Shaolin Shi

Evaluation of MicroRNAs miR-196a, miR-30a-5P, and miR-490 as Biomarkers of Disease Activity among Patients with FSGS

BACKGROUND AND OBJECTIVES This study aimed to identify urinary microRNAs (miRNAs) as biomarkers for FSGS disease activity. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Candidate urinary miRNAs were identified in pooled urine samples from patients with active FSGS (FSGS-A) and FSGS in remission (FSGS-CR), and were then validated using individual samples. Their levels were compared both under different treatment responses in a prospective study of FSGS and in patients with different membranous nephropathy (MN) and diabetic nephropathy (DN) disease activity. The prediction of these miRNAs for treatment responses was further analyzed in both retrospective and prospective cohorts of patients with FSGS. RESULTS All 54 miRNAs were included as candidate biomarkers, including those with high levels in patients with FSGS-A (n=9) under the TaqMan Low Density Array as well as those with conserved expression in kidneys and involved in immune response. TaqMan probe-based quantitative RT-PCR confirmed the higher levels of four miRNAs in patients with FSGS-A in two independent cohorts (n=18 and n=80). Urinary miR-196a, miR-30a-5p, and miR-490 discriminated FSGS-A from FSGS-CR, with an area under the curve of ≥ 0.80. After steroid treatment, their levels were lower in steroid-responsive patients with FSGS (all P<0.001), but were unchanged in steroid-resistant patients. The levels of miRNAs were similar between active MN and MN in remission as well as active DN and incipient DN (all P>0.05). Urinary miR-30a-5p marginally predicted the response to steroid treatment in patients with FSGS-A, with an area under the curve of 0.63 (P=0.03). CONCLUSIONS The levels of urinary miR-196a, miR-30a-5p, and miR-490 are associated with FSGS disease activity.

MicroRNA-30 family members regulate calcium/calcineurin signaling in podocytes.

Calcium/calcineurin signaling is critical for normal cellular physiology. Abnormalities in this pathway cause many diseases, including podocytopathy; therefore, understanding the mechanisms that underlie the regulation of calcium/calcineurin signaling is essential. Here, we showed that critical components of calcium/calcineurin signaling, including TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3, are the targets of the microRNA-30 family (miR-30s). We found that these 5 genes are highly expressed as mRNA, but the level of the proteins is low in normal podocytes. Conversely, protein levels were markedly elevated in podocytes from rats treated with puromycin aminonucleoside (PAN) and from patients with focal segmental glomerulosclerosis (FSGS). In both FSGS patients and PAN-treated rats, miR-30s were downregulated in podocytes. In cultured podocytes, PAN or a miR-30 sponge increased TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3 expression; calcium influx; intracellular Ca2+ concentration; and calcineurin activity. Moreover, NFATC3 nuclear translocation, synaptopodin degradation, integrin β3 (ITGB3) activation, and actin fiber loss, which are downstream of calcium/calcineurin signaling, were induced by miR-30 reduction but blocked by the calcineurin inhibitor FK506. Podocyte-specific expression of the miR-30 sponge in mice increased calcium/calcineurin pathway component protein expression and calcineurin activity. The mice developed podocyte foot process effacement and proteinuria, which were prevented by FK506. miR-30s also regulated calcium/calcineurin signaling in cardiomyocytes. Together, our results identify miR-30s as essential regulators of calcium/calcineurin signaling.

Rhein protects pancreatic β-cells from dynamin-related protein-1-mediated mitochondrial fission and cell apoptosis under hyperglycemia

Rhein, an anthraquinone compound isolated from rhubarb, has been shown to improve glucose metabolism disorders in diabetic mice. The mechanism underlying the protective effect of rhein, however, remains unknown. Here, we demonstrate that rhein can protect the pancreatic β-cells against hyperglycemia-induced cell apoptosis through stabilizing mitochondrial morphology. Oral administration of rhein for 8 or 16 weeks in db/db mice significantly reduced fasting blood glucose (FBG) level and improved glucose tolerance. Cell apoptosis assay using both pancreatic sections and cultured pancreatic β-cells indicated that rhein strongly inhibited β-cell apoptosis. Morphological study showed that rhein was mainly localized at β-cell mitochondria and rhein could preserve mitochondrial ultrastructure by abolishing hyperglycemia-induced mitochondrial fission protein dynamin-related protein 1 (Drp1) expression. Western blot and functional analysis confirmed that rhein protected the pancreatic β-cells against hyperglycemia-induced apoptosis via suppressing mitochondrial Drp1 level. Finally, mechanistic study further suggested that decreased Drp1 level by rhein might be due to its effect on reducing cellular reactive oxygen species. Taken together, our study demonstrates for the first time that rhein can serve as a novel therapeutic agent for hyperglycemia treatment and rhein protects pancreatic β-cells from apoptosis by blocking the hyperglycemia-induced Drp1 expression.

Inhibition of miRNA-21 prevents fibrogenic activation in podocytes and tubular cells in IgA nephropathy.

Podocytopathy and tubular interstitial fibrosis impact on renal outcomes of IgA nephropathy (IgAN). We found that level of miR-21 was up regulated in both glomerular and tubular-interstitial tissues of patients with IgAN. Enhanced expression of miR-21 mainly located in podocytes and tubular cells. Mesangial cell derived cytokines contributed to the increase of miR-21 in podocytes and HK2 cells. IgA-HMC medium prepared with pIgA from IgAN, lead to obvious fibrogenic activation, evidenced by the loss of Podocin and CD2AP in podocytes, loss of E-cadherin and Megalin in HK2 cells and increase of FN and Col I in both cells. miR-21 targeted PTEN in these cells. Expression of PTEN was decreased and phosphorylation of Akt was increased in podocytes and HK2 cells exposed to the medium prepared with pIgA from IgAN. Inhibition of miR-21 preserved the expression of PTEN, prevented the activation of Akt and inhibited the fibrogenic activation in podocytes and HK2 cells exposed to the IgA-HMC medium prepared with pIgA from IgAN. In conclusion, our study suggests that inhibition of miR-21 prevents fibrogenic activation in podocytes and tubular cells by preventing PTEN/Akt pathway activation in IgAN.

Podocyte autophagic activity plays a protective role in renal injury and delays the progression of podocytopathies.

The progression of podocytopathies is quite variable among patients and the underlying reason for this remains unclear. Here, we report that autophagic activity in podocytes plays a critical role in controlling the progression of podocytopathies. Morphological and biochemical studies on renal biopsies from patients with minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS) showed that glomeruli, and in particular podocytes, from MCD patients had higher levels of Beclin1‐mediated autophagic activity than glomeruli from FSGS patients. Repeat renal biopsies of MCD patients enabled tracking of podocyte autophagic activity and confirmed that patients maintaining high podocyte autophagic activity retained MCD status, whereas patients with decreased podocyte autophagic activity progressed to FSGS. Inhibition of autophagic activity, by knocking down Beclin1 or by treating with 3‐methyladenine (3‐MA) or chloroquine, enhanced puromycin aminonucleoside (PAN)‐induced apoptosis of podocytes. In contrast, rapamycin‐mediated promotion of autophagic activity decreased this apoptosis. In PAN‐treated rats, inhibition of autophagy with 3‐MA or chloroquine resulted in earlier onset and greater proteinuria, more extensive foot‐process effacement, and reduction in podocyte markers, whereas rapamycin‐mediated stimulation of autophagy led to decreased proteinuria and less severe foot‐process effacement, but higher expression of podocyte markers. This study demonstrates that podocyte autophagic activity plays a critical protective role in renal injury and that maintaining podocyte autophagic activity represents a potential therapeutic strategy for controlling the progression of podocytopathies. Copyright

Plasma MicroRNA-186 and Proteinuria in Focal Segmental Glomerulosclerosis

BACKGROUND MicroRNAs (miRNAs) are stable in circulation, and their unique expression profiles can serve as fingerprints for various diseases. This study explored whether plasma miRNAs could be used as biomarkers to evaluate disease activity in patients with focal segmental glomerulosclerosis (FSGS). STUDY DESIGN Retrospective and prospective cohorts. SETTING & PARTICIPANTS 78 patients with FSGS with nephrotic proteinuria (protein excretion > 3.5g/24 h), 35 patients with FSGS in complete remission, 63 patients with membranous nephropathy, 59 patients with diabetic nephropathy, and 69 apparently healthy controls were recruited. Plasma samples from 51 other patients with FSGS with nephrotic proteinuria were collected prospectively before and after steroid treatment. PREDICTORS Plasma miRNA concentration. OUTCOMES Complete remission (protein excretion < 0.4g/24 h), or no response (sustained protein excretion > 3.5g/24 h after 8 weeks of steroid treatment). MEASUREMENTS Quantitative reverse transcription-polymerase chain reaction analysis of plasma miRNAs. RESULTS Increases in miR-125b, miR-186, and miR-193a-3p levels were identified in a pooled plasma sample of 9 patients with FSGS compared with that of 9 healthy controls and were confirmed with individual samples from patients with FSGS (n=32) and healthy controls (n=30). Areas under the receiver operating characteristic curves of miR-125b, miR-186, miR-193a-3p, and the 3 miRNAs in combination were 0.882, 0.789, 0.910, and 0.963, respectively. miR-125b and miR-186 concentrations were significantly lower in patients with FSGS in complete remission (n=35) than those with nephrotic proteinuria (n=37). In a prospective study, miR-125b and miR-186 levels declined markedly in patients with FSGS with complete remission (n=29), but not those with no response (n=22), after steroid treatment. Plasma miR-125b and miR-186 levels were not elevated in patients with membranous nephropathy (n=63) and diabetic nephropathy (n=59) regardless of degree of proteinuria. Last, plasma miR-186, but not miR-125b, level was correlated with degree of proteinuria in patients with FSGS (151 samples). LIMITATIONS Relatively small cohort size. CONCLUSIONS Plasma miR-186 may be a biomarker for FSGS with nephrotic proteinuria.

GADD45B mediates podocyte injury in zebrafish by activating the ROS-GADD45B-p38 pathway.

GADD45 gene has been implicated in cell cycle arrest, cell survival or apoptosis in a cell type specific and context-dependent manner. Members of GADD45 gene family have been found differentially expressed in several podocyte injury models, but their roles in podocytes are unclear. Using an in vivo zebrafish model of inducible podocyte injury that we have previously established, we found that zebrafish orthologs of gadd45b were induced upon the induction of podocyte injury. Podocyte-specific overexpression of zebrafish gadd45b exacerbated edema, proteinuria and foot-process effacement, whereas knockdown of gadd45b by morpholino-oligos in zebrafish larvae ameliorated podocyte injury. We then explored the role of GADD45B induction in podocyte injury using in vitro podocyte culture. We confirmed that GADD45B was significantly upregulated during the early phase of podocyte injury in cultured human podocytes and that podocyte apoptosis induced by TGF-β and puromycin aminonucleoside (PAN) was aggravated by GADD45B overexpression but ameliorated by shRNA-mediated GADD45B knockdown. We also showed that ROS inhibitor NAC suppressed PAN-induced GADD45B expression and subsequent activation of p38 MAPK pathway in podocytes and that inhibition of GADD45B diminished PAN-induced p38 MAPK activation. Taken together, our findings demonstrated that GADD45B has an important role in podocyte injury and may be a therapeutic target for the management of podocyte injury in glomerular diseases.