Eldho Paul

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

BackgroundCalcium 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.ResultsRecombinants 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.ConclusionThe 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.

Molecular analysis of oxalate-induced endoplasmic reticulum stress mediated apoptosis in the pathogenesis of kidney stone disease

Oxalate, a non-essential end product of metabolism, causes hyperoxaluria and eventually calcium oxalate (CaOx) stone disease. Kidney cells exposed to oxalate stress results in generation of reactive oxygen species (ROS) and progression of stone formation. Perturbations in endoplasmic reticulum (ER) result in accumulation of misfolded proteins and Ca2+ ions homeostasis imbalance and serve as a common pathway for various diseases, including kidney disorders. ER stress induces up-regulation of pro-survival protein glucose-regulated protein 78 (GRP78) and pro-apoptotic signaling protein C/EBP homologous protein (CHOP). Since the association of oxalate toxicity and ER stress on renal cell damage is uncertain, the present study is an attempt to elucidate the interaction of GRP78 with oxalate by computational analysis and study the role of ER stress in oxalate-mediated apoptosis in vitro and in vivo. Molecular docking results showed that GRP78-oxalate/CaOx interaction takes place. Oxalate stress significantly up-regulated expression of ER stress markers GRP78 and CHOP both in vitro and in vivo. Exposure of oxalate increased ROS generation and altered antioxidant enzyme activities. N-Acetyl cysteine treatment significantly ameliorated oxalate-mediated oxidative stress and moderately attenuated ER stress marker expression. The result indicates oxalate toxicity initiated oxidative stress-induced ER stress and also activating ER stress mediated apoptosis directly. In addition, the up-regulation of transforming growth factor β-1 revealed oxalate may induce kidney fibrosis through ER stress-mediated mechanisms. The present study provide insights into the pathogenic role of oxidative and ER stress by oxalate exposure in the formation of calcium oxalate stone.

Expression of heterologous oxalate decarboxylase in HEK293 cells confers protection against oxalate induced oxidative stress as a therapeutic approach for calcium oxalate stone disease

Abstract Oxalates stimulate alterations in renal epithelial cells and thereby induce calcium oxalate (CaOx) stone formation. Bacillus subtilis YvrK gene encodes for oxalate decarboxylase (OxdC) which degrades oxalate to formate and CO2. The present work is aimed to clone the oxdC gene in a mammalian expression vector pcDNA and transfect into Human Embryonic Kidney 293 (HEK293) cells and evaluate the oxdC expression, cell survival rate and oxalate degrading efficiency. The results indicate cell survival rate of HEK293/pcDNAOXDC cells pre-incubated with oxalate was enhanced by 28%. HEK293/pcDNAOXDC cells expressing OxdC treated with oxalate, significantly restored antioxidant activity, mitochondrial membrane potential and intracellular reactive oxygen species (ROS) generation compared with HEK293/pcDNA. Apoptotic marker caspase 3 downregulation illustrates HEK293/pcDNAOXDC cells were able to survive under oxalate-mediated oxidative stress. The findings suggest HEK293 cells expressing oxdC capable of degrading oxalate protect cells from oxidative damage and thus serve as a therapeutic option for prevention of CaOx stone disease.

Mobile group II intron based gene targeting in Lactobacillus plantarum WCFS1.

The usage of recombinant lactic acid bacteria for delivery of therapeutic proteins to the mucosa has been emerging. In the present study, an attempt was made to engineer a thyA mutant of Lactobacillus plantarum (L. plantarum) using lactococcal group II intron Ll.LtrB for the development of biologically contained recombinant L. plantarum for prevention of calcium oxalate stone disease. The 3 kb Ll.LtrB intron donor cassettes from the source vector pACD4C was PCR amplified, ligated into pSIP series of lactobacillus vector pLp_3050sAmyA, yielding a novel vector pLpACD4C (8.6 kb). The quantitative real‐time PCR experiment shows 94‐fold increased expression of Ll.LtrB intron and 14‐fold increased expression of ltrA gene in recombinant L. plantarum containing pLpACD4C. In order to target the thyA gene, the potential intron RNA binding sites in the thyA gene of L. plantarum was predicted with help of computer algorithm. The insertion location 188|189s of thyA gene (lowest E‐0.134) was chosen and the wild type intron Ll.LtrB was PCR modified, yielding a retargeted intron of pLpACDthyA. The retargeted intron was expressed by using induction peptide (sppIP), subsequently the integration of intron in thyA gene was identified by PCR screening and finally ThyA− mutant of L. plantarum (ThyA18) was detected. In vitro growth curve result showed that in the absence of thymidine, colony forming units of mutant ThyA18 was decreased, whereas high thymidine concentration (10 μM) supported the growth of the culture until saturation. In conclusion, ThyA− mutant of L. plantarum (ThyA18) constructed in this study will be used as a biologically contained recombinant probiotic to deliver oxalate decarboxylase into the lumen for treatment of hyperoxaluria and calcium oxalate stone deposition.

Oral administration of oxalate-enriched spinach extract as an improved methodology for the induction of dietary hyperoxaluric nephrocalcinosis in experimental rats

Abstract Experimental induction of hyperoxaluria by ethylene glycol (EG) administration is disapproved as it causes metabolic acidosis while the oral administration of chemically synthesized potassium oxalate (KOx) diet does not mimic our natural system. Since existing models comprise limitations, this study is aimed to develop an improved model for the induction of dietary hyperoxaluria, and nephrocalcinosis in experimental rats by administration of naturally available oxalate rich diet. Male albino Wistar rats were divided into five groups. Group I, control; group II rats received 0.75% EG, group III rats fed with 5% KOx diet and group IV and V rats were administered with spinach extract of 250 and 500 mg soluble oxalate/day respectively, for 28 d. Urine and serum biochemistry were analyzed. After the experimental period, rats were sacrificed, liver and kidney tissue homogenates were used for antioxidant and lipid peroxidation assay. Relative change in expression of kidney injury molecule-1 (KIM-1) and crystal modulators genes in kidney tissues were evaluated. Tissue damage was assessed by histology studies of liver and kidney. Experimental group rats developed hyperoxaluria and crystalluria. Urine parameters, serum biochemistry, antioxidant profile, lipid peroxidation levels and gene expression analysis of experimental group II and III rats reflected acute kidney damage compared to group V rats. Histopathology results showed moderate hyperplasia in liver and severe interstitial inflammation in kidneys of group II and III than group V rats. Ingestion of naturally available oxalate enriched spinach extract successfully induced dietary hyperoxaluria and nephrocalcinosis in rats with minimal kidney damage.

Antioxidant activity of phenolic compounds from extracts of Eucalyptus globulus and Melaleuca styphelioides and their protective role on D-glucose-induced hyperglycemic stress and oxalate stress in NRK-49Fcells

Abstract Phytochemicals serve as potential therapeutic agents for the prevention and treatment of diseases. In this study, we elucidate the renoprotective activity of compounds isolated from Eucalyptus globulus and Melaleuca styphelioides extracts in glucose- and oxalate-challenged NRK-49F cell model. The antioxidant potential of isolated compounds was evaluated based on their effect on antioxidant enzyme activities and lipid peroxidation levels. The results demonstrated that exposure of NRK-49F cells to glucose and oxalate stress augmented cell damage and attenuated antioxidant enzyme activities. The phytochemicals 2,2,8-trimethyl-6-formyl-chrom-3-ene-7-O-β-D-glucopyranoside, Cornusiin B and tellimagrandin I treatment restored antioxidant enzyme activity, significantly lowered lipid peroxidation levels and effectively protected cells from glucose and oxalate stress equivalent to the known antioxidant, N-acetyl cysteine. Pterocarinin A significantly reversed cellular damage owing to glucose stress. In conclusion, the compounds isolated from E. globulus and M. styphelioides showed potential cytoprotective and anti-oxidative property against glucose- and oxalate-induced oxidative stress in NRK-49F cells.

Designer probiotic Lactobacillus plantarum expressing oxalate decarboxylase developed using group II intron degrades intestinal oxalate in hyperoxaluric rats

Increased intestinal absorption of oxalate causes hyperoxaluria, a major risk factor for kidney stone disease. Intestinal colonization of recombinant probiotic bacteria expressing oxalate-degrading gene (OxdC) is an effective therapeutic option for recurrent calcium oxalate (CaOx) stone disease. Therefore, we aimed to develop food-grade probiotic L. plantarum secreting OxdC using lactococcal group II intron, Ll.LtrB and evaluate its oxalate degradation ability in vivo. Male Wistar albino rats were divided into four groups. The rats of group I received normal rat chow and drinking water. Groups II, III and IV rats received 5% potassium oxalate containing diet for 28 days. Groups III and IV rats received L. plantarum and food-grade recombinant L. plantarum respectively from 15 to 28 days. Biochemical parameters and crystalluria were analysed in 24 h urine samples. At the end of experimental period, rats were sacrificed; intestine and kidneys were dissected out for colonization studies and histopathological analysis. Herein, we found that the administration of recombinant probiotics significantly reduced the urinary oxalate, calcium, urea, and creatinine levels in rats of group IV compared to group II. Furthermore, colonization studies indicated that recombinant probiotics have gastrointestinal transit and intestinal colonization ability similar to that of wild-type bacteria. In addition, gene expression studies revealed down-regulation of OPN and KIM-1 among group IV rats. Histopathological analysis showed less evidence of nephrocalcinosis in group IV rats. In conclusion, the study demonstrates that food-grade L. plantarum secreting OxdC is capable of degrading intestinal oxalate and thereby prevent CaOx stone formation in experimental rats.

Recombinant Lactic Acid Bacteria Secreting OxdC as a Novel Therapeutic Tool for the Prevention of Kidney Stone Disease

Abstract Kidney stone disease is a dreadful pathological condition, and affects 20% of the population worldwide. The majority of renal stones contain calcium oxalate, and oxalate has vital role in the pathogenesis of renal stone. Humans derive oxalate from endogenous production and increased intake of dietary oxalate. Elevated oxalate levels lead to hyperoxaluria, a major risk factor for recurrent nephrolithiasis. Current treatment options in patients with primary and secondary hyperoxaluria are inadequate, and do not always lead to significant reduction in urinary oxalate excretion. Manipulation of gut flora with the potential probiotic bacteria may have a positive impact on gut oxalate levels, and may decrease oxalate absorption. Several reports documented gut microbes capable to degrade luminal oxalate, and reduce the risk of hyperoxaluria. In addition, the oxalate decarboxylase gene from Bacillus subtilis degrades oxalate into CO2 and formate, and studies have proven that administration of recombinant lactic acid bacteria (LAB) expressing oxalate decarboxylase (OxdC) decreased urinary oxalate excretion, and prevented calcium oxalate stone formation. Thus, potential oxalate degrading probiotic recombinant LAB expressing heterologous oxalate decarboxylase could be beneficial to mitigate hyperoxaluria efficiently by intestinal degradation of dietary oxalate.

Combination of ramipril and rutin alleviate alloxan induced diabetic nephropathy targeting multiple stress pathways in vivo

Diabetic nephropathy (DN) is considered as one of the major microvascular complications of diabetes mellitus (DM) which leads to end stage renal disease (ESRD). Even though existing therapeutic options are effective in decreasing albuminuria, drugs targeting the preservation of GFR and prevention of ESRD may provide better strategy for the treatment. Since metabolic disorders are multifactorial, poly-herbal medications, and drug-herbal combination are in demand. Therefore, the present work is focused on the combinatorial renoprotective effect of rutin and ramipril on alloxan induced DN in experimental rats. Male Wistar rats were divided into five groups, group I-control, group II-diabetic rats, group III-diabetic rats treated with ramipril, group IV-diabetic rats treated with rutin, group V-diabetic rats treated with ramipril and rutin for a period of six weeks. Results revealed administration of alloxan induced hyperglycemia and alteration in antioxidant profile. However, combination of a bioflavonoid with an Angiotensin converting enzyme (ACE) inhibitor administration restored the antioxidant status in experimental DN rats. Over-expression of ACE, TGF-β1 and decreased podocin expression in diabetic rats was significantly reversed in rats administered with both ramipril and rutin. In addition to attentuating oxidative stress and fibrosis, combinatorial therapy significantly down-regulated endoplasmic reticulum stress markers GRP78 and CHOP. Notably, combination of both ramipril and rutin in low doses reduced the side effects than the administration of monotherapy alone. Histopathological results revealed that combinatorial therapy was associated with a reduction in tubulointerstitial injury. The current study contributes the understanding of the multifactorial nature of DN and implies combinatorial treatment of ACE inhibitor with an antioxidant will be a promising therapeutic strategy for DN by their mechanism of action targeting various pathophysiological changes and stress pathways.