Catheter-based renal denervation in Chinese patients with chronic kidney disease and uncontrolled hypertension.

Sympathetic activation contributes to the progression of hypertension and chronic kidney disease (CKD). Ablation of renal sympathetic nerves lowers blood pressure (BP) and preserves renal function in patients with CKD and uncontrolled hypertension by reducing sympathetic nerve activity. But whether this approach is safe and effective in Chinese patients with CKD is unknown. We performed an observational study of eight patients with CKD stages from 1 to 5, office BP ≥150/90 mmHg, while on at least three antihypertensive drug classes including a diuretic, and diagnosis confirmed by 24 h ambulatory systolic BP measurement ≥135 mmHg. All patients underwent catheter-based renal denervation (RDN) using a newly designed RDN System (Golden Leaf Medtech, Shanghai, China). For up to 6 months after RDN, BP was monitored and renal function was assessed. Mean baseline office BP was 165.0 ± 13.9/97.8 ± 5.5 mmHg, despite treatment with three antihypertensive drugs. Six months after RDN, office BP was reduced by 22.1 ± 12.0 (P = .002)/11.0 ± 8.8 mmHg (P = .012) and average 24 h ambulatory BP by 18 ± 13.7 (P = .01)/9.3 ± 7.7 mmHg (P = .016). After RDN, heart rate and estimated glomerular filtration rate (GFR) had no significant change compared with before RDN. In Chinese patients with CKD, our observational pilot study found that treating hypertension with RDN lowers BP while not affecting renal function. Brief Abstract: We performed RDN in eight Chinese patients with hypertension and CKD. The results showed that RDN lowered blood pressure of these patients significantly and eGFR was stable. No obvious adverse event was observed.

Proximal Tubular Vacuolization and Hypersensitivity to Drug-Induced Nephrotoxicity in Male Mice With Decreased Expression of the NADPH-Cytochrome P450 Reductase.

The effect of variations in the expression of cytochrome P450 reductase (CPR or POR) is determined in mice with decreased POR expression to identify potential vulnerabilities in people with low POR expression. There is an age-dependent appearance of increasing vacuolization in the proximal tubules of the renal cortex in 4- to 9-month-old male (but not female) Cpr-low (CL) mice. These mice have low POR expression in all cells of the body and upregulation of lysosome-associated membrane protein 1 expression in the renal cortex. Vacuolization is also seen in extrahepatic CL and extrarenal CL male mice, but not in mice with tissue-specific Por deletion in liver, intestinal epithelium, or kidney. The occurrence of vacuolization is accompanied by increases in serum blood-urea-nitrogen levels. Male CL mice are hypersensitive to cisplatin- and gentamicin-induced renal toxicity at 3 months of age, before proximal tubular (PT) vacuoles are detectable. At doses that do not cause renal toxicity in wild-type mice, both drugs cause substantial increases in serum blood-urea-nitrogen levels and PT vacuolization in male but not female CL mice. The hypersensitivity to drug-induced renal toxicity is accompanied by increases in circulating drug levels. These novel findings demonstrate deficiency of renal function in mice with globally reduced POR expression and suggest that low POR expression may be a risk factor for drug-induced nephrotoxicity in humans.

Suppression of cytochrome P450 reductase expression promotes astrocytosis in subventricular zone of adult mice.

The aim of this study was to determine the role of NADPH-cytochrome P450 reductase (CPR) and CPR-dependent enzymes in neural stem cell (NSC) genesis in the brain. A mouse model with globally suppressed Cpr gene expression (Cpr-low mouse) was studied for this purpose. Cpr-low and wild-type (WT) mice were compared immunohistochemically for the expression of markers of cell proliferation (Ki67), immature neurons (doublecortin, DCX), oligodendrocytes (oligodendrocyte transcription factor 2, OLIG2), and astrocytes (glial fibrillary acidic protein, GFAP) in the SVZ, and for the in vitro capability of their SVZ cells to form neurospheres and differentiate into astrocytes. We found that the abundance of SVZ cells that are positive for Ki67 or GFAP expression, but not the abundance of SVZ cells that are positive for DCX and OLIG2 expression, was significantly increased in Cpr-low mice, at various ages, compared with WT mice. Furthermore, extents of astrocyte differentiation and growth, but not neurosphere formation, from SVZ cells of the Cpr-low mice were significantly increased, compared with WT mice. These results suggest that CPR and CPR-dependent enzymes play a role in suppressing astrocytosis in the SVZ of adult mice.

The role of renal proximal tubule P450 enzymes in chloroform-induced nephrotoxicity: utility of renal specific P450 reductase knockout mouse models.

The kidney is a primary target for numerous toxic compounds. Cytochrome P450 enzymes (P450) are responsible for the metabolic activation of various chemical compounds, and in the kidney are predominantly expressed in proximal tubules. The aim of this study was to test the hypothesis that renal proximal tubular P450s are critical for nephrotoxicity caused by chemicals such as chloroform. We developed two new mouse models, one having proximal tubule-specific deletion of the cytochrome P450 reductase (Cpr) gene (the enzyme required for all microsomal P450 activities), designated proximal tubule-Cpr-null (PTCN), and the other having proximal tubule-specific rescue of CPR activity with the global suppression of CPR activity in all extra-proximal tubular tissues, designated extra-proximal tubule-Cpr-low (XPT-CL). The PTCN, XPT-CL, Cpr-low (CL), and wild-type (WT) mice were treated with a single oral dose of chloroform at 200mg/kg. Blood, liver and kidney samples were obtained at 24h after the treatment. Renal toxicity was assessed by measuring BUN and creatinine levels, and by pathological examination. The blood and tissue levels of chloroform were determined. The severity of toxicity was less in PTCN and CL mice, compared with that of WT and XPT-CL mice. There were no significant differences in chloroform levels in the blood, liver, or kidney, between PTCN and WT mice, or between XPT-CL and CL mice. These findings indicate that local P450-dependent activities play an important role in the nephrotoxicity induced by chloroform. Our results also demonstrate the usefulness of these novel mouse models for studies of chemical-induced kidney toxicity.

Role of mammary epithelial and stromal P450 enzymes in the clearance and metabolic activation of 7,12-dimethylbenz(a)anthracene in mice.

Microsomal cytochrome P450 (P450) enzymes, which are important in the metabolism of carcinogens, are expressed in both epithelial and stromal cells in the mammary gland. The aim of this study was to investigate the roles of mammary epithelial P450 enzymes in the bioactivation and disposition of 7,12-dimethylbenz(a)anthracene (DMBA), a breast carcinogen, in the mammary gland. A new mouse model (named MEpi-Cpr-null) was produced, wherein P450 activities in the mammary epithelial cells are suppressed through tissue-specific deletion of the gene for P450 reductase (Cpr), an enzyme required for the activities of all microsomal P450 enzymes. Comparisons between wild-type and MEpi-Cpr-null mice showed that the tissue-specific deletion of Cpr in the mammary epithelial cells was accompanied by significant increases in the levels of DMBA and DMBA-DNA adduct in the mammary gland following a single intraperitoneal injection of DMBA at 50mg/kg. Immunohistochemical and immunoblot analysis further revealed greater induction of CYP1B1 expression by the DMBA treatment in the mammary stroma of the MEpi-Cpr-null mice than in that of the WT mice. These findings not only demonstrate that the epithelial P450 enzymes play important roles in the clearance of DMBA, but also suggest that P450 enzymes in both mammary epithelial and stromal cells contribute to carcinogen-mediated DNA damage.

Metabonomic investigation on the protective effects of rosiglitazone and caloric restriction for renal senescence in a rat model.

BACKGROUND AND AIMS: A liquid chromatography coupled with mass spectrometry based metabonomics approach was applied to investigate the protective effects of rosiglitazone (RGTZ) and caloric restriction (CR) for renal senescence in a rat model. METHODS: Kidney tissues and serum samples were collected from four groups of rats, including 12- month and 24-month ad libitum fed rats, RGTZ and CR treated 24-month rats. Multivariate data analysis was performed on the mass data of metabonomic profiles to detect the differences among the groups. RESULTS: By metabolite profiling and partial least squares discriminate analysis, 23 renal senescence-related endogenous metabolites were discovered, including phospholipids, carnitine, acetylcarnitine, and creatinine, most of which were related to the oxidative stress and lipid metabolism. CONCLUSION: Renal senescence is characterized by oxidative stress and changes in lipid metabolism, and RGTZ administration and CR treatment may have similar protective effects for renal senescence via restraining oxidative stress and lipid metabolism.

High ambient glucose levels modulates the production of MMP-9 and alpha5(IV) collagen by cultured podocytes.

Recent evidences have demonstrated an important role for glomerular visceral epithelial cell (podocyte) in the development and progression of diabetic nephropathy. We investigated the high-glucose (HG)-triggered signaling pathway and its role in matrix metalloproteinase (MMP) production in murine podocytes. The activity of 92-kDa (MMP-9) gelatinase, but not of 72 kDa (MMP-2), in an HG medium significantly increased during incubation of 2 to 3 days and decreased during incubation of more than 5 days revealed by Gelatin zymography. Opposite to the increases in MMP-9 activity, HG medium produced significant decreases in the protein levels of alpha5(IV) collagen. Changes in MMP-9 activity were associated with the same pattern as MMP-9 mRNA levels in podocytes exposed to HG media. HG medium rapidly activated ERK1/2 MAPK in podocytes. Moreover, ERK1/2 activation was required for HG-induced enhancement of MMP-9 activity and a decrease in the level of alpha5(IV) collagen. HG incubation rapidly induced an increase in the nuclear accumulation of Ets-1 protein. Blocking the ERK pathway suppressed HG-induced expression and nuclear accumulation of transcriptional factor Ets-1, and MMP-9 mRNA expression. We suggest that short- or long-term exposure to HG concentrations increases or decreases MMP-9 production and alpha5(IV) collagen expression in podocytes, this may contribute to the GBM abnormality caused by an imbalance in extracellular matrix (ECM) synthesis and degradation, and may play a critical role in the pathogenesis of proteinuria in diabetic nephropathy.

Increase in extracellular cross-linking by tissue transglutaminase and reduction in expression of MMP-9 contribute differentially to focal segmental glomerulosclerosis in rats.

Tissue transglutaminase (tTG) is a Ca(2+)-dependent enzyme which stabilizes the extracellular matrix (ECM) through post-translational modification, and may play an important role in the pathogenesis of focal and segmental glomerulosclerosis (FSGS). Here, we have investigated whether tTG contributes to the glomerular ECM expansion in the puromycin aminonucleoside (PAN)-injection-induced experimental rat model of FSGS. The localization and expression of tTG, MMP-9 gelatinase, and the ECM component fibronectin (FN) in kidneys was determined by immunohistochemistry and measured by semi-quantitative analysis. Protein levels of tTG and MMP-9 were also analyzed by Western blotting.In situtransglutaminase activity was assayed by measurement of incorporated substrate and the immunofluorescence staining for the cross-linking product, epsilon-(gamma-glutamyl) lysine. Prominent proteinuria, a typical pathological feature of FSGS, was observed in PAN injection group rats. tTG immunoreactivity was located markedly in glomeruli and the levels of this protein in whole-kidney homogenates of PAN injection group rats were significantly increased (361+/- 106% control, P< 0.05). Similarly, transglutaminase activity and epsilon-(gamma-glutamyl) lysine were also predominately located within glomeruli and were much more intense in the PAN-injected group than that in control animals. MMP-9 was also located primarily within glomeruli. In PAN-injected kidneys, protein levels of active MMP-9 were significantly reduced (59+/- 27% control, P< 0.01), while pro-MMP-9 levels increased (148+/- 42% control, P< 0.05). Remarkable expression of glomerular fibronectin (FN) was found in PAN injection group rats. Semi-quantitative analysis demonstrated this increased intensity of FN staining in the PAN-injected rats was 149+/- 23% of the control values (P< 0.05). Enhanced cross-linking of ECM by tissue transglutaminase and decreased degradation due to reduced active MMP-9 expression may be at least partially responsible for the deposition of FN within injured glomeruli in experimental FSGS.

ERK-dependent signaling pathway and transcriptional factor Ets-1 regulate matrix metalloproteinase-9 production in transforming growth factor-beta1 stimulated glomerular podocytes.

The unregulated synthesis of glomerular basement membrane (GBM) components, extracelluar matrix (ECM) proteins, or the secretion of ECM-degradation enzymes, matrix metalloproteinases (MMPs), by podocytes under pathological conditions might be major factors in GBM damage. The present study examined the effects and the underlying molecular mechanism of transforming growth factor beta1 (TGFbeta1) on the production of gelatinase in cultured murine podocytes. Our results showed that TGFbeta1 is the most potent inducer of MMP-9 secretion in both a dose- and time-dependent manner, but has very little effect on MMP-2 secretion. TGFbeta1 upregulated MMP-9 mRNA levels, but did not affect the expression of matrix mettaloproteinases TIMP-1 mRNA. TGFbeta1 induced activation of both Smad2 and extracellular signal-regulated kinases (ERK1/2). However, blockade of Smad2 signaling pathway by Staurosporine did not affect the TGFbeta1-stimulated secretion of MMP-9, whereas inhibition of activation of ERK1/2 by PD98059 abolished TGFbeta1-stimulated secretion of MMP-9 and expression of MMP-9 mRNA. Protein levels of the transcriptional factor Ets-1 increased and were sustained for 12 h by TGFbeta1-stimulation. Our data also showed that blockage of ERK1/2 activation by PD98059 led to a reduction in the level of Ets-1 protein and to a consequent decrease in MMP-9 mRNA levels. These results demonstrate that TGFbeta1 can induce the production of MMP-9 in podocytes through the ERK1/2 MAPK pathway, and suggested that an increase in MMP-9 enzymatic activities may be involved in the damage of the GBM in response to inflammatory factors, ultimately leading to glomerulosclerosis.

[Transforming growth factor-beta1 stimulates matrix metalloproteinase-9 production through ERK activation pathway and upregulation of Ets-1 protein].

OBJECTIVE: To investigate the molecular mechanism of transforming growth factor-beta1 (TGF-beta1) in regulating the production of matrix metalloproteinase-9 (MMP-9) protein. METHODS: Mouse immortal podocyte cells were cultured. Different concentrations (1, 2, and 5 ng/l) of TGF-beta1 were added into the culture medium. Cell culture without TGF-beta1 stimulation was used as control group. The activity of MMP-9 in the supernatant of the culture medion was assayed by gelatin zymography, expression of MMP-9 mRNA was assessed by RT-PCR; the activation of ERK pathway and the level of a transcriptional factor Ets-1 protein was analyzed by Western blotting. PD98059, a specific inhibitor of ERK1/2 activation, was added into the culture fluid of the podocytes for 30 minutes, than 2 ng/ml TGF-beta1 was added. The above mentioned tested were conducted to observe the influence of the inhibitor of ERK1/2 activation. RESULTS: The MMP-9 activity was very week in the supernatant of culture fluid of the control group and was increased in the TGF-beta1 groups dose-dependently. After the podocytes were co-incubated with 1 ng/ml TGF-beta1 for 24 hours, the MMP-9 activity was 26.86 times that of the control group (P < 0.01). Since the 12th hour after co-incubation with 2 ng/ml TGF-beta1 the MMP-9 activity in the supernatant of culture fluid began to be significantly increased and remained at high level till the 48 th hour. RT-PCR showed that low-level MMP-9 mRNA expression in the control group. After stimulation of 2 ng/ml TGF-beta1 for 6 hours the MMP-9 mRNA expression was 2.71 times that of the control group (P < 0.01) and the high-level expression lasted 24 hours. Western blotting showed low-level Ets-1 protein in the control group. At the time point of 12 th hour after stimulation of TGF-beta1 the Ets-1 protein expression was increased in all the three TGF-beta1 groups. After stimulation with 2 ng/ml TGF-beta1 for 4 hours the Ets-1 protein expression was 2.71 times that of the control group (P <0.01). After pretreatment of the podocyte with PD98059 for 30 minutes, the added 2 ng/ml TGF-beta1 failed to increase the MMP-9 activity and up-regulate the MMP-9 mRNA expression. CONCLUSION: TGF-beta1 stimulates the production of MMP-9 by activation of cytoplasmic ERK signaling pathway and upregulation of Ets-1 expression.

[Tissue transglutaminase and renal fibrosis].

Tissue transglutaminase (tTG), an enzyme which can catalyze the posttranslational modification of proteins, is widely distributed and plays an important role in both physiological and pathological processes. More recently, tissue transglutaminase has been believed to participate in tissue fibrosis. Cross-linked extracellular matrix proteins by tissue transglutaminase are stable and highly resistant to proteolytic degradation, thus lead to accumulation of the ECM proteins in tissue fibrosis. This review summarizes the important features of this multifunctional enzyme and particularly introduces the correlation between tTG and renal fibrosis.