Physical, X-ray diffraction and scanning electron microscopic studies of uroliths

Identification of chemical constituents of calculus is important in the diagnosis and management of urolithiasis. The compositional variability of uroliths has different etiologies and requires various modes of treatment and prophylaxis. In the present study, we report the chemical compositional analyses of calculi recovered from buck and bullock by X-ray diffraction (XRD) and energy dispersive X-ray (EDX) techniques and ultra-structure examination by scanning electron microscopy (SEM). XRD and EDX investigations conclusively established the chemical compositions of urinary calculi under investigation. The calculus from buck (sample I) had calcium oxalate monohydrate, a dominant salt phase and magnesium compound in significant amount. The calculus from bullock (sample II) had magnesium ammonium phosphate phase, with significant amount of calcium in apatite form and K+ ions. SEM study at higher magnification (X 1000) showed bipyramidal crystals in external zones of urolith (sample I). The struvite apatite calculus showed that basic unit of structure was lamination and the laminitis appeared to be made up of fine granules and high porosity. The bio-mineralization process of calculus formation was also studied, with a view to take preventive and therapeutic measures for amelioration of urinary stone diseases in animals and humans.     

Struvite calculus in the vagina of a bottlenose dolphin (Tursiops truncatus)

On 27 January 2000, a struvite calculus was observed in the vagina during necropsy of a 138-cm-long female bottlenose dolphin (Tursiops truncatus) collected from the Stono River, South Carolina (USA). Vaginal calculi have been reported in other species of cetaceans but not in bottlenose dolphins. Urinary tract infection might have been an underlying cause of the calculus. While urinary tract inflammation was not detected by light microscopic evaluation of sections of the urinary tract, it is conceivable that sufficient time had lapsed following voiding of the calculus through the urethra for urinary tract infection to have resolved. To further define the prevalence and significance of urolithiasis, prosectors of dead stranded marine mammals are encouraged to closely observe their urinary and genital tracts for calculi and to submit them for quantitative analysis.     

Asian small-clawed otter (Aonyx cinerea) urolithiasis prevalence in North America

The prevalence of urolithiasis in the North American Asian small-clawed otter (Aonyx cinerea) population was determined through a retrospective survey. A questionnaire regarding diet and radiographic or necropsy evidence of urolithiasis was sent to the 16 North American institutions or individuals listed in the Asian Small-Clawed Otter North American Regional Studbook. Completed forms were returned by 75% of questionnaire recipients. Individual surveys or necropsy reports were received for 79.8% of the living or dead animals listed as comprising the North American population. Renal calculi were detected in 66.1%, and cystic calculi in 23.2%, of the captive adult population that had been radiographed or necropsied. All otters with cystic calculi also had renal calculi. Bilateral renal calculi occurred in 83.8% of affected otters. Both males (61.2%) and females (72%) were frequently affected. The prevalence of urolithiasis in wild-born otters was 76.7%, and in adult (≥1 year) captive-born otters it was 53.8%. The higher prevalence in wild-born otters may be a reflection of the older mean age at which they were first evaluated. In the overwhelming majority of otters with renal calculi, the calculi were multiple and diffusely distributed throughout the renal parenchyma. Renal and cystic calculi analyzed were primarily composed of calcium oxalate or urates. Glucosuria was infrequently reported. Necropsies of dependent neonates (≤ 2 months old) that died revealed cystic calculi in one animal. Renal disease was the cause of, or a contributing factor in, the deaths of all older animals whose necropsy reports were reviewed. The captive diet may be a contributing factor to urolith formation and progression.     

Struvite Urolithiasis in Long–Evans Rats

Struvite urinary calculi, which are composed of magnesium, ammonium, and phosphate, can cause complications including sepsis and renal failure. Struvite calculi were identified within the urinary bladder and renal pelvis of 2 Long-Evans rats that died within days after arrival from a commercial vendor. The remaining rats in the shipment were screened by physical examination, radiography, and ultrasonography, revealing an additional 2 animals that were clinically affected. These rats were euthanized, necropsied, and yielded similar findings to those from the first 2 rats. In addition, urine samples had an alkaline pH and contained numerous bacteria (predominantly Proteus mirabilis), leukocytes, and crystals. All calculi were composed completely of struvite. Another 7 rats in the shipment had alkaline urine with the presence of blood cells; 6 of these rats also had abundant struvite crystals, and P. mirabilis was cultured from the urine of 3 rats. Further investigation by the vendor identified 2 of 100 rats with struvite calculi from the same colony. Although no specific cause could be implicated, the fact that all the affected rats came from the same breeding area suggests a genetic or environmental triggering event; a contribution due to diet cannot be ruled out. Our findings suggest that the affected rats had metabolic disturbances coupled with bacterial infection that predisposed them to develop struvite calculi. During sudden increases of struvite urinary calculi cases in rats, urine cultures followed by appropriate surgical intervention and antibiotic therapy is warranted. Additional factors, including diet, merit attention as well.Struvite, also known as triple phosphate, is a crystalline substance composed of magnesium ammonium phosphate (MgNH₄PO₄• 6 H₂0)¹⁰ that was first identified in the 18th century. This mineral is what consolidates into urinary calculi, or stones, both in humans and animals. Other frequently encountered types of urinary stones include calcium oxalate, calcium phosphate, uric acid, and cysteine.¹⁷ Although only 2% to 3% of stones from humans are composed of struvite,⁴³ struvite calculi are important clinically because they can lead to sepsis and renal failure.⁴³In both humans and animals, stones in the urinary tract can obstruct the urine outflow, with subsequent extreme pain, hydronephrosis, and (possibly) rupture of the urinary bladder. In addition, disruption of kidney function can lead to metabolic imbalances, such as uremia, seizures, depression, anorexia, dehydration, even coma and death.^1,8 Here we present a case study describing the spontaneous presentation of struvite urolithiasis in a recently imported cohort of rats and related significant clinical findings.     

Urogenital sinus calculi in a sand tiger shark (Odontaspis taurus)

Two calculi were found in the urogenital sinus of a 70 kg female sand tiger shark (Odontaspis taurus). The calculi were white in color, rough surfaced, and spherical in shape. Crystallographic examination revealed that they were composed of magnesium ammonium phosphate hexahydrate (80% struvite) and calcium phosphate (15% carbonate apatite). Approximately 5% of the stone matrix consisted of blood and protein and a distinct bacterial nidus was not present microscopically.     

Expression of the androgen receptor and 5α-reductase type 2 in the developing human fetal penis and urethra

Normal penile development is dependent on testosterone, its conversion via steroid 5 alpha-reductase type 2 to dihydrotestosterone, and a functional androgen receptor (AR). The goal of this study was to investigate the distribution of AR and 5 alpha-reductase type 2 in the developing human fetal external genitalia with special emphasis on urethra formation. Twenty fetal genital specimens from normal human males (12-20 weeks gestation) were sectioned serially and stained by avidin-biotinylated peroxidase complex method with antigen retrieval. Stained sections throughout male genital development documented the expression of AR and 5 alpha-reductase type 2 in the phallus. Between 12 and 14 weeks of gestation, AR was localized to epithelial cells of the urethral plate in the glans, the tubular urethra of the penile shaft, and stromal tissue surrounding the urethral epithelium. In the fetal penis between 16 and 20 weeks gestation, the density of AR expression was greatest in urethral epithelial cells versus the surrounding stromal tissues. There was a characteristic pattern of AR expression in the glandular urethral epithelium between 16 and 20 weeks gestation. AR expression was greater along the ventral aspect of the glandular urethra than along the dorsal aspect of the urethral epithelium. The expression of 5 alpha-reductase type 2 was localized to the stroma surrounding the urethra, especially along the urethral seam area in the ventral portion of the remodeling urethra. These anatomical studies support the hypothesis that androgens are essential for the formation of the ventral portion of the urethra and that abnormalities in either the AR or 5 alpha-reductase type 2 can explain the occurrence of hypospadias.     

Normal development of the male anterior urethra

A histological study was performed on serially sectioned human and mouse embryos to study the influences of programmed cell death (PCD) during morphogenesis for clarifying the existing controversies on the morphology and basic processes involved in the embryonic development of the male anterior urethra. The following new insights into the development of the anterior urethra could be established. The formation of the urethra starts with the early adhesion of the arms of the genital tubercle. In this way an epithelial plate is formed, located in the ventral midline, that is in continuity with the cloacal membrane. Male sex differentiation takes place following rupture of this cloacal membrane through programmed cell death. Fusion of the urogenital swellings with primary luminization gives rise to the penile urethra, whereas the glandular part of the urethra is formed through secondary luminization of the epithelial cord that is formed during fusion of the arms of the genital tubercle, i.e., the glans. In both fusion processes, apoptosis plays a key role. The consequence of fusion of the urogenital swellings is that their mesodermal cores unite on the ventral aspect of the penile urethra, where they differentiate into the integumental structures. The prepuce starts to develop as a fold of ectoderm with a mesodermal core after complete fusion of the entire urethra. Finally, the scrotum was found to develop through merging of the labioscrotal swellings and not by fusion.     

The Occurrence of Biogenic Calcian Struvite, (Mg, Ca)NH4PO4.6H2O, as Intracellular Crystals in Paramecium

ABSTRACT. Intracellular crystals are conspicuous refractile "inclusion bodies" commonly found in many protozoans, but very few have been identified mineralogically. We have isolated crystals from axenically grown mass cultures of Paramecium tetraurelia , and purified them using differential centrifugation. The crystals' structure and chemistry were analyzed using x-ray powder diffraction and energy-dispersive electron microprobe techniques. The morphology was studied by means of scanning electron microscopy. The crystals were identified as the orthorhombic mineral, calcian struvite, (Mg, Ca)NH₄PO₄.6H₂O. Struvite from P. tetraurelia exhibited a variety of crystal habits, including hemimorphic forms, epitactic overgrowths and several types of twins. A linear correlation between computed hydration number and Mg content suggests that the crystal composition may reflect the range of conditions under which struvite nucleation and growth occur. The mineral struvite also occurs in association with guano and other rich organic products, and can be biologically induced to precipitate extracellularly. Extracellular struvite has been well characterized in pathogenic calculi (kidney stones) of humans and cats, where precipitation is enhanced by bacterial urease activity that produces ammonia in the urinary tract. This is the first study demonstrating that struvite is also biologically controlled to form as an intracellular mineral. These crystals may have formed within lipid-rich, membrane-bound vesicles in Paramecium .     

Recovery of phosphorous from swine wastewater through crystallization

All the phosphate rock Japan needs must be presently imported from abroad because the country has no subterranean phosphorous resources. Therefore, there is a need to accelerate the development of and establish the technologies for phosphorous recovery from waste and wastewater. Swine wastewater has a high potential for phosphorous recovery in Japan. A reactor for removing and recovering phosphorous from swine wastewater was designed with dual functions, crystallization through aeration and separation of formed struvite by settling. However, a dehydration, composting and characterization process was first needed before using sediment sludge, including struvite, on farmland, since the struvite will settle along with huge amounts of other suspended solids (organic matter). For the recovery of pure struvite, an accumulation device was designed and its efficiency examined. The device has a struvite-accumulation face made of stainless steel wire mesh (1 mm in diameter, 1 cm(2) square) to reduce its total weight. During submergence in the aeration column of the demonstration reactor, struvite cross-bridged and accumulated on the face of the device. The struvite could be scraped off easily with only a light brushing, and was found to be approximately 95% pure. Because this device is a very simple structure, it is thought to be acceptable to swine farmers.     

Urolithiasis in ferrets (Mustela putorius).

Urinary calculi was observed frequently in ferrets which were from a group used for influenza research. They were submitted for necropsy with various clinical signs. The calculi were composed of magnesium ammonium phosphate hexahydrate and were found in the pelvis of the kidney, urinary bladder and urethra. Crystals of undetermined nature occasionally were observed in the kidneys.     

Urethral seam formation and hypospadias

Knowledge of the formation of the normal male urethra may elucidate the etiology of hypospadias. We describe urethral formation in the mouse, show the similarities and relevance to human urethral development, and introduce the concept of the epithelial seam formation and remodeling during urethral formation. Three mechanisms may account for epithelial seam formation: (1) epithelial-mesenchymal transformation similar to that described in the fusion of the palatal shelves, (2) apoptosis, and/or (3) tissue remodeling via cellular migration. Urethral development in the embryonic mouse (14-21 days of gestation) was compared with urethral formation in embryonic human specimens (8-16 weeks of gestation) by using histology, immunohistochemistry, and three-dimensional reconstruction. The urethra forms by fusion of the epithelial edges of the urethral folds, giving a midline epithelial seam. The epithelial seam is remodeled via cellular migration into a centrally located urethra and ventrally displaced remnant of epithelial cells. The epithelial seam is remodeled by narrowing approximately at its midpoint, with subsequent epithelial migration into the urethra or penile skin. The epithelial cells are replaced by mesenchymal cells. This remodeling seam displays a narrow band (approximately 30 microns wide) of apoptotic activity corresponding to the mesenchymal cells and not to epithelial cells. No evidence was seen of the co-expression of cytokeratin and mesenchymal markers (actin or vimentin). Urethral seam formation occurs in both the mouse and the human. Our data in the mouse support the hypothesis that seam transformation occurs via cellular migration and not by epithelial mesenchymal transformation or epithelial apoptosis. We postulate that disruption of epithelial fusion remodeling, and cellular migration leads to hypospadias.     

Struvite urolithiasis in a B6C3F1 mouse.

In a 2 year carcinogenicity bioassay using B6C3F1 mice, one male mouse developed clinical signs near termination of the study, comprising skin sores around the prepuce, penile prolapse and urine scalding. The predominant finding at necropsy was a markedly distended urinary bladder filled with numerous crystallized particles. Microscopically, there was subacute cystitis with marked hyperplasia of the transitional epithelium. X-ray diffraction analysis of the crystals showed a diffraction pattern characteristic of struvite (ammonium magnesium phosphate). The implications of the spontaneous occurrence of bladder stones in rodents on long-term toxicology studies are discussed.     

Control of struvite precipitation by selective removal of $${\text{NH}}^{{\text{ + }}}_{{\text{4}}} $$ with dialyzer/zeolite in an anaerobic membrane bioreactor

This study focused on the mitigation of membrane fouling caused by struvite precipitation using a dialyzer/zeolite (D/Z) unit in an anaerobic membrane bioreactor. The D/Z unit was designed to selectively remove NH(4)+ ions, one of the main components of the inorganic foulant, struvite. The maximum mass transfer coefficient for NH(4)+ through the dialyzer was estimated to be 0.92 l m(-2)h(-1), whereas the Na-substituted zeolite had the highest ion exchange capacity with respect to ammonium among intact or differently pretreated zeolites. During a single passage of dialysate through the zeolite column, substantial NH(4)+ removal (in excess of 90%) was achieved, leading to the reduction in struvite precipitation in the digester. The D/Z unit played a significant role in controlling struvite precipitation, thereby enhancing permeate flux for the case of the ceramic membrane, in which struvite fouling would be more pronounced compared to the polymeric membrane. For the polymeric membrane, however, no significant improvement in flux was observed even with the D/Z unit because the fouling of the polymeric membrane was mainly due to the deposition of biomass rather than the struvite precipitation.     

Microcalcifications,calcium-sensing receptor,and cancer

Calcium stones and calculi are observed in numerous human tissues. They are the result of deposition of calcium salts and are due to high local calcium concentrations. Prostatic calculi are usually classified as endogenous or extrinsic stones. Endogenous stones are commonly caused by obstruction of the prostatic ducts around an enlarged prostate resulting from benign prostatic hyperplasia or from chronic inflammation. The latter occurs mainly around the urethra and is generally caused by reflux of urine into the prostate. Calcium concentrations higher than in the plasma at sites of infection may induce the chemotactic response that eventually leads to recruitment of inflammatory cells. The calcium sensing receptor (CaSR) may be crucial for this recruitment as its expression and activity are increased by cytokines such as IL-6 and high extracellular calcium concentrations, respectively. The links between calcium calculi, inflammation, calcium supplementation, and CaSR functions in prostate cancer patients will be discussed in this review.     

Application of B-mode ultrasonography in the assessment of the dog penis

With the purpose to characterize the ultrasonographic features of the dog penis, 20 healthy dogs aged from 2 to 11 years old were used. Ultrasonography was performed using a 5-9MHz linear probe and a Philips HD3 scanner. Ultrasonograms were obtained at the penile body (pre- and post-scrotal sections) and at both the bulbus glandis and the pars longa glandis, with the dog placed in dorsal recumbency. The typical anatomy of the dog penis is easily demonstrated in ultrasonographic images, as revealed in this study. On ultrasonograms taken at the body level, the two corpora cavernosa showed a homogeneous, hypoechoic structure; ventrally, the corpus spongiosum appears as a slightly hyperechoic structure surrounding the urethra. In glans ultrasonograms, the most typical feature is the os penis, observed as a strong hyperechoic structure, whose acoustic shadow impairs the screening of adjacent tissues. Lining on its concavity, the corpus spongiosum is observed as a slightly hyperechoic tissue around the urethra. An ovoid, regularly hypoechoic tissue corresponding to the erectile tissue of the pars longa, surrounds these structures. The bulbus glandis is visualized as a regular hypoechoic structure with small anechoic areas, corresponding to sinusoidal spaces. The albuginea is evidenced as a well-defined, hyperechoic external layer. The preputial cavity appears as a thin anechoic layer surrounding the albuginea. The results of this study demonstrate that ultrasonographic assessment of the dog penis is easily performed and accurate enough for penile examination. Furthermore, ultrasonography of the dog penis can become an interesting diagnostic tool to explore in the canine practice, during breeding soundness evaluation and also whenever penile exposition is impossible to achieve.     

Effect of mixing on spontaneous struvite precipitation from semiconductor wastewater

The objective of this study was to investigate on the effect of mixing intensity (G) and mixing duration (t(d)) on struvite precipitation in the chemical mechanical polishing (CMP) wastewater generated from the semiconductor manufacturing process. Batch-scale experiments revealed that struvite crystallization was affected by both G and t(d). The mixing effect was to enhance the mass transfer of solute to the crystals in the process, resulting in the improvement of struvite crystallization and growth. By forming struvite, removal efficiencies of N and P increased logarithmic with the multiple values of G and t(d), i.e., Gt(d). Insufficient mixing energy with the Gt(d) value less than 10(5) caused an increase in the formation potential of unexpected precipitate unlike to pure struvite, causing a decrease in removal efficiencies of N and P in the process. At the Gt(d) value over 10(6), struvite precipitation was not restricted by fluoride, of which high level inherently contained in the CMP wastewater. The study results can be taken into consideration in the design and operation of the struvite precipitation process for both nutrient (N and P) removal and recovery.     

Isolation and metagenomic characterization of bacteria associated with calcium carbonate and struvite precipitation in a pure moving bed biofilm reactor-membrane bioreactor

A bench-scale pure moving bed bioreactor-membrane bioreactor (MBBR-MBR) used for the treatment of urban wastewater was analyzed for the identification of bacterial strains with the potential capacity for calcium carbonate and struvite biomineral formation. Isolation of mineral-forming strains on calcium carbonate and struvite media revealed six major colonies with a carbonate or struvite precipitation capacity in the biofouling on the membrane surface and showed that heterotrophic bacteria with the ability to precipitate calcium carbonate and struvite constituted ~7.5% of the total platable bacteria. These belonged to the genera Lysinibacillus, Trichococcus, Comamomas and Bacillus. Pyrosequencing analysis of the microbial communities in the suspended cells and membrane biofouling showed a high degree of similarity in all the samples collected with respect to bacterial assemblage. The study of operational taxonomic units (OTUs) identified through pyrosequencing suggested that ~21% of the total bacterial community identified in the biofouling could potentially form calcium carbonate or struvite crystals in the pure MBBR-MBR system used for the treatment of urban wastewater.     

T-incision technique in distal hypospadias: a modification of meatal advancement and glanuloplasty

Hypospadias is among the most common of the congenital anomalies. Distal hypospadias refers to an orifice in the distal third of the penile shaft. Correction of distal hypospadias requires different techniques, depending on the location of the meatus. Simple advancement techniques can be used for most distal hypospadias, whereas hypospadias with chordee requires reconstruction of a urethra. The meatoplasty and glanuloplasty procedures developed by Duckett have become standard operations to correct these lesions. Complications such as meatal stenosis, meatal retraction, and fish mouth-like meatus can be seen after meatal advancement and glanuloplasty ("MAGPI"), though it usually yields good results. In an attempt to avoid the complications associated with the meatal advancement and glanuloplasty procedure, the authors added a modification to the procedure for those hypospadias cases located in the coronal sulcus or its distal part. As an addition to conventional meatal advancement and glanuloplasty, a transverse incision on top of the vertical incision was made so that the urethra was supported by lateral triangle flaps created on the glans. Lateral triangle flaps of the glans were sutured to the dorsal aspect of the urethra advanced from the previous position. Thus, stress on the urethra was lessened and meatal retraction was prevented. When closure was performed with a T incision, an M-shaped, zigzag incision line was placed instead of a circular incision line. Therefore, stenosis was prevented and a vertical meatus with good cosmetic appearance was obtained. Fifty-three boys aged 4 to 7 years were operated on with this technique and were followed for 2.4 years. Good functional and cosmetic results were achieved in most of the cases.     

Urolithiasis associated with experimental lymphocytic choriomeningitis virus inoculation in Lewis rats

A high frequency of struvite urolithiasis, hydronephrosis, and other urinary tract lesions developed in a group of Lewis rats inoculated intracranially with lymphocytic choriomeningitis virus (LCMV). Initially, clinically ill rats were referred to necropsy: 30 rats over 3 years. These rats had high frequency of urolithiasis (8/30, 27%), hydronephrosis (12/30, 40%), cystitis (9/30, 30%), transitional cell carcinoma (4/30, 13%), and pyelonephritis (19/30, 63%). Lesions were more common in LCMV-inoculated rats. After this trend was noted, all rats on this protocol were necropsied as part of a cohort study (n = 144). Although the apparent frequency of disease was lower due to increased sampling, there still was a high number of urolithiasis (9/144, 6%) and hydronephrosis (40/144, 28%) cases. All cases of urolithiasis developed in rats inoculated with LCMV (9/44, 20%), as did most cases of hydronephrosis (31/44, 70%). Although sham-injected and uninoculated control rats also had high frequency of hydronephrosis (6/57 [11%] and 3/43 [7%], respectively), LCMV-inoculated rats had a significantly higher frequency of disease than did sham inoculated (P < 0.0001) and uninoculated (P < 0.0001) controls. These results suggest that Lewis rats may be predisposed to developing lesions of the urinary tract, and that intracranial inoculation of rats with LCMV augments this tendency, leading to formation of struvite calculi and associated urinary tract disease.