Sufang Shi

Pathologic Predictors of Renal Outcome and Therapeutic Efficacy in IgA Nephropathy: Validation of the Oxford Classification

BACKGROUND AND OBJECTIVES The Oxford classification of IgA nephropathy (IgAN) may aid in predicting prognosis and providing therapeutic strategy but must be validated in different ancestry. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS A total of 410 patients with IgAN, enrolled from one of the largest renal centers in China, were evaluated for the predictive value of the Oxford classification to prognosis defined as end stage renal disease. A total of 294 of these patients were prospectively treated with renin-angiotensin system blockade and immunosuppressants sequentially and were evaluated separately to assess the predictive value to therapeutic efficacy (defined as time-averaged proteinuria <1 g/d). Three pathologists reviewed specimens independently according to the Oxford classification and were blinded to clinical data. RESULTS Segmental glomerulosclerosis and tubular atrophy and interstitial fibrosis were independent predictive factors of end stage renal disease. Patients who had >25% of glomeruli with endocapillary hypercellularity showed higher proteinuria, lower estimated GFR, and higher mean BP than patients with less endocapillary hypercellularity. Immunosuppressive therapy showed a protective effect to prognosis of endocapillary hypercellularity in patients with endoncapillary hypercellularity could benefit from immunosuppressive therapy. Mesangial hypercellularity and tubular atrophy and interstitial fibrosis were independent factors of inefficiency of renin-angiotensin system blockade alone. Crescents were not significant in predicting prognosis or in therapeutic efficacy. CONCLUSIONS The Oxford classification may aid in predicting prognosis and providing a therapeutic strategy in Chinese patients with IgAN.

Variants in Complement Factor H and Complement Factor H-Related Protein Genes, CFHR3 and CFHR1, Affect Complement Activation in IgA Nephropathy

Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1-5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3-1 deletion (CFHR3-1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype-phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3-1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN.

Prediction of Outcomes in Crescentic IgA Nephropathy in a Multicenter Cohort Study

Crescentic IgA nephropathy (IgAN), defined as >50% crescentic glomeruli on kidney biopsy, is one of the most common causes of rapidly progressive GN. However, few studies have characterized this condition. To identify risk factors and develop a prediction model, we assessed data from patients ≥ 14 years old with crescentic IgAN who were followed ≥ 12 months. The discovery cohort comprised 52 patients from one kidney center, and the validation cohort comprised 61 patients from multiple centers. At biopsy, the mean serum creatinine (SCr) level ± SD was 4.3 ± 3.4 mg/dl, and the mean percentage of crescents was 66.4%± 15.8%. The kidney survival rates at years 1, 3, and 5 after biopsy were 57.4%± 4.7%, 45.8%± 5.1%, and 30.4%± 6.6%, respectively. Multivariate Cox regression revealed initial SCr as the only independent risk factor for ESRD (hazard ratio [HR], 1.32; 95% confidence interval [CI], 1.10 to 1.57; P=0.002). Notably, the percentage of crescents did not associate independently with ESRD. Logistic regression showed that the risk of ESRD at 1 year after biopsy increased rapidly at SCr>2.7 mg/dl and reached 90% at SCr>6.8 mg/dl (specificity=98.5%, sensitivity=64.6% for combined cohorts). In both cohorts, patients with SCr>6.8 mg/dl were less likely to recover from dialysis. Analyses in additional cohorts revealed a similar association between initial SCr and ESRD in patients with antiglomerular basement membrane disease but not ANCA-associated systemic vasculitis. In conclusion, crescentic IgAN has a poor prognosis, and initial SCr concentration may predict kidney failure in patients with this disease.

Rare Variants in the Complement Factor H–Related Protein 5 Gene Contribute to Genetic Susceptibility to IgA Nephropathy

A recent genome-wide association study of IgA nephropathy (IgAN) identified 1q32, which contains multiple complement regulatory genes, including the complement factor H (CFH) gene and the complement factor H-related (CFHRs) genes, as an IgAN susceptibility locus. Abnormal complement activation caused by a mutation in CFHR5 was shown to cause CFHR5 nephropathy, which shares many characteristics with IgAN. To explore the genetic effect of variants in CFHR5 on IgAN susceptibility, we recruited 500 patients with IgAN and 576 healthy controls for genetic analysis. We sequenced all exons and their intronic flanking regions as well as the untranslated regions of CFHR5 and compared the frequencies of identified variants using the sequence kernel association test. We identified 32 variants in CFHR5, including 28 rare and four common variants. The distribution of rare variants in CFHR5 in patients with IgAN differed significantly from that in controls (P=0.002). Among the rare variants, in silico programs predicted nine as potential functional variants, which we then assessed in functional assays. Compared with wild-type CFHR5, three recombinant CFHR5 proteins, CFHR5-M (c.508G>A/p.Val170Met), CFHR5-S (c.533A>G/p.Asn178Ser), and CFHR5-D (c.822A>T/p.Glu274Asp), showed significantly higher C3b binding capacity (CFHR5-M: 109.67%±3.54%; P=0.02; CFHR5-S: 174.27%±9.78%; P<0.001; CFHR5-D: 127.25%±1.75%; P<0.001), whereas another recombinant CFHR5 (c.776T>A/p.Leu259Termination) showed less C3b binding (56.89%±0.57%; P<0.001). Our study found that rare variants in CFHR5 may contribute to the genetic susceptibility to IgAN, which suggests that CFHR5 is an IgAN susceptibility gene.

Elevated Soluble VEGF Receptor sFlt-1 Correlates with Endothelial Injury in IgA Nephropathy

Background Endothelial injury, which may present clinically as hypertension, proteinuria and increased von Willebrand Factor (vWF) level, is a common manifestation in IgA nephropathy (IgAN). However, causal factors for endothelial injury in IgAN are not completely understood. An imbalance of vascular endothelial growth factor/Soluble fms-like tyrosine kinase-1 (VEGF/sFlt-1) has been observed in many diseases with endothelial dysfunction, including pre-eclampsia and diabetic retinopathy, but whether it contributes to endothelial injury in IgAN requires further exploration. Methods Initially, 96 IgAN patients and 22 healthy volunteers were enrolled as a discovery cohort. VEGF/sFlt-1, sFlt-1 and VEGF levels were compared between patients with IgAN and healthy volunteers to explore the underlying factors that contribute to endothelial injury in IgAN. The identified contributor (sFlt-1) was further confirmed in a replication cohort, which included 109 IgAN patients and 30 healthy volunteers. Correlations of sFlt-1 with hypertension, proteinuria, Oxford-E score and plasma vWF were further evaluated in the combined 205 patients with IgAN. Results VEGF/sFlt-1 levels were significantly lower in IgAN patients than healthy volunteers (0.33±0.27 vs. 0.43±0.22, p = 0.02) in the discovery cohort. Within the ratio, plasma sFlt-1 levels were significantly elevated (101.18±25.19 vs. 79.73±18.85 pg/ml, p<0.001), but plasma VEGF levels showed no significant differences. Elevated sFlt-1 levels in the replication cohort were confirmed in IgAN patients (93.40±39.78 vs. 71.92±15.78 pg/ml, p<0.001). Plasma sFlt-1 levels in IgAN patients correlated with proteinuria (severe (>3.5 g/d) vs. moderate (1–3.5 g/d) vs. mild (<1 g/d) proteinuria: 115.95±39.09 vs. 99.89±28.55 vs. 83.24±33.92 pg/ml; severe vs. mild: p<0.001, moderate vs. mild p = 0.001, severe vs. moderate: p = 0.014), hypertension (with vs. without hypertension: 107.87±31.94 vs. 87.32±32.76 pg/ml, p = 0.015) and vWF levels (r = 0.161, p = 0.021). Conclusions The present study found elevated sFlt-1 in IgAN patients and further identified its correlation with proteinuria, hypertension and vWF levels. These results suggested that elevated sFlt-1 contributes to endothelial injury in IgAN.

Progression of IgA Nephropathy under Current Therapy Regimen in a Chinese Population

BACKGROUND AND OBJECTIVES Current therapy for IgA nephropathy mainly includes renin-angiotensin system inhibitors and adding steroids for patients with persistent proteinuria. This study aimed to evaluate kidney disease progression and its risk factors in a Chinese cohort under current therapy. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Patients with IgA nephropathy followed up for at least 12 months from a prospective database were involved. Renal survival and the relationship between clinical parameters and composite kidney failure events (defined as end stage kidney failure or eGFR halving) were assessed. RESULTS Overall, 703 patients between 2003 and 2011 were enrolled in this study, with a mean follow-up time of 45 months. Mean eGFR was 84.0 ml/min per 1.73 m(2), systolic BP was 124 mmHg, and time-averaged mean arterial pressure was 90.0 mmHg. Median proteinuria at baseline was 1.60 g/d, and time-averaged proteinuria was 0.80 g/d. The mean rate of eGFR decline was -3.12 ml/min per 1.73 m(2) per year (95% confidence interval, -19.07 to 11.80), and annual end stage kidney failure rate was 2.3%. Multivariate Cox regression analyses revealed that baseline eGFR (hazard ratio, 0.76 per 10 ml/min per 1.73 m(2); 95% confidence interval, 0.66 to 0.91), proteinuria at 6 months (hazard ratio, 1.53 per g/d; 95% confidence interval, 1.27 to 1.84), and systolic BP control at 6 months (hazard ratio, 1.36 per 10 mmHg; 95% confidence interval, 1.05 to 1.77) were associated with composite kidney failure events. Baseline eGFR (regression coefficient, -0.06; 95% confidence interval, -0.07 to -0.04), time-averaged proteinuria (regression coefficient, -0.21; 95% confidence interval, -0.25 to -0.16), and time-averaged mean arterial pressure (regression coefficient, -0.15; 95% confidence interval, -0.21 to -0.09) were independent predictors of the slope of eGFR by linear regression. CONCLUSION Lower proteinuria and lower BP were associated with slower eGFR decline and lower risk of end stage kidney failure in patients currently being treated for IgA nephropathy.

Synergistic Effect of Mesangial Cell-Induced CXCL1 and TGF-β1 in Promoting Podocyte Loss in IgA Nephropathy

Podocyte loss has been reported to relate to disease severity and progression in IgA nephropathy (IgAN). However, the underlying mechanism for its role in IgAN remain unclear. Recent evidence has shown that IgA1 complexes from patients with IgAN could activate mesangial cells to induce soluble mediator excretion, and further injure podocytes through mesangial-podocytic cross-talk. In the present study, we explored the underlying mechanism of mesangial cell-induced podocyte loss in IgAN. We found that IgA1 complexes from IgAN patients significantly up-regulated the expression of CXCL1 and TGF-β1 in mesangial cells compared with healthy controls. Significantly higher urinary levels of CXCL1 and TGF-β1 were also observed in patients with IgAN compared to healthy controls. Moreover, IgAN patients with higher urinary CXCL1 and TGF-β1 presented with severe clinical and pathological manifestations, including higher 24-hour urine protein excretion, lower eGFR and higher cresentic glomeruli proportion. Further in vitro experiments showed that increased podocyte death and reduced podocyte adhesion were induced by mesangial cell conditional medium from IgAN (IgAN-HMCM), as well as rhCXCL1 together with rhTGF-β1. In addition, the over-expression of CXCR2, the receptor for CXCL1, by podocytes was induced by IgAN-HMCM and rhTGF-β1, but not by rhCXCL1. Furthermore, the effect of increased podocyte death and reduced podocyte adhesion induced by IgAN-HMCM and rhCXCL1 and rhTGF-β1 was rescued partially by a blocking antibody against CXCR2. Moreover, we observed the expression of CXCR2 in urine exfoliated podocytes in IgAN patients. Our present study implied that IgA1 complexes from IgAN patients could up-regulate the secretion of CXCL1 and TGF-β1 in mesangial cells. Additionally, the synergistic effect of CXCL1 and TGF-β1 further induced podocyte death and adhesion dysfunction in podocytes via CXCR2. This might be a potential mechanism for podocyte loss observed in IgAN.

Cumulative Effects of Variants Identified by Genome-wide Association Studies in IgA Nephropathy

The effect of genetic markers associated with IgA nephropathy on risk of disease in sub-phenotype and progression is uncertain. Data from 2096 Chinese patients were used to create both un-weighted (uw) and weighted (w) genetic risk score (GRS). The association between GRS with disease susceptibility and clinical parameters were assessed. All nine selected single nucleotide polymorphisms (SNPs) were associated with susceptibility to IgAN. uwGRS and wGRS showed a similar fit in disease associations. With every 1-unit increase in the uwGRS, the disease risk increased by approximately 20%; whereas every one standard deviation increase in the wGRS, disease risk increased by approximately 40% ~ 60%. Association between rs3803800 and serum IgA was replicated, and risk groups in GRSs were associated with increased IgA/IgA1 levels. uwGRS9 ≥ 16 was an independent predictor for end stage renal disease (ESRD) in IgAN, with a relative risk of 2.52 (p = 6.68 × 10−3). In conclusion, we observed that GRSs comprising nine SNPs identified in a GWAS of IgAN were strongly associated with susceptibility to IgAN. The high risk GRS9 group had a high risk of ESRD in follow-up.

Measures of Urinary Protein and Albumin in the Prediction of Progression of IgA Nephropathy

BACKGROUND AND OBJECTIVES Proteinuria is an independent predictor for IgA nephropathy (IgAN) progression. Urine albumin-to-creatinine ratio (ACR), protein-to-creatinine ratio, and 24-hour urine protein excretion (UPE) are widely used for proteinuria evaluation in clinical practice. Here, we evaluated the association of these measurements with clinical and histologic findings of IgAN and explored which was the best predictor of IgAN prognosis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Patients with IgAN were followed up for ≥12 months, were diagnosed between 2003 and 2012, and had urine samples available (438 patients). Spot urine ACR, protein-to-creatinine ratio, and 24-hour UPE at the time of renal biopsy were measured on a Hitachi Automatic Biochemical Analyzer 7180 (Hitachi, Yokohama, Japan). RESULTS In our patients, ACR, protein-to-creatinine ratio, and 24-hour UPE were highly correlated (correlation coefficients: 0.71-0.87). They showed good relationships with acknowledged markers reflecting IgAN severity, including eGFR, hypertension, and the biopsy parameter (Oxford severity of tubular atrophy/interstitial fibrosis parameter). However, only ACR presented with positive association with the Oxford segmental glomerulosclerosis/adhesion parameter and extracapillary proliferation lesions. The follow-up time was 37.0 (22.0-58.0) months, with the last follow-up on April 18, 2014. In total, 124 patients reached the composite end point (30% eGFR decline, ESRD, or death). In univariate survival analysis, ACR consistently had better performance than protein-to-creatinine ratio and 24-hour UPE as represented by higher area under the curve using time-dependent survival analysis. When adjusted for well known risk factors for IgAN progression, ACR was most significantly associated with the composite end point (hazard ratio, 1.56 per 1-SD change of standard normalized square root-transformed ACR; 95% confidence interval, 1.29 to 1.89; P<0.001). Compared with protein-to-creatinine ratio and 24-hour UPE, addition of ACR to traditional risk factors resulted in more improvement in the predictive ability of IgAN progression (c statistic: ACR=0.70; protein-to-creatinine ratio =0.68; 24-hour UPE =0.69; Akaike information criterion: ACR=1217.85; protein-to-creatinine ratio =1229.28; 24-hour UPE =1234.96; P<0.001). CONCLUSIONS In IgAN, ACR, protein-to-creatinine ratio, and 24-hour UPE had comparable association with severe clinical and histologic findings. Compared with protein-to-creatinine ratio and 24-hour UPE, ACR showed slightly better performance in predicting IgAN progression.

Plasma Soluble Urokinase Receptor Level Is Correlated with Podocytes Damage in Patients with IgA Nephropathy

Background Focal segmental glomerulosclerosis (FSGS) lesions are similar in characteristics to S lesions of the Oxford classification of IgA nephropathy (IgAN) and may predict poor prognosis. In the present study, we aimed to explore the association between plasma soluble urokinase receptor (suPAR) levels and S lesions and podocytes damage in IgAN patients. Methods We enrolled 569 IgAN patients with follow-up data and detected plasma suPAR levels at renal biopsy by enzyme-linked immunosorbent assay. Results Plasma suPAR levels in IgAN patients with or without S lesions did not differ significantly (P = 0.411). However, suPAR levels were positively correlated with proteinuria (r = 0.202, P < 0.001), and negatively correlated with estimated glomerular filtration rate (eGFR, r = –0.236, P < 0.001). In the partial correlation to adjust for eGFR, plasma suPAR levels remained positively correlated with proteinuria (r = 0.112, P = 0.023). In a Cox proportional hazards model, higher levels of plasma suPAR were not associated with poor renal outcome. Plasma suPAR levels of IgAN and primary FSGS patients with nephrotic syndrome were not significantly different (P = 0.306). Plasma suPAR levels in patients with extensive effacement of the epithelial cell foot processes of glomerular podocytes were significantly higher than those with segmental effacement on the basis of comparable eGFR (P = 0.036). Conclusions In IgAN patients, plasma suPAR levels were not associated with S lesions. However, they were positively associated with proteinuria and negatively associated with eGFR. In addition, plasma suPAR levels were positively associated with the effacement degree of the foot processes, which might partially contribute to the development of proteinuria in patients with IgAN.