Tauroursodeoxycholic acid ameliorates renal injury induced by COL4A3 mutation.

COL4A3/A4/A5 mutations have been identified as critical causes of Alport syndrome and other genetic chronic kidney diseases. However, the underlying pathogenesis remains unclear, and specific treatments are lacking. Here, we constructed a transgenic Alport syndrome mouse model by generating a mutation (Col4a3 p.G799R) identified previously from one large Alport syndrome family into mice. We observed that the mutation caused a pathological decrease in intracellular and secreted collagen IV α3α4α5 heterotrimers. The mutant collagen IV α3 chains abnormally accumulated in the endoplasmic reticulum and exhibited defective secretion, leading to persistent endoplasmic reticulum stress in vivo and in vitro. RNA-seq analysis revealed that the MyD88/p38 MAPK pathway plays key roles in mediating subsequent inflammation and apoptosis signaling activation. Treatment with tauroursodeoxycholic acid, a chemical chaperone drug that functions as an endoplasmic reticulum stress inhibitor, effectively suppressed endoplasmic reticulum stress, promoted secretion of the α3 chains, and inhibited the activation of the MyD88/p38 MAPK pathway. Tauroursodeoxycholic acid treatment significantly improved kidney function in vivo. These results partly clarified the pathogenesis of kidney injuries associated with Alport syndrome, especially in glomeruli, and suggested that tauroursodeoxycholic acid might be useful for the early clinical treatment of Alport syndrome.

Develop and Validate a Risk Score in Predicting Renal Failure in Focal Segmental Glomerulosclerosis.

Introduction: The aim of this study was to develop and validate a risk score (RS) for end-stage kidney disease (ESKD) in patients with focal segmental glomerulosclerosis (FSGS). Methods: Patient with biopsy-proven FSGS was enrolled. All the patients were allocated 1:1 to the two groups according to their baseline gender, age, and baseline creatinine level by using a stratified randomization method. ESKD was the primary endpoint. Results: We recruited 359 FSGS patients, and 177 subjects were assigned to group 1 and 182 to group 2. The clinicopathological variables were similar between two groups. There were 23 (13%) subjects reached to ESKD in group 1 and 22 (12.1%) in group 2. By multivariate Cox regression analyses, we established RS 1 and RS 2 in groups 1 and 2, respectively. RS 1 consists of five parameters including lower eGFR, higher urine protein, MAP, IgG level, and tubulointerstitial lesion (TIL) score; RS 2 also consists of five predictors including lower C3, higher MAP, IgG level, hemoglobin, and TIL score. RS 1 and RS 2 were cross-validated between these two groups, showing RS 1 had better performance in predicting 5-year ESKD in group 1 (c statics, 0.86 [0.74-0.98] vs. 0.82 [0.69-0.95]) and group 2 (c statics, 0.91 [0.83-0.99] vs. 0.89 [0.79-0.99]) compared to RS 2. We then stratified the risk factors into four groups, and Kaplan-Meier survival curve revealed that patients progressed to ESKD increased as risk levels increased. Conclusions: A predictive model incorporated clinicopathological feature was developed and validated for the prediction of ESKD in FSGS patients.

Adult-Onset Focal Segmental Glomerulosclerosis With Steroid-Dependent Nephrotic Syndrome Caused by a Novel TBC1D8B Variant: A Case Report and Literature Review.

Focal segmental glomerulosclerosis (FSGS) is a histological lesion with a variety of potential causes, including rare variants of podocyte-related genes. Recently, it has been found that variants in the TBC1D8B gene on the X chromosome can lead to early-onset focal segmental glomerulosclerosis and steroid-resistant nephrotic syndrome by affecting endocytosis and recycling of nephrin. Here, we report a 19-year-old Chinese patient with nephrotic syndrome and normal kidney function. He had a complete remission of nephrotic syndrome after full-dose prednisone and cyclosporine treatment. Unfortunately, a relapse of nephrotic syndrome occurred during prednisone tapering. Focal segmental glomerulosclerosis was proven by a kidney biopsy, and a hemizygous pathogenic variant located in the TBC (Tre-2-Bub2-Cdc16) domain of TBC1D8B was detected by whole-exome sequencing. By comparing our case with reports of other patients with TBC1D8B variants, we suggest possible genotype-phenotype correlations. To our knowledge, this is the first report identifying a pathogenetic variant in the TBC domain of TBC1D8B in an adult-onset focal segmental glomerulosclerosis patient with steroid-dependent NS. With this report, we broaden the clinical and genetic spectrum of X-linked genetic FSGS.

Mutation in XPO5 causes adult-onset autosomal dominant familial focal segmental glomerulosclerosis.

BACKGROUND: Focal and segmental glomerulosclerosis (FSGS) is a histological pathology that characterizes a wide spectrum of diseases. Many genes associated with FSGS have been studied previously, but there are still some FSGS families reported in the literature without the identification of known gene mutations. The aim of this study was to investigate the new genetic cause of adult-onset FSGS. METHODS: This study included 40 FSGS families, 77 sporadic FSGS cases, 157 non-FSGS chronic kidney disease (CKD) families and 195 healthy controls for analyses. Whole-exome sequencing (WES) and Sanger sequencing were performed on probands and family members of all recruited families and sporadic FSGS cases. RESULTS: Using WES, we have identified a novel heterozygous missense variant (c.T1655C:p.V552A) in exportin 5 gene (XPO5) in two families (FS-133 and CKD-05) affected with FSGS and CKD. Sanger sequencing has confirmed the co-segregation of this identified variant in an autosomal dominant pattern within two families, while this variant was absent in healthy controls. Furthermore, the identified mutation was absent in 195 ethnically matched healthy controls by Sanger sequencing. Subsequently, in silico analysis demonstrated that the identified variant was highly conservative in evolution and likely to be pathogenic. CONCLUSIONS: Our study reports an adult-onset autosomal dominant inheritance of the XPO5 variant in familial FSGS for the first time. Our study expanded the understanding of the genotypic, phenotypic and ethnical spectrum of mutation in this gene.

Impact of fluid overload on blood pressure variability in patients on peritoneal dialysis.

Fluid overload is a common complication in patients who are on continuous ambulatory peritoneal dialysis (CAPD). Blood pressure is the traditional indicator of fluid status in these patients. However, it has poor sensitivity. Blood pressure variability (BPV) can detect fluctuations in blood pressure sooner and more accurately and be useful for the assessment of fluid volume in hemodialysis patient. However, there are limited relevant studies involving patients on CAPD. This retrospective study included 175 patients on CAPD who underwent regular assessment of the peritoneal dialysis adequacy at 2-3-month intervals at our center from January 2018 to December 2020. The overhydration (OH) value was measured using a body composition monitor. Blood pressure measurements obtained in each visit over one-year follow-up were used to determine each patient's BPV under peritoneal dialysis. The patients were divided into the normal volume (OH ≤2 L), mild volume overload (OH 2-4 L), and severe volume overload groups (OH ≥4 L). There was no significant between-group difference in the mean blood pressure (p > 0.05). However, the systolic and diastolic BPV values were significantly higher in the severe volume overload group than in the other groups (p < 0.05). Further correlation and regression analysis showed volume overload severity and BPV existed positive association, and BPV was also significantly associated with PD volume related indexes, including diabetes mellitus, blood parathyroid hormone levels, Kt/V and subjective global assessment scores in the volume overloaded group (p < 0.05). All these results suggested BPV may be a useful indicator for fluid status in PD patients.

Particulate matter of air pollution may increase risk of kidney failure in IgA nephropathy.

IgA nephropathy (IgAN) is characterized by deposition of galactose-deficient IgA1 (Gd-IgA1) in glomerular mesangium associated with mucosal immune disorders. Since environmental pollution has been associated with the progression of chronic kidney disease in the general population, we specifically investigated the influence of exposure to fine particulate matter less than 2.5 µm in diameter (PM2.5) on IgAN progression. Patients with biopsy-proven primary IgAN were recruited from seven Chinese kidney centers. PM2.5 exposure from 1998 to 2016 was derived from satellite aerosol optical depth data and a total of 1,979 patients with IgAN, including 994 males were enrolled. The PM2.5 exposure levels for patients from different provinces varied but, in general, the PM2.5 exposure levels among patients from the north were higher than those among patients from the south. The severity of PM2.5 exposure in different regions was correlated with regional kidney failure burden. In addition, each 10 µg/m3 increase in annual average concentration of PM2.5 exposure before study entry (Hazard Ratio, 1.14; 95% confidence interval, 1.06-1.22) or time-varying PM2.5 exposure after study entry (1.10; 1.01-1.18) were associated with increased kidney failure risk after adjustment for age, gender, estimated glomerular filtration rate, urine protein, uric acid, hemoglobin, mean arterial pressure, Oxford classification, glucocorticoid and renin-angiotensin system blocker therapy. The associations were robust when the time period, risk factors of cardiovascular diseases or city size were further adjusted on the basis of the above model. Thus, our results suggest that PM2.5 is an independent risk factor for kidney failure in patients with IgAN, but these findings will require validation in more diverse populations and other geographic regions.

A Validation Study Comparing Risk Prediction Models of IgA Nephropathy.

We aimed to validate three IgAN risk models proposed by an international collaborative study and another CKD risk model generated by an extended CKD cohort with our multicenter Chinese IgAN cohort. Biopsy-proven IgAN patients with an eGFR ≥15 ml/min/1.73 m2 at baseline and a minimum follow-up of 6 months were enrolled. The primary outcomes were a composite outcome (50% decline in eGFR or ESRD) and ESRD. The performance of those models was assessed using discrimination, calibration, and reclassification. A total of 2,300 eligible cases were enrolled. Of them, 288 (12.5%) patients reached composite outcome and 214 (9.3%) patients reached ESRD during a median follow-up period of 30 months. Using the composite outcome for analysis, the Clinical, Limited, Full, and CKD models had relatively good performance with similar C statistics (0.81, 0.81, 0.82, and 0.82, respectively). While using ESRD as the end point, the four prediction models had better performance (all C statistics > 0.9). Furthermore, subgroup analysis showed that the models containing clinical and pathological variables (Full model and Limited model) had better discriminatory abilities than the models including only clinical indicators (Clinical model and CKD model) in low-risk patients characterized by higher baseline eGFR (≥60 ml/min/1.73 m2). In conclusion, we validated recently reported IgAN and CKD risk models in our Chinese IgAN cohort. Compared to pure clinical models, adding pathological variables will increase performance in predicting ESRD in low-risk IgAN patients with baseline eGFR ≥60 ml/min/1.73 m2.

Increased Lifetime Risk of ESRD in Familial IgA Nephropathy.

INTRODUCTION: Familial IgA nephropathy (IgAN) has been widely reported. However, its clinicohistologic characteristics and long-term prognosis are not clear. METHODS: A total of 348 familial IgAN cases from 167 independent families were recruited and their clinicohistologic characteristics as well as lifetime risk of end-stage renal disease (ESRD) were compared to 1116 sporadic IgAN patients from the same geographic region. RESULTS: Of all familial IgAN patients, 60 (17%) came from 32 single-generation (SG; all affected individuals are siblings) families, whereas 286 (82%) came from 134 multiple-generation (MG; affected individuals were present in at least 2 consecutive generations) families. The lifetime ESRD risk was significantly higher in familial patients than sporadic ones after adjusting by gender (hazard ratio [HR]=1.40, 95% confidence interval [CI]: 1.12-1.74, P = 0.004), with 5 years younger in median ESRD age (60 years vs. 65 years in familial and sporadic cases separately). Interestingly, among familial patients, we found cases from SG families (vs. MG families: HR = 2.62, 95% CI: 1.59-4.31, P < 0.001) or with early onset (onset age <30 years) (vs. late onset: HR = 4.79, 95% CI: 3.16-7.26, P < 0.001) had higher lifetime ESRD risk. Furthermore, among sporadic patients, men had lower estimated glomerular filtration rate (eGFR), higher urine protein, higher Oxford T score, and higher risk for life span ESRD compared with women (male vs. female, 25% vs. 17%, P = 0.003) whereas these gender differences were not seen in familial patients. CONCLUSION: Familial IgAN cases had poorer renal outcomes and less gender differences compared with sporadic cases. These findings provide evidence that familial disease represent a distinct subtype of more progressive IgAN. Early diagnosis could improve the prognosis of cases with familial IgAN.

Tubular HIPK2 is a key contributor to renal fibrosis.

We previously used global Hipk2-null mice in various models of kidney disease to demonstrate the central role of homeodomain-interacting protein kinase 2 (HIPK2) in renal fibrosis development. However, renal tubular epithelial cell-specific (RTEC-specific) HIPK2 function in renal fibrogenesis has yet to be determined. Here, we show that modulation of tubular HIPK2 expression and activity affects renal fibrosis development in vivo. The loss of HIPK2 expression in RTECs resulted in a marked diminution of renal fibrosis in unilateral ureteral obstruction (UUO) mouse models and HIV-associated nephropathy (HIVAN) mouse models, which was associated with the reduction of Smad3 activation and downstream expression of profibrotic markers. Conversely, WT HIPK2 overexpression in RTECs accentuated the extent of renal fibrosis in the setting of UUO, HIVAN, and folic acid-induced nephropathy in mice. Notably, kinase-dead HIPK2 mutant overexpression or administration of BT173, an allosteric inhibitor of HIPK2-Smad3 interaction, markedly attenuated the renal fibrosis in these mouse models of kidney disease, indicating that HIPK2 requires both the kinase activity and its interaction with Smad3 to promote TGF-ß-mediated renal fibrosis. Together, these results establish an important RTEC-specific role of HIPK2 in kidney fibrosis and further substantiate the inhibition of HIPK2 as a therapeutic approach against renal fibrosis.

New risk score for predicting steroid resistance in patients with focal segmental glomerulosclerosis or minimal change disease.

BACKGROUND: Glucocorticosteroid is used for patients with primary nephrotic syndrome. This study aims to identify and validate that biomarkers can be used to predict steroid resistance. METHODS: Our study contained two stages, discovery and validation stage. In discovery stage, we enrolled 51 minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS) patients treated with full dose steroid. Five urinary biomarkers including ß2-microglobulin (ß2-MG) and α1-microglobulin (α1-MG) were tested and candidates' biomarkers were selected based on their associations with steroid response. In validation stage, candidates' biomarkers were validated in two prospectively enrolled cohorts. Validation cohort A included 157 FSGS/MCD patients. Validation cohort B included 59 membranous nephropathy (MN) patients. Patients were classified into response group (RG) or non-response group (NRG) based on their responses to steroid treatment. RESULTS: In discovery stage, higher urinary ß2-MG was independently associated with response to corticosteroid treatment in MCD/FSGS patients [OR = 1.89, 95% CI 1.02-3.53] after adjusted by age and gender. In validation cohort A, patients in NRG had a significant higher urinary ß2-MG [Ln (ß2-MG/uCr): 4.6 ± 1.7 vs 3.2 ± 1.5] compared to patients in RG. We then developed a 3-variable risk score in predicting steroid resistance in FSGS/MCD patients based on the best predictive model including Ln(ß2-MG/uCr) [OR = 1.76, 95% CI 1.30-2.37], age [OR = 1.005, 95% CI 0.98-1.03] and pathology [MCD vs FSGS, OR = 0.20, 95% CI 0.09-0.46]. The area under the ROC curves of the risk score in predicting steroid response was 0.80 (95% CI 0.65-0.85). However, no such association was found in MN patients. CONCLUSIONS: Our study identified a 3-variable risk score in predicting steroid resistance in patients with FSGS or MCD.

Clinical and genetic analysis of lipoprotein glomerulopathy patients caused by APOE mutations.

BACKGROUND: Lipoprotein glomerulopathy (LPG) is a rare kidney disease caused by APOE mutations. The aim of this study was to correlate the genetic and clinical features of LPG. METHODS: Totally eight LPG patients were recruited in this study and Sanger sequencing of APOE was performed for all available family members. Clinical and histological features were analyzed. A literature review of LPG was also conducted. RESULTS: Genetic analysis revealed five patients with APOE-Kyoto, two with APOE-Osaka/Kurashiki, and one with APOE-Chicago mutations. LPG patients with urine protein reduced more than 50% had a slower decrease in renal function than those with less urine protein reduction (estimated glomerular filtration rate reduction rate -5.0 ± 0.8 vs. 1.5 ± 0.7 ml/min per 1.73 m2 ⋅month-1 , p = .03). We then enrolled 95 LPG patients from previous studies and this study. LPG patients had higher blood pressure (mean arterial pressure: 109.4 ± 19.4 vs. 94.4 ± 11.1 mmHg, p < .001) than the control group. Interestingly, patients with APOE mutations in the LDL receptor binding region had higher serum apolipoprotein E (apoE) levels [ln(apoE): 2.7 ± 0.4 vs. 2.0 ± 0.5 mg/dl, p < .001] in comparison to other domains. CONCLUSION: Here, we report for the first time APOE-Osaka/Kurashiki and APOE-Chicago mutations in the Chinese population. LPG was associated with higher blood pressure and serum apoE levels were higher in patients with mutations in LDL receptor binding region. In addition, the findings further indicated that treatment of proteinuria might slow down renal function progression in these patients.

Glomerular Transcriptome Profiles in Focal Glomerulosclerosis: New Genes and Pathways for Steroid Resistance.

BACKGROUND: Patients with focal segmental glomerulosclerosis (FSGS) characterized by steroid-resistant nephrotic syndrome (SRNS) are prone to progress to ESRD. Mechanism for the FSGS patients' response to steroid treatment is still unknown and currently, it is impossible to predict the steroid resistance before treatment of patients with FSGS. METHODS: To identify biomarkers and potential therapeutic targets of FSGS patients with SRNS, patients diagnosed as kidney biopsy-proven FSGS and nephrotic syndrome (NS) were prospectively enrolled. They were divided into 2 groups, steroid-sensitive NS and SRNS based on their treatment response. Cortical regions were selected from biopsied renal tissues, and glomeruli were isolated under an inverted microscope. RNA was prepared from the isolated glomeruli and further used for microarray analysis. Followed by multiple analyses, the top 6 highest and lowest, and a selected panel of differentially expressed genes obtained and their related pathways were validated via real-time PCR, western blot, and measurement of reactive oxygen species (ROS). RESULTS: In SRNS group, we discovered that the most significant up-regulated pathway was primarily related to cellular amino acid and derivative metabolic process. Meanwhile, the most significant down-regulated pathway was primarily involved in anatomical structure morphogenesis. Moreover, we found NADPH oxidase 4 (NOX4), one of the key regulators of renal ROS, at a much higher level in SRNS both at transcriptomic and proteomic levels. We also found the levels of ROS, p-p38 MAPK and matrix metalloproteinase (MMP)-2, which were all regulated by NOX4, were also higher in glomeruli isolated from SRNS patients. At last, we detected stimulated by retinoic acid gene 6 homolog (STRA6), a cell surface receptor formerly known as a gene preventing podocytes from over-proliferative lesion induced by HIV infection and was up-regulated by retinoic acid, expressed at a much higher level in SRNS kidneys. CONCLUSION: We found 2 potential mechanisms underline the SRNS, NOX4/ROS/P38 MAPK/MMP-2 pathway and STRA6. Our findings provided new insights into the steroid resistance.

Proteasome inhibitor MG132 inhibits the process of renal interstitial fibrosis.

The proteasome inhibitor pathway serves a crucial role in cell cycle progression and apoptosis, and in the activation of transcription factors and cytokines in tumor cells. The aim of the current study was to investigate the effect of the proteasome inhibitor, MG132, on transforming growth factor (TGF)-ß1-induced expression of extracellular matrix proteins in rat renal interstitial fibroblasts (NRK-49F cells) and to better elucidate the mechanism by which MG132 functions. The level of connective tissue growth factor (CTGF), α-smooth muscle actin (SMA), fibronectin (FN) and collagen type III (Col III) in the MG132-pretreated groups was significantly decreased compared with groups treated with TGF-ß1 alone. MG132 significantly decreased mRNA and the protein levels of fibrosis-associated factors induced by TGF-ß1 treatment. The MG132-pretreated groups exhibited lower phosphorylated-mothers against decapentaplegic homolog (p-Smad)2, p-Smad3 and FN protein expression compared with the groups treated with TGF-ß1 alone. In conclusion, MG132 reduced mRNA and protein expression of fibrosis-associated factors. It can successfully inhibit the inflammatory reaction induced by TGF-ß via the Smad signaling pathway. These results indicate that MG132 appears to have a potent effect in counteracting renal fibrosis. MG132 may be applied in the treatment of patients with chronic kidney disease.

A Rare Genetic Defect of MBL2 Increased the Risk for Progression of IgA Nephropathy.

The aim of this study was to investigate the association between lectin pathway-related genetic variations and progression in IgA nephropathy. Biopsy-proven IgAN patients with eGFR ≥15 ml/min/1.73 m2 at baseline and a minimum follow-up of 12-months were enrolled. A total of 1,007 patients and 121 healthy controls were enrolled from two Chinese renal centers. The discovery cohort consisted of 606 patients, and the validation cohort consisted of 401 patients. First, promoters, all exons and their boundary regions of MBL2 and FCN2 were sequenced in 50 patients, and then 37 variations were identified. Of these variations, 7 expression-associated variations were selected and genotyped in the whole discovery cohort. We found that rs1800450 in MBL2 and rs7851696 in FCN2 were associated with an increased risk for ESRD as well as serum MBL or L-ficolin levels. However, only rs1800450 was successively validated for its association with ESRD (HR, 15.91; 3.27-77.34; P = 0.001) in the fully adjusted model in the validation cohort. In addition, 2.7% of patients, and 2.5% of healthy controls carried rs1800450-AA. IgAN patients with rs1800450-AA lacked expression of MBL in both serum and renal tissue and had more severe tubulointerstitial damage. Furthermore, a combined effect of rs1800450-AA with a previously reported clinical risk score was observed in which patients with both a high clinical risk score (≥1%) and rs1800450-AA had a strikingly increased 10-years ESRD risk by 37.1-fold (7.17 to 192.13-fold). In summary, IgAN patients carrying MBL2 rs1800450-AA have a high risk for renal function deterioration, probably due to inactivation of the complement MBL pathway.

Interleukin-10 deficiency aggravates kidney inflammation and fibrosis in the unilateral ureteral obstruction mouse model.

Interleukin-10 functions as a general immunosuppressive cytokine, which also negatively regulates inflammatory responses through complex mechanisms. Recent studies suggested that IL-10 may also inhibit fibrosis in various diseased models. However, the role of IL-10 in renal fibrosis has not been demonstrated. Here, we investigated the effects of IL-10 in the development of renal tubulointerstitial fibrosis by creating the unilateral ureteral obstruction (UUO) model in IL-10 knockout (-/-) mice. We performed sham or unilateral ureteral obstruction surgery in 8-week-old IL-10-/- male mice and age and sex-matched wild type littermates. Mice were killed at 7 days or 14 days post surgery and renal tissues were obtained for RNA, protein, and immunohistochemical analysis. Our results found IL-10 deficiency resulted in enhanced renal fibrosis demonstrated by more severe tubular injury and collagen deposition and higher expression of pro-fibrotic genes (including α-SMA, MMP-2, fibronectin, FSP-1 and vimentin). Our results also found IL-10-/- UUO mice developed more severe renal inflammation with a significant increase in inflammatory cells infiltration, and upregulation of inflammatory chemokines (MCP-1 and RANTES), and cytokines (TNF-α, IL-6, IL-8, and M-CSF). Further study revealed that enhanced renal inflammation and fibrosis was associated with significantly increased activation of both TGF-ß/Smad3 and NF-κB signaling pathways. In summary, our study provides the direct evidence that IL-10 is an endogenous cytokine that has a key role in protecting against development of renal inflammation and fibrosis. Enhancement of IL-10 expression could be a potential anti-fibrosis therapy for patients with chronic kidney diseases.

Proteasome inhibitor inhibits proliferation and induces apoptosis in renal interstitial fibroblasts.

BACKGROUND: The ubiquitin proteasome pathway plays a pivotal role in controlling cell proliferation, apoptosis and differentiation in a variety of normal and tumor cells. This study aimed to investigate the role of a proteasome inhibitor on proliferation, apoptosis and related proteins in renal interstitial fibroblasts (NRK-49F). METHODS: NRK-49F cells were induced using transforming growth factor-ß1 (TGF-ß1) and pretreated with the proteasome inhibitor MG-132. Cell proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The cell cycle and apoptosis were analyzed using flow cytometry. Apoptosis was also analyzed using a DNA ladder. The protein expression of p53, p27, p21, caspase-3, Bcl-2 and Bax was examined using western blots. RESULTS: The results showed that TGF-ß1 (5 ng/ml) can stimulate the proliferation of NRK-49F cells.MG-132 (0.25-5 µM) inhibited TGF-ß1-induced proliferation in a dose-dependent manner through G1-arrest; TGF-ß1 alone did not induce apoptosis (3.8 ± 0.4% vs. 4.7 ± 1.6%). However, pretreatment with MG-132 significantly induced apoptosis in TGF-b1-stimulated NRK-49F cells in a dose-dependent manner. A typical DNA ladder was also confirmed in these two groups. Western blot analysis showed that MG-132 activated p53, p21, caspase-3 and Bax, and inhibited Bcl-2 in a dose-dependent manner, while p27 expression remained unchanged. CONCLUSIONS: A proteasome inhibitor inhibited proliferation and induced apoptosis in renal interstitial fibroblasts stimulated by TGF-ß1. The mechanism may relate to the p53, p21, caspase-3, Bcl-2 and Bax pathways. Our results suggest that a proteasome inhibitor could be a new strategy to treat renal interstitial fibrosis.

Analysis of the gene expression profile of curcumin-treated kidney on endotoxin-induced renal inflammation.

Acute or chronic kidney inflammation is closely related to the progress of kidney diseases. Curcumin, a yellow pigment present in the rhizome of turmeric (Curcuma longa L. Zingiberaceae), was found to be a potential anti-inflammatory agent. The present study aimed to investigate the effects and explore the protective mechanism of curcumin on lipopolysaccharide (LPS)-induced kidney inflammation in mice using gene chip and pathological technology. Nine SPF Kunming mice (aged 6-8 weeks, weighing 20-25 g) were divided into three groups. Saline and LPS were injected intraperitoneally in a normal control group and a model group, respectively. Mice in the treatment group were first injected with curcumin (5 mg/kg) for 3 days before being injected with LPS (5 mg/kg). Kidney tissues were harvested at 6 h after treatment. Parts of kidney were fixed with 10 % formaldehyde for HE, Periodic acid-Schiff staining, and immunohistochemistry. Affymetrix gene chips (mouse 430 chip) were used to detect the renal gene expression profile, and the results were analyzed using bioinformatics methods. The renal gene expression profile showed that there are 148 Affy IDs (up-down group) whose levels of gene expression were increased after LPS stimulation and decreased by curcumin treatment and that there are 133 Affy IDs (down-up group) exhibiting the opposite trend. In the differentially expressed genes of the up-down group, 21 Gene Ontology (GO) genes were selected by screening function (P ≤ 0.01). In the biological processes, most of the genes were found to be related to the genes of regulation of macrophage activation and macrophage activation-associated genes. In the cellular localization, there were four functional GO genes (P ≤ 0.01); in the molecular structure, there were seven functional GO genes (P ≤ 0.01). In the down-up group, there were functional GO genes (P ≤ 0.01) and one functional GO gene (P ≤ 0.01) in the biological process and the cellular localization, respectively. Macrophage infiltration could be observed as early as 6 h after LPS stimulation. Pretreatment with 5 mg/kg of curcumin significantly decreased the macrophage infiltration. At 6 h after LPS injection, significant decreased expression of M6PRBP-1 and NEDD-4 was observed in renal tissue. On the other hand, pretreatment with curcumin significantly increased renal M6PRBP-1 and NEDD-4 expression. In this study, we also found the signaling pathway and the possible target gene of the protective effects of curcumin on endotoxin-induced renal inflammation. The kidney gene expression profile in the inflammatory state was clarified by using gene chip technology. Furthermore, we confirmed that curcumin treatment can change the gene expression profile.

A systems approach identifies HIPK2 as a key regulator of kidney fibrosis.

Kidney fibrosis is a common process that leads to the progression of various types of kidney disease. We used an integrated computational and experimental systems biology approach to identify protein kinases that regulate gene expression changes in the kidneys of human immunodeficiency virus (HIV) transgenic mice (Tg26 mice), which have both tubulointerstitial fibrosis and glomerulosclerosis. We identified homeo-domain interacting protein kinase 2 (HIPK2) as a key regulator of kidney fibrosis. HIPK2 was upregulated in the kidneys of Tg26 mice and in those of patients with various kidney diseases. HIV infection increased the protein concentrations of HIPK2 by promoting oxidative stress, which inhibited the seven in absentia homolog 1 (SIAH1)-mediated proteasomal degradation of HIPK2. HIPK2 induced apoptosis and the expression of epithelial-to-mesenchymal transition markers in kidney epithelial cells by activating the p53, transforming growth factor ß (TGF-ß)-SMAD family member 3 (Smad3) and Wnt-Notch pathways. Knockout of HIPK2 improved renal function and attenuated proteinuria and kidney fibrosis in Tg26 mice, as well as in other murine models of kidney fibrosis. We therefore conclude that HIPK2 is a potential target for anti-fibrosis therapy.

Curcumin attenuates lipopolysaccharide-induced renal inflammation.

Renal inflammation is the main pathological change in many acute and chronic kidney diseases. Curcumin, a yellow pigment present in the rhizome of turmeric (Curcuma longa L. Zingiberaceae), was found to be a potential anti-inflammatory agent. The present study aimed to investigate the effects of curcumin on the inflammation of mice kidney and cultured renal tubular epithelial cells (HK-2 cells) induced by lipopolysaccharide (LPS) and to explore the mechanism. Curcumin was injected intraperitoneally before LPS administration. Renal inflammation was assessed by evaluating monocyte chemoattractant protein-1 (MCP-1) expression and macrophage infiltration in renal tissue using immunohistochemical methods, and also by measuring renal MCP-1 mRNA level using Real-Time polymerase chain reaction (PCR). HK-2 cells were cultured to investigate the in vitro effect of curcumin against LPS-induced renal inflammation. The expression of MCP-1 and interleukin-8 (IL-8) mRNA was measured by Real-Time PCR. The expression of MCP-1 and IL-8 protein in supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The activity of nuclear factor (NF)-κB was detected by electrophoretic mobility shift assay (EMSA). The results demonstrated that curcumin could inhibit LPS-induced renal MCP-1 mRNA expression. Curcumin also significantly inhibited the expression of MCP-1 and IL-2 mRNA in HK-2 cells, and partially inhibited the secretion of MCP-1 and IL-8. Furthermore, curcumin was found to inhibit the DNA-binding activity of NF-κB. The present study demonstrated that curcumin has a protective effect on LPS-induced experimental renal inflammation, and this effect might be attributed to its inhibitory effects on MCP-1 mRNA expression and DNA-binding activity of NF-κB. Hence, curcumin might be potentially useful in some kidney diseases by preventing renal inflammation.