The significance of tubular and glomerular proteinuria in critically ill patients with severe acute kidney injury.

OBJECTIVE: Critically ill patients with acute kidney injury (AKI) frequently need acute renal replacement therapy (aRRT). We evaluated an inexpensive, rapid quantitative and qualitative analysis of proteinuria on the course of AKI patients requiring aRRT in intensive care. METHOD: This was a prospective, observational study of critically ill patients with severe established AKI or Acute on Chronic Kidney Injury (AoCKI) requiring aRRT. Urine samples were analyzed using Sodium-Dodecyl-Sulphate-Polyacryamide Gel Electrophoresis (SDS-PAGE). RESULTS: A total of 30 critically ill patients were studied. Those who died have higher APACHE II (29 ± 6 vs. 20 ± 5, p<0.001), multi-organ failure (0.7 ± 0.5 vs. 0.2 ± 0.4, p < 0.02) and Tubular/Glomerular ratio (114 ± 60 vs. 75± 37, p < 0.05).The renal non-recoverers have higher baseline creatinine (415 ± 328 vs. 125± 19 umol/l, p < 0.01), urinary Dipstick value (1.8±0.8 vs. 0.5±0, p <0.05) and Glomerular score (3.0 ± 1.8 vs. 0.6 ± 0.2, p < 0.02).Heavy tubular proteinuria also predicts a longer duration of interim dialysis support and mortality whereas glomerular proteinuria correlates with development of chronicity and End Stage Renal Disease (ESRD). CONCLUSIONS: The dominant presence of tubular proteinuria is associated with poor survival in patients who have high APACHE II score and multi-organ failure. It also correlates with a longer duration of dialysis support in survivals. Renal Non-recoverers had heavy dominant presence of glomerular proteinuria. SDS-PAGE proteinuria analysis offers a reliable and inexpensive method to prognosticate proteinuria in this group of critically ill patients.

Genomics and disease progression in IgA nephritis.

Apart from clinical, histological and biochemical indices, genomics are now being employed to unravel the pathogenetic mechanisms in the disease progression of IgA nephritis (IgAN). The results of angiotensin converting enzyme (ACE) gene polymorphism have been controversial. Those patients with the DD genotype seem to have a poorer prognosis. However, with high dose angiotensin receptor blocker (ARB) therapy, the ACE gene polymorphism status of a patient may no longer be a matter for concern as those with the DD genotype would also respond favourably to high dose ARB therapy. Association studies with gene sequencing and haplotypes have suggested that multiple genes are involved in the pathogenesis of IgAN. Some workers have reported a synergistic effect in the combined analysis of AGT-M235T and ACE I/D polymorphism. With the use of deoxyribo nucleic acid (DNA) microarray, tens of thousands of gene expressions genome-wide can be examined together simultaneously. A locus of familial IgAN has been described with strong evidence of linkage to IgAN1 on chromosome 6q22-23. Two other loci were reported at 4q26-31 and 17q12-22. DNA microarray techniques could also help in the identification of specific pathogenic genes that are up- or down-regulated and this may allow genome wide analyses of these genes and their role in the pathogenesis and progression of IgAN. Recently, using genome-wide association studies (GWAS) more loci for disease susceptibility for IgAN have been identified at 17p13, 8p23, 22q12, 1q32 and 6p21.

Urotensin 2 and retinoic acid receptor alpha (RARA) gene expression in IgA nephropathy.

INTRODUCTION: IgA nephropathy is a disease where the pathogenesis is still poorly understood. Deoxyribonucleic acid (DNA) microarray technique allows tens of thousands of gene expressions to be examined at the same time. Commercial availability of microarray genechips has made this powerful tool accessible for wider utilisation in the study of diseases. MATERIALS AND METHODS: Seven patients with IgA nephropathy, 6 with minimal change nephrotic syndrome (MCNS) as patient controls and 7 normal healthy subjects were screened for the differential expression of genes, genome-wide. The Human Genome U133 Plus 2.0 Arrays (Affymetrix, USA) were used to quantitate the differential expression of 38,500 well-characterised human genes. RESULTS: A total of 7761 gene expressions were identified that have an IgAN/Normal gene expression ratio of 0.06-fold to 5.58-fold. About 35% of the altered gene expressions have no gene title or just a hypothetical protein label such as FLJ30679. Most of the remaining 65% are identified proteins where their importance to IgAN is not immediately apparent at this time. Among the 30 most upregulated and 30 most downregulated genes are Urotensin 2 (upregulated 3.09-fold, P <0.05) and Fatty-acid binding protein 6 (downregulated to 0.12-fold, P <0.05). Retinoic acid receptor alpha (vitamin A receptor) was also found downregulated to 0.41-fold (P <0.005). Taqman realtime polymerase chain reaction (PCR) for urotensin 2 and retinoic acid receptor alpha (RARA) were performed on 20 patients with IgA nephropathy and 11 with Minimal Change Disease and the data correlated with various clinical indices. CONCLUSIONS: The findings suggest that there may be a therapeutic role for retinoic acid receptor alpha (RARA) in IgA nephropathy and a clinical monitoring role for Urotensin 2 in Minimal Change Disease.

Global evolutionary trend of the prevalence of primary glomerulonephritis over the past three decades.

OBJECTIVE: The prevalence of primary glomerulonephritis in Singapore is compared with that of 28 other countries to review changing trends in the evolution of primary glomerulonephritis in Asia and other countries. METHOD: 2,586 renal biopsies in Singapore over the past 3 decades were reviewed and compared with data from 28 other countries. RESULTS: In the 1st decade most Asian countries have mesangial proliferative glomerulonephritis as the most common form of primary glomerulonephritis, and in the 3rd decade there has been a dramatic increase in focal and segmental glomerulosclerosis reflecting aging and obesity in keeping with more developed countries. IgA nephritis remains the commonest glomerulonephritis in many countries. Membranous glomerulonephritis continues to be more prevalent in Western countries while mesangial proliferative glomerulonephritis remains prevalent in many Asian countries. CONCLUSION: Apart from geographical and genetic influences, socioeconomic factors may play a role in the evolution of the biopsy pattern in some countries. Worldwide, the prevalence of focal segmental glomerulosclerosis continues to increase. In third world countries some of the commoner forms of glomerulonephritis are related to infections, in contrast to developed countries where the antigenic exposure may be related to diet, allergens and other industrial agents.

Parallel genotyping of 10,204 single nucleotide polymorphisms to screen for susceptible genes for IgA nephropathy.

INTRODUCTION: IgA nephritis (IgAN) is the most common glomerulonephritis worldwide. We aim to genotype SNPs (single nucleotide polymorphisms) genomewide in patients with IgAN to search for genetic clues to its aetiology. MATERIALS AND METHODS: Genotyping for 10,204 SNPs genomewide was done with the Gene Chip Human Mapping 10K Microarray (Affymetrix). Twenty-eight patients with IgAN and 30 normal subjects were screened and analysed for differences in genotype frequency, allele frequency and heterozygosity reduction. RESULTS: Among the most significantly associated SNPs, 48 SNPs were found mapping directly to the intron of 42 genes that localised in 13 somatic chromosomes and chromosome X. Genotype distribution of these SNPs did not deviate from the Hardy-Weinberg equilibrium in normal subjects. The most significantly associated gene, glial cells missing homolog 1 (GCM, 2 =13.05, P = 0.000) is a transcription factor mapped to 6p12.2. GCM1 reported decreased in placenta of patients with pre-eclampsia. The second gene, Tenascin-R (TNR, 2 = 9.85, P = 0.002) is a glycoprotein and extra-cellular matrix component mapped to 1q25.1. Tenascin-R was associated with motor coordination impairment and enhanced anxiety profile in deficient mice. Interestingly, Triadin (TRDN, 2 = 9.16, P = 0.01) is an integral membrane protein mapped to 6q22.31 within the IgAN1 locus. Triadin was shown to participate in cardiac myocyte arrhythemia. However, there is no published study of these genes in IgAN. CONCLUSION: Forty-two associated genes (particularly GCM1, TNR and TRDN) are identified as possible susceptibility or marker genes for IgAN. Knowledge of their mesangial expression and binding capacity for IgA-containing complexes may help elucidate the pathogenesis of IgAN.

Angiotensin-converting enzyme inhibitor versus angiotensin 2 receptor antagonist therapy and the influence of angiotensin-converting enzyme gene polymorphism in IgA nephritis.

INTRODUCTION: In this study of 109 patients with IgA nephritis (IgAN), we compared the longterm effects on patients treated with angiotensin converting enzyme inhibitor (ACEI) or angiotensin receptor antagonist (ATRA) alone with respect to renal outcome in terms of ESRF from 1995 to 2006. The renal outcome is also correlated with the ACE gene ID polymorphism to study its influence on response to ACEI/ATRA therapy. MATERIALS AND METHODS: Seventy-seven patients were on treatment with ACEI/ATRA (22 on ACEI alone, 47 on ATRA alone and 8 on both). The other 32 patients were on no treatment (control group). RESULTS: Compared to controls, treated patients had lower serum creatinine (P <0.001), lower proteinuria (P <0.001) and fewer number progressing to ESRF (P <0.001). For those with the II and ID genotype there were significantly fewer patients with ESRF in the treatment group. With the DD genotype, treatment did not change the poor renal outcome with regard to ESRF. Patients on ACEI therapy had a higher incidence of ESRF compared to those on ATRA (P <0.001). For the control group, the projected number of years-to-ESRF was 10 years. For those on ACEI therapy it was 11 years, and for those on ATRA therapy it was 24 years. Among patients with the II genotype, those treated with ATRA had significantly less incidence of ESRF compared to those treated with ACEI (P <0.001). CONCLUSION: ATRA therapy was found to be effective in retarding disease progression to ESRF in IgAN compared to ACEI therapy. Genotyping showed better response to ATRA therapy only for those with the II genotype.

Disease progression, response to ACEI/ATRA therapy and influence of ACE gene in IgA nephritis.

Various studies have shown that angiotensin-converting enzyme (ACE) gene insertion/deletion (ID) polymorphism may play a role in the progression to end stage renal failure (ESRF) in patients with IgA nephritis (IgAN). In this randomized controlled trial, patients were followed up for 5 years to determine their long-term renal outcome to ACEI/ATRA therapy and to ascertain if their ACE gene profile could play a role in determining their response to therapy. Seventy-five patients with IgAN were enlisted. Thirty-seven were on ACEI/ATRA therapy for 62+/-5 months and thirty-eight were untreated and served as controls. All patients had their ACE gene ID polymorphism genotyped. Compared to controls, treated patients had lower serum creatinine (p < 0.001), lower proteinuria (p < 0.002) and fewer numbers progressing to ESRF (p < 0.002). Among patients with genotype II, there were less ESRF in the treatment group when compared to the untreated control group (p < 0.02). The advantage of therapy was not seen in patients with ID or DD genotypes. ACEI/ATRA therapy was found to be effective in retarding disease progression in IgAN with years to ESRF significantly extended in patients at all levels of renal function, including patients whose outcome were ESRF. Genotyping showed better response to therapy only for those with genotype II. The common mechanism is probably through lower levels of ACE, glomerular pressure and proteinuria resulting in reduced renal damage and retardation of progression to ESRF.

3rd College of Physicians' lecture--translational research: From bench to bedside and from bedside to bench; incorporating a clinical research journey in IgA nephritis (1976 to 2006).

Translational research (TR) can be defined as research where a discovery made in the laboratory (bench) can be applied in the diagnosis, treatment or prevention of a disease. Examples of medical discoveries contributing to translational medicine (TM) include the isolation of insulin by Banting (Nobel Laureate, 1923), the discovery of penicillin by Alexander Fleming (Nobel Laureate, 1945) and recently the discovery of the role of bacterium Helicobacter pylori in the causation of gastritis and peptic ulcer by Marshall and Warren (Nobel Laureates, 2005). Clinical research (CR) would be a more appropriate term for the bulk of research work undertaken by doctors. CR embraces both clinical based and laboratory-based research. The terminology "bedside to bench" applies more to CR as opposed to "bench to bedside" in the case of TR. But regardless of who does it, as long as the discovery can be translated to the bedside and results in improvement in patient care it can be considered a contribution to TM. Our work spans a 30-year period, involving laboratory-based research, clinical trials and genomics of IgA nephritis (Nx). This is a series of work to elucidate the pathogensis and therapy of IgANx. Plasma beta-thromboglobulin (BTG) an in-vivo index of platelet aggregation and anti-thrombin III increase due to a constant thrombogenecity resulting from platelet degranulation formed the basis for anti-platelet and low-dose warfarin therapy. A study of the natural history of IgANx revealed 2 courses, a slowly progressive course with end-stage renal failure (ESRF) at 7.7 years and a more rapid course at 3.3 years. Triple therapy (cyclophosphamide, persantin and low-dose warfarin) delayed progression to ESRF by about 8 years and for some patients up to 20 years. Documentation of abnormal suppressor T cell function provided the basis for immune therapy. Four patterns of proteinuria were present in IgANx and it is the quality and not so much the quantity of proteinuria which determined the prognosis. Low molecular weight proteinuria was a bad prognostic marker. A controlled therapeutic trial using ACEI/ATRA showed that therapy decreases proteinuria, improves renal function and converts non-selective to selective proteinuria. Subsequent work confirmed that it was the ATRA, not the ACEI which contributed to improved renal function. Individual anti proteinuria response to ATRA varies depending on ACE gene polymorphism. We found that the II genotype of the ACE gene was renoprotective and patients with this genotype had significantly reduced incidence of ESRF compared to those with the DD genotype. Patients responsive to ATRA therapy can retard progression to ESRF by up to 32 years. Mild renal failure can be reversed with possible regression of glomerulosclerosis because of glomerular remodelling by ATRA.

Polymorphism of renin-angiotensin system genes in IgA nephropathy.

BACKGROUND AND AIMS: Individuals are prone to disease because of certain disease-susceptible genes. The angiotensin I-converting enzyme (ACE) gene insertion/deletion (I/D), the angiotensinogen (AGT) gene, M235T, and the angiotensin II type 1 receptor (ATR) gene, A1166C, polymorphisms have been associated with IgA nephropathy (IgAN) and its progression. Several studies on Caucasians and Japanese patients have reported contradictory results. We determined these polymorphisms in 118 Chinese patients with IgAN and 94 healthy Chinese subjects to assess their clinical impact. METHODS: Genotyping was performed with DNA isolated from peripheral leucocytes, polymerase chain reaction amplification of the polymorphic sequence, restriction enzymes digestion, and separation and identification of DNA fragments. Clinical data at renal biopsy and final status on renal function were determined from patients' records. RESULTS: Comparing all IgAN patients with controls, AGT and ATR genotype distributions were similar, whereas there was a significant increase in the ACE DD genotype (P < 0.05). When comparing patients with end-stage renal failure (IgAN-ESRF) and those without (IgAN-nonESRF), there was no difference among the three gene polymorphisms. In contrast, there were significant differences in higher male prevalence (P < 0.05), increased serum creatinine at presentation (P < 0.05), more sclerosis (P < 0.01) and higher tubulointerstitial lesion score (P < 0.001) in the IgAN-ESRF group. CONCLUSION: Among the ACE, AGT and ATR gene polymorphisms, only the DD genotype may predispose the individual to IgAN in the Chinese population. None are significant for prognosticating ESRF.

Renin-angiotensin system gene polymorphisms: its impact on IgAN and its progression to end-stage renal failure among Chinese in Singapore.

BACKGROUND: Gene polymorphisms in angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II type 1 receptor (ATR) had been associated with IgA nephropathy (IgAN) and its progression. Several studies on Caucasian and Japanese had reported contradicting results. We determined these polymorphisms in 118 Chinese patients with IgAN and 94 healthy Chinese to assess their clinical impact. METHODS: Genotyping was performed with DNA from peripheral leukocytes, PCR amplification of the polymorphic sequence, restriction enzymes digestion, separation and identification of DNA fragments. Clinical data at renal biopsy and final status on renal function were determined from patients' records. RESULTS: Among controls, genotype distributions were in Hardy-Weinberg equilibrium. Comparing all IgAN patients with controls, AGT and ATR genotype distributions were similar whereas there was significant increase in the ACE DD genotype (p < 0.05). Comparing patients with end-stage renal failure (IgAN-ESRF) and without (IgAN-nonESRF), there was no difference in any of the three gene polymorphisms. But in contrast, there were significant differences in higher male prevalence (p < 0.05), increased serum creatinine at presentation (p < 0.05), more sclerosis (p < 0.01) and higher tubulointerstitial lesion score (p < 0.001) in the IgAN-ESRF group. CONCLUSION: Among the ACE, AGT and ATR gene polymorphisms, only the DD genotype may predispose the individual to IgAN in our Chinese population. In contrast to clinical and histological risk factors, these genetic variations showed no impact on disease progression to ESRF. It is unlikely that genotyping more patients will prove these genes useful. Nevertheless, preclinically determined genetic markers are very useful as risk factors for disease occurrence and as prognostic indices for disease progression. Therefore, continuing efforts should be made to look at other genes to find those with significance.