Bone marrow-derived exosomes promote inflammation and osteoclast differentiation in high-turnover renal osteodystrophy.

Introduction: Renal osteodystrophy (ROD) is a type of bone metabolic disorder in patients with chronic kidney disease (CKD). Inflammation is associated with bone loss in ROD. However, its precise mechanism has not yet been elucidated. The present study was conducted to investigate whether exosomes (Exos) in bone marrow (BM) are involved in the pathogenesis of high-turnover ROD.Methods: Bone mass, osteoclast number, and pro-inflammatory cytokines levels of BM supernatant were detected in adenine-induced ROD rats. The effect of Exos derived from BM (BM-Exos) of ROD (ROD-Exos) on inflammatory genes and osteoclast differentiation of BM-derived macrophages (BMMs) were further examined. Then, exosomal miRNA sequencing was performed and an miRNA-mRNA-pathway network was constructed.Results: we found increased osteoclasts and decreased bone mass in ROD rats, as well as inflammatory activation in the BM niche. Furthermore, BMMs from ROD rats displayed overproduction of proinflammatory cytokines and increased osteoclast differentiation, accompanied by nuclear factor κB (NF-κB) signaling activation. Mechanistically, we found that ROD-Exos activates NF-κB signaling to promote the release of proinflammatory cytokines and increase osteoclast differentiation of BMMs. Meanwhile, a total of 24 differentially expressed miRNAs were identified between BM-Exos from ROD and normal control (NC). The miRNA-mRNA-pathway network suggests that rno-miR-9a-5p, rno-miR-133a-3p, rno-miR-30c-5p, rno-miR-206-3p, and rno-miR-17-5p might play pivotal roles in inflammation and osteoclast differentiation. Additionally, we validated that the expression of miR-9a-5p is upregulated in ROD-Exos.Conclusion: The BM niche of ROD alters the miRNA cargo of BM-Exos to promote inflammation and osteoclast differentiation of BMMs, at least partially contributing to the pathogenesis of high-turnover ROD.

Glomerulosclerosis is a prognostic risk factor in patients with membranous nephropathy and non-nephrotic proteinuria.

OBJECTIVE: To explore the predictive value of the proportion of glomerulosclerosis (GS) incidences on the progression of membranous nephropathy with non-nephrotic proteinuria (NNP). METHODS: This study was a single-center, retrospective, cohort study. Patients with biopsy-proven idiopathic membranous nephropathy were divided into three groups based on the proportion of glomerular sclerosis, and their demographic, clinical, and pathological data were compared. The proportions of primary and secondary endpoints were recorded, and the relationship between GS and primary outcomes (progression to nephrotic syndrome, complete remission, and persistent NNP) and the renal composite endpoint was analyzed. RESULTS: A total of 112 patients were divided into three groups according to the proportions of glomerulosclerosis. The median follow-up time was 26.5 (13-51) months. There were significant differences in blood pressure (p < 0.01), renal interstitial lesions (p < 0.0001), and primary endpoints (p = 0.005). The survival analysis showed that prognosis was significantly worse in patients with a high proportion of GS than in those patients with a middle and low proportion of GS (p < 0.001). The Cox multivariate analysis showed that after adjusting for age, sex, BP, 24-h urinary protein, serum creatinine, treatment scheme, and pathological factors, the risk of renal composite outcome in the low proportion group was 0.076 times higher than that in the high proportion group (p = 0.009, HR = 0.076, 95% CI: 0.011-0.532). CONCLUSION: A high level of glomerulosclerosis was an independent risk factor for the prognosis of patients with membranous nephropathy with non-nephrotic proteinuria.

Association of gut microbiota with idiopathic membranous nephropathy.

BACKGROUND: The prevalence of idiopathic membranous nephropathy (IMN) is increasing worldwide and the gut microbiota is recognized to play a role in its pathology. The aim of this study was to understand the involvement of the gut-kidney axis in IMN by analyzing the composition of the gut microbiota of biopsy-proven IMN patients compared with healthy controls (HC). METHODS: Fecal samples from 30 patients with IMN diagnosed by renal biopsy and 30 healthy co-residents (control group) were collected for analysis in the Nephrology Department of the Second Affiliated Hospital of Harbin Medical University. The microbiota composition was analyzed by a 16S rRNA microbial profiling approach. RESULTS: The results indicated that the α- and ß-diversity of IMN patients differed significantly from those of the HC groups (P < 0.05). At the phylum level, IMN patients showed an increased abundance of Proteobacteria but a reduced abundance of Bacteroidota compared with the HC group. Actinobacteriota abundance showed a strong negative correlation with the estimated glomerular filtration rate. At the genus level, Faecalibacterium, Agathobacter, and Bacteroides were less abundant in the IMN group than in the HC group (LDA score > 2). Abundant bacterial functions related to lipid metabolism were observed among IMN group. CONCLUSION: Patients with IMN appear to have an altered gut microbiome, which could provide reference for future research on the interaction mechanism between the intestinal flora and IMN.

Identification of miRNA-mRNA network and immune-related gene signatures in IgA nephropathy by integrated bioinformatics analysis.

BACKGROUND: IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide, and its diagnosis depends mainly on renal biopsy. However, there is no specific treatment for IgAN. Moreover, its causes and underlying molecular events require further exploration. METHODS: The expression profiles of GSE64306 and GSE93798 were downloaded from the Gene Expression Omnibus (GEO) database and used to identify the differential expression of miRNAs and genes, respectively. The StarBase and TransmiR databases were employed to predict target genes and transcription factors of the differentially expressed miRNAs (DE-miRNAs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to predict biological functions. A comprehensive analysis of the miRNA-mRNA regulatory network was constructed, and protein-protein interaction (PPI) networks and hub genes were identified. CIBERSORT was used to examine the immune cells in IgAN, and correlation analyses were performed between the hub genes and infiltrating immune cells. RESULTS: Four downregulated miRNAs and 16 upregulated miRNAs were identified. Forty-five and twelve target genes were identified for the upregulated and downregulated DE-miRNAs, respectively. CDKN1A, CDC23, EGR1, HIF1A, and TRIM28 were the hub genes with the highest degrees of connectivity. CIBERSORT revealed increases in the numbers of activated NK cells, M1 and M2 macrophages, CD4 naive T cells, and regulatory T cells in IgAN. Additionally, HIF1A, CDC23, TRIM28, and CDKN1A in IgAN patients were associated with immune cell infiltration. CONCLUSIONS: A potential miRNA-mRNA regulatory network contributing to IgAN onset and progression was successfully established. The results of the present study may facilitate the diagnosis and treatment of IgAN by targeting established miRNA-mRNA interaction networks. Infiltrating immune cells may play significant roles in IgAN pathogenesis. Future studies on these immune cells may help guide immunotherapy for IgAN patients.

Effect of parathyroid hormone-related protein on intracellular calcium ion and cyclic adenosine monophosphate concentrations in cardiac fibroblasts.

OBJECTIVE: This study aimed to determine the effect of parathyroid hormone-related protein (PTHrP) on proliferation of cardiac fibroblasts (CFs) in primary cultures of neonatal Wistar rats. METHODS: Different PTHrP concentrations were added to CFs of neonatal Wistar rats and the cells were grouped according to the concentrations added. A verapamil (VPL) group and a calcitriol (CAL) group were also established. Changes in cell proliferation and in cyclic adenosine monophosphate and calcium ion levels were identified and recorded. RESULTS: We found that as the concentration of PTHrP increased, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, a tetrazolium salt) colorimetric absorbance values (A values) decreased. These values in the PTHrP groups were significantly lower than those in the control group. MTT colorimetric A values and 3H-thymidine deoxyribose intake were lower in the VPL group, low-dose CAL group, and the PTHrP 10-7 mol/L group compared with the control group. However, MTT colorimetric A values and 3H-thymidine deoxyribose intake were higher in the high-dose CAL group than in the PTHrP 10-7 mol/L group. As PTHrP concentrations increased, intracellular cyclic adenosine monophosphate concentrations also increased. CONCLUSION: PTHrp, VPL, and low-dose CAL inhibit proliferation of CFs, while high-dose CAL promotes proliferation of CFs.

Impact of epigallocatechin­3­gallate on expression of nuclear factor erythroid 2­related factor 2 and γ­glutamyl cysteine synthetase genes in oxidative stress­induced mouse renal tubular epithelial cells.

The aim of the present study was to investigate the antioxidant response mechanism of epigallocatechin­3­gallate (EGCG) in H2O2­induced mouse renal tubular epithelial cells (MRTECs). The cultured MRTECs were divided into normal, H2O2 (control) and EGCG treatment groups. The MTT assay was used to assess cell viability, and reverse transcription­quantitative polymerase chain reaction (RT­qPCR), immunocytochemical and western blot analyses were performed to detect the expression of nuclear factor erythroid 2­related factor 2 (Nrf2) and γ­glutamyl cysteine synthetase (γ­GCS). EGCG was able to mitigate H2O2­mediated cell damage. The RT­qPCR results demonstrated that EGCG was able to upregulate the gene expression of Nrf2 and γ­GCS in MRTECs in a dose­dependent manner. The immunocytochemistry and western blot analyses demonstrated that EGCG was able to increase the protein expression of Nrf2 and γ­GCS in MRTECs in a dose­dependent manner. Oxidative stress may lead to a decrease in the viability of MRTECs, while EGCG was able to promote the expression of Nrf2 and γ­GCS in MRTECs, thereby improving the antioxidant capacity of the cells and promoting the repair of oxidative stress injury.

Co-administration of Wuzhi capsules and tacrolimus in patients with idiopathic membranous nephropathy: clinical efficacy and pharmacoeconomics.

PURPOSE: Tacrolimus is an effective (but relatively expensive) immunosuppressant that is used widely in patients with membranous nephropathy. To reduce the tacrolimus dose while maintaining an equivalent therapeutic effect, we studied the clinical efficacy and pharmacoeconomic impact of co-administration of Wuzhi capsules (WZC that protects against damage to liver cells) and tacrolimus. METHODS: Sixty patients with membranous nephropathy were divided randomly into two groups: experimental (tacrolimus + WZC + corticosteroids) and control (tacrolimus + corticosteroids). Each group received treatments continuously for >6 months. Liver function; renal function; and whole-blood concentrations of tacrolimus, sugars, lipids, as well as 24-h urinary protein levels were used in the clinical evaluation. The cost of drugs was calculated, and the pharmacoeconomic cost-effectiveness analyses were carried out to compare indices between the two groups. RESULTS: Doses and costs of tacrolimus differed significantly between experimental and control groups (p < 0.01 or p < 0.05). Costs in the experimental group were 13,702.62 ± 1,458.6 CNY (2,194.10 ± 233.56 USD) and those in the control group were 17,796.87 ± 2,469.27 CNY (2,849.69 ± 395.39 USD), with clinical efficacy of 93.3 and 90.0 %, respectively. The cost-effectiveness ratios were 146.86 ± 15.63 and 197.73 ± 27.44, respectively. Compared with the experimental group, the control group showed an incremental cost-effectiveness ratio of 1,240.68 ± 306.25 CNY (198.66 ± 49.04 USD), whereas remission between the two groups was similar. CONCLUSION: Co-administration of WZCs and tacrolimus can reduce the dose of tacrolimus and decrease the costs incurred by patients within the same therapeutic window to that seen for treatment with tacrolimus alone.

Involvement of matrix metalloproteinase-2 in the development of renal interstitial fibrosis in mouse obstructive nephropathy.

Renal fibrosis is a common finding in progressive renal diseases. Matrix metalloproteinases (MMPs) are involved in epithelial-to-mesenchymal transition (EMT). We investigated the role of MMP-2 and the effect of inhibition of MMPs on the development of renal fibrosis. Renal fibrosis was induced in MMP-2 wild-type (MMP-2⁺/⁺) mice by unilateral ureteral obstruction (UUO). Renal histopathology, EMT-associated molecules, and activity of MMP-2 and MMP-9 were examined during the development of interstitial fibrosis. UUO-renal fibrosis was also induced in MMP-2 deficient (MMP-2⁻/⁻) and MMP-2⁺/⁺ mice treated with minocycline (inhibitor of MMPs). In MMP-2⁺/⁺ mice, MMP-2 and MMP-9 were expressed in damaged tubules, and their activities increased in a time-dependent manner after UUO. Interstitial fibrosis was noted at day 14, with deposition of types III and I collagens and expression of markers of mesenchymal cells (S100A4, vimentin, α-smooth muscle actin, and heat shock protein-47) in damaged tubular epithelial cells, together with F4/80+ macrophage infiltration. Fibrotic kidneys expressed EMT-associated molecules (ILK, TGF-ß1, Smad, Wnt, ß-catenin, and Snail). In contrast, the kidneys of MMP-2⁻/⁻ mice and minocycline-treated MMP-2⁺/⁺ mice showed amelioration of renal fibrosis with reduced expression of markers of mesenchymal cells in tubular epithelial cells, inhibition of upregulated EMT-associated molecules, and suppression of macrophage infiltration. The results suggested that MMP-2 have a pathogenic role in renal interstitial fibrosis, possibly through the induction of EMT and macrophage infiltration. Inhibition of MMPs may be beneficial therapeutically in renal fibrosis.

Role of matrix metalloproteinase-2 in recovery after tubular damage in acute kidney injury in mice.

BACKGROUND/AIMS: Matrix metalloproteinases (MMPs) are zinc endopeptidases that degrade extracellular matrix and are involved in the pathogenesis of ischemic damage in acute kidney injury (AKI). In the present study, we analyzed the role of MMP-2 in the repair process in ischemic AKI. METHODS: AKI was induced in MMP-2 wild-type (MMP-2(+/+)) and MMP-2-deficient (MMP-2(-/-)) mice by 90-min renal artery clamping followed by reperfusion. Renal histology and the activity and distribution of MMP-2 were examined from day 1 to day 14. During the recovery from AKI, MMP-2(+/+) mice were also treated with MMP-2/MMP-9 inhibitor. RESULTS: In both MMP-2(+/+) and MMP-2(-/-) mice, AKI developed on day 1 after ischemia/reperfusion with widespread acute tubular injury, but subsequent epithelial cell proliferation was evident on days 3-7. During the repair process, active MMP-2 and MMP-9 increased in regenerating tubular epithelial cells in MMP-2(+/+) mice on days 7-14, and the tubular repair process was almost complete by day 14. On the other hand, in MMP-2(-/-) mice, less prominent proliferation of tubular epithelial cells was evident on days 3 and 7, and damaged tubules that were covered with elongated and immature regenerated epithelial cells were identified on days 7 and 14. Incomplete recovery of injured microvasculature was also noted with persistent macrophage infiltration. Similarly, treatment with MMP-2/MMP-9 inhibitor resulted in impaired recovery in MMP-2(+/+) mice. CONCLUSION: MMP-2 is involved in tubular repair after AKI. The use of the MMP-2/MMP-9 inhibitor was a disadvantage when it was administered during the repair stage of ischemic AKI. Treatment with MMP inhibitor for AKI needs to be modified to enhance recovery from AKI.

Inhibition of capillary repair in proliferative glomerulonephritis results in persistent glomerular inflammation with glomerular sclerosis.

The pathological process of glomerulonephritis (GN) includes glomerular capillary damage, and vascular endothelial growth factor (VEGF) has an important role in glomerular capillary repair in GN. We examined the effect of inhibition of glomerular capillary repair after capillary injury in GN. Experimental Thy-1 GN was induced in rats that were divided into two groups: rats that received anti-VEGF neutralizing antibody (50 µg per 100 g body weight per day) and those treated with the vehicle from day 2 to day 9. We assessed the renal function and histopathology serially until week 6. Rats of the Thy-1 GN group showed diffuse glomerular mesangiolysis with ballooning destruction of the capillary network by day 3. VEGF(164) protein levels increased in the damaged glomeruli during days 5 to 10, and endothelial-cell proliferation increased with capillary repair in the vehicle-injected group. Proliferative GN resolved subsequently with decreased mesangial hypercellularity, and recovery of most of the glomeruli to the normal structure was evident by week 6. In contrast, administration of anti-VEGF antibody significantly decreased endothelial-cell proliferation and capillary repair in glomeruli by week 2. Thereafter, glomerular mesangial-cell proliferation and activation continued with persistent infiltration of macrophages. At week 6, segmental glomerular sclerosis developed with mesangial matrix accumulation and proteinuria. Deposition of type I collagen was also noted in sclerotic lesions. We conclude that impaired capillary repair was the underlying mechanism in the prolongation of glomerular inflammation in proliferative GN and in the development of glomerular sclerosis. Capillary repair has an important role in the recovery of glomerular damage and in the resolution of proliferative GN.