Separating the effects of life course adiposity on diabetic nephropathy: a comprehensive multivariable Mendelian randomization study.

Aims: Previous Mendelian randomization (MR) of obesity and diabetic nephropathy (DN) risk used small sample sizes or focused on a single adiposity metric. We explored the independent causal connection between obesity-related factors and DN risk using the most extensive GWAS summary data available, considering the distribution of adiposity across childhood and adulthood. Methods: To evaluate the overall effect of each obesity-related exposure on DN (Ncase = 3,676, Ncontrol = 283,456), a two-sample univariate MR (UVMR) analysis was performed. The independent causal influence of each obesity-related feature on DN was estimated using multivariable MR (MVMR) when accounting for confounding variables. It was also used to examine the independent effects of adult and pediatric obesity, adjusting for their interrelationships. We used data from genome-wide association studies, including overall general (body mass index, BMI) and abdominal obesity (waist-to-hip ratio with and without adjustment for BMI, i.e., WHR and WHRadjBMI), along with childhood obesity (childhood BMI). Results: UVMR revealed a significant association between adult BMI (OR=1.24, 95%CI=1.03-1.49, P=2.06×10-2) and pediatric BMI (OR=1.97, 95%CI=1.59-2.45, P=8.55×10-10) with DN risk. At the same time, adult WHR showed a marginally significant increase in DN (OR =1.27, 95%CI = 1.01-1.60, P=3.80×10-2). However, the outcomes were adverse when the influence of BMI was taken out of the WHR (WHRadjBMI). After adjusting for childhood BMI, the causal effects of adult BMI and adult abdominal obesity (WHR) on DN were significantly attenuated and became nonsignificant in MVMR models. In contrast, childhood BMI had a constant and robust independent effect on DN risk(adjusted for adult BMI: IVW, OR=1.90, 95% CI=1.60-2.25, P=2.03×10-13; LASSO, OR=1.91, 95% CI=1.65-2.21, P=3.80×10-18; adjusted for adult WHR: IVW, OR=1.80, 95% CI=1.40-2.31, P=4.20×10-6; LASSO, OR=1.90, 95% CI=1.56-2.32, P=2.76×10-10). Interpretation: Our comprehensive analysis illustrated the hazard effect of obesity-related exposures for DN. In addition, we showed that childhood obesity plays a separate function in influencing the risk of DN and that the adverse effects of adult obesity (adult BMI and adult WHR) can be substantially attributed to it. Thus, several obesity-related traits deserve more attention and may become a new target for the prevention and treatment of DN and warrant further clinical investigation, especially in childhood obesity.

GPR56 Promotes Diabetic Kidney Disease Through eNOS Regulation in Glomerular Endothelial Cells.

Although glomerular endothelial dysfunction is well recognized as contributing to the pathogenesis of diabetic kidney disease (DKD), the molecular pathways contributing to DKD pathogenesis in glomerular endothelial cells (GECs) are only partially understood. To uncover pathways that are differentially regulated in early DKD that may contribute to disease pathogenesis, we recently conducted a transcriptomic analysis of isolated GECs from diabetic NOS3-null mice. The analysis identified several potential mediators of early DKD pathogenesis, one of which encoded an adhesion G protein-coupled receptor-56 (GPR56), also known as ADGRG1. Enhanced glomerular expression of GPR56 was observed in human diabetic kidneys, which was negatively associated with kidney function. Using cultured mouse GECs, we observed that GPR56 expression was induced with exposure to advanced glycation end products, as well as in high-glucose conditions, and its overexpression resulted in decreased phosphorylation and expression of endothelial nitric oxide synthase (eNOS). This effect on eNOS by GPR56 was mediated by coupling of Gα12/13-RhoA pathway activation and Gαi-mediated cAMP/PKA pathway inhibition. The loss of GPR56 in mice led to a significant reduction in diabetes-induced albuminuria and glomerular injury, which was associated with reduced oxidative stress and restoration of eNOS expression in GECs. These findings suggest that GPR56 promotes DKD progression mediated, in part, through enhancing glomerular endothelial injury and dysfunction.

Preliminary study on 24p3 / neutrophil gelatinase-associated lipocalin (NGAL) ferroptosis inhibition in renal tubular epithelial cells.

The 24p3/neutrophil gelatinase-associated lipocalin (NGAL) protein plays an important protective role in acute kidney injury (AKI), but the exact mechanism remains unclear. Therefore, we have made a preliminary exploration of its mechanism. The experimental group was formed by constructing and transfecting 24P3 overexpressed plasmid into renal tubular epithelial cells. Western Bolt was used to detect NGAL expression. Cell proliferation was detected by CCK8 kit, cell death was detected by Hoechst 33342 and PI kit, mitochondrial morphology was observed under light microscope, reactive oxygen species (ROS) content was detected by fluorescence probe, and iron level and glutathione peroxidase 4 (GPX4) activity were detected by kit. Furthermore, the mechanism of NGAL action was further demonstrated by adding ferrostein-1 (Fer-1), an ferroptosis inhibitor, and erastin (containing DMSO),an ferroptosis inductor. We found that ferroptosis-related indicators were lower in the NGAL overexpression group than in the control group. At the same time, we found that NGAL alleviated ferroptosis induced by erastin and coordinated with Fer-1 to alleviate ferroptosis. In conclusion, NGAL inhibits ferroptosis in renal tubular epithelial cells, which may be associated with the progression of AKI and may provide a new therapeutic target for the transition from acute kidney injury to chronic kidney injury.

A case report of mitochondrial myopathy with membranous nephropathy.

BACKGROUND: MtDNA 3243 A > G mutation leads to mitochondrial myopathies with predominant hyperlactatemia. Given the ubiquitous nature of mitochondria, cellular dysfunction can also appear in tissues with high metabolic turnover; thus, there can be cardiac, digestive, ophthalmologic, and kidney complications. MtDNA 3243 A > G mutation has been shown to be with renal involvement in the previous cases of which are FSGS and tubularinterstitial nephritis. CASE PRESENTATION: We report a case of patient who had the mitochondrial myopathy with mitochondrial DNA (mtDNA) 3243 A > G mutation diagnosed membranous nephropathy by kidney biopsy, which was never reported before. Our patient was found to have chest tightness and shortness of breath with hyperlactatemia and was diagnosed mitochondrial myopathy with mtDNA 3243 A > G mutation 11 months ago. Acute kidney injury occurred with hyperuricemia (urid acid 1011umol/L) which may be associated with mtDNA mutation. Since then, persistent proteinuria was also found and the 24-h urine protein quantitative was around 2 g. Kidney biopsy was performed and the result was consistent with membranous nephropathy, with abnormal mitochondria seen in renal tubules by electron microscopy. CONCLUSIONS: Patients with mitochondrial myopathy could also have renal presentation of membranous nephropathy. Patients with mtDNA mutation may have various renal manifestations so that more attention should be paid on their kidneys.

Global transcriptomic changes in glomerular endothelial cells in mice with podocyte depletion and glomerulosclerosis.

Podocytes are a key component of the glomerular filtration barrier, and its dysfunction and eventual loss drive glomerular disease progression. Recent research has demonstrated the importance of podocyte cross-talk with other glomerular cells, such as glomerular endothelial cells (GECs), in both glomerular homeostasis and in disease settings. However, how GECs are affected globally by podocyte injury and loss in disease settings remains unclear. Therefore, to characterize the molecular changes occurring in GECs in response to the podocyte loss, we performed the transcriptomic profiling of isolated GECs after diphtheria toxin (DT)-mediated podocyte depletion in transgenic mice with podocyte-specific human DT receptor and endothelial-specific enhanced yellow fluorescent protein (EYFP) expression. DT administration led to nearly 40% of podocyte loss with the development of glomerulosclerosis. Differential gene expression analysis of isolated GECs in the diseased mice showed significant changes in pathways related to cell adhesion and actin cytoskeleton, proliferation, and angiogenesis, as well as apoptosis and cell death. However, quantification of EYFP + GECs indicated that there was a reduction in GECs in the diseased mice, suggesting that despite the ongoing proliferation, the concomitant injury and the activation of cell death program results in their overall net loss. The upstream regulator analysis strongly indicated the involvement of p53, TGF-ß1, and TNF-α as key mediators of the molecular changes occurring in GECs in the diseased mice. Our findings demonstrate significant molecular changes in GECs as a secondary consequence of podocyte loss and provide a valuable resource for further in-depth analysis of potential glomerular cross-talk mediators.

Gene expression profiles of glomerular endothelial cells support their role in the glomerulopathy of diabetic mice.

Endothelial dysfunction promotes the pathogenesis of diabetic nephropathy (DN), which is considered to be an early event in disease progression. However, the molecular changes associated with glomerular endothelial cell (GEC) injury in early DN are not well defined. Most gene expression studies have relied on the indirect assessment of GEC injury from isolated glomeruli or renal cortices. Here, we present transcriptomic analysis of isolated GECs, using streptozotocin-induced diabetic wildtype (STZ-WT) and diabetic eNOS-null (STZ-eNOS-/-) mice as models of mild and advanced DN, respectively. GECs of both models in comparison to their respective nondiabetic controls showed significant alterations in the regulation of apoptosis, oxidative stress, and proliferation. The extent of these changes was greater in STZ-eNOS-/- than in STZ-WT GECs. Additionally, genes in STZ-eNOS-/- GECs indicated further dysregulation in angiogenesis and epigenetic regulation. Moreover, a biphasic change in the number of GECs, characterized by an initial increase and subsequent decrease over time, was observed only in STZ-eNOS-/- mice. This is consistent with an early compensatory angiogenic process followed by increased apoptosis, leading to an overall decrease in GEC survival in DN progression. From the genes altered in angiogenesis in STZ-eNOS-/- GECs, we identified potential candidate genes, Lrg1 and Gpr56, whose function may augment diabetes-induced angiogenesis. Thus, our results support a role for GEC in DN by providing direct evidence for alterations of GEC gene expression and molecular pathways. Candidate genes of specific pathways, such as Lrg1 and Gpr56, can be further explored for potential therapeutic targeting to mitigate the initiation and progression of DN.

Transcriptomic analysis uncovers novel synergistic mechanisms in combination therapy for lupus nephritis.

A recent clinical study showed that combination therapy consisting of mycophenolate mofetil, tacrolimus and steroids was shown to be more effective in achieving complete remission in patients with severe forms of lupus nephritis than conventional therapy consisting of intravenous cyclophosphamide and steroids. To explore the underlying molecular and cellular mechanisms of increased efficacy of the combination therapy regimen, we employed a mouse model of lupus nephritis, MRL/lpr mice, and treated them with monotherapies of prednisone, mycophenolate mofetil, or tacrolimus, or with their combination. Consistent with previous clinical findings, combination therapy markedly improved renal outcome compared to the monotherapies in mice with lupus nephritis. Transcriptomic analysis of their kidneys revealed distinct molecular pathways that were differentially regulated in combination therapy versus monotherapies. Combination therapy not only provided additive immunosuppressive effects, but also induced gene expression and molecular pathways to confer enhanced renoprotection. Specifically, combination therapy inhibited TLR7 expression in the kidneys of mice with lupus nephritis; combination of tacrolimus and mycophenolate mofetil led to better stabilization of the podocyte actin cytoskeleton through the reciprocal regulation of RhoA and Rac1 activities. Combination therapy strongly suppressed the IL-6/Stat3 pathway. These findings were further validated in renal biopsy samples from patients with lupus nephritis before and after treatments with mycophenolate mofetil, tacrolimus or combination therapy. Thus, our study further supports the earlier clinical finding and further provides insights into the molecular basis for increased efficacy of combination therapy.

DC-SIGN expression on podocytes and its role in inflammatory immune response of lupus nephritis.

Podocytes, the main target of immune complex, participate actively in the development of glomerular injury as immune cells. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is an innate immune molecular that has an immune recognition function, and is involved in mediation of cell adhesion and immunoregulation. Here we explored the expression of DC-SIGN on podocytes and its role in immune and inflammatory responses in lupus nephritis (LN). Expression of DC-SIGN and immunoglobulin (Ig)G1 was observed in glomeruli of LN patients. DC-SIGN was co-expressed with nephrin on podocytes. Accompanied by increased proteinuria of LN mice, DC-SIGN and IgG1 expressions were observed in the glomeruli from 20 weeks, and the renal function deteriorated up to 24 weeks. Mice with anti-DC-SIGN antibody showed reduced proteinuria and remission of renal function. After the podocytes were stimulated by serum of LN mice in vitro, the expression of DC-SIGN, major histocompatibility complex (MHC) class II and CD80 was up-regulated, stimulation of T cell proliferation was enhanced and the interferon (IFN)-γ/interleukin (IL)-4 ratio increased. However, anti-DC-SIGN antibody treatment reversed these events. These results suggested that podocytes in LN can exert DC-like function through their expression of DC-SIGN, which may be involved in immune and inflammatory responses of renal tissues. However, blockage of DC-SIGN can inhibit immune functions of podocytes, which may have preventive and therapeutic effects.

Effects of rapamycin on DC-SIGN expression and biological functions in DC.

Rapamycin, a macrolide antibiotic, has potent immunosuppressive properties as an antirejection therapy in organ transplantation. Studies show that dendritic cells (DC) are important targets for rapamycin, which can inhibit DC maturation and DC-induced allogeneic T cell proliferation. In this study, we investigated the effects of rapamycin on the expressions of DC-SIGN and transcription factor PU.1 and the function of DC. Treatment with rapamycin significantly reduced the expression of DC-SIGN in a dose-dependent manner associated with suppression of PU.1 gene expression and the ability of DC to migrate and stimulate T cell proliferation. The expression of DC-SIGN was significantly suppressed using PU.1 siRNA. Intriguingly, rapamycin treatment largely decreased the expressions of PU.1 and DC-SIGN in THP-1 cells. In addition, treatment with rapamycin down-regulated the promoter activity of DC-SIGN. In conclusion, rapamycin inhibits DC-SIGN expression and suppresses the ability of DC to migrate and stimulate T cell proliferation through the PU.1 gene transcription pathway.

A Lectin-EGF antibody promotes regulatory T cells and attenuates nephrotoxic nephritis via DC-SIGN on dendritic cells.

BACKGROUND: Interactions between dendritic cells (DCs) and T cells play a critical role in the development of glomerulonephritis, which is a common cause of chronic kidney disease. DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), an immune-regulating molecule of the C-type lectin family, is mainly expressed on DCs and mediates DC adhesion and migration, inflammation, activation of primary T cells. DC-SIGN triggers immune responses and is involved in the immune escape of pathogens and tumours. In addition, ligation of DC-SIGN on DCs actively primes DCs to induce Tregs. Under certain conditions, DC-SIGN signalling may result in inhibition of DC maturation, by promoting regulatory T cell (Treg) function and affecting Th1/Th2 bias. METHODS: A rat model of nephrotoxic nephritis was used to investigate the therapeutic effects of an anti-lectin-epidermal growth factor (EGF) antibody on glomerulonephritis. DCs were induced by human peripheral blood mononuclear cells in vitro. The expression of DC surface antigens were detected using flow cytometry; the levels of cytokines were detected by ELISA and qPCR, respectively; the capability of DCs to stimulate T cell proliferation was examined by mixed lymphocyte reaction; PsL-EGFmAb targeting to DC-SIGN on DCs was identified by immunoprecipitation. RESULTS: Anti-Lectin-EGF antibody significantly reduced global crescent formation, tubulointerstitial injury and improved renal function impairment through inhibiting DC maturation and modulating Foxp3 expression and the Th1/Th2 cytokine balance in kidney. Binding of anti-Lectin-EGF antibody to DC-SIGN on human DCs inhibited DC maturation, increased IL-10 production from DCs and enhanced CD4+CD25+ Treg functions. CONCLUSIONS: Our results suggest that treatment with anti-Lectin-EGF antibody modulates DCs to suppressive DCs and enhances Treg functions, contributing to the attenuation of renal injury in a rat model of nephrotoxic nephritis.

DC-SIGN modulates DC maturation and function in rat renal tubulointerstitial lesions.

The role of DC-SIGN in tubulointerstitial lesions (TILs) and the effect of anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb) were investigated in rat nephrotoxic nephritis (NTN). On Day 4, immature DC-SIGN+DCs infiltrated into renal tubulointerstitium and matured by Day 14, showing increased migratory capacity and ability to induce T cell proliferation. The distribution of DC-SIGN+ DC significantly correlated with crescent formation, TIL severity, and changes in renal function. RANTES and TNF-alpha mRNA were continuously up-regulated from Day 4, while IL-10 mRNA was down-regulated after a marked increase on Day 4. Expression of IFN-gamma and IL-4 mRNA increased on Day 14 due to DC maturation. PsL-EGFmAb suppressed DC maturation, migration and ability to activate T cells. It also down-regulated TNF-alpha and up-regulated IL-10, resulting in a Th1/Th2 bias. The number of crescents decreased and TILs and renal function improved. These results suggest that DC-SIGN mediates DC tubulointerstitial infiltration and is an important regulator of local immune reactions and TILs. PsL-EGFmAb inhibited DC migration, maturation and function by targeting DC-SIGN, and may therefore be a potential treatment for NTN.

Effects of DC-SIGN expression on renal tubulointerstitial fibrosis in nephritis.

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is important for dendritic cell (DC) in migrating, recognizing, capturing, presenting antigens and in initiating T cell responses. In the present study, we investigated the role of DC-SIGN in renal tubulointerstitial inflammation and fibrosis. DC-SIGN was mainly expressed in tubular epithelial cells and DC-SIGN+ DCs were primarily distributed in renal tubulointerstitial areas during the early stage of nephritis, which was correlated with the degree of renal tubular interstitial lesions and fibrosis. In vitro, DC-SIGN expression in cultured human renal tubular epithelial cells was elevated when treated by tumor necrosis factor-alpha, and was inhibited by anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb). In a rat model of chronic renal interstitial fibrosis, there was a significant correlation of DC-SIGN expression with DC-SIGN+ DC distribution and the degree of tubulointerstitial lesion. PsL-EGFmAb reduced DC-SIGN expression and DC-SIGN+ DC accumulation in renal tissues in this rat model. These results suggest that DC-SIGN plays an important role in DC-mediated renal tubular interstitial lesions induced by immuno-inflammatory responses.

Effects of DC-SIGN expression on renal tubulointerstitial fibrosis in nephritis.

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is important for dendritic cell (DC) in migrating, recognizing, capturing, presenting antigens and in initiating T cell responses. In the present study, we investigated the role of DC-SIGN in renal tubulointerstitial inflammation and fibrosis. DC-SIGN was mainly expressed in tubular epithelial cells and DC-SIGN+ DCs were primarily distributed in renal tubulointerstitial areas during the early stage of nephritis, which was correlated with the degree of renal tubular interstitial lesions and fibrosis. In vitro, DC-SIGN expression in cultured human renal tubular epithelial cells was elevated when treated by tumor necrosis factor-alpha, and was inhibited by anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb). In a rat model of chronic renal interstitial fibrosis, there was a significant correlation of DC-SIGN expression with DC-SIGN+ DC distribution and the degree of tubulointerstitial lesion. PsL-EGFmAb reduced DC-SIGN expression and DC-SIGN+ DC accumulation in renal tissues in this rat model. These results suggest that DC-SIGN plays an important role in DC-mediated renal tubular interstitial lesions induced by immuno-inflammatory responses.

PsL-EGFmAb inhibits the stimulatory functions of human dendritic cells via DC-SIGN.

Dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is a DC-specific C-type lectin that plays an important role in recognizing and capturing pathogens, DC migration and initiation of T cell responses. Here, we show that anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb), originally prepared for blockade of the adhesive molecule P-selectin, significantly down-regulated DC-SIGN expression as well as expression of mature DC-related molecules including CD83, CD86 and CD80 on human DCs. This PsL-EGFmAb treatment of DCs resulted in impaired allogeneic T cell proliferation and IL-12 production. Furthermore, we show that PsL-EGFmAb-induced down-regulation of DC-SIGN may inhibit NF-kappaB expression in DCs, which accounts for the inhibition of DC maturation and stimulatory function. Our present studies indicate that PsL-EGFmAb may be a useful reagent for regulating DC-SIGN expression and DC function.