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Recombinant Lactobacillus plantarum expressing and secreting heterologous oxalate decarboxylase prevents renal calcium oxalate stone deposition in experimental rats.

Sasikumar P, Gomathi S, Anbazhagan K, Abhishek A, Paul E, Vasudevan V, Sasikumar S, Selvam GS - J. Biomed. Sci. (2014)

Bottom Line: Oxalate degrading bacteria might have beneficial effects on urinary oxalate excretion resulting from decreased intestinal oxalate concentration and absorption.Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V.The present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary oxalate excretion and CaOx crystal deposition by increased intestinal oxalate degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Centre for Advanced Studies in Organismal and Functional Genomics, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India. drselvamgsbiochem@rediffmail.com.

ABSTRACT

Background: Calcium oxalate (CaOx) is the major constituent of about 75% of all urinary stone and the secondary hyperoxaluria is a primary risk factor. Current treatment options for the patients with hyperoxaluria and CaOx stone diseases are limited. Oxalate degrading bacteria might have beneficial effects on urinary oxalate excretion resulting from decreased intestinal oxalate concentration and absorption. Thus, the aim of the present study is to examine the in vivo oxalate degrading ability of genetically engineered Lactobacillus plantarum (L. plantarum) that constitutively expressing and secreting heterologous oxalate decarboxylase (OxdC) for prevention of CaOx stone formation in rats. The recombinants strain of L. plantarum that constitutively secreting (WCFS1OxdC) and non-secreting (NC8OxdC) OxdC has been developed by using expression vector pSIP401. The in vivo oxalate degradation ability for this recombinants strain was carried out in a male wistar albino rats. The group I control; groups II, III, IV and V rats were fed with 5% potassium oxalate diet and 14th day onwards group II, III, IV and V were received esophageal gavage of L. plantarum WCFS1, WCFS1OxdC and NC8OxdC respectively for 2-week period. The urinary and serum biochemistry and histopathology of the kidney were carried out. The experimental data were analyzed using one-way ANOVA followed by Duncan's multiple-range test.

Results: Recombinants L. plantarum constitutively express and secretes the functional OxdC and could degrade the oxalate up to 70-77% under in vitro. The recombinant bacterial treated rats in groups IV and V showed significant reduction of urinary oxalate, calcium, uric acid, creatinine and serum uric acid, BUN/creatinine ratio compared to group II and III rats (P < 0.05). Oxalate levels in kidney homogenate of groups IV and V were showed significant reduction than group II and III rats (P < 0.05). Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V.

Conclusion: The present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary oxalate excretion and CaOx crystal deposition by increased intestinal oxalate degradation.

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Microscopy examinations of kidney tissue and CaOx crystals in experimental rat at 20X magnification. I, II, III, IV and V represent the respective group of rat. A, B and C represents H&E stained section, pizzolato methods stained section for CaOx crystal and polarized microscopy examination of CaOx crystal respectively. Arrow indicates the CaOx crystal in kidney section of respective group.
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Fig8: Microscopy examinations of kidney tissue and CaOx crystals in experimental rat at 20X magnification. I, II, III, IV and V represent the respective group of rat. A, B and C represents H&E stained section, pizzolato methods stained section for CaOx crystal and polarized microscopy examination of CaOx crystal respectively. Arrow indicates the CaOx crystal in kidney section of respective group.

Mentions: Renal function was examined by using semi-quantitative PCR for renin, ACE and OPN expression. The up-regulation of renin mRNA was observed in groups II and III when compared to group I rats. While the recombinant bacterial treated group IV and V shows significant reduction in mRNA level compared to group II and III. The down regulations of ACE, OPN mRNA were seen in groups II, III, IV and V rats (Figure 7A, B). Histopathological examination of kidney sections of group I rats showed normal histological structures. Group II and III rats showed a reduced number of glomeruli and large areas of red blood cell casts with dialated tubules. Stroma showed hemorrhage and blood vessels were congested and thickened. Sections obtained from rats in the group IV administered with WCFS1OxdC revealed normal glomeruli with no red blood cast, but slight tubular necrosis. Examination of stroma shows areas of hemorrhage. Similarly, group V rats that received NC8OxdC showed normal glomeruli, but high tubular necrosis and congested blood vessels. The CaOx crystals were examined by pizzolato staining and also by using polarized microscopy. It revealed no incidence of CaOx crystal deposition in group I whereas as high score (4+) of CaOx crystals in groups II and III rats. However, group IV showed no identifiable crystal deposits in the kidneys and group V showed significantly lower score (1+) (Figure 8).Figure 7


Recombinant Lactobacillus plantarum expressing and secreting heterologous oxalate decarboxylase prevents renal calcium oxalate stone deposition in experimental rats.

Sasikumar P, Gomathi S, Anbazhagan K, Abhishek A, Paul E, Vasudevan V, Sasikumar S, Selvam GS - J. Biomed. Sci. (2014)

Microscopy examinations of kidney tissue and CaOx crystals in experimental rat at 20X magnification. I, II, III, IV and V represent the respective group of rat. A, B and C represents H&E stained section, pizzolato methods stained section for CaOx crystal and polarized microscopy examination of CaOx crystal respectively. Arrow indicates the CaOx crystal in kidney section of respective group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4256919&req=5

Fig8: Microscopy examinations of kidney tissue and CaOx crystals in experimental rat at 20X magnification. I, II, III, IV and V represent the respective group of rat. A, B and C represents H&E stained section, pizzolato methods stained section for CaOx crystal and polarized microscopy examination of CaOx crystal respectively. Arrow indicates the CaOx crystal in kidney section of respective group.
Mentions: Renal function was examined by using semi-quantitative PCR for renin, ACE and OPN expression. The up-regulation of renin mRNA was observed in groups II and III when compared to group I rats. While the recombinant bacterial treated group IV and V shows significant reduction in mRNA level compared to group II and III. The down regulations of ACE, OPN mRNA were seen in groups II, III, IV and V rats (Figure 7A, B). Histopathological examination of kidney sections of group I rats showed normal histological structures. Group II and III rats showed a reduced number of glomeruli and large areas of red blood cell casts with dialated tubules. Stroma showed hemorrhage and blood vessels were congested and thickened. Sections obtained from rats in the group IV administered with WCFS1OxdC revealed normal glomeruli with no red blood cast, but slight tubular necrosis. Examination of stroma shows areas of hemorrhage. Similarly, group V rats that received NC8OxdC showed normal glomeruli, but high tubular necrosis and congested blood vessels. The CaOx crystals were examined by pizzolato staining and also by using polarized microscopy. It revealed no incidence of CaOx crystal deposition in group I whereas as high score (4+) of CaOx crystals in groups II and III rats. However, group IV showed no identifiable crystal deposits in the kidneys and group V showed significantly lower score (1+) (Figure 8).Figure 7

Bottom Line: Oxalate degrading bacteria might have beneficial effects on urinary oxalate excretion resulting from decreased intestinal oxalate concentration and absorption.Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V.The present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary oxalate excretion and CaOx crystal deposition by increased intestinal oxalate degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Centre for Advanced Studies in Organismal and Functional Genomics, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India. drselvamgsbiochem@rediffmail.com.

ABSTRACT

Background: Calcium oxalate (CaOx) is the major constituent of about 75% of all urinary stone and the secondary hyperoxaluria is a primary risk factor. Current treatment options for the patients with hyperoxaluria and CaOx stone diseases are limited. Oxalate degrading bacteria might have beneficial effects on urinary oxalate excretion resulting from decreased intestinal oxalate concentration and absorption. Thus, the aim of the present study is to examine the in vivo oxalate degrading ability of genetically engineered Lactobacillus plantarum (L. plantarum) that constitutively expressing and secreting heterologous oxalate decarboxylase (OxdC) for prevention of CaOx stone formation in rats. The recombinants strain of L. plantarum that constitutively secreting (WCFS1OxdC) and non-secreting (NC8OxdC) OxdC has been developed by using expression vector pSIP401. The in vivo oxalate degradation ability for this recombinants strain was carried out in a male wistar albino rats. The group I control; groups II, III, IV and V rats were fed with 5% potassium oxalate diet and 14th day onwards group II, III, IV and V were received esophageal gavage of L. plantarum WCFS1, WCFS1OxdC and NC8OxdC respectively for 2-week period. The urinary and serum biochemistry and histopathology of the kidney were carried out. The experimental data were analyzed using one-way ANOVA followed by Duncan's multiple-range test.

Results: Recombinants L. plantarum constitutively express and secretes the functional OxdC and could degrade the oxalate up to 70-77% under in vitro. The recombinant bacterial treated rats in groups IV and V showed significant reduction of urinary oxalate, calcium, uric acid, creatinine and serum uric acid, BUN/creatinine ratio compared to group II and III rats (P < 0.05). Oxalate levels in kidney homogenate of groups IV and V were showed significant reduction than group II and III rats (P < 0.05). Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V.

Conclusion: The present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary oxalate excretion and CaOx crystal deposition by increased intestinal oxalate degradation.

Show MeSH
Related in: MedlinePlus