Identification of stable reference genes for relative quantification of long RNA expression in urinary extracellular vesicles.

Urinary extracellular vesicles (uEVs) are rich in valuable biomolecule information which are increasingly recognized as potential biomarkers for various diseases. uEV long RNAs are among the critical cargos capable of providing unique transcriptome information of the source cells. However, consensus regarding ideal reference genes for relative long RNAs quantification in uEVs is not available as of date. Here we explored stable reference genes through profiling the long RNA expression by RNA-seq following unsupervised analysis and validation studies. Candidate reference genes were identified using four algorithms: NormFinder, GeNorm, BestKeeper and the Delta Ct method, followed by validation. RNA profile showed uEVs contained abundant long RNAs information and the core transcriptome was related to cellular structures, especially ribosome which functions mainly as translation, protein and RNA binding molecules. Analysis of RNA-seq data identified RPL18A, RPL11, RPL27, RACK1, RPSA, RPL41, H1-2, RPL4, GAPDH, RPS27A as candidate reference genes. RT-qPCR validation revealed that RPL41, RPSA and RPL18A were reliable reference genes for long RNA quantification in uEVs from patients with diabetes mellitus (DM), diabetic nephropathy (DN), IgA nephropathy (IgAN) and prostate cancer (PCA). Interestingly, RPL41 also outperformed traditional reference genes in renal tissues of DN and IgAN, as well as in plasma EVs of several types of cancers. The stable reference genes identified in this study may facilitate development of uEVs as novel biomarkers and increase the accuracy and comparability of biomarker studies.

Podocyte-derived soluble RARRES1 drives kidney disease progression through direct podocyte and proximal tubular injury.

Retinoic acid receptor responder protein-1 (RARRES1) is a podocyte-enriched transmembrane protein whose increased expression correlates with human glomerular disease progression. RARRES1 promotes podocytopenia and glomerulosclerosis via p53-mediated podocyte apoptosis. Importantly, the cytopathic actions of RARRES1 are entirely dependent on its proteolytic cleavage into a soluble protein (sRARRES1) and subsequent podocyte uptake by endocytosis, as a cleavage mutant RARRES1 exerted no effects in vitro or in vivo. As RARRES1 expression is upregulated in human glomerular diseases, here we investigated the functional consequence of podocyte-specific overexpression of RARRES1 in mice in the experimental focal segmental glomerulosclerosis and diabetic kidney disease. We also examined the effects of long-term RARRES1 overexpression on slowly developing aging-induced kidney injury. As anticipated, the induction of podocyte overexpression of RARRES1 (Pod-RARRES1WT) significantly worsened glomerular injuries and worsened kidney function in all three models, while overexpression of RARRES1 cleavage mutant (Pod-RARRES1MT) did not. Remarkably, direct uptake of sRARRES1 was also seen in proximal tubules of injured Pod-RARRES1WT mice and associated with exacerbated tubular injuries, vacuolation, and lipid accumulation. Single-cell RNA sequence analysis of mouse kidneys demonstrated RARRES1 led to a marked deregulation of lipid metabolism in proximal tubule subsets. We further identified matrix metalloproteinase 23 (MMP23) as a highly podocyte-specific metalloproteinase and responsible for RARRES1 cleavage in disease settings, as adeno-associated virus 9-mediated knockdown of MMP23 abrogated sRARRES1 uptake in tubular cells in vivo. Thus, our study delineates a previously unrecognized mechanism by which a podocyte-derived protein directly facilitates podocyte and tubular injury in glomerular diseases and suggests that podocyte-specific functions of RARRES1 and MMP23 may be targeted to ameliorate glomerular disease progression in vivo.

Therapeutic role of miR-26a on cardiaorenal injury in mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway.

BACKGROUND: Chronic kidney disease (CKD) is associated with common pathophysiological processes, such as inflammation and fibrosis, in both the heart and the kidney. However, the underlying molecular mechanisms that drive these processes are not yet fully understood. Therefore, this study focused on the molecular mechanism of heart and kidney injury in CKD. METHODS: We generated a microRNA (miR)-26a knockout (KO) mouse model to investigate the role of miR-26a in angiotensin (Ang)-II-induced cardiac and renal injury. We performed Ang-II modeling in wild type (WT) mice and miR-26a KO mice, with six mice in each group. In addition, Ang-II-treated AC16 cells and HK2 cells were used as in vitro models of cardiac and renal injury in the context of CKD. Histological staining, immunohistochemistry, quantitative real-time polymerase chain reaction (PCR), and Western blotting were applied to study the regulation of miR-26a on Ang-II-induced cardiac and renal injury. Immunofluorescence reporter assays were used to detect downstream genes of miR-26a, and immunoprecipitation was employed to identify the interacting protein of LIM and senescent cell antigen-like domain 1 (LIMS1). We also used an adeno-associated virus (AAV) to supplement LIMS1 and explored the specific regulatory mechanism of miR-26a on Ang-II-induced cardiac and renal injury. Dunnett's multiple comparison and t-test were used to analyze the data. RESULTS: Compared with the control mice, miR-26a expression was significantly downregulated in both the kidney and the heart after Ang-II infusion. Our study identified LIMS1 as a novel target gene of miR-26a in both heart and kidney tissues. Downregulation of miR-26a activated the LIMS1/integrin-linked kinase (ILK) signaling pathway in the heart and kidney, which represents a common molecular mechanism underlying inflammation and fibrosis in heart and kidney tissues during CKD. Furthermore, knockout of miR-26a worsened inflammation and fibrosis in the heart and kidney by inhibiting the LIMS1/ILK signaling pathway; on the contrary, supplementation with exogenous miR-26a reversed all these changes. CONCLUSIONS: Our findings suggest that miR-26a could be a promising therapeutic target for the treatment of cardiorenal injury in CKD. This is attributed to its ability to regulate the LIMS1/ILK signaling pathway, which represents a common molecular mechanism in both heart and kidney tissues.

Tubule-specific cyclin-dependent kinase 12 knockdown potentiates kidney injury through transcriptional elongation defects.

Direct tubular injury caused by several medications, especially chemotherapeutic drugs, is a common cause of AKI. Inhibition or loss of cyclin-dependent kinase 12 (CDK12) triggers a transcriptional elongation defect that results in deficiencies in DNA damage repair, producing genomic instability in a variety of cancers. Notably, 10-25% of individuals developed AKI after treatment with a CDK12 inhibitor, and the potential mechanism is not well understood. Here, we found that CDK12 was downregulated in the renal tubular epithelial cells in both patients with AKI and murine AKI models. Moreover, tubular cell-specific knockdown of CDK12 in mice enhanced cisplatin-induced AKI through promotion of genome instability, apoptosis, and proliferative inhibition, whereas CDK12 overexpression protected against AKI. Using the single molecule real-time (SMRT) platform on the kidneys of CDK12RTEC+/- mice, we found that CDK12 knockdown targeted Fgf1 and Cast through transcriptional elongation defects, thereby enhancing genome instability and apoptosis. Overall, these data demonstrated that CDK12 knockdown could potentiate the development of AKI by altering the transcriptional elongation defect of the Fgf1 and Cast genes, and more attention should be given to patients treated with CDK12 inhibitors to prevent AKI.

HIPK2 C-terminal domain inhibits NF-κB signaling and renal inflammation in kidney injury.

HIPK2 is a multifunctional kinase that acts as a key pathogenic mediator of chronic kidney disease and fibrosis. It acts as a central effector of multiple signaling pathways implicated in kidney injury, such as TGF-ß/Smad3-mediated extracellular matrix accumulation, NF-κB-mediated inflammation, and p53-mediated apoptosis. Thus, a better understanding of the specific HIPK2 regions necessary for distinct downstream pathway activation is critical for optimal drug development for CKD. Our study now shows that caspase-6-mediated removal of the C-terminal region of HIPK2 (HIPK2-CT) lead to hyperactive p65 NF-κB transcriptional response in kidney cells. In contrast, the expression of cleaved HIPK2-CT fragment could restrain the NF-κB transcriptional activity by cytoplasmic sequestration of p65 and the attenuation of IκBα degradation. Therefore, we examined whether HIPK2-CT expression can be exploited to restrain renal inflammation in vivo. The induction of HIPK2-CT overexpression in kidney tubular cells attenuated p65 nuclear translocation, expression of inflammatory cytokines, and macrophage infiltration in the kidneys of mice with unilateral ureteral obstruction and LPS-induced acute kidney injury. Collectively, our findings indicate that the HIPK2-CT is involved in the regulation of nuclear NF-κB transcriptional activity and that HIPK2-CT or its analogs could be further exploited as potential antiinflammatory agents to treat kidney disease.

Measurement of urinary exosomal phospholipase A2 receptor is a sensitive method for diagnosis of PLA2R-associated membranous nephropathy.

Background: The discovery of phospholipase A2 receptor (PLA2R) and its antibody (aPLA2Rab) has paved the way for diagnosing PLA2R-associated membranous nephropathy (PLA2R-MN) with a high specificity of 98%. However, the sensitivity was only 40% to 83.9%, and there is ongoing discussion around determining the optimal threshold for diagnosis. Recent advancements in the use of exosomes, a novel form of "liquid biopsy," have shown great promise in identifying markers for various medical conditions. Methods: Protein mass spectrometry and western blot were applied to verify the existence of PLA2R antigen in the urine exosome. We then evaluated the efficacy of urinary exosomal PLA2R antigen alone or combined with serum aPLA2Rab level to diagnose PLA2R-MN. Results: The urinary exosomes contained a high abundance of PLA2R antigen as evidenced by protein mass spectrometry and western blot in 85 PLA2R-MN patients vs the disease controls (14 secondary MN patients, 22 non-MN patients and 4 PLA2R-negative MN patients) and 20 healthy controls. Of note, urinary exosomal PLA2R antigen abundance also had a good consistency with the PLA2R antigen level in the renal specimens of PLA2R-MN patients. The sensitivity of urinary exosomal PLA2R for diagnosing PLA2R-MN reached 95.4%, whereas the specificity was 63.3%. Combining detection of the urinary exosomal PLA2R and serum aPLA2Rab could develop a more sensitive diagnostic method for PLA2R-MN, especially for patients with serum aPLA2Rab ranging from 2 to 20 RU/mL. Conclusions: Measurement of urinary exosomal PLA2R could be a sensitive method for the diagnosis of PLA2R-MN.

Computed tomography-determined skeletal muscle density predicts 3-year mortality in initial-dialysis patients in China.

BACKGROUND: Skeletal muscle mass and quality assessed by computed tomography (CT) images of the third lumbar vertebra (L3) level have been established as risk factors for poor clinical outcomes in several illnesses, but the relevance for dialysis patients is unclear. A few studies have suggested a correlation between CT-determined skeletal muscle mass and quality at the first lumbar vertebra (L1) level and adverse outcomes. Generally, chest CT does not reach beyond L1. We aimed to determine whether opportunistic CT scan (chest CT)-determined skeletal muscle mass and quality at L1 are associated with mortality in initial-dialysis patients. METHODS: This 3-year multicentric retrospective study included initial-dialysis patients from four centres between 2014 and 2017 in China. Unenhanced CT images of the L1 and L3 levels were obtained to assess skeletal muscle mass [by skeletal muscle index, (SMI), cm2 /m2 ] and quality [by skeletal muscle density (SMD), HU]. Skeletal muscle measures at L1 were compared with those at L3. The sex-specific optimal cutoff values of L1 SMI and L1 SMD were determined in relation to all-cause mortality. The outcomes were all-cause death and cardiac death. Cox regression models were applied to investigate the risk factors for death. RESULTS: A total of 485 patients were enrolled, of whom 257 had both L1 and L3 images. Pearson's correlation coefficient between L1 and L3 SMI was 0.84 (P < 0.001), and that between L1 and L3 SMD was 0.90 (P < 0.001). No significant association between L1 SMI and mortality was observed (P > 0.05). Low L1 SMD (n = 280, 57.73%) was diagnosed based on the optimal cutoff value (<39.56 HU for males and <33.06 HU for females). Multivariate regression analysis revealed that the low L1 SMD group had higher risks of all-cause death (hazard ratio 1.80; 95% confidence interval 1.05-3.11, P = 0.034) and cardiac death (hazard ratio 3.74; 95% confidence interval 1.43-9.79, P = 0.007). CONCLUSIONS: In initial-dialysis patients, there is high agreement between the L1 and L3 measures for SMI and SMD. Low SMD measured at L1, but not low SMI, is an independent predictor of both all-cause death and cardiac death.

Quercetin alleviates tubulointerstitial inflammation by inhibiting exosomes-mediated crosstalk between tubular epithelial cells and macrophages.

BACKGROUND: Tubulointerstitial inflammation (TII) is a critical pathological feature of kidney disease leading to renal fibrosis, and its treatment remains a major clinical challenge. We sought to explore the role of quercetin, a potential exosomes inhibitor, in exosomes release and TII. METHODS: The effects of quercetin on exosomes release and TII were examined by two TII mouse models: the unilateral ureteral obstruction (UUO) models and the LPS-induced mouse models. In vitro, exosomes-mediated crosstalk between tubular epithelial cells (TECs) and macrophages was performed to investigate the mechanisms by which quercetin inhibited exosomes and TII. RESULTS: In this study, we found that exosomes-mediated crosstalk between TECs and macrophages contributed to the development of TII. In vitro, exosomes released from LPS-stimulated TECs induced increased expression of inflammatory cytokines and fibrotic markers in Raw264·7 cells and vice versa. Interestingly, heat shock protein 70 (Hsp70) or Hsp90 proteins could control exosomes release from TECs and macrophages both in vivo and in vitro. Importantly, quercetin, a previously recognized heat shock protein inhibitor, could significantly reduce exosomes release in TII models by down-regulating Hsp70 or Hsp90. Quercetin abrogated exosomes-mediated intercellular communication, which attenuated TII and renal fibrosis accordingly. CONCLUSION: Quercetin could serve as a novel strategy for treatment of tubulointerstitial inflammation by inhibiting the exosomes-mediated crosstalk between tubules and macrophages.

Comprehensive analysis of the roles of fatty acid transport related proteins in clear cell renal cell carcinoma.

OBJECTIVE: This study aimed to explore the clinical significance of fatty acid transport-related protein (FATRP) in patients with clear cell renal cell carcinoma(ccRCC). METHODS: RNA-seq data and corresponding clinical data of ccRCC were obtained from TCGA data portal. Seventeen key FATRP genes were comprehensively investigated using bioinformatics approaches to systematically investigate their expression patterns in ccRCC. In addition, the correlation between the expression levels of these genes and clinicopathological features in ccRCC was further explored. RESULTS: Among the 17 key FATRP genes, only FABP5, FABP6, and FABP7 could be regarded as ideal biomarkers for ccRCC, as they were highly expressed in ccRCC tumor tissues, and positively correlates with tumor progression and poor prognosis. FABP6 had the highest copy number variations (CNV) events (63.07 %), and ccRCC patients with FABP6 amplification had a better prognosis than the unaltered group. DNA methylation levels of FABP6 and FABP7 were downregulated in ccRCC tumor tissues compared to those in normal tissues. FABP5 showed the opposite results. Moreover, a novel four FATRP gene (FABP1, FABP5, FABP7, FATP2) and three clinical parameter (age, stage, and grade) prediction model was constructed and that comprised a significant independent prognostic signature. CONCLUSIONS: Only a few FATRP genes are upregulated in ccRCC tumor tissue, and positively correlate with tumor progression and poor prognosis. The accuracy of a single gene of these FATRP genes as predictors of progression and prognosis of ccRCC is limited. The performance of the novel prediction model proposed by this study was much better than that of any single gene.

KIM-1 augments hypoxia-induced tubulointerstitial inflammation through uptake of small extracellular vesicles by tubular epithelial cells.

Tubular epithelial cells (TECs) exposed to hypoxia incite tubulointerstitial inflammation (TII), while the exact mechanism is unclear. In this study, we identified that hypoxia evoked tubule injury as evidenced by tubular hypoxia-inducible factor-1α and kidney injury molecule-1 (KIM-1) expression and that renal small extracellular vesicle (sEV) production was increased with the development of TII after ischemia-reperfusion injury (IRI). Intriguingly, KIM-1-positive tubules were surrounded by macrophages and co-localized with sEVs. In vitro, KIM-1 expression and sEV release were increased in hypoxic TECs and the hypoxia-induced inflammatory response was ameliorated when KIM-1 or Rab27a, a master regulator of sEV secretion, was silenced. Furthermore, KIM-1 was identified to mediate hypoxic TEC-derived sEV (Hypo-sEV) uptake by TECs. Phosphatidylserine (PS), a ligand of KIM-1, was present in Hypo-sEVs as detected by nanoflow cytometry. Correspondingly, the inflammatory response induced by exogenous Hypo-sEVs was attenuated when KIM-1 was knocked down. In vivo, exogenous-applied Hypo-sEVs localized to KIM-1-positive tubules and exacerbated TII in IRI mice. Our study demonstrated that KIM-1 expressed by injured tubules mediated sEV uptake via recognizing PS, which participated in the amplification of tubule inflammation induced by hypoxia, leading to the development of TII in ischemic acute kidney injury.

The analysis of low-dose glucocorticoid maintenance therapy in patients with primary nephrotic syndrome suffering from COVID-19.

Objectives: This study aimed to describe the effects of low-dose (prednisolone acetate 2.5-7.5 mg/day) glucocorticoids (GCs) maintenance therapy in patients with primary nephrotic syndrome (NS) suffering from coronavirus disease 2019 (COVID-19). Methods: A single-center retrospective study of NS patients with COVID-19 infection in Zhongda Hospital Affiliated to Southeast University from 1 February 2022 to 31 March 2023 was conducted. All enrolled patients underwent renal biopsy for the pathological diagnosis and reached complete remission (CR) or near-CR before COVID-19 infection. According to the maintained therapy regimen, patients were divided into low-dose GCs group and non-GCs group. Results: A total of 125 patients were enrolled in the study. Their median age was 46.0 ± 15.6 years, and the median value of 24-h urine protein was 0.77 g. The majority of these patients received treatment for more than 6 months, with a significant portion achieving CR (29.6%) or near-CR (43.2%). The leading cause of NS was membranous nephropathy (52%). There were no significant differences in the baseline characteristics between low-dose GCs and non-GCs group. As compared to those in the non-GCs group, patients receiving low-dose GCs treatment showed less fatigue or muscle weakness, smell disorder, palpitations, decreased appetite, taste disorder, dizziness, sore throat or difficult to swallow and fever (p < 0.05). Moreover, patients in the low-dose GCs group were with higher median quality of life scores (85.0) than in the non-GCs group (p = 0.001). Further serum inflammatory factor analysis indicated that interleukin-6 (IL-6) levels in the non-GCs group were significantly higher than that in the low-dose GCs group (p < 0.05). Conclusion: Patients with NS in low-dose GCs maintenance therapy stage showed milder symptom, higher quality of life and decreased serum IL-6 levels compared to those, who were not on GCs maintenance therapy. These results suggest the beneficial effect of low-dose GCs therapy in NS patients with CR/near-CR suffering from COVID-19 infection.

Exosomal Vaccine Loading T Cell Epitope Peptides of SARS-CoV-2 Induces Robust CD8+ T Cell Response in HLA-A Transgenic Mice.

Purpose: Current vaccines for the SARS-CoV-2 virus mainly induce neutralizing antibodies but overlook the T cell responses. This study aims to generate an exosomal vaccine carrying T cell epitope peptides of SARS-CoV-2 for the induction of CD8+ T cell response. Methods: Thirty-one peptides presented by HLA-A0201 molecule were conjugated to the DMPE-PEG-NHS molecules, and mixed with DSPE-PEG to form the peptide-PEG-lipid micelles, then fused with exosomes to generate the exosomal vaccine, followed by purification using size-exclusion chromatography and validation by Western blotting, liquid nuclear magnetic resonance (NMR) test and transmission electron microscopy. Furthermore, the exosomal vaccine was mixed with Poly (I:C) adjuvant and subcutaneously administered for three times into the hybrid mice of HLA-A0201/DR1 transgenic mice with wild-type mice. Then, the epitope-specific T cell responses were detected by ex vivo ELISPOT assay and intracellular cytokine staining. Results: The exosomal vaccine was purified from the Peak 2 fraction of FPLC and injected into the hybrid mice for three times. The IFN-γ spot forming units and the frequencies of IFN-γ+/CD8+ T cells were 10-82-fold and 13-65-fold, respectively, higher in the exosomal vaccine group compared to the Poly (I:C) control group, without visible organ toxicity. In comparison with the peptides cocktail vaccine generated in our recent work, the exosomal vaccine induced significantly stronger T cell response. Conclusion: Exosomal vaccine loading T cell epitope peptides of SARS-CoV-2 virus was initially generated without pre-modification for both peptides and exosomes, and elicited robust CD8+ T cell response in HLA-A transgenic mice.

MiR-155 deficiency protects renal tubular epithelial cells from telomeric and genomic DNA damage in cisplatin-induced acute kidney injury.

Rationale: Cisplatin nephrotoxicity is an important cause of acute kidney injury (AKI), limiting cisplatin application in cancer therapy. Growing evidence has suggested that genome instability, telomeric dysfunction, and DNA damage were involved in the tubular epithelial cells (TECs) damage in cisplatin-induced AKI (cAKI). However, the exact mechanism is largely unknown. Methods: We subjected miR-155-/- mice and wild-type controls, as well as HK-2 cells, to cAKI models. We assessed kidney function and injury with standard techniques. The cell apoptosis and DNA damage of TECs were evaluated both in vivo and in vitro. Telomeres were measured by the fluorescence in situ hybridization. Results: The expression level of miR-155 was upregulated in cAKI. Inhibition of miR-155 expression protected cisplatin-induced AKI both in vivo and in vitro. Compared with wild-type mice, miR-155-/- mice had reduced mortality, improved renal function and pathological damage after cisplatin intervention. Moreover, inhibition of miR-155 expression attenuated TECs apoptosis and DNA damage. These protective effects were caused by increasing expression of telomeric repeat binding factor 1 (TRF1) and cyclin-dependent kinase 12 (CDK12), thereby limiting the telomeric dysfunction and the genomic DNA damage in cAKI. Conclusion: We demonstrated that miR-155 deficiency could significantly attenuate pathological damage and mortality in cAKI through inhibition of TECs apoptosis, genome instability, and telomeric dysfunction, which is possibly regulated by the increasing expression of TRF1 and CDK12. This study will provide a new molecular strategy for the prevention of cAKI.

Tubular-specific CDK12 knockout causes a defect in urine concentration due to premature cleavage of the slc12a1 gene.

Cyclin-dependent kinase 12 (CDK12) plays a critical role in regulating gene transcription. CDK12 inhibition is a potential anticancer therapeutic strategy. However, several clinical trials have shown that CDK inhibitors might cause renal dysfunction and electrolyte disorders. CDK12 is abundant in renal tubular epithelial cells (RTECs), but the exact role of CDK12 in renal physiology remains unclear. Genetic knockout of CDK12 in mouse RTECs causes polydipsia, polyuria, and hydronephrosis. This phenotype is caused by defects in water reabsorption that are the result of reduced Na-K-2Cl cotransporter 2 (NKCC2) levels in the kidney. In addition, CKD12 knockout causes an increase in Slc12a1 (which encodes NKCC2) intronic polyadenylation events, which results in Slc12a1 truncated transcript production and NKCC2 downregulation. These findings provide novel insight into CDK12 being necessary for maintaining renal homeostasis by regulating NKCC2 transcription, which explains the critical water and electrolyte disturbance that occurs during the application of CDK12 inhibitors for cancer treatment. Therefore, there are safety concerns about the clinical use of these new anticancer drugs.

[Research progress of vascular endothelial growth factor-A and its isoforms in kidney disease].

Vascular endothelial growth factor-A (VEGF-A) is a critical angiogenic factor which is mainly secreted from podocytes and epithelial cells in kidney and plays an important role in renal pathophysiology. In recent years, functions of different isoforms of VEGF-A and the new secretion approach via extracellular vesicles (EVs) have been identified. Thus, further understanding are needed for the role of VEGF-A and its isoforms in renal injury and repair. In this review, we summarized the expression, secretion and regulation of VEGF-A, its biological function, and the role of different isoforms of VEGF-A in the development of different renal diseases. Meanwhile, the research progress of VEGF-A as diagnostic marker and therapeutic target for renal diseases were discussed.

Urinary sediment CCL5 messenger RNA as a potential prognostic biomarker of diabetic nephropathy.

BACKGROUND: Urinary sediment messenger RNAs (mRNAs) have been shown as novel biomarkers of kidney disease. We aimed to identify targeted urinary mRNAs in diabetic nephropathy (DN) based on bioinformatics analysis and clinical validation. METHODS: Microarray studies of DN were searched in the GEO database and Nephroseq platform. Gene modules negatively correlated with estimated glomerular filtration rate (eGFR) were identified by informatics methods. Hub genes were screened within the selected modules. In validation cohorts, a quantitative polymerase chain reaction assay was used to compare the expression levels of candidate mRNAs. Patients with renal biopsy-confirmed DN were then followed up for a median time of 21 months. End-stage renal disease (ESRD) was defined as the primary endpoint. Multivariate Cox proportional hazards regression was developed to evaluate the prognostic values of candidate mRNAs. RESULTS: Bioinformatics analysis revealed four chemokines (CCL5, CXCL1, CXLC6 and CXCL12) as candidate mRNAs negatively correlated with eGFR, of which CCL5 and CXCL1 mRNA levels were upregulated in the urinary sediment of patients with DN. In addition, urinary sediment mRNA of CXCL1 was negatively correlated with eGFR (r = -0.2275, P = 0.0301) and CCL5 level was negatively correlated with eGFR (r = -0.4388, P < 0.0001) and positively correlated with urinary albumin:creatinine ratio (r = 0.2693, P = 0.0098); also, CCL5 and CXCL1 were upregulated in patients with severe renal interstitial fibrosis. Urinary sediment CCL5 mRNA was an independent predictor of ESRD [hazard ratio 1.350 (95% confidence interval 1.045-1.745)]. CONCLUSIONS: Urinary sediment CCL5 and CXCL1 mRNAs were upregulated in DN patients and associated with a decline in renal function and degree of renal interstitial fibrosis. Urinary sediment CCL5 mRNA could be used as a potential prognostic biomarker of DN.

SAP130 released by damaged tubule drives necroinflammation via miRNA-219c/Mincle signaling in acute kidney injury.

Tubules injury and immune cell activation are the common pathogenic mechanisms in acute kidney injury (AKI). However, the exact modes of immune cell activation following tubule damage are not fully understood. Here we uncovered that the release of cytoplasmic spliceosome associated protein 130 (SAP130) from the damaged tubular cells mediated necroinflammation by triggering macrophage activation via miRNA-219c(miR-219c)/Mincle-dependent mechanism in unilateral ureteral obstruction (UUO) and cisplatin-induced AKI mouse models, and in patients with acute tubule necrosis (ATN). In the AKI kidneys, we found that Mincle expression was tightly correlated to the necrotic tubular epithelial cells (TECs) with higher expression of SAP130, a damaged associated molecule pattern (DAMP), suggesting that SAP130 released from damaged tubular cells may trigger macrophage activation and necroinflammation. This was confirmed in vivo in which administration of SAP130-rich supernatant from dead TECs or recombinant SAP130 promoted Mincle expression and macrophage accumulation which became worsen with profound tubulointerstitial inflammation in LPS-primed Mincle WT mice but not in Mincle deficient mice. Further studies identified that Mincle was negatively regulated via miR-219c-3p in macrophages as miR-219c-3p bound Mincle 3'-UTR to inhibit Mincle translation. Besides, lentivirus-mediated renal miR-219c-3p overexpression blunted Mincle and proinflammatory cytokine expression as well as macrophage infiltration in the inflamed kidney of UUO mice. In conclusion, SAP130 is released by damaged tubules which elicit Mincle activation on macrophages and renal necroinflammation via the miR-219c-3p-dependent mechanism. Results from this study suggest that targeting miR-219c-3p/Mincle signaling may represent a novel therapy for AKI.

VDR/Atg3 Axis Regulates Slit Diaphragm to Tight Junction Transition via p62-Mediated Autophagy Pathway in Diabetic Nephropathy.

Foot process effacement is an important feature of early diabetic nephropathy (DN), which is closely related to the development of albuminuria. Under certain nephrotic conditions, the integrity and function of the glomerular slit diaphragm (SD) structure were impaired and replaced by the tight junction (TJ) structure, resulting in so-called SD-TJ transition, which could partially explain the effacement of foot processes at the molecular level. However, the mechanism underlying the SD-TJ transition has not been described in DN. Here, we demonstrated that impaired autophagic flux blocked p62-mediated degradation of ZO-1 (TJ protein) and promoted podocytes injury via activation of caspase3 and caspase8. Interestingly, the expression of VDR in podocytes was decreased under diabetes conditions, which impaired autophagic flux through downregulating Atg3. Of note, we also found that VDR abundance was negatively associated with impaired autophagic flux and SD-TJ transition in the glomeruli from human renal biopsy samples with DN. Furthermore, VDR activation improved autophagic flux and attenuated SD-TJ transition in the glomeruli of diabetic animal models. In conclusion, our data provided the novel insight that VDR/Atg3 axis deficiency resulted in SD-TJ transition and foot processes effacement via blocking the p62-mediated autophagy pathway in DN.

Urinary small extracellular vesicles derived CCL21 mRNA as biomarker linked with pathogenesis for diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnostic biomarkers are still lacking. METHODS: Fourteen cytokines and chemokines mRNA were detected in urinary extracellular vesicles (EVs) from the screening cohort including 4 healthy controls (HC), 4 diabetes mellitus (DM) and 4 biopsy-proven DN patients, and was validated in another 16 HC and 15 DM and 28 DN patients. Correlation analysis was performed between the candidate biomarkers and clinic parameters as well as kidney histological changes. The findings were also confirmed in DN rat model with single injection of STZ. RESULTS: The number of small EVs secreted in urine was increased in DN patients compared to DM patients and healthy controls, with expression of AQP1 (a marker of proximal tubules) and AQP2 (a marker of distal/collecting tubules). Small EVs derived CCL21 mRNA increased significantly in DN patients and correlated with level of proteinuria and eGFR. Interestingly, elevated CCL21 mRNA from urine small EVs was observed in DN patients with normal renal function and could discriminate early DN patients from DM more efficiently compared to eGFR and proteinuria. CCL21 also showed an accurate diagnostic ability in distinguishing incipient from overt DN. Histologically, CCL21 mRNA expression increased progressively with the deterioration of tubulointerstitial inflammation and showed the highest level in nodular sclerosis group (class III) in DN patients. Remarkable infiltration of CD3 positive T cells including both CD4 and CD8 positive T cell population were observed in DN patients with high-CCL21 expression. Besides, accumulation of CD3 positive T cells correlated with level of urinary small EVs derived CCL21 and co-localized with CCL21 in the tubulointerstitium in DN patients. Finally, the correlation of CCL21 expression in renal cortex and urinary small EVs was confirmed in STZ-induced DN rat model. CONCLUSIONS: Urinary small EVs derived CCL21 mRNA may serve as early biomarker for identifying DN linked with pathogenesis. CCL21 mRNA mediated T cell infiltration may constitute the key mechanism of chronic inflammation in DN.