Cryoglobulinemic vasculitis and glomerulonephritis: concerns in clinical practice / 中华医学杂志(英文版)

Objective@#Cryoglobulinemia often causes systemic vasculitis, thereby damaging to skin and internal organs including kidneys, even life-threatening. This review aimed to introduce the advances in understanding, detection, and treatment of this disease in recent years, with a particular concern to clinical practice.@*Data sources@#All the data in this review were from the English or Chinese literature in the PubMed and China National Knowledge Infrastructure databases as of March 2019.@*Study selection@#This review selected important original articles, meaningful reviews, and some reports on cryoglobulinemia published in recent years and in history, as well as the guidelines for treatment of underlying diseases which lead to cryoglobulinemia.@*Results@#Diagnosis of cryoglobulinemia relies on serum cryoglobulin test, in which to ensure that the blood sample temperature is not less than 37°C in the entire pre-analysis phase is the key to avoid false negative results. Cryoglobulinemic vasculitis (Cryo Vas), including cryoglobulinemic glomerulonephritis (Cryo GN), usually occurs in types II and III mixed cryoglobulinemia, and can also be seen in type I cryoglobulinemia caused by monoclonal IgG3 or IgG1. Skin purpura, positive serum rheumatoid factor, and decreased serum levels of C4 and C3 are important clues for prompting types II and III Cryo Vas. Renal biopsy is an important means for diagnosis of Cryo GN, while membranous proliferative GN is the most common pathological type of Cryo GN. In recent years, great advances have been made in the treatment of Cryo Vas and its underlying diseases, and this review has briefly introduced these advances.@*Conclusions@#Laboratory examinations of serum cryoglobulins urgently need standardization. The recent advances in the diagnosis and treatment of Cryo Vas and GN need to be popularized among the clinicians in related disciplines.

Cryoglobulinemic vasculitis and glomerulonephritis: concerns in clinical practice / 中华医学杂志(英文版)

OBJECTIVE@#Cryoglobulinemia often causes systemic vasculitis, thereby damaging to skin and internal organs including kidneys, even life-threatening. This review aimed to introduce the advances in understanding, detection, and treatment of this disease in recent years, with a particular concern to clinical practice.@*DATA SOURCES@#All the data in this review were from the English or Chinese literature in the PubMed and China National Knowledge Infrastructure databases as of March 2019.@*STUDY SELECTION@#This review selected important original articles, meaningful reviews, and some reports on cryoglobulinemia published in recent years and in history, as well as the guidelines for treatment of underlying diseases which lead to cryoglobulinemia.@*RESULTS@#Diagnosis of cryoglobulinemia relies on serum cryoglobulin test, in which to ensure that the blood sample temperature is not less than 37°C in the entire pre-analysis phase is the key to avoid false negative results. Cryoglobulinemic vasculitis (Cryo Vas), including cryoglobulinemic glomerulonephritis (Cryo GN), usually occurs in types II and III mixed cryoglobulinemia, and can also be seen in type I cryoglobulinemia caused by monoclonal IgG3 or IgG1. Skin purpura, positive serum rheumatoid factor, and decreased serum levels of C4 and C3 are important clues for prompting types II and III Cryo Vas. Renal biopsy is an important means for diagnosis of Cryo GN, while membranous proliferative GN is the most common pathological type of Cryo GN. In recent years, great advances have been made in the treatment of Cryo Vas and its underlying diseases, and this review has briefly introduced these advances.@*CONCLUSIONS@#Laboratory examinations of serum cryoglobulins urgently need standardization. The recent advances in the diagnosis and treatment of Cryo Vas and GN need to be popularized among the clinicians in related disciplines.

Clinical and pathological features of idiopathic membranous nephropathy with phospholipase A2 receptor negative and serum anti PLA2R antibody negative: 40 cases analysis / 中国实用内科杂志

OBJECTIVE: To investigate the clinicopathological features of phospholipase A2 receptor(PLA2R) negative patents with idiopathic membranous nephropathy(IMN). METHODS: IMN patients diagnosed by renal biopsy were enrolled in this study. Glomerular PLA2 R deposition(GAg) and serum anti-PLA2 R antibodies(SAbs) were detected by immunohistochemical staining and enzyme linked immunosorbent assay, respectively. Patients were divided into two groups. Both GAg and SAbs were negative in patients of Group A. Patients of group B were selected from patients who were positive for GAg and SAbs and were matched with group A in gender and age. The clinical and laboratory data of the two groups were collected. Glomerular thrombospondin type-1 domaincontaining 7A(THSD7A) deposition and serum anti-THSD7 A antibody were also measured by immunohistochemical staining and indirect immunofluorescence in the two groups, respectively. RESULTS:(1) Compared with group B, patients in group A had lower levels of proteinuria, lower proportion of microscopic hematuria, higher remission rate(P<0.05). The positive rate of IgG4 in group A(45.0%) was significantly lower than that in group B(85.0%)(P<0.01).(2) The positive rate of glomerular THSD7 A deposition and serum anti-THSD7 A antibody of group A were 17.5% and 7.5%. Patients in group B showed negative THSD7 A tissue staining and antiTHSD7 A antibodies. CONCLUSION: Compared with patients who were positive for GAg and SAbs, patients who were negative for GAg and SAbs exhibited lower levels of proteinuria and higher remission rate. The positive rate of glomerular THSD7 A deposition and serum anti-THSD7 A antibody was low in patients with IMN.

Purinergic 2X7 Receptor is Involved in the Podocyte Damage of Obesity-Related Glomerulopathy via Activating Nucleotide-Binding and Oligomerization Domain-Like Receptor Protein 3 Inflammasome / 中华医学杂志(英文版)

Background@#The nucleotide-binding and oligomerization domain-like receptor protein 3 (NLRP3) inflammasome composed of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), and caspase-1 is engaged in the inflammatory response of many kidney diseases and can be activated by purinergic 2X7 receptor (P2X7R). This study was conducted to explore whether P2X7R plays a pathogenic role in the podocyte damage of obesity-related glomerulopathy (ORG) and whether this role is mediated by the activation of NLRP3 inflammasome.@*Methods@#A mouse model of ORG was established by high-fat diet feeding. The conditionally immortalized mouse podocytes were cultured with leptin or with leptin and P2X7R antagonist (KN-62 or A438079). The mRNA and protein expression of the P2X7R and NLRP3 inflammasome components including NLRP3, ASC, and caspase-1, as well as the podocyte-associated molecules including nephrin, podocin, and desmin in mouse renal cortex or cultured mouse podocytes were tested by real-time-polymerase chain reaction and Western blot analysis, respectively.@*Results@#The significantly upregulated expression of P2X7R and NLRP3 inflammasome components and the NLRP3 inflammasome activation were observed in the renal cortex (in fact their location in podocytes was proved by confocal microscopy) of ORG mice in vivo, which were accompanied with the morphological changes of podocyte damage and the expression changes of podocyte-associated molecules. Similar changes in the expression of P2X7R and NLRP3 inflammasome components as well as in the expression of podocyte-associated molecules were also observed in the cultured podocyte studies treated by leptin in vitro, and all of the above changes were significantly attenuated by the P2X7R antagonist KN-62 or A438079.@*Conclusions@#P2X7R could trigger the activation of NLRP3 inflammasome, and the activated P2X7R/NLRP3 inflammasome in podocytes might be involved in the podocyte damage of ORG.

Application of Immunohistochemistry and Immunofluorescence Staining in Detection of Phospholipase A2 Receptor on Paraffin Section of Renal Biopsy Tissue / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate the application of immunohistochemistry and fluorescence staining method in the detection of phospholipase A2 receptor (PLA2R) on paraffin section of renal biopsy tissue,and to find an accurate and fast method for the detection of PLA2R in renal tissue.</p><p><b>METHODS</b>The PLA2R of 193 cases were detected by immunohistochemical staining,and the antigen was repaired by the method of high pressure cooker (HPC) hot repair plus trypsin repair. The 193 samples including 139 cases of idiopathic membranous nephropathy (IMN), 15 cases of membranous lupus nephritis, 8 cases of hepatitis B virus associated membranous nephropathy, 18 cases of IgA nephropathy, and 13 cases of minimal change diseases. To compare the dyeing effects, 22 paraffin sections of renal biopsy tissue of IMN cases with positive PLA2R were stained by using 4 different.</p><p><b>METHODS</b>of antigen repairing,which included HPC hot repair, HPC hot repair plus trypsin repair, water bath heat repair, and water bath heat repair plus trypsin repair. To compare the dyeing effects, 15 paraffin sections of renal biopsy tissue of IMN cases with positive PLA2R were stained by using 3 different.</p><p><b>METHODS</b>of antigen repairing,which included water bath heat repair plus trypsin repair, protease K digestion repair, and pepsin digestion repair.</p><p><b>RESULTS</b>In 193 cases, the positive rate of PLA2R in IMN cases was 90.6% (126/139), and the other 54 patients without IMN were negative. Twenty-two IMN patients were positive for PLA2R by using the HPC heat repair plus trypsin repaire or the water bath heat repair plus trypsin repair;while only a few cases of 22 IMN cases were positive by using the HPC hot repair alone or water bath heat repair alone. Fifteen IMN patients were positive for PLA2R by using water bath heat repair plus trypsin repair,protease K digestion repair,and pepsin digestion repair, but the distribution of positive deposits and the background were different.</p><p><b>CONCLUSIONS</b>PLA2R immunohistochemical staining can effectively identify IMN and secondary MN. For immunohistochemical staining and immunofluorescence staining, the preferred method of antigen repair is water bath heat repair plus trypsin repair.</p>

Establishment of a new rat model of chronic cyclosporine A nephrotoxicity / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a new rat model of chronic cyclosporine A nephrotoxicity and explore its features.</p><p><b>METHODS</b>Totally 24 male SD rats were equally randomized divided into 3 groups: sham-adrenalectomized (sham-ADX) group, ADX group and ADX plus cyclosporine A (CsA) group. Rats in ADX and CsA group first underwent adrenalectomy, followed by the administration of placebo or dexamethasone, respectively. Rats in sham-ADX group received sham adrenalectomy and distilled water as control. Six weeks later, all rats were sacrificed and the following indicators were evaluated: urine protein excretion, creatinine clearance, aldosterone level in serum and urine, aldosterone level and its synthase CYP11B2 gene expression in kidney, serum natrium and potassium, urine natrium and potassium excretion, and tubulointerstitial fibrosis by masson trichrome stain.</p><p><b>RESULTS</b>In ADX and CsA group, serum and urine aldosterone were undetectable on the second post-operative day, with other observations including natriuresis, hyponatremia, decreased urine potassium excretion, and hyperpotassemia, suggesting that adrenals were removed intact and the adrenalectomy was successful. Rats in CsA group showed increased urine protein, decreased creatinine clearance and tubulointerstitial fibrosis, suggesting that a model of chronic CsA nephrotoxicity was successfully established. At the endpoint, serum potassium, serum aldosterone, urine potassium and urine aldosterone excretion partially retrieved. Natrium in serum and urine was not significant different between ADX group/CsA group and sham-ADX group. Local renal aldosterone and its gene expression were remarkably upregulated.</p><p><b>CONCLUSIONS</b>We successfully established a new rat model of chronic CsA nephrotoxicity by adrenalectomy without low sodium diet. After adrenalectomy, local renal aldosterone in kidney may compensate for circulatory aldosterone deficit to maintain electrolyte balance.</p>

Effects of Hirsutella sinensis on TGF-beta1 and Snail expressions and transdifferentiation of tubular epithelial-myofibroblast in renal tissue of rats with chronic aristolochic acid nephropathy / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the antagonizing effect of Hirsutella sinensis (HS) on renal tubular epithelial-myofibroblast transdifferentiation (TEMT) and its possible pathogenic mechanism in rats with chronic aristolochic acid nephropathy (CAAN).</p><p><b>METHODS</b>Eighteen male Sprague-Dawley rats were equally divided into 3 groups, the model (M) group, the intervention (I) group and the control (C) group. The 24 h urinary protein (UP) in rats was measured before intervention and at the end of the 1st, 4th, 8th, and 12th week, and creatinine clearance rate (CCr) was measured before intervention and at the end of the 12th week respectively. All rats were sacrificed at the end of the 12th week, their kidney was taken for examining the degree of fibrosis in renal interstitial with Masson's stain and determining mRNA and protein expressions of transforming growth factor-beta1 (TGF-beta1), Snail, alpha-smooth muscle actin (alpha-SMA) and cytokeratin in renal tissue by Real-time RT-PCR and immunohistochemistry staining, respectively.</p><p><b>RESULTS</b>Compared with the C group, CCr was significantly lower, while 24 h UP was higher; the relative area of interstitial fibrosis was significantly larger in the M group; besides, the mRNA and protein expressions of TGF-beta1, Snail and alpha-SMA were significantly up-regulated (P < 0.01 or P < 0.05), and those of cytokeratin were significantly down-regulated (P < 0.01) in renal tissue of the M group. While in the I group, all the above-mentioned abnormalities were restored to some extent (P < 0.05) and showed significant difference (all P < 0.05) as compared with those in the M group.</p><p><b>CONCLUSION</b>HS can downregulate TGF-beta1 and Snail expressions in renal tissue, antagonize TEMT and renal interstitial fibrosis, and improve renal function in CAAN rats.</p>

Organic anion transporter system mediates the transport of aristolochic acid in tubular epithelial cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate whether aristolochic acid can be transported into human kidney proximal tubular cell (HKC) and its potential mechanism.</p><p><b>METHODS</b>Intracellular aristolochic acid was measured by liquid chromatography-tandem mass spectrometry. The release of lactate dehydrogenase (LDH) induced by aristolochic acid in the presence of organic anion transporter inhibitor (probenecid) or organic cation transporter inhibitor (tetraethylammonium) was evaluated. The effects of probenecid on aristolochic acid induced connective tissue growth factor (CTGF) mRNA and protein expression were also examined by real time polymerase chain reaction and Western blot, respectively.</p><p><b>RESULTS</b>Aristolochic acid was detected in the suspension of the denatured HKC after incubation with aristolochic acid sodium salt. The release of LDH from HKC, which was induced by 60 mg/L aristolochic acid sodium salt, was significantly inhibited by 1 mmol/L probenecid (P < 0.01), but not by 1 mmol/L tetraethylammonium. The increased CTGF mRNA and protein expression in HKC stimulated by 40 mg/L aristolochic acid sodium salt was significantly down-regulated by 1 mmol/L probenecid (P < 0.05), with an inhibition rate of 16% and 21%, respectively.</p><p><b>CONCLUSION</b>Aristolochic acid can be transported into HKC by organic anion transport system, and then exerts its biological effects.</p>

Synthesis and mass spectrometric analysis of aristolochic acid-deoxyguanosine adducts / 药学学报

To synthesize aristolochic acid (AA)-2'-deoxyguanosine 5'-monophosphate (dGp) adducts in vitro and develop a novel method for the characterization of the adducts using multiple mass spectrometric techniques. AA was incubated with dGp in vitro using either enzymatic activation (by xanthine oxidase) or chemical activation (by zinc) to synthesize AA-dGp adducts, and the reaction conditions were optimized. Crude extracts were analyzed by techniques of liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-MS/MS) and high accuracy mass data and isotope pattern of super high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICRMS). The quasi-molecular ion peaks of the AA-dGp adducts were obtained in the negative ion mode. Analysis by electrospray ionization/tandem mass spectrometry (ESI-MS/MS) provided useful structural information about AA-dGp adducts. AA can bind covalently to the exocyclic amino group of deoxyguanosine to form AA-dGp adducts. MS analysis is a powerful tool to detect and identify AA-dGp adducts simply, rapidly and accurately.

The medicinal serum of yi-shen ruan-jian san antagonized the effect of aristolochic acid on human proximal tubular epithelial cells in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To evaluate whether the medicinal serum of Yi-shen Ruan-jian san can antagonize the fibrogenic effect of human proximal tubular epithelial cell line (HKC) activated by aristolochic acid (AA) in vitro.</p><p><b>METHOD</b>The HKC was incubated in the media containing 40 mg x L(-1) aristolochic acid sodium salt (AA-Na) with or without 10% concentration of Yi-shen Ruan-jian san medicinal serum. Then the cell proliferation and cytotoxicity of HKC were determined by MTF and lactate dehydrogenase (LDH) release assay respectively, the antigen expression of cytokeratin and alpha-smooth muscle actin on HKC was detected by immunocytochemistry, the mRNA expression of transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type I Collagen (Col I) of HKC was measured by RT-PCR, and their protein expression was measured by ELISA or Western blot.</p><p><b>RESULT</b>No cytotoxic effect was found in HKC after stimulation of AA-Na with or without the medicinal serum of Yi-shen Ruan-jian san (P > 0.05). No epithelial-myofibroblast transdifferentiation was found in HKC after AA-Na stimulation. The mRNA and protein expression of TGF-beta1, CTGF, PAI-1 and TIMP-1 of HKC was significantly upregulated by AA-Na (P < 0.05). The above-mentioned enhanced mRNA and protein expression, except for PAI-1, was significantly downregulated by the medicinal serum of Yi-shen Ruan-jian san, compared with the control (normal rat serum in the same concentration) (P < 0.05).</p><p><b>CONCLUSION</b>The fibrogenic effects of HKC activated by AA are antagonized by Yi-shen Ruan-jian san, through downregulating the expression of promoting excellular matrix (ECM) synthesis factors (TGF-beta1, CTGF) and inhibiting ECM degradation factor (TIMP-1).</p>

Protective effects of yishen ruanjian power on renal interstitial fribrosis in chronic aristolochic acid induced nephropathy rat model / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the protective effects of Yishen Ruanjian Power (YRP) on renal interstitial fibrosis in rats with chronic aristolochic acid induced nephropathy (CAAN).</p><p><b>METHODS</b>Eighteen male SD rats were divided into 3 groups, 6 in each group. Water solution of Caulis Aristolochia Manshuriensis (CAM) Liquid Extract were given to the mice in the model group by gastrogavage to make CAAN animal model. For those in the TCM group, decocted water solution of YRP was given by gastrogavage after the mice being modeled with the above-mentioned method. Tap water was given by gastrogavage to the mice in the control group. Body weight, 24-hr urinary protein excretion and beta2 microglobulin (beta2-MG), and serum creatinine (r) were determined at the end of the 1st, 4th, 8th, 12th and 16th week. At the end of the 16th week, the rats were sacrificed and the pathological figure of their kidneys were observed by Masson staining. Transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type I collagen (Col I ) in kidney tissue were determined by RT-PCR and immunohistochemical method, respectively.</p><p><b>RESULTS</b>At end of the 1st week, urinary protein excretion, urinary beta2-MG and SCr in the model group were significantly increased to the levels higher than those in the control group (P < 0.01 or 0.05). Relative area of interstitial fibrosis was significantly enlarged in the model group at the end of the 16th week (P<0.01), and at the same time, the mRNA and protein expression of TCF-beta1, CTGF, PAI-1, TIMP-1 and Col I in kidney tissue were significant up-regulated (P<0.01). After intervention with YRP, the above-mentioned up-regulated parameters, except 24-hr urinary protein excretion, were all significantly inhibited (P <0.01 or 0.05).</p><p><b>CONCLUSION</b>YRP could inhibit the accumulation of extracellular matrix in renal interstitial tissue, so as to alleviate the renal interstitial fibrosis and improve the renal function.</p>

Multicenter prospective study on relationship of TCM syndrome type and renal pathology in 286 patients with IgA nephropathy / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the distribution pattern of TCM Syndrome type and its relationship with renal pathology in patients with IgA nephropathy.</p><p><b>METHODS</b>Apopting multicenter coordinated method, patients' TCM Syndrome type was differentiated according to their clinical manifestations, data concerning laboratory examination and renal pathology were collected to establish a database for analyzing the relationship between TCM Syndrome type and renal pathology in 286 patients.</p><p><b>RESULTS</b>Patients of Pi-Fei Qi-deficiency type (type 1) and both Qi-Yin deficiency type (type 2) showed rather milder pathological changes, by Lee classification, most of them belonged to grade I-III (72.3%, 70.2%); patients of Gan-Shen Yin deficiency type (type 3) had severe pathological change and majority of them belonged to grade III-IV (84.6%); and the most severe pathological change was shown in patients of Pi-Shen Yang-deficiency type (type 4), and the Lee's grade IV-V was dominant (88.0%) in them. Syndrome type of patients was significantly correlated with their Lee's grade (r = 0.26, P < 0.01). Percentage of glomerular sclerosis in patients of type 4 was higher than that in patients of the other three types. Semi-quantitative scoring upon pathological changes showed that the total, glomerular, tubulo-interstitial and vascular scores were significantly higher in patients of type 4 than in those of the other three types; those scores were higher in patients of type 3 than in type 1; the total, glomerular and vascular scores were higher in patients of type 3 than in type 2; and scores in patients of type 1 and type 2 showed insignificant difference.</p><p><b>CONCLUSION</b>Multicentric prospective study proves that the TCM Syndrome typeof patients with IgA nephropathy is significantly correlated with the grade and severity of their renal pathological changes, thus, the TCM Syndrome typing shows definite referential importance to conclude the severity of renal pathological change in patients with IgA nephropathy.</p>

Antagonistic effect of yishen ruanjian san contained serum against aristolochic acid in antagonizing human renal interstitial fibroblasts / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study whether yishen ruanjian san contained serum (S-YRS) could intervene the action of aristolochic acid (AA) in antagonizing human renal interstitial fibroblasts (hRIFs) to induce extracellular matrix (ECM) accumulation.</p><p><b>METHODS</b>AA-Na 40 microg/ml, with or without 10% S-YRS, was co-cultured with hRIFs, then the hRIFs mRNA of transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type I collagen (Col I) in the cultured cells were detected by RT-PCR, and their protein expression monitored with ELISA and Western blot respectively.</p><p><b>RESULTS</b>The mRNA and protein expression of all the above-mentioned factors were significantly up-regulated by AA-Na (P < 0.05). Excepting PAI-1, the enhanced mRNA and protein expression were significantly down-regulated by S-YRS (P < 0.05).</p><p><b>CONCLUSION</b>S-YRS could down-regulate the hRIF to promote the expression of ECM synthesis factors and inhibit the ECM degradation factors in hRIFs, so as to antagonize the AA stimulated accumulation of ECM such as Col I.</p>