Ashok Kumar Yadav

Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen

Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored.

Vascular function and cholecalciferol supplementation in CKD: A self-controlled case series.

Vitamin D deficiency is common and associated with mortality in chronic kidney disease (CKD) patients. Cardiovascular disease (CVD) is the commonest cause of mortality in CKD patients. In a randomized, double blind, placebo controlled trial, we have recently reported favorable effects of vitamin D supplementation on vascular & endothelial function and inflammatory biomarkers in vitamin D deficient patients with non-diabetic stage 3-4 CKD (J Am Soc Nephrol 28: 3100-3108, 2017). Subjects in the placebo group who had still not received vitamin D after completion of the trial received two oral doses 300,000 IU of oral cholecalciferol at 8 weeks interval followed by flow mediated dilatation (FMD), pulse wave velocity (PWV), circulating endothelial and inflammatory markers (E-Selectin, vWF, hsCRP and IL-6), 125 (OH)2D, iPTH and iFGF-23 assessment at 16 weeks. 31 subjects completed this phase of the study. Last values recorded in the preceding clinical trial were taken as baseline values. Serum 25(OH)D and 1,25(OH)2D increased and FMD significantly improved after cholecalciferol supplementation [mean change in FMD%: 5.8% (95% CI: 4.0-7.5%, pu202f<u202f0.001]. Endothelium independent nitroglycerine mediated dilatation, PWV, iPTH, iFGF-23 and IL-6 also showed favorable changes. The data further cement the findings of beneficial effects of correction of vitamin D deficiency on vascular function.

SUMO4 163 G>A variation is associated with kidney disease in Indian subjects with type 2 diabetes

Genetic susceptibility probably plays a role in the development and/or progression of diabetic kidney disease. Small ubiquitin-related modifier 4 (SUMO4) mRNA is expressed in human kidney. Substitution of methionine with valine at codon 55 (M55V) of SUMO4 gene induces higher nuclear factor-kB activity, which is known to mediate the development of kidney disease in individuals with diabetes. We investigated the association between the SUMO4 M55V (rs237025, c.163 G>A) and kidney disease in north Indian subjects with diabetes. A case–control analysis was performed using genomic DNA samples from 216 diabetic patients without nephropathy (DM) and 201 diabetic with nephropathy (DN). The SUMO4 c.163 G>A polymorphism was genotyped using polymerase chain reaction amplification followed by restriction digestion. The duration of diabetes was significantly greater in DN. The genotypic and allelic frequencies were different in DM and DN groups: GG genotype was significantly more frequent in DN as compared to DM (pxa0=xa00.018, OR 1.72, 95xa0% CI 1.1–2.7). Similarly the G allele was more frequent in DN compared to DM (pxa0=xa00.017, OR 1.4, 95xa0% CI 1.1–1.8). This study suggests that SUMO4 c.163 G>A polymorphism is associated with the susceptibility to diabetic nephropathy in north Indian subjects with type 2 diabetes.

Effect of vitamin D supplementation on serum sclerostin levels in chronic kidney disease.

Vitamin D deficiency, cardiovascular disease and abnormal bone mineral metabolism are common in chronic kidney disease (CKD). Abnormal bone mineral metabolism has been linked to vascular calcification in CKD. Sclerostin has emerged as an important messenger in cross talk between bone-vascular axis. We analyzed sclerostin in subjects who participated in the randomized, double blind, placebo controlled trial investigating the effect of cholecalciferol supplementation on vascular function in non-diabetic CKD stage G3-4 and vitamin Du202f≤u202f20u202fng/ml [CTRI/2013/05/003648]. Patients were randomized (1:1) to receive either two directly observed oral doses of 300,000 IU of cholecalciferol or matching placebo at baseline and 8 weeks. Of the 120 subjects enrolled, 58 in the cholecalciferol group and 59 in the placebo group completed the study. At baseline, serum levels of sclerostin were similar in both groups (cholecalciferol - median;190pg/ml, IQR;140-260u202fpg/ml and placebo - median;180u202fpg/ml, IQR; 140-240u202fpg/ml, pu202f=u202f0.67). 16 weeks after cholecalciferol supplementation, there was no change in level of sclerostin (mean change;1.10u202fpg/ml, 95%CI; -27.34 to 29.34u202fpg/ml, pu202f=u202f0.25). However, a significant decrease in sclerostin level was noted in the placebo group (mean change; -31.94u202fpg/ml, 95%CI; -54.76 to -9.13u202fpg/ml, pu202f=u202f0.002). Change (Δ) in sclerostin level at 16 weeks correlated negatively with Δ eGFR (ru202f=u202f-0.20, pu202f=u202f0.03) and positively with Δuric acid (ru202f=u202f0.37, pu202f<u202f0.001) but not with Δ25(OH) D (ru202f=u202f0.06, pu202f=u202f0.54), Δ iPTH (ru202f=u202f-u202f0.03, pu202f=u202f0.78) ΔFGF23 (ru202f=u202f-u202f0.08, pu202f=u202f0.38) and Δ125 (OH)2 D (ru202f=u202f-u202f0.04, pu202f=u202f0.65). In conclusion, high dose cholecalciferol supplementation did not change sclerostin levels in non-diabetic stage 3-4 CKD subjects.

PLA2R related primary membranous nephropathy in a hepatitis C positive patient.

A 58-year-old non-diabetic female with history of hypertension presented with swelling of both lower limbs and facial puffiness of 5-month duration. Evaluation revealed nephrotic syndrome (proteinuria 2.8 g/day, bland urinary sediment, serum albumin 1.6 g/dL) and normal kidney function (serum creatinine 0.63 mg/dL). The kidney biopsy was suggestive of membranous nephropathy with enhanced staining for M-type phospholipase A2 receptor (PLA2R). Anti-hepatitis C virus (HCV) antibody was positive, with 38,90,243 IU/mL of HCV RNA (genotype 3). The serum antibody to PLA2R (aPLA2R) was 236 RU/mL. The patient was started on telmisartan, atorvastatin, diuretics, warfarin, sofosbuvir and ribavirin. The HCV RNA became undetectable (< 50 IU/mL) at 2 months. The aPLA2R after 2 months of antiviral therapy came down to 4.67 RU/mL. However, the nephrotic syndrome persisted. A repeat aPLA2R 9 months after initial presentation showed a rise (117.62 RU/mL). At this time, a diagnosis of aPLA2R related MN was made, cyclical cyclophosphamide and steroids were started and the patient achieved complete clinical remission. The proteinuria became undetectable; serum albumin was 3.69 g/dL and repeat aPLA2R 0.0019 RU/mL 8 months after starting the therapy. The patient remains in remission after 5 months of stopping immunosuppressive therapy, and the HCV viral load remains undetectable (13 months after stopping antivirals). The present case highlights the fact that not all MN in hepatitis C positive patients are virus related. Larsen et al. reported enhanced staining for PLA2R in 64% of HCV associated MN. The studies describing PLA2R in MN with hepatitis B and/or C infection are limited by lack of sequential antibody monitoring after treating infection. In the index case, the NS failed to abate despite successful treatment of hepatitis C infection. Interestingly, the aPLA2R came down during the anti-viral treatment but reappeared, perhaps due to the serum-glomerular dynamics, which has been well described in the past. The clinical and serological remission with immunosuppressive treatment clearly demonstrates the primary nature of the disease. Alternatively, the virus could be acting as a trigger for producing antibodies.

Uromodulin rs4293393 T>C variation is associated with kidney disease in patients with type 2 diabetes

Background & objectives: Uromodulin, a UMOD gene encoded glycoprotein is synthesized exclusively in renal tubular cells and released into urine. Mutations lead to uromodulin misfolding and retention in the kidney, where it might stimulate cells of immune system to cause inflammation and progression of kidney disease. Genome-wide association studies (GWAS) have identified UMOD locus to be associated with hypertension and diabetic nephropathy (DN). In this study, we investigated the association between rs4293393 variation in UMOD gene and susceptibility to kidney disease in individuals with type 2 diabetes mellitus (T2DM). Methods: A total of 646 individuals, 208 with T2DM without evidence of kidney disease (DM), 221 with DN and 217 healthy controls (HC) were genotyped for UMOD variant rs4293393T>C by restriction fragment length polymorphism. Serum uromodulin levels were quantified by enzyme-linked immunosorbent assay. Results: A significant difference was found in genotype and allelic frequency among DM, DN and HC. TC+CC genotype and C allele were found more frequently in DN compared to HC (33.9 vs 23.0%, P=0.011 and 20.1 vs 12.9%, P=0.004, respectively). Compared to DM, C allele was found to be more frequent in individuals with DN (20.1 vs 14.7%, P=0.034). Those with DN had higher serum uromodulin levels compared to those with DM (P=0.001). Serum uromodulin levels showed a positive correlation with serum creatinine (r=0.431; P<0.001) and negative correlation with estimated glomerular filtration rate (r=−0.423; P<0.001). Interpretation & conclusions: The frequency of UMOD rs4293393 variant with C allele was significantly higher in individuals with DN. UMOD rs4293393 T>C variation might have a bearing on susceptibility to nephropathy in north Indian individuals with type 2 diabetes.

A prospective study of collapsing focal segmental glomerulosclerosis.

Abstract Collapsing focal segmental glomerulosclerosis (cFSGS) is characterized by rapid progression to end-stage renal disease (ESRD). We evaluated the clinicopathological spectrum of cFSGS and compared its clinical behavior to steroid and tacrolimus (TAC)-resistant noncollapsing focal segmental glomerulosclerosis (FSGS). All patients (>14 years) diagnosed with cFSGS were enrolled in the study. Staining for differentiated podocyte markers such as WT 1, PAX and KI67 were performed in all patients. The outcome and histological features of cFSGS was compared with a prospectively followed cohort of steroid and TAC-resistant noncollapsing FSGS. The study included 22 cFSGS patients and 19 cases of steroid and TAC-resistant FSGS. Complete remission, partial remission, steroid resistance, progression to ESRD and death were observed in 13.6%, 4.5%, 27.3%, 36.4% and 18.2% patients, respectively. Patients with cFSGS had higher serum creatinine and more advanced tubulointerstitial changes compared to resistant FSGS. Twenty-six percent of therapy resistant noncollapsing FSGS progressed to ESRD after two years of stopping TAC. However, there was no difference in progression to ESRD between cFSGS and therapy-resistant noncollapsing FSGS at the end of two years. Glomerular collapse in the setting of FSGS is poorly responsive to treatment and has a high rate of progression to ESRD. The long-term prognosis of cFSGS and steroid and TAC-resistant FSGS are similar.