Phulwinder K. Grover

Intracrystalline Proteins and Urolithiasis: A Synchrotron X‐ray Diffraction Study of Calcium Oxalate Monohydrate

The existence of intracrystalline proteins and amino acids in calcium oxalate monohydrate was demonstrated by X‐ray synchrotron diffraction studies. Their presence has implications for the destruction of calcium oxalate crystals formed in the urinary tract and the prevention of kidney stones.

Intracrystalline proteins and urolithiasis : a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals

To compare the ultrastructure and protein content, particularly prothrombin fragment 1 and osteopontin, of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals precipitated from human urine, and their susceptibility to proteolysis, to try to clarify the role of intracrystalline proteins in urolithiasis, as differences between these types of crystal may determine whether calcium oxalate crystals nucleated in urine progress to stone formation.

Does Tamm-Horsfall mucoprotein inhibit or promote calcium oxalate crystallization in human urine?

Using two different experimental techniques, Tamm-Horsfall mucoprotein (THM) has been reported both to inhibit and to promote calcium oxalate (CaOx) crystallization in ultrafiltered human urine. In this study, these two techniques were used to compare the effects of THM on CaOx crystallization in the same ultrafiltered urine samples. Urine was collected from 10 healthy men and ultrafiltered (10,000 Da). Each sample was divided and to one half was added sufficient human THM to give a final concentration of 35 mg/L. CaOx crystallization was induced in the samples by addition of an oxalate load and by evaporation. Using the evaporation technique THM significantly increased the deposition of CaOx determined as 14C-oxalate, from 9,772 cpm to 43,652 cpm (P less than 0.01). Using the oxalate load method THM had no effect on the metastable limits of the urine with respect to CaOx, and significantly increased the volume of particulate material deposited from 26,000 to 39,995 microns 3/microliters - an increase of 54%. This increase was reduced to 21% when values were corrected for the volume of THM particles recorded in control samples to which no oxalate load was added. Using 14C-oxalate, it was shown that this increase in volume could not be attributed to an enhanced deposition of crystalline CaOx, but was probably the result of an increased polymerization of THM in the presence of CaOx crystals. Despite this, the average size of the particles precipitated in the presence of THM (6.5 microns) was significantly (P less than 0.01) less than that observed in the absence of THM (12.1 microns). It was concluded that the effect of THM on CaOx crystallization in urine depends upon the methodology used to assess it and that promotion would only be expected in vivo in cases of extreme dehydration. Under usual physiological conditions THM would be expected to inhibit CaOx crystal aggregation and to have little effect, if any, on the amount of crystalline material deposited.

Dissolved Urate Salts Out Calcium Oxalate in Undiluted Human Urine In Vitro: Implications for Calcium Oxalate Stone Genesis

Hyperuricosuria has long been documented as a predisposing factor to calcium oxalate (CaOx) stone pathogenesis. However, its mechanism is still without sound scientific foundation. Previously, we showed that hyperuricosuria, simulated by the addition of dissolved sodium urate, promotes the crystallization of CaOx. In the present study, we demonstrate that the urates effect on the crystallization is attributable to its salting out CaOx from solution. Furthermore, analysis of urines revealed that their metastable limit decreased with increases in the product of the prevailing concentrations of calcium and urate: this has implications for CaOx stone genesis. We also outline anti-salting out strategies for future research for the prevention and/or treatment of CaOx calculi.

Detection and Clinical Significance of Circulating Tumor Cells in Colorectal Cancer—20 Years of Progress

Circulating tumor cells (CTC) may be defined as tumor- or metastasis-derived cells that are present in the bloodstream. The CTC pool in colorectal cancer (CRC) patients may include not only epithelial tumor cells, but also tumor cells undergoing epithelial-mesenchymal transition (EMT) and tumor stem cells. A significant number of patients diagnosed with early stage CRC subsequently relapse with recurrent or metastatic disease despite undergoing “curative” resection of their primary tumor. This suggests that an occult metastatic disease process was already underway, with viable tumor cells being shed from the primary tumor site, at least some of which have proliferative and metastatic potential and the ability to survive in the bloodstream. Such tumor cells are considered to be responsible for disease relapse in these patients. Their detection in peripheral blood at the time of diagnosis or after resection of the primary tumor may identify those early-stage patients who are at risk of developing recurrent or metastatic disease and who would benefit from adjuvant therapy. CTC may also be a useful adjunct to radiological assessment of tumor response to therapy. Over the last 20 years many approaches have been developed for the isolation and characterization of CTC. However, none of these methods can be considered the gold standard for detection of the entire pool of CTC. Recently our group has developed novel unbiased inertial microfluidics to enrich for CTC, followed by identification of CTC by imaging flow cytometry. Here, we provide a review of progress on CTC detection and clinical significance over the last 20 years.

The importance of a clean face: the effect of different washing procedures on the association of Tamm–Horsfall glycoprotein and other urinary proteins with calcium oxalate crystals

This study was undertaken to determine whether the use of different washing procedures could explain dissident findings in published studies examining the role of urinary macromolecules in urolithiasis. Calcium oxalate monohydrate (COM) crystals were deposited from or added to the same sieved urine, washed with copious or limited amounts of distilled water, or with methanol, and examined by field emission scanning electron microscopy (FESEM). Demineralized extracts were analysed by SDS-PAGE and Western blotting for Tamm–Horsfall glycoprotein (THG), human serum albumin (HSA), osteopontin (OPN) and prothrombin fragment 1 (PTF1). Synchrotron X-ray diffraction (SXRD) with Rietveld whole-pattern peak fitting and profile analysis was used to determine non-uniform crystal strain and crystallite size in crystals generated from inorganic solutions in the presence of increasing concentrations of THG and prothrombin (PT). HSA and PTF1 were present in all demineralized crystal extracts, confirming their inclusion within COM. OPN was present in all extracts except those derived from pure inorganic COM crystals, because of its occlusion within small numbers of calcium oxalate dihydrate (COD) crystals contaminating the COM population. THG was absent from the demineralized extracts of all crystals washed copiously with water, but present in those washed with methanol or limited amounts of water. FESEM showed extraneous organic material associated only with crystals whose extracts contained THG, confirming that the protein does not bind permanently to the COM crystal surface and is not occluded within the mineral bulk. This was confirmed by SXRD, which showed that non-uniform strain and crystallite size remained unaltered in crystals grown in the presence of increasing THG concentrations. However, non-uniform strain increased and crystallite size decreased with increasing PT concentrations, demonstrating unambiguously that PT is included in COM crystals. It was concluded that scrupulous care must be taken to ensure the complete removal of extraneous THG adventitiously associated with CaOx crystals in order to avoid inaccurate analysis of crystal matrix protein content and possible misinterpretation of experimental data.

Urate and Calcium Stones — Picking Up a Drop of Mercury With One's Fingers?*

The evidence invariably cited to support the suspicion that urinary urate is a predisposing factor in calcium oxalate (CaOx) stone formation is critically reviewed. Analysis of the relevant literature shows that speculation is based on the clinical impression that CaOx stone-formers appear to excrete more urate than do normal subjects, and that allopurinol reduces the rate of CaOx stone recurrences. On balance, this is sufficient to suggest that a high urinary excretion of urate promotes CaOx stone formation. However, in the past, evidence to disclose the mechanism by which urate could exert this effect has been largely shrouded in confusion and controversy. The evidence for two theories that have dominated thinking in this area are reviewed and new findings are reported that indicate that neither can account for the purported effect of urate. It is concluded that dissolved urate in urine, at normal physiological pH values, directly provokes CaOx crystal nucleation by the phenomenon of salting-out. The possibility that urate promotes CaOx stone formation is further strengthened by its ability to increase significantly the amount of CaOx precipitated from solution and to cause the aggregation of individual crystals into large clusters. Future avenues of investigation that should assist in the formulation of diagnostic and therapeutic guidelines are presented.

The effects of intracrystalline and surface-bound proteins on the attachment of calcium oxalate monohydrate crystals to renal cells in undiluted human urine

To compare the binding to Madin‐Darby canine kidney (MDCK)‐II cells of: (i) inorganic calcium oxalate monohydrate (iCOM) crystals and COM crystals precipitated from urine containing different concentrations of protein; and (ii) urinary COM crystals containing intracrystalline and intracrystalline + surface‐bound protein.

Face‐specific binding of prothrombin fragment 1 and human serum albumin to inorganic and urinary calcium oxalate monohydrate crystals

To compare the intracrystalline distributions of prothrombin fragment 1 (PTF1) and human serum albumin (HSA) within inorganic and urinary calcium oxalate (CaOx) monohydrate (COM) crystals and to determine whether binding of PTF1 can be explained by interactions between particular γ‐carboxyglutamic (Gla) residues and atomic arrays on individual faces of the COM crystal.

A comparison of the binding of urinary calcium oxalate monohydrate and dihydrate crystals to human kidney cells in urine.

To compare the binding kinetics of urinary calcium oxalate monohydrate (COM) and dihydrate (COD) crystals to human kidney (HK‐2) cells in ultra‐filtered (UF), and centrifuged and filtered (CF) human urine; and to quantify the binding of COM and COD crystals to cultured HK‐2 cells in UF and CF urine samples collected from different individuals.