Chronic Kidney Disease of Unknown Etiology in India: What Do We Know and Where We Need to Go.

Chronic kidney disease (CKD) not associated with known risk factors has been reported from parts of India and is presumed to be similar to CKD of unknown etiology (CKDu) that has been described from Central America. The reports from India have been fragmented without clear description of the disease phenotype or its determinants. This paper summarizes the current state of knowledge around CKDu in India based on a review of literature, multi-stakeholder consultation, and a survey of Indian nephrologists. We also contacted individual research groups to solicit data. Our findings suggest that that CKDu is reported from most regions in India; however, it is interpreted differently from the phenotype described from Central America and Sri Lanka. The differences include lack of a clear demographic or occupation group, older age of affected participants, and presence of mild hypertension and low-grade proteinuria. Well-designed prospective field studies with appropriate diagnostic workup are needed to establish the disease burden and identify etiologies, along with socioeconomic and health consequences, the intersection with the environment, and the public health response. Community-based research should phenotype the entire CKD population rather than be restricted to cases with presumed CKDu based on predefined criteria. Guidelines are needed for clinical evaluation, referral, management, and harmonization of clinical documentation and health records. More data are needed to support the existence of a unique CKDu phenotype in India.

A Systematic Study of the Prevalence and Risk Factors of CKD in Uddanam, India.

INTRODUCTION: Despite reports of a high prevalence of chronic kidney disease (CKD) from the coastal Uddanam region of Andhra Pradesh, India, there are no accurate data on the distribution of kidney function abnormalities and CKD risk factors in this region. METHODS: A total of 2419 participants were recruited through multistage cluster random sampling from 67 villages. Serum creatinine and urine protein creatinine ratio were measured using validated methodologies. All abnormal estimated glomerular filtration rate (eGFR) and urine protein creatinine ratio values were reconfirmed after 3 months. A range of sociodemographic factors were evaluated for their association with CKD using Poisson regression. RESULTS: Of 2402 eligible subjects (mean ± SD age, 45.67 ± 13.29 years; 51% female), 506 (21.07%) had CKD (mean ± SD age, 51.79 ± 13.12 years; 41.3% female). A total of 246 (10.24%) had eGFR <60 ml/min/1.73 m2, whereas 371 (15.45%) had an elevated urine protein creatinine ratio (>0.15 g/g). The poststratified estimates, adjusted for age and sex distribution of the region for CKD prevalence, are 18.7% (range, 16.4%-21.0%) overall and 21.3% (range, 18.2%-24.4% ) and 16.2% (range, 13.7%-18.8%) in men and women, respectively. Older age, male sex, tobacco use, hypertension, and family history of CKD were independently associated with CKD. Compared with those with higher eGFR, those with eGFR <60 ml/min/1.73m2 were older, were more likely to be uneducated, manual laborers/farmers, or tobacco users, and were more likely to have hypertension, a family history of CKD, a diagnosis of heart disease, and a lower body mass index. Among those with low eGFR, there was no difference between those with urine protein creatinine ratio <0.15 or >0.15, except a lower frequency of males in the former. CONCLUSION: We confirmed the high prevalence of CKD in the adult population of Uddanam. The cause was not apparent in a majority. Subjects with a low eGFR with or without elevated proteinuria were phenotypically distinct from those with proteinuria and preserved eGFR. Our data suggest the need to apply a population-based approach to screening and prevention and studies to understand the causes of CKD in this region.

Study to Test and Operationalize Preventive Approaches for CKD of Undetermined Etiology in Andhra Pradesh, India.

INTRODUCTION: High prevalence of chronic kidney disease (CKD) not associated with known risk factors has been reported from coastal districts of Andhra Pradesh. The Study to Test and Operationalize Preventive Approaches for Chronic Kidney Disease of Undetermined Etiology in Andhra Pradesh (STOP CKDu AP) aims to ascertain the burden (prevalence and incidence) of CKD, the risk factor profile, and the community perceptions about the disease in the Uddanam area of Andhra Pradesh. METHODS: Study participants will be sampled from the Uddanam area using multistage cluster random sampling. Information will be collected on the demographic profile, occupational history, and presence of conventional as well as nonconventional risk factors. Glomerular filtration rate (GFR) will be estimated using the Chronic Kidney Disease Epidemiology Collaboration equation, and proteinuria will be measured. All abnormal values will be confirmed by repeat testing after 3 months. Cases of CKD not associated with identified etiologies will be identified. Biospecimens will be stored to explore future hypotheses. The entire cohort will be followed up every 6 months to determine the incidence of CKD and to identify risk factors for decline in kidney function. Qualitative studies will be performed to understand the community perceptions and expectations with respect to the interventions. IMPLICATIONS: CKD is an important public health challenge in low- and middle-income countries. This study will establish the prevalence and determine the incidence of CKD not associated with known risk factors in a reported high-burden region, and will provide insights to help design targeted health systems responses. The findings will contribute to the policy development to tackle CKD in the region and will permit international comparisons with other regions with similar high prevalence.

Association of NFKB1 gene polymorphism (rs28362491) with levels of inflammatory biomarkers and susceptibility to diabetic nephropathy in Asian Indians.

AIM: To investigate the association of NFKB1 gene -94 ATTG insertion/deletion (rs28362491) polymorphism with inflammatory markers and risk of diabetic nephropathy in Asian Indians. METHODS: A total of 300 subjects were recruited (100 each), normoglycemic, (NG); type 2 diabetes mellitus (T2DM) without any complications (DM) and T2DM with diabetic nephropathy [DM-chronic renal disease (CRD)]. Analysis was carried out by polymerase chain reaction-restriction fragment length polymorphism and ELISA. Pearson's correlation, analysis of variance and logistic regression were used for statistical analysis. RESULTS: The allelic frequencies of -94 ATTG insertion/deletion were 0.655/0.345 (NG), 0.62/0.38 (DM) and 0.775/0.225 (DM-CRD). The -94 ATTG ins allele was associated with significantly increased levels of urinary monocyte chemoattractant protein-1 (uMCP-1); uMCP-1 (P = 0.026) and plasma tumor necrosis factor-alpha (TNF-α); TNF-α (P = 0.030) and almost doubled the risk of diabetic nephropathy (OR = 1.91, 95%CI: 1.080-3.386, P = 0.025). CONCLUSION: -94 ATTG ins/ins polymorphism might be associated with increased risk of developing nephropathy in Asian Indian subjects with diabetes mellitus.

Association of glutathione-S-transferase with patients of type 2 diabetes mellitus with and without nephropathy.

STATEMENTS OF THE PROBLEM: Hyperglycemia induced oxidative stress is implicated as a contributor to the onset and progression of type 2 diabetes mellitus (T2DM) and its complications like diabetic nephropathy (DN). Glutathione-S-transferase (GST) is primarily involved in the neutralization of reactive oxygen species (ROS) by enzymatic conjugation with the scavenger peptide glutathione (GSH). Therefore, present study was aimed to evaluate the role of GST along with oxidative stress markers and their correlation in patients with Type 2 diabetes mellitus with and without nephropathy. METHODS: This study comprised of 300 participants divided into three groups of 100 each: healthy controls (HC), T2DM without complications and DN. Plasma GST, malondialdehyde (MDA), reduced GSH levels and ferric reducing ability of plasma (FRAP) were estimated spectrophotometrically. RESULTS: Highest GST levels was observed in T2DM which was significantly higher (p<0.05) as compared to DN and HC. However, GSH and FRAP levels were found to be significantly lowest whereas MDA levels were significantly highest in DN as compared to T2DM and HC. GST showed a significant negative correlation with GSH, FRAP and positive correlation with MDA in both patients groups. CONCLUSIONS: Highest activity of GST in T2DM might be as a compensatory mechanism in response to oxidative stress. GST is found to have significant negative association with decreased GSH. Altered redox milieu in DN collectively conspire to increase the risk of renal damage in T2DM.

Genetic association of NAD(P)H quinone oxidoreductase (NQO1*2) polymorphism with NQO1 levels and risk of diabetic nephropathy.

NAD(P)H quinone oxidoreductase 1 (NQO1) catalyzes reactions having a cyto-protective effect against redox cycling and oxidative stress. A single base polymorphism (C/T) at nucleotide 609 of the NQO1 gene impairs the stability and function of its protein. Its role in the development of diabetic nephropathy (DN) has not been deciphered. Therefore, this study aimed to evaluate the association of NQO1*2 (rs1800566) polymorphism with plasma NQO1 levels and DN. This study screened 600 participants including healthy controls (HC), type 2 diabetes mellitus without complications (T2DM) and diabetic nephropathy (DN): 200 each for studying NQO1*2 gene polymorphism using the PCR-RFLP. Plasma NQO1 levels were measured by ELISA. Analysis of variance and logistic regression were used to evaluate the association of NQO1 polymorphism with plasma NQO1 levels and DN. The allelic frequencies of NQO1*1/NQO1*2 were 0.88/0.12 in HC, 0.765/0.235 in T2DM and 0.65/0.35 in DN. Carriers of the NQO1*2 allele had significantly lower plasma NQO1 levels (p<0.05) and revealed higher risk towards the development of DN (OR=1.717, p=0.010). NQO1*2 SNP is a functional polymorphism having a significant effect on NQO1 levels. Our results indicate that NQO1*2 genotype may increase susceptibility to DN in north Indian subjects with T2DM.

Association of tumor necrosis factor (TNF) promoter polymorphisms with plasma TNF-α levels and susceptibility to diabetic nephropathy in North Indian population.

AIM: The concept of diabetic nephropathy (DN) as a metabolic disease is now being replaced by chronic low-grade inflammatory disease. Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine which plays an important role in the pathogenesis and clinical outcome of DN. Therefore, this work was planned to evaluate the association of -863C/A (rs1800630) and -1031T/C (rs1799964) polymorphisms in TNF gene with plasma TNF-α levels and DN among subjects with type 2 diabetes (T2DM) in a population from North India. METHODS: Age and sex matched 100 healthy controls (HC), 100 T2DM subjects without nephropathy (DM) and 100 subjects with DN were screened for above polymorphisms using the PCR-RFLP methods. Plasma TNF-α levels were measured by ELISA. Analysis of variance and logistic regression were used to associate individual polymorphisms with plasma TNF-α levels and DN. RESULTS: The allelic frequencies of -863C/A were 0.86/0.14 in HC, 0.72/0.23 in DM and 0.84/0.16 in DN, and that of -1031T/C were 0.89/0.11 in HC, 0.95/0.05 in DM and 0.80/0.20 in DN. The carriers of -863A allele had significantly lower plasma TNF-α levels (p<0.05). The -863C/A (OR=0.439, 95% CI=0.244-0.789, p=0.006) and -1031T/C (OR=3.0, 95% CI=1.355-6.642, p=0.007) were strongly associated with risk of development of DN. CONCLUSIONS: -863C/A was associated with low whereas -1031T/C with high TNF-α levels. The, results suggest that -863C/A polymorphism might be protective whereas -1031T/C may be associated with increased risk for DN in subjects with T2DM from North India.

Increased level of organochlorine pesticides in chronic kidney disease patients of unknown etiology: role of GSTM1/GSTT1 polymorphism.

Chronic kidney disease (CKD) of unknown etiology represents about 16% of CKD patients in Indian subcontinents and 10% worldwide. The aetiology of CKD of unknown etiology remains unclear though epidemiological studies indicate the involvement of the environmental toxins. Organochlorine pesticides (OCPs) have been detected in general population in India. It is possible that polymorphism of xenobiotic metabolizing enzymes (XMEs) may play an important role in this process. In this we intend to find out blood levels of OCPs in CKD patients of unknown etiology and to evaluate the consequence of glutathione S-transferase (GST) gene polymorphism on the same. We have assessed 270 CKD patients and 270 age-sex-matched healthy controls for this study. The blood OCP levels were analyzed by gas chromatograph. GSTM1, GSTT1 genotyping were carried out by multiplex PCR. Blood levels of HCH, endosulfan and total pesticides were significantly higher in CKD patients and negatively correlated with eGFR. The combined frequency of GSTM1(-)/GSTT1(-) genotype increased the risk of CKD by 1.8-fold as compared to healthy controls. To find out the dependence of blood OCPs level on genotype, we carried out logistic regression analysis and results revealed that GSTM1(-)/GSTT1(-) genotype associated significantly with a number of OCPs namely γ-HCH, p,p'-DDT and total pesticides. Polymorphism of XMEs not only increased accumulation of pesticides but also aggravates kidney dysfunction as evident from significant decrease in eGFR.

Association of CYP1A1 gene polymorphism with chronic kidney disease: a case control study.

CYP1A1 is an important xenobiotic metabolizing enzyme, present in liver and kidney. Expression of CYP1A1 enzyme increases manifold when kidney cells are exposed to nephrotoxins/chemicals leading to oxidative stress-induced cell damage. To study the association of CYP1A1 gene polymorphism in patients of chronic kidney disease with unknown etiology (CKDU), we recruited 334 CKDU patients and 334 age and sex matched healthy controls. CYP1A1*2A and *2C polymorphisms were studied by PCR-RFLP and allele specific-PCR respectively. Subjects carrying at least one mutant allele of CYP1A1*2A (TC, CC) and *2C (AG, GG) were shown to be associated with 1.4-2-fold increased risk of CKDU. Also, genotypic combinations of hetero-/homozygous mutants of CYP1A1*2A (TC, CC) with hetero-/homozygous mutant genotypes of CYP1A1*2C (AG, GG) i.e. TC/AG (p<0.01), TC/GG (p<0.05), CC/AG (p<0.05) and CC/GG (p<0.01) were associated with CKDU with an odd ratio ranging 1.8-3.3 times approximately. This study demonstrates association of CYP1A1 polymorphisms with CKDU.

Study on organochlorine pesticide levels in chronic kidney disease patients: association with estimated glomerular filtration rate and oxidative stress.

Nephrotoxicity of organochlorine pesticides (OCPs) has been established in experimental animal models. This study was designed to evaluate the relationship of the blood OCPs level with the estimated glomerular filtration rate (eGFR) and oxidative stress (OS) in chronic kidney disease (CKD) patients. Patients in different stages of CKD (n = 150) and age, sex matched healthy controls (n = 96) were recruited. The blood OCPs level were analyzed by gas chromatography, and plasma levels of several OS parameters such as malondialdehyde (MDA), protein carbonyl, advanced oxidation protein products (AOPP), and total thiols were quantified by standard spectrophotometric methods. We observed significantly higher levels of hexachlorocyclohexane (α, γ), endosulfan, aldrin, p,p'-dichlorodiphenyldichloroethylene (DDE), and total pesticides in CKD patients. Negative correlation was also observed for aldrin, p,p'-DDE and total pesticides (p < 0.05) with eGFR. Plasma levels of MDA and AOPP showed significant positive association with the total pesticides level, indicating augmentation of OS with increased accumulation of OCPs in CKD patients.

Association of glutathione S-transferase M1 and T1 gene polymorphism with oxidative stress in diabetic and nondiabetic chronic kidney disease.

BACKGROUND AND OBJECTIVE: Glutathione S-transferases (GSTs) belong to a family of ubiquitous and multifunctional enzymes that work as one of the endogenous antioxidants in our body. This study was designed to look into the association of GST polymorphism with oxidative stress in both diabetic and nondiabetic chronic kidney disease (CKD). DESIGN AND METHODS: Three groups of patients (50 in each): diabetics without CKD (DM), diabetic CKD (DM-CKD), and nondiabetic CKD (NDM-CKD) and 50 age- and sex-matched healthy controls were recruited. Genotyping was done for GSTM1 and GSTT1 genes using a multiplex polymerase chain reaction. Serum GST and malondialdehyde (MDA) as a marker of oxidative stress were measured spectrophotometrically. RESULTS: Based on genotyping, subjects were categorized as GSTM1+/GSTT1+, GSTM1-/GSTT1+, GSTM1+/GSTT1-, and GSTM1-/GSTT1-. Serum GST levels were lower among subjects with deletion in one/both GST genes, whereas MDA levels were found to be correspondingly raised. A negative correlation for MDA versus GST levels was observed among genotypes with one/both gene deletions. Presence of GSTM1+/GSTT1- and GSTM1-/GSTT1- was significantly higher among patients with CKD in both diabetics and nondiabetics. INTERPRETATIONS AND CONCLUSIONS: GSTM1 and GSTT1 deletions singly or together were associated with lower GST levels and higher oxidative stress in both diabetic and nondiabetic CKD. Interestingly, GSTT1 deletion appears to be associated with both diabetic and nondiabetic CKD irrespective of the GSTM1 status.

The structural insights of stem cell factor receptor (c-Kit) interaction with tyrosine phosphatase-2 (Shp-2): an in silico analysis.

BACKGROUND: Stem cell factor (SCF) receptor c-Kit is recognized as a key signaling molecule, which transduces signals for the proliferation, differentiation and survival of stem cells. Binding of SCF to its receptor triggers transactivation, leading to the recruitment of kinases and phosphatases to the docking platforms of c-Kit catalytic domain. Tyrosine phosphatase-1 (Shp-1) deactivates/attenuates 'Kit' kinase activity. Whereas, Asp816Val mutation in the Kit activation loop transforms kinase domain to a constitutively activated state (switch off-to-on state), in a ligand-independent manner. This phenomenon completely abrogates negative regulation of Shp-1. To predict the possible molecular basis of interaction between c-Kit and Shp-1, we have performed an in silico protein-protein docking study between crystal structure of activated c-Kit (phosphorylated c-Kit) and full length crystal structure of Shp-2, a close structural counterpart of Shp-1. FINDINGS: Study revealed a stretch of conserved amino acids (Lys818 to Ser821) in the Kit activation domain, which makes decisive H-bonds with N-sh2 and phosphotyrosine binding pocket residues of the phosphatase. These H-bonds may impose an inhibitory steric hindrance to the catalytic domain of c-Kit, there by blocking further interaction of the activation loop molecules with incoming kinases. We have also predicted a phosphotyrosine binding pocket in SH2 domains of Shp-1, which is found to be predominantly closer to a catalytic groove like structure in c-Kit kinase domain. CONCLUSIONS: This study predicts that crucial hydrogen bonding between N-sh2 domain of Shp-1 and Kit activation loop can modulate the negative regulation of c-Kit kinase by Shp-1. Thus, this finding is expected to play a significant role in designing suitable gain-of-function c-Kit mutants for inducing conditional proliferation of hematopoietic stem cells.

Dual genetic abnormality in the coagulation pathway as a cause of familial thrombophilia.

Inherited deficiency of certain factors is responsible for increased tendency to vascular thrombosis; however two genetic defects in the coagulation pathway may coexist and cause recurrent thrombosis. Previously studies of thrombophilia have focused on the identification of single gene defects with the concept that familial thrombophilia is a single gene disorder. Now it has become accepted that familial thrombosis in protein C-deficient families is caused by co-segregation of one or more additional genetic factors that increase the risk of thrombosis. Co-existence of two or more genetic abnormalities increases the risk of thrombotic tendencies in affected persons. Simultaneous presence of factor V Leiden and deficiency of protein C results in higher risk of thrombosis. We report two such cases with additional analysis of the family tree highlighting that dual abnormality results in higher penetrance of the disease among family members.

Effect of hemodialysis on cognitive function in ESRD patients.

BACKGROUND: Uremia is associated with impairment of different cognitive functions. However the pathogenesis of this cognitive dysfunction is unknown. OBJECTIVE: In this study, long-latency event related potentials (ERPs) were used to assess changes in cortical function due to hemodialysis treatment. METHODS: In this cross-sectional study, we measured event related potentials in 15 end stage renal disease (ESRD) patients maintained on hemodialysis, two hours before and two hours after they underwent hemodialysis and compared their data with a strictly age and sex matched healthy control group. The P3 was elicited by using standard auditory "odd-ball" paradigm and the data obtained was statistically analyzed (Wilcoxon signed ranks, Mann Whitney). RESULTS: Before hemodialysis, the patients' P3 latency (347.73 +/- 39.47 ms) was significantly increased as compared with that of healthy control group (308.4 +/- 13.73 ms) (p = 0.001). After hemodialysis, P3 latency of the patients showed a significant decrease (347.73 +/- 39.47 ms to 325.20 +/- 37.15 ms, p = 0.001). P3 latency after dialysis was not significantly different from controls. No significant correlation was noted between various biochemical parameters (hemoglobin, blood urea, creatinine, uric acid and calcium) and P3 latency or amplitude. CONCLUSIONS: Removal of uremic toxins by hemodialysis leads to an improvement in cognitive processing.

Cognitive impairment in chronic kidney disease.

BACKGROUND: Although end-stage renal disease (ESRD) has been associated with cognitive impairment, the relation between lesser degrees of chronic kidney disease (CKD) and cognitive impairment is less well understood. The objective of this study was to assess the cognitive function in patients with varying severity of CKD using P3 event-related potentials (P3ERPs). METHODS: In this cross-sectional study, 15 neurologically asymptomatic (Mini Mental State Examination >24) patients each of CKD stage 3, 4 and 5 (undialysed) were enrolled. Besides this, 15 healthy controls were also studied. All groups were age and sex matched. Glomerular filtration rate (GFR) was estimated using the Modification of Diet in Renal Disease equation. The P300 was studied in all subjects by using standard auditory 'odd-ball' paradigm and the data obtained were statistically analysed. RESULTS: We noted significant prolongation of P300 latencies as severity of CKD increased from stage 3 (318.8 +/- 28.98 ms) to stage 4 (357 +/- 24.65 ms) (P < 0.002) and from stage 4 to 5 (388.47 +/- 31.67 ms) (P < 0.01). P300 latency in CKD stage 3 was not found to be significantly different from controls (308.4 +/- 13.73 ms). Significant positive correlation was noted between serum creatinine, blood urea and uric acid with P3 latency. Significant negative correlation was noted between GFR, serum calcium and haemoglobin with P3 latency. CONCLUSIONS: Increasing severity of CKD is associated with progressive cognitive decline and this may have important clinical consequences.

Phagocytic polymorphonuclear function in patients with progressive uremia and the effect of acute hemodialysis.

BACKGROUND: Susceptibility to infections is a major cause of high morbidity and mortality in patients with chronic kidney disease (CKD). Polymorphonuclear leukocytes (PMNs) play a major role in the host defense against various infections. Although the phagocytic function of PMNs in uremic patients has been subjected to repeated evaluation, results have been inconsistent. Also, the effects of progressive uremia and acute hemodialysis (HD) on polymorphonuclear function have been scarcely evaluated. METHODS: In this study, we assessed the phagocytic index (PI) of PMNs following their uptake of IgG-coated sheep erythrocytes in patients with varying severity of CKD and in healthy controls. In patients with advanced renal failure, the effects of acute HD on PI pre- and post-HD were also studied. RESULTS: A total of 30 patients with CKD and 15 healthy controls were studied. Patients with CKD were categorized into two groups: mild to moderate renal failure (S. Creatinine 2-6 mg/dL) and advanced renal failure (S. Creatinine >6 mg/dL) (n=15 for each). All three groups were age and sex matched. Patients with advanced renal failure had significantly impaired PI (4.03 +/- 1.15) as compared to patients with mild to moderate renal failure (7.17 +/- 2.62) and normal controls (8.13 +/- 3.31) (p < 0.001 for both). There was a statistically insignificant decline in the PI in patients with mild to moderate RF when compared to controls (7.17 +/- 2.62 versus 8.13 +/- 3.31). In patients with advanced renal failure, acute HD led to a significant improvement in PI (5.52 +/- 1.55 versus 4.03 +/- 1.15, p < 0.01). CONCLUSION: In patients with CKD, there is a progressive decline in the phagocytic index of the PMNs with increasing severity of uremia. Significant improvement in the phagocytic index following acute hemodialysis suggests the role of a circulating uremic toxin in this PMN dysfunction.