The effects of human urine on the adhesion of calcium oxalate crystal to Madin–Darby canine kidney cells

Ebisuno,; Umehara,; Kohjimoto,; Ohkawa, None

doi:10.1046/j.1464-410x.1999.00103.x

Cited by 10 publications

(5 citation statements)

References 21 publications

(35 reference statements)

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“…However, most of these studies have been performed in aqueous inorganic buffers and not in urine, the real medium in which stones form. In addition, of the studies that used urine as the proxy for tubular fluid (10,(17)(18), they too pretreated urine in different ways to remove cellular debris or concentrate the macromolecules. This is very perplexing because prior treatment of urine has been documented to significantly alter its macromolecular content in general and THG, HSA, and inter-␣-inhibitor in particular (6,32).…”

Section: Discussionmentioning

confidence: 99%

“…Since urine contains a spectrum of low-and high-molecular-weight constituents, the ratios of which vary from sample to sample even when collected from the same subject, it can be reasonably argued that no synthetic solution can possibly mimic the likely effects in urine. In only a handful of previous studies urine samples were used as a proxy for tubular fluid (10,17,18), but the samples were pretreated in different ways to remove cellular debris or concentrate the macromolecules. In one case (10), the urine was diluted to final concentrations of only 1 or 10%.…”

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confidence: 99%

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Effect of urine fractionation on attachment of calcium oxalate crystals to renal epithelial cells: implications for studying renal calculogenesis

Grover

Thurgood²,

Ryall³

2007

American Journal of Physiology-Renal Physiology

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Our aim was to determine whether fractionation of human urine affects the attachment of calcium oxalate monohydrate (COM) crystals to renal cells. Urine collected from six healthy subjects was fractionated into sieved (S), centrifuged (C), centrifuged and filtered (CF), or ultrafiltered (UF). Attachment of [(14)C]COM crystals to Madin-Darby canine kidney (MDCK) cells was studied after precoating the crystals or the cells with the urine fractions and by using the same fractions as the binding medium. Protein content of the fractions and precoated crystals was analyzed with SDS-PAGE and Western blotting. All urine fractions inhibited crystal attachment. When fractions from the six urine samples were used to precoat the cells, the median inhibitions of crystal adhesion ( approximately 40%) were not significantly different. Median inhibition after preincubation of crystals was the same for the S, C, and CF fractions ( approximately 40%) but significantly greater than for the UF fraction ( approximately 28%). When fractions were used as the binding medium, median inhibitions decreased from 64% in the S fraction to 47 (C), 42 (CF), and to 29% (UF). SDS-PAGE analysis showed that centrifugation and filtration reduced the amount of Tamm-Horsfall glycoprotein (THG), which was confirmed by Western blotting. Human serum albumin, urinary prothrombin fragment 1, and osteopontin, but not THG, were present in demineralized extracts of the precoated crystals. Fractionation of human urine affects the attachment of COM crystals to MDCK cells. Hence future studies investigating regulation of crystal-cell interactions should be carried out in untreated urine as the binding medium.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Effect of urine fractionation on attachment of calcium oxalate crystals to renal epithelial cells: implications for studying renal calculogenesis

Grover

Thurgood²,

Ryall³

2007

American Journal of Physiology-Renal Physiology

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“…The non-adherent crystals were removed by rapidly washing the cells three times with PBS, and the rudimental crystals were counted using fluorescence microscope. Then rudimental crystals were dissolved by 2 ml of HCl (5 mol/L), and the calcium concentration of the solution was assessed by flame atomic absorption spectrophotometry (AA-6300, Shimadzu, Japan), to quantitative analyze adherent COM crystals [16].…”

Section: Com Crystal Adhesionmentioning

confidence: 99%

L-Carnitine Protects Renal Tubular Cells Against Calcium Oxalate Monohydrate Crystals Adhesion Through Preventing Cells From Dedifferentiation

Li¹,

Wu²,

Duan³

et al. 2016

Kidney Blood Press Res

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Background/Aims: The interactions between calcium oxalate monohydrate (COM) crystals and renal tubular epithelial cells are important for renal stone formation but still unclear. This study aimed to investigate changes of epithelial cell phenotype after COM attachment and whether L-carnitine could protect cells against subsequent COM crystals adhesion. Methods: Cultured MDCK cells were employed and E-cadherin and Vimentin were used as markers to estimate the differentiate state. AlexaFluor-488-tagged COM crystals were used in crystals adhesion experiment to distinguish from the previous COM attachment, and adhesive crystals were counted under fluorescence microscope, which were also dissolved and the calcium concentration was assessed by flame atomic absorption spectrophotometry. Results: Dedifferentiated MDCK cells induced by transforming growth factor β1 (TGF-β1) shown higher affinity to COM crystals. After exposure to COM for 48 hours, cell dedifferentiation were observed and more subsequent COM crystals could bind onto, mediated by Akt/GSK-3β/Snail signaling. L-carnitine attenuated this signaling, resulted in inhibition of cell dedifferentiation and reduction of subsequent COM crystals adhesion. Conclusions: COM attachment promotes subsequent COM crystals adhesion, by inducing cell dedifferentiation via Akt/GSK-3β/Snail signaling. L-carnitine partially abolishes cell dedifferentiation and resists COM crystals adhesion. L-carnitine, may be used as a potential therapeutic strategy against recurrence of urolithiasis.

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“…The harvested solutions were allowed to stand for at least 1 h and then centrifuged at 300 g for 10 min. Quantitative analysis of the adherent COM crystals was accomplished by measuring the calcium concentration of the cell lysate by atomic absorption analysis …”

Section: Methodsmentioning

confidence: 99%

“…Quantitative analysis of the adherent COM crystals was accomplished by measuring the calcium concentration of the cell lysate by atomic absorption analysis. 9 Various Kampo drugs were added to the COM crystal suspension just before exposure of the cells and their inhibitory effects on crystal adhesion were calculated as follows; inhibitory activity (%) = (ab)/a ¥ 100, where a is the calcium concentration after exposure of the COM crystals in Tris-HCl buffer as a control, and b is the calcium concentration after exposure to Tris-HCl buffer containing Kampo drugs.…”

Section: Measurement Of Crystal Adhesion To Mdck Cellsmentioning

confidence: 99%

Effect of Kampo extracts on urinary stone formation: An experimental investigation

2013

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Abbreviations & AcronymsObjective: To examine the effects of Kampo extracts on the process of urinary stone formation. Methods: A total of 14 Kampo extracts (10 mg/mL) were examined in vitro by assessing whether they could inhibit two critical steps in the early process of calcium oxalate stone formation, namely crystal aggregation and crystal adhesion to the renal tubular epithelium of Madin-Darby canine kidney cells. The inhibitory effect of the extracts on stone formation was examined by using a rat model. Results: Sanshishi and Takusha showed a strong inhibitory effect on the calcium oxalate monohydrate crystal aggregation (84.5% and 64.2%, respectively) and on the crystal adhesion to Madin-Darby canine kidney cells (88.2% and 54.6%, respectively). As pretreatment of Madin-Darby canine kidney cells with these two Kampo extracts did not show any inhibitory effect on crystal adhesion, they were considered to mainly affect the crystal surface to block crystal adhesion to Madin-Darby canine kidney cells. Gorin-san, containing both Sanshishi and Takusha, showed significantly stronger inhibitory activities at lower concentrations compared with Chorei-to, which contains Takusha. Finally, Sanshishi showed a prophylactic effect on calcium oxalate crystal deposition in the rat model. Conclusions: These findings suggest that Gorin-san, which contains Sanshishi and Takusha, might be used for the prophylaxis of urolithiasis in humans.

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The effects of human urine on the adhesion of calcium oxalate crystal to Madin–Darby canine kidney cells

Cited by 10 publications

References 21 publications

Effect of urine fractionation on attachment of calcium oxalate crystals to renal epithelial cells: implications for studying renal calculogenesis

Effect of urine fractionation on attachment of calcium oxalate crystals to renal epithelial cells: implications for studying renal calculogenesis

L-Carnitine Protects Renal Tubular Cells Against Calcium Oxalate Monohydrate Crystals Adhesion Through Preventing Cells From Dedifferentiation

Effect of Kampo extracts on urinary stone formation: An experimental investigation

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