Association of podocyte ultrastructural changes with proteinuria and pathological classification in type 2 diabetic nephropathy.

AIMS: Podocyte injury plays an essential role in the progression of diabetic nephropathy (DN). The associations between the ultrastructural changes of podocyte with proteinuria and the pathological classification of DN proposed by Renal Pathology Society (RPS) have not been clarified in patients with type 2 diabetic nephropathy (T2DN). METHODS: We collected 110 patients with kidney biopsy-confirmed T2DN at Peking University First Hospital from 2017 to 2022. The morphometric analysis on the podocyte foot process width (FPW) and podocyte detachment (PD) as markers of podocyte injury was performed, and the correlations between the ultrastructural changes of podocytes with severity of proteinuria and the RPS pathological classification of DN were analyzed. RESULTS: Mean FPW was significantly broader in the group of T2DN patients with nephrotic proteinuria (565.1 nm) than those with microalbuminuria (437.4 nm) or overt proteinuria (494.6 nm). The cut-off value of FPW (> 506 nm) could differentiate nephrotic proteinuria from non-nephrotic proteinuria with a sensitivity of 75.3% and a specificity of 75.8%. Percentage of PD was significantly higher in group of nephrotic proteinuria (3.2%) than that in microalbuminuria (0%) or overt proteinuria (0.2%). FPW and PD significantly correlated with proteinuria in T2DN (r = 0.473, p < 0.001 and r = 0.656, P < 0.001). FPW and PD correlated with RPS pathological classification of T2DN (r = 0.179, P = 0.014 and r = 0.250, P = 0.001). FPW value was increased significantly with more severe DN classification (P for trend =0.007). The percentage of PD tended to increase with more severe DN classification (P for trend = 0.017). CONCLUSIONS: Podocyte injury, characterized by FPW broadening and PD, was associated with the severity of proteinuria and the pathological classification of DN.

Clinical and pathological characteristics of DKD patients with early-onset type 2 diabetes.

AIMS: In diabetic kidney disease (DKD) patients, early-onset T2DM effects on renal disease severity and outcomes remain uncertain. Herein, we aim to investigate the clinicopathological characteristics and renal outcomes in DKD patients with early-onset T2DM. METHODS: 489 patients with T2DM and DKD were retrospectively recruited and classified as having early (age at onset of T2DM < 40 years) and late (age at onset of T2DM ≥ 40 years) T2DM onset, analyzing the clinical and histopathological data. The predictive value of early-onset T2DM to renal outcomes in DKD patients was analyzed by Cox's regression. RESULTS: Among 489 DKD patients, 142 and 347 were classified as early and late T2DM onset, respectively. Early-onset T2DM patients exhibited worse glycaemic control (7.36 % ± 1.80 % vs. 6.86 % ± 1.57 %, P = 0.007) and more severe proteinuria (3.69 [1.55 to 7.03] vs. 1.81 [0.50 to 4.33] g/24 h, P < 0.001). Those with early-onset T2DM presented more severe glomerular lesions. In univariable Cox regression, early-onset T2DM showed a significant correlation with renal composite endpoint (HR [95%CI]: 0.56 [0.43 to 0.73], P < 0.001). However, after adjusting for potential confounders, early-onset T2DM was not independently correlated with renal composite endpoint (HR [95%CI]: 0.74 [0.46 to 1.21], P = 0.232). CONCLUSIONS: In DKD patients with early-onset T2DM, renal clinicopathological manifestations were severe. Age at onset in T2DM was significantly correlated with eGFR slope (r = 0.211, P < 0.001).

Glutathione Peroxidase 4 Is a Predictor of Diabetic Kidney Disease Progression in Type 2 Diabetes Mellitus.

Background: Diabetic kidney disease (DKD) represents a heavy burden in type 2 diabetes mellitus (T2DM). Ferroptosis plays an important role in DKD, and it thus provides new perspectives to pursue more related biomarkers to assess the disease severity and prognosis. Glutathione peroxidase 4 (GPX4) is the mainstay in regulating ferroptosis. The current study investigated the predictive value of kidney GPX4 expression level in DKD progression. Methods: We measured GPX4 levels in kidney paraffin sections of 85 biopsy-proven DKD patients by immunohistochemistry staining. The associations between the GPX4 level and clinicopathological parameters as well as renal outcomes were analyzed. Results: GPX4 is mainly expressed in kidney tubulointerstitium, especially in tubular epithelial cells of DKD patients. The GPX4 expression level was significantly lower in DKD patients than healthy controls. Besides, GPX4 level significantly correlated with proteinuria (r = -0.42, p < 0.001), urinary albumin-to-creatinine ratio (uACR) (r = -0.40, p < 0.01), serum creatinine (Scr) (r = -0.59, p < 0.001), estimated glomerular filtration rate (eGFR) (r = 0.66, p < 0.001), and the percentage of sclerosed glomeruli (r = -0.42, p < 0.001) in renal specimens. During follow-up, the GPX4 level positively correlated with eGFR slope (r = 0.48, p < 0.001), and GPX4-low patients showed a significantly higher probability of developing end-stage kidney disease (ESKD) compared with GPX4-high patients (p < 0.01). Moreover, after adjusting for other potential predictors, the GPX4 level was still an independent predictor of developing ESKD (HR 2.15, 95% CI 1.08 to 4.28, p < 0.05). Conclusions: Kidney tubulointerstitial GPX4 expression level was associated with the disease severity and progression of DKD.

C3c deposition predicts worse renal outcomes in patients with biopsy-proven diabetic kidney disease in type 2 diabetes mellitus.

BACKGROUND: Although extensive efforts have been paid to identify reliable predictors for renal outcomes of diabetic kidney disease (DKD) patients in type 2 diabetes mellitus (T2DM), there are still only a limited number of predictive factors for DKD progression. Increasing evidence reported the role of the overactivated complement system in the pathogenesis of DKD. Whether renal complement depositions are associated with renal outcomes of DKD in T2DM is of interest. METHODS: A total of 213 biopsy-proven DKD patients with T2DM were retrospectively recruited. Clinical and pathological data of the patients were analyzed. Kaplan-Meier analysis and Cox regression analysis were performed to explore predictors of end-stage renal disease (ESRD). RESULTS: During a median follow-up of 23.0 (12.0, 39.0) months, 100/213 (46.9%) patients progressed to ESRD. C3c and C1q deposition were observed in 133/213 (62.4%) and 45/213 (21.1%) patients, respectively. Kaplan-Meier analysis revealed patients with C3c or C1q deposition had significantly worse renal outcomes compared with those without C3c or C1q deposition (p = .001 and p < .001, respectively). Univariate and multivariate Cox regression analysis demonstrated proteinuria (per 1 g/24 h increase, hazard ratio [HR] 1.134, 95% confidence interval [CI] [1.079, 1.191], p < .001), interstitial fibrosis and tubular atrophy score (score 2 and 3 vs. 0 and 1, HR 3.925, 95% CI [1.855, 8.304], p < .001), and C3c deposition (per 1+ increase, HR 1.299, 95% CI [1.073, 1.573], p = .007) were independent predictors for ESRD in DKD patients with T2DM. CONCLUSIONS: C3c deposition in the kidney was associated with worse renal outcomes and was an independent predictor for ESRD in DKD patients with T2DM.

Clinical and Pathological Characteristics of Patients With Nonproteinuric Diabetic Nephropathy.

Introduction: As the most common complication of diabetes mellitus (DM), diabetic nephropathy (DN) was initially considered to begin with proteinuria preceding the progression of renal insufficiency. This clinical paradigm has been questioned in the late decades, as many DM patients without proteinuria have progressive renal insufficiency. However, the characteristics of nonproteinuric DN were not fully clear yet. Patients and Methods: A total of 390 patients with renal biopsy-proven DN in our center were retrospectively recruited in the current study. Clinical and histopathological data of the patients were analyzed. We used propensity score-matching methods to address the imbalance of age, sex, and diabetes duration for comparative analyses. Results: Among all the renal biopsy-proven DN patients with renal biopsy proven DN, 18 patients were classified as nonproteinuric DN. Compared with 36 propensity score-matched proteinuric DN patients, diabetic retinopathy (DR) was less frequent in nonproteinuric DN patients (38.9% vs. 66.4%, p<0.05). During the follow-up of 24.0 (12.0-42.0) months, the probability of developing the end-stage renal disease (ESRD) was significantly lower in nonproteinuric DN patients than in proteinuric ones in both the propensity score-matched cohort and overall cohort (log-rank test, p<0.001 and p<0.001, respectively). Conclusions: Compared with proteinuric DN patients, DR was less frequent in nonproteinuric DN patients. Nonproteinuric DN patients had better renal outcomes than proteinuric DN patients.

Association between serum uric acid level and mortality in China.

BACKGROUND: Whether there is an association between serum uric acid (SUA) level and risk of mortality in the general population remains unclear. Based on the China National Survey of Chronic Kidney Disease linked to mortality data, a population-based cohort study was performed to investigate the association between SUA level and all-cause mortality, cardiovascular disease (CVD) mortality, and cancer mortality in China. METHODS: The survival status of participants in the cross-sectional survey was identified from January 1, 2006 to December 31, 2017. Only 33,268 individuals with complete SUA data among the 47,204 participants were included in the analysis. We determined the rates of all-cause mortality, CVD mortality, and cancer mortality. We used Cox proportional hazards regression models to evaluate the effect of the SUA level on mortality. RESULTS: During a total of 297,538.4 person-years of follow-up, 1282 deaths occurred. In the Cox proportional hazards regression model, the rate of all-cause mortality, CVD mortality, and cancer mortality had a U-shaped association with SUA levels only in men, whereas no significant associations were detected in women. For all-cause mortality in men, the multivariable-adjusted hazard ratios (HRs) in the first, second, and fourth quartiles compared with the third quartile were 1.31 (95% confidence interval [CI] 1.04-1.67), 1.17 (95% CI 0.92-1.47), and 1.55 (95% CI 1.24-1.93), respectively. For CVD mortality, the corresponding HRs were 1.47 (95% CI 1.00-2.18), 1.17 (95% CI 0.79-1.75), and 1.67 (95% CI 1.16-2.43), respectively. For the cancer mortality rate, only a marginally significant association was detected in the fourth quartile compared with the third quartile with an HR of 1.43 (95% CI 0.99-2.08). CONCLUSIONS: The association between SUA and mortality differed by sex. We demonstrated a U-shaped association with SUA levels for all-cause and CVD mortalities among men in China.

Myeloperoxidase-ANCA-positive granulomatosis with polyangiitis is a distinct subset of ANCA-associated vasculitis: A retrospective analysis of 455 patients from a single center in China.

OBJECTIVE: Antineutrophil cytoplasmic antibody (ANCA) directed to proteinase 3 (PR3) used to be considered the serologic marker for granulomatosis with polyangiitis (GPA). However, patients with myeloperoxidase (MPO)-ANCA positive GPA have been increasingly reported. The aim of this study was to analyze the clinical and pathological characteristics and outcome of Chinese patients with MPO-ANCA positive GPA. METHODS: The clinical and renal histology data, renal outcomes, response to treatment, relapse and mortality were compared between patients with MPO-ANCA positive GPA and MPO-ANCA positive microscopic polyangiitis (MPA) as well as proteinase 3 (PR3)-ANCA positive GPA. RESULTS: 455 patients with ANCA-associated vasculitis (AAV) were recruited in this study. 276/455 patients were classified as MPO-ANCA positive MPA, 4/455 patients were classified as PR3-ANCA positive MPA, 124/455 were MPO-ANCA positive GPA and 51/455 were PR3-ANCA positive GPA. Compared with MPO-ANCA positive MPA patients, MPO-ANCA positive GPA patients had significantly higher level of BVAS and milder renal lesion at diagnosis. The probability of developing ESRD was significantly higher in patients with MPO-ANCA positive MPA than MPO-ANCA positive GPA. MPO-ANCA positive GPA patients were likely to have relapse than MPO-ANCA positive MPA patients. Compared with PR3-ANCA positive GPA patients, MPO-ANCA positive GPA patients had significantly higher proportion of female, less constitutional symptoms and milder renal lesion at diagnosis. CONCLUSIONS: Patients with MPO-ANCA positive GPA should be regarded as a unique subset of AAV. This subset of AAV patients had relatively milder renal injury. Although ANCA specificities play an important role in differentiating AAV, taking the disease type together to classify AAV may be more rational.

Persistent hematuria in patients with antineutrophil cytoplasmic antibody-associated vasculitis during clinical remission: chronic glomerular lesion or low-grade active renal vasculitis?

BACKGROUND: Whether persistent hematuria in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) during clinical remission reflects active disease or chronic glomerular injury is uncertain. This study aimed to investigate the significance of persistent hematuria during clinical remission in a large cohort of AAV patients. METHODS: A cohort of 219 AAV patients in complete clinical remission after induction therapy at our center was retrospectively studied, and their clinical and laboratory data as well as long-term outcomes were analyzed. RESULTS: A total of 80 out of 219 patients had persistent hematuria during clinical remission of AAV. Compared with patients without hematuria during remission, the slope of eGFR decline in patients with persistent hematuria was significantly higher during the long-term follow-up [3.6 (IQR 1.2, 7.2) vs. 1.5 (IQR 0.2, 4.0) mL/min/1.73 m2/year, P < 0.001]. Among the 80 patients with persistent hematuria during remission, there was little difference between those with fast and slow decline of eGFR, as divided by either median or interquartile range of the slope of eGFR decline. We also compared patients without hematuria who had a slope of eGFR decline that was lower than the median level of the slope of eGFR decline with those with persistent hematuria, and found that patients with hematuria had significantly lower levels of CRP and ESR at baseline and higher levels of ANCA at remission. CONCLUSIONS: Among the AAV patients who achieved clinical remission after immunosuppressive therapy, those with persistent hematuria are not rare and may reflect either chronic renal damage or low-grade active renal disease.

HMGB1 contributes to glomerular endothelial cell injury in ANCA-associated vasculitis through enhancing endothelium-neutrophil interactions.

Our previous studies demonstrated that high mobility group box-1 (HMGB1), a typical damage-associated molecular pattern (DAMP) protein, is associated with the disease activity of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Moreover, HMGB1 participates in ANCA-induced neutrophil activation. The current study aimed to investigate whether HMGB1 regulated the interaction between neutrophils and glomerular endothelial cells (GEnC) in the presence of ANCA. Correlation analysis on HMGB1 levels in AAV patients and soluble intercellular cell adhesion molecule-1 (sICAM-1) levels or vascular endothelial growth factor (VEGF) levels, which are markers of endothelial cell activation, was performed. The effect of HMGB1 on neutrophil migration towards GEnC, respiratory burst and degranulation of neutrophils in coculture conditions with GEnC was measured. The activation of neutrophils, the activation and injury of GEnC, and the consequent pathogenic role of injured GEnC were evaluated. Plasma levels of HMGB1 correlated with sICAM-1 and VEGF (r = 0.73, P < 0.01; r = 0.41, P = 0.04) in AAV patients. HMGB1 increased neutrophil migration towards GEnC, as well as respiratory burst and degranulation of neutrophils in the presence of ANCA in the coculture system. In the presence of robust neutrophil activation, GEnC were further activated and injured in the coculture system of GEnC and neutrophils. In addition, injured GEnC could produce TF-positive leuco-endothelial microparticles and endothelin-1 (ET-1), while NF-κB was phosphorylated (S529) in the injured GEnC. Plasma levels of HMGB1 correlated with endothelial cell activation in AAV patients. HMGB1 amplified neutrophil activation and the activation and injury of GEnC in the presence of ANCA.

Urinary levels of high mobility group box-1 are associated with disease activity in antineutrophil cytoplasmic autoantibody-associated vasculitis.

BACKGROUND: High mobility group box-1 (HMGB1), a kind of pro-inflammatory mediator, is associated with inflammatory conditions and tissue damage. Our previous study demonstrated that the circulating levels of HMGB1 correlated with disease activity of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In the current study, we aimed to measure urinary levels of HMGB1 in AAV patients, correlated them to clinical activity index and analysed the immunohistochemical HMGB1 staining in kidney specimens. METHODS: 50 patients with AAV in active stage and 56 patients with AAV in remission were recruited. The urinary levels of HMGB1 were determined by enzyme-linked immunosorbent assay. Moreover, renal biopsy specimens from 27 patients with active AAV were randomly collected to evaluate the deposition of HMGB1. RESULTS: Urinary HMGB1 levels in AAV patients in active stage were significantly higher than those in AAV patients in remission and healthy controls (1.46 [0.56-3.43] versus 0.38 [0.10-1.35] mg/µmolCr, P=0.001; 1.46 [0.56-3.43] versus 0.48 [0.40-0.60] mg/µmolCr, P=0.000, respectively). Further analysis found that urinary levels of HMGB1 correlated with erythrocyte sedimentation rate (r=0.354, p=0.012), C-reactive protein (r=0.289, p=0.042), and Birmingham Vasculitis Activity Score (r=0.350, p=0.013). Renal tissue of active AAV patients showed HMGB1 was mainly expressed in the cytoplasm and the extracellular space. The percentage of HMGB1-negative nuclei in renal tissue of patients with active AAV was significantly higher than that in normal controls (60.6±20.2 % versus 2.7±0.6 %, p<0.01). CONCLUSION: Urinary levels of HMGB1 may be associated with the disease activity in AAV patients.

Predictors of treatment resistance and relapse in antineutrophil cytoplasmic antibody-associated vasculitis: a study of 439 cases in a single Chinese center.

OBJECTIVE: Treatment resistance and relapse in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) are major challenges for physicians. The aim of this study was to assess the risk factors for treatment resistance and relapse in a single-center cohort of Chinese patients with AAV. METHODS: Four hundred thirty-nine consecutive patients with AAV were recruited for inclusion in this study. The value of various clinical and pathologic parameters for the prediction of treatment resistance and relapse was analyzed. RESULTS: Treatment resistance occurred in 47 (10.7%) of 439 patients and was independently associated with a higher serum creatinine level (odds ratio [OR] 1.087, 95% confidence interval [95% CI] 1.001-1.180, P = 0.047), a higher erythrocyte sedimentation rate (OR 1.009, 95% CI 1.001-1.018, P = 0.025), therapy with corticosteroids plus cyclophosphamide (OR 0.115, 95% CI 0.051-0.256, P = 0.000), and the presence of muscle pain (OR 0.249, 95% CI 0.083-0.747, P = 0.013). Relapse occurred in 128 (32.7%) of 392 patients in whom remission was achieved and was independently associated with lung involvement (hazard ratio [HR] 1.768, 95% CI 1.088-2.872, P = 0.021) and a lower serum creatinine level (HR 0.925, 95% CI 0.872-0.981, P = 0.009). CONCLUSION: In Chinese patients with AAV, lung involvement and lower serum creatinine levels were independently associated with an increased risk of relapse. Elevated serum creatinine levels were associated with treatment resistance.

Association of circulating level of high mobility group box 1 with disease activity in antineutrophil cytoplasmic autoantibody-associated vasculitis.

OBJECTIVE: High mobility group box 1 (HMGB-1), a kind of proinflammatory mediator, is associated with inflammatory conditions and tissue damage. Previous studies have reported that circulating HMGB-1 levels in patients with active antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) were associated with renal manifestations and burdens of granulomatous inflammation. The current study aimed to investigate whether circulating HMGB-1 levels were associated with disease activity in AAV. METHODS: Plasma samples from 74 patients with AAV in active stage and 65 patients with AAV in remission were collected. The plasma levels of HMGB-1 were determined by enzyme-linked immunosorbent assay. Associations between plasma levels of HMGB-1 with clinical and pathologic parameters were analyzed. RESULTS: Plasma levels of HMGB-1 in active AAV patients were significantly higher than those in normal controls and AAV patients in remission (median 6.11 [interquartile range (IQR) 3.25-12.79] ng/ml versus median 1.12 [IQR 0.53-1.39] ng/ml, P< 0.001; median 6.11 [IQR 3.25-12.79] ng/ml versus median 3.04 [IQR 1.97-4.63] ng/ml, P < 0.001, respectively). Correlation analysis showed that plasma levels of HMGB-1 correlated with initial serum creatinine (r = 0.275, P = 0.018), estimated glomerular filtration rate (r = -0.277, P = 0.017), the Birmingham Vasculitis Activity Score (r = 0.308, P = 0.008), and C-reactive protein level (r = 0.309, P = 0.008). Among the patients with myeloperoxidase (MPO)-ANCA, those within the first quartile of plasma HMGB-1 levels had a significantly lower level of MPO-ANCA than those within the other 3 quartiles. CONCLUSION: Circulating HMGB-1 levels might reflect the disease activity and renal involvement of AAV vasculitis.

C-reactive protein enhances the respiratory burst of neutrophils-induced by antineutrophil cytoplasmic antibody.

Serum C-reactive protein (CRP) was one of the useful biomarkers for evaluating the disease activity in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Cumulating studies proved that CRP was pathogenic in a variety of diseases. In the current study, the in vitro effects of CRP to prime neutrophils for ANCA-induced respiratory burst were investigated with flow cytometry. Without TNF-α in the reactive system, ANCA could only induce a slight level of respiratory burst of neutrophils. CRP could enhance the respiratory burst of neutrophils induced by ANCA against myeloperoxidse [mean fluorescence intensity (MFI, 68.45 ± 16.87 vs. 58.65 ± 15.09, P < 0.05) or by ANCA against proteinase 3 (MFI, 79.51 ± 15.90 vs. 61.73 ± 14.89, P < 0.05). Although CRP (50 µg/mL, incubating for 30 min) could not active neutrophils alone, after incubation with neutrophils for 10 min, CRP (50 µg/mL) could increase the expression of membrane proteinase 3 of neutrophils (MFI, 365.27 ± 143.50 vs. 235.32 ± 124.65, P < 0.05). Heat-treated CRP could not enhance the levels of neutrophils respiratory burst induced by ANCA or increase the expression of membrane proteinase 3 of neutrophils. So CRP can prime neutrophils and enhance the respiratory burst induced by ANCA and might be pathogenic in AAV.

Re-evaluation of the histopathologic classification of ANCA-associated glomerulonephritis: a study of 121 patients in a single center.

BACKGROUND: The recently published histopathologic classification of antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis might greatly aid in the prognostication of patients at the time of diagnosis. This study aims to re-evaluate the new classification for its prognostic capacity in an independent Chinese series. METHODS: One hundred and twenty-one consecutive patients with ANCA-associated glomerulonephritis, diagnosed in our center from 1997 to 2010, were included in this retrospective study. The renal specimens were reviewed according to the proposed histopathologic classification. The predictive value of the classification for renal outcome and renal response to treatment was analyzed. RESULTS: Thirty-three (27.3%), 24 (19.8%), 53 (43.8%) and 11 (9.1%) patients were classified as focal, mixed, crescentic and sclerotic ANCA-associated glomerulonephritis, respectively. The renal biopsy categories correlated with initial serum creatinine and the renal response to treatment (P < 0.001, P < 0.01, respectively). The probability of progressing to end-stage renal disease (ESRD) increased with ascending categories of focal, mixed, crescentic and sclerotic glomerulonephritis (P < 0.01). The patients with focal, mixed and crescentic ANCA-associated glomerulonephritis were all at decreased risk for developing ESRD compared with the patients with in the sclerotic category (P < 0.05). CONCLUSIONS: The proposed classification system was re-evaluated for the first time in a relatively large and independent series of patients. This system reflects the severity of the initial renal impairment and can predict, at least to some extent, the renal response to treatment. More importantly, it can independently predict renal outcome, in particular development of ESRD.