ray analysis (Edax) showed that the crystals consisted primarily of calcium, oxygen, and carbon, with little to no phosphate (Fig. 3C and 3D), strongly suggesting that the crystals were of calcium oxalate in nature. Overall, 13 of 17 knockout mice that had been pretreated with ethylene glycol and vitamin D 3 developed renal calcium crystals. In contrast, none of the 16 similarly treated wild-type littermates developed crystals (P < 0.001) (Table 1). These results indicate that THP is a critical inhibitor of calcium crystal formation and that its deficiency predisposes the urinary system to developing calcium crystals. Compensatory induction of OPN in THP-deficient mice Because both OPN and THP are expressed by the ascending limb of Henle’s loop and both exhibit calcium crystal inhibition activity, we wished to know whether the lack of THP would provoke an adaptive increase of OPN. Exposure of wild-type mice to calcium and oxalate induced the expression of OPN RNA and protein over baseline levels, albeit to a moderate degree (Fig. 4). In the absence of a calcium/oxalate overload, THP deficiency did not induce OPN expression over baseline levels (Fig. 4A), but calcium/oxalate treatment dramatically induced OPN expression in THP knockout mice (Fig. 4). Virtually all renal tubules, including those normally expressing no OPN (Fig. 5A to C), expressed large amounts of OPN (Fig. 5D to F). Even nontubular structures, such as Bowman’s capsules, were seen to express OPN (Fig. 5D, inset). Induction of OPN was also seen in renal papillary epithelium (Fig. 5F), which only expresses

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A

B

C

D

E

Fig. 1. Formation of spontaneous calcium crystals in murine kidneys of Tamm-Horsfall protein (THP) absent mice. Four micron paraffin sections from kidneys of wild-type (A) and THP knockout mice (B to D) were subjected to the von Kossa stain, which specifically detects calcium deposits, and were counterstained by neutral red. There were no reactions with the von Kossa stain in kidneys of wild-type mice. In the kidneys of knockout mice, intensely stained, dark-colored calcium crystals (arrows) were detected within the lumen (inset) of the kidney tubules in the renal papilla (B) and deep medulla (C and D) [magnifications are 50× (A and B); 150× (C and D); 200 × (inset)].

small amounts of OPN under normal conditions (Fig. 5C). The fact that there was a significantly greater induction of OPN, upon calcium/oxalate overload, in THPdeficient mice than in wild-type mice further supports the role of OPN as an inducible crystal inhibitor [16], and suggests that under normal conditions THP and OPN act synergistically in preventing urinary stone formation. It should be pointed out, however, that in the absence of THP, OPN induction is insufficient to suppress renal crystal formation. In contrast to OPN, THP remains unchanged during calcium/oxalate overload in wild-type mice (Fig. 4A), suggesting that THP, which is produced in 25-fold greater amounts than OPN, acts as a constitutive inhibitor. DISCUSSION A central functional requirement in the maintenance of renal homeostasis is dealing effectively with the supersaturation of urine by mineral salts. Clearly a powerful set of

F

G

Fig. 2. Experimentally induced renal calcium crystal formation. Wildtype and Tamm-Horsfall protein (THP) knockout mice were fed 1% ethylene glycol and 4 IU/mL Vitamin D 3 in the drinking water for 1 month. The kidney sections were stained either with von Kossa stain and viewed by light microscopy (A to D) or they were stained with hematoxylin and eosin (E to G) and viewed under dark field with polarized light illumination (E and F) or by light microcopy (G). Note that the THP knockout mice (B to D, F and G) developed numerous renal stones that were distinctively reactive with von Kossa stain (B, boxed area, C and D, arrowheads) and that exhibited strong birefringence in dark field (F, arrowheads). Wild-type mice were completely unaffected (A, boxed area, and E). The induced calcium crystals are concentrated at the outer medullary zone where THP is normally synthesized (B, boxed area). (F and G) The same sections viewed with dark field or bright field microscopy [magnifications are 50× (A and B); 400× (C to G)].

Table 1. Renal calcium crystal formation in wild-type and Tamm-Horsfall protein (THP)–deficient mice Without Ca2+ overload

With Ca2+ overload

Wild-type

Knockout

Wild-type

0/25 (0%)

4/25 (16%) P < 0.05

0/16 (0%)

Knockout 13/17 (76%) P < 0.001

defense mechanisms is required to keep the urinary tract free of harmful stones, but these mechanisms have been only partially defined to date. By inactivating the murine THP gene, we have provided the first in vivo evidence that THP deficiency can result in renal calcium crystal formation. These results, in conjunction with our recent finding that THP knockout mice are prone to urinary tract infections by type 1 fimbriated Escherichia coli [24], indicate that THP, a kidney-specific glycoprotein, serves as a critical first line of defense protecting against urinary tract colonization by bacterial pathogens and formation of renal stones. The results also suggest that a deficiency

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A

B

A THP +/+ +/+ +/+ +/+ +/+ −/− −/− −/− −/− −/− −/− EG/D3 − − + + + − − − + + + OPN

Ca

C

THP

Ca

D C

Actin

O Ca

P

3.50 4.00

1.00

Na

3.00

Ca

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

P

0.50

O

1.50 2.00 2.50

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1

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3

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9 10 11

B THP +/+ +/+ −/− −/− −/− −/− −/− EG/D3 − + − + + + +

Fig. 3. Scanning electron microscopic and x-ray elemental analyses of experimentally induced renal crystals. (A) Scanning electron microscopy showing a 25 lm × 25 lm intraluminal crystal aggregate composed primarily of calcium (Ca), carbon (C) and oxygen (O) with very little phosphate (P). (C) This profile strongly suggests that the crystals are of calcium oxalate type. (B) An independent renal section showing several smaller crystal aggregates that contained similar chemical components (D).

OPN

MAPK

of this major urinary protein may predispose humans to urinary stone diseases and urinary tract infections. Our results also provide experimental evidence suggesting that, under some circumstances, THP and OPN can function together to keep renal stones from forming and are not merely redundant systems. OPN-deficient mice, which presumably expressed normal amounts of THP, did not develop renal calcium crystals unless subjected to oxalate overload [15, 16]. Thus, under normal conditions, THP is apparently a sufficient inhibitor. In contrast, significant numbers of THP-deficient mice developed renal calcium crystals even when drinking water. Increased calcium/oxalate uptake by wild-type mice, which did not develop any renal calcium crystals, only elicited a slight increase of OPN, suggesting that constitutively expressed THP and slightly induced OPN can sufficiently counter the adverse effects of mineral overload. In striking contrast, increased calcium/oxalate uptake by THP-deficient mice led to dramatically increased expression of OPN, even stimulating OPN production by renal cell types heretofore not known to produce this glycoprotein. However, this compensatory increase was not sufficient to keep calcium crystals from forming. While our data suggest that THP is the more potent of these two inhibitors, it is also not sufficient to keep calcium crystals from forming when animals are exposed to increased levels of oxalate, as occurring in OPN-deficient

1

2

3

4

5

6

7

Fig. 4. Induction of osteopontin (OPN) in Tamm-Horsfall (THP) knockout mice. Wild-type and THP knockout mice were fed with regular drinking water or that supplemented with ethylene glycol (EG) and vitamin D 3 (D3). (A) Total RNA was extracted from the whole kidneys, electrophoresed, transferred to a nylon membrane, and hybridized with a mouse OPN cDNA probe. Following the striping of the probe, the same membrane was hybridized with a mouse THP probe followed by a b-actin probe. Note that EG/D3 treatment led to a moderate induction of OPN in wild-type mice (lanes 3 to 5) and a strong induction of OPN in THP knockout mice (lanes 9 to 11), but that THP knockout without EG/D3 treatment did not induce OPN expression (lanes 6 to 8). Also note that EG/D3 treatment failed to induce THP in wild-type mice (lanes 3 to 5). (B) Total kidney proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with an anti-OPN antibody followed by an antibody against mitogen activated protein kinase (MAPK), which was used to normalize protein loading. Note that EG/D3 treatment triggers a slight increase of OPN protein in wild-type mice and a marked increase of OPN in THP knockout mice.

mice [16]. Thus, it appears that both OPN and THP are required to maintain a healthy kidney. The precise mechanism by which THP and OPN exert their inhibitory effects on calcium crystal formation is under intensive investigation, but it appears that direct binding between these macromolecules and calcium ions or calcium crystals may be crucial. Both proteins contain calcium-binding motifs, with OPN having one and THP having two in its epidermal growth factor (EGF)like repeats [12] [Mo and Wu, unpublished observations,

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A

D

B

E

C

F

Fig. 5. Immunolocalization of osteopontin (OPN) in wild-type (A to C) and knockout (D to F) mouse kidneys. Sections of wild-type and THP knockout mouse kidneys were immunohistochemically stained with an anti-OPN antibody. Note that while OPN expression is normally at low levels and is restricted to thick-ascending limb of Henle’s loop (A), outer medulla (B), and papillary epithelium (C), its expression is dramatically induced in almost all tubular epithelial cells in THP knockout mice pretreated with ethylene glycol (EG)/vitamin D 3 (D3) (D to F), as well as in the epithelium of Bowman’s capsule (magnifications are 200× for all panels).

2004]. Alternatively, THP and OPN could bind to calcium via their abundant negatively charged groups. THP is heavily sialylated, whereas OPN is both sialylated and highly phosphorylated at its clusters of serine and threonine residues [26, 27]. There may be multiple consequences from the binding of these macromolecules to calcium, such as (1) reducing the intratubular calcium concentration to below the threshold of crystallization; (2) inhibiting calcium crystal aggregation and growth; and (3) blocking crystals from adhering to the renal epithelial cells, a critical step in stone retention [5]. It has been established that THP is a GPI-linked surface protein covering the luminal membrane of the thick ascending limb of Henle’s loop [19]. While OPN is a secretory protein, it is also found on the membrane surface, particularly that of the renal papillary epithelium, presumably through the interaction of OPN’s arginine-glycline-aspartate (RGD) sequence with surface membrane receptors (Fig. 5) [9, 13]. It is conceivable, therefore, that both proteins may act in certain ways to prevent crystal-epithelial interaction. Alternatively, the crystal inhibitory activity of OPN and THP may be indirect. It is known that ethylene glycol, which has been widely used to induce renal stone formation in rats, causes renal tubular cell injury [28]. It cannot be ruled out, therefore, that THP is involved in modulating renal cell damage, necrosis and apoptosis, thus indirectly preventing calcium crystal formation. Regard-

less of whether the role of THP is direct or indirect, the fact that ethylene glycol and vitamin D 3 treatment causes severe calcium crystal formation in THP knockout mice, but none in wild-type mice, unequivocally demonstrates THP as a potent inhibitor of calcium crystal formation. Naturally occurring mutations of the THP gene have recently been linked to familial juvenile hyperuricemic nephropathy and medullary cystic kidney disease 2 [29]. It has been shown that the mutations lead to misfolding and faulty delivery of THP to the luminal surface, resulting in lower-than-normal urinary THP level [30, 31]. Of note, renal stone diseases have not been described as a common feature, at least in the reported patient families [32]. It is possible that the limited number of patients do not have other stone-forming risk factors. It is also possible that gene ablation and gene mutations can have different biologic consequences on renal physiology, thus presenting different phenotypes. Further investigation is needed to clarify these issues. Renal stone disease and urinary tract infection are both exceedingly common conditions in humans. More than 10% of the United States population will suffer from a kidney stone during their lifetime. Approximately 1 million cases of renal stones were diagnosed in 1996 and the incidence has been rising in recent years due to as yet unknown reasons [33]. Urinary tract infection is the second most common infectious disease in developed countries,

Mo et al: Tamm-Horsfall protein in renal calcium crystal formation

afflicting 150 million people annually on a global basis [34]. Both diseases occur in otherwise healthy individuals and are frequently recurrent, raising the possibility that deficiency in innate defenses of the urinary tract plays critical roles in an individual’s susceptibility to these disease conditions. By ablating the murine THP gene, we have provided the first in vivo evidence clearly establishing that THP is on the first line of host defenses against both renal stone formation and bacterial infection [24]. These results from mice may also reflect conditions in humans because quantitative and qualitative defects of THP have been frequently observed in human diseases. For instance, reduced urinary excretion of THP has been associated with recurrent kidney stone formers in several cohorts [35, 36]. In addition, insufficient sialylation of THP has been shown to cause a functional defect in the inhibition of stone formation in vitro [37, 38]. THP abnormalities have also been found in type I diabetics, who frequently have either a profound reduction in urinary THP or an altered THP glycosylation pattern [39, 40]. Since diabetics are prone to developing urinary tract infections, it would be interesting to determine whether a THP defect contributes to their propensity for urinary tract infections. The majority of recurrent urinary tract infections occur in anatomically normal individuals. Our studies draw attention to the possibility that physiologic defects in the THP component of innate urinary tract defenses may contribute to the predisposition of some individuals to recurrent urinary tract infections. Finally, it is known that recurrent urinary tract infections are associated with an increased risk of renal stone formation [41]. Our findings raise the possibility that a THP defect provides a common link between these two seemingly unrelated diseases.

ACKNOWLEDGMENTS This work was supported in part by grants from National Institutes of Health (DK56903) to X.-R. Wu, (AI42886) to D.L. Hasty, and a Merit Review award from the Veterans Administration’s Medical Research Service to X.-R. Wu. Repint requests to Xue-Ru Wu, M.D., Department of Urology, New York University School of Medicine, 423 East 23 Street, 18th Floor, Room 18064 South, New York, New York 10010 E-mail: [email protected]

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