David Sheikh-Hamad

Uric Acid Stimulates Monocyte Chemoattractant Protein-1 Production in Vascular Smooth Muscle Cells Via Mitogen-Activated Protein Kinase and Cyclooxygenase-2

Abstract— Previous studies have reported that uric acid stimulates vascular smooth muscle cell (VSMC) proliferation in vitro. We hypothesized that uric acid may also have direct proinflammatory effects on VSMCs. Crystal‐ and endotoxin‐free uric acid was found to increase VSMC monocyte chemoattractant protein‐1 (MCP‐1) expression in a time‐ and dose‐dependent manner, peaking at 24 hours. Increased mRNA and protein expression occurred as early as 3 hours after uric acid incubation and was partially dependent on posttranscriptional modification of MCP‐1 mRNA. In addition, uric acid activated the transcription factors nuclear factor‐[kappa]B and activator protein‐1, as well as the MAPK signaling molecules ERK p44/42 and p38, and increased cyclooxygenase‐2 (COX‐2) mRNA expression. Inhibition of p38 (with SB 203580), ERK 44/42 (with UO126 or PD 98059), or COX‐2 (with NS398) each significantly suppressed uric acid–induced MCP‐1 expression at 24 hours, implicating these pathways in the response to uric acid. The ability of both n‐acetyl‐cysteine and diphenyleneionium (antioxidants) to inhibit uric acid–induced MCP‐1 production suggested involvement of intracellular redox pathways. Uric acid regulates critical proinflammatory pathways in VSMCs, suggesting it may have a role in the vascular changes associated with hypertension and vascular disease.

p38 Kinase Activity Is Essential for Osmotic Induction of mRNAs for HSP70 and Transporter for Organic Solute Betaine in Madin-Darby Canine Kidney Cells

In renal cells, hypertonicity induces genes for heat shock proteins (HSP70, αB-crystallin), as well as enzymes and transporters directly involved in the metabolism and transport of protective organic osmolytes. While heat shock proteins are induced by many stresses including osmotic stress, the induction of the osmolytes genes appears to be specific to osmotic stress. These two adaptive mechanisms allow kidney cells to survive and function in the hypertonic environment that exists on routine basis in kidney medulla. In mammalian cells, hypertonicity induces three mitogen-activated protein kinase pathways: ERK (extracellular regulated kinase), JNK (Jun N-terminal kinase), and p38. ERK activation by osmotic stress is a consistent finding in many cells, but it is not essential for transcriptional regulation of mRNA for transporter of organic osmolyte betaine. While the growth of yeast cells on NaCl-supplemented medium is dependent on HOG1 pathway, it is still unclear which pathway mediates the adaptation to osmotic stress in mammalian cells. Here, we show that inhibition of p38 kinase activity, using the specific inhibitor SB203580 (4-(fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl) imidazole), abolishes the hypertonicity-mediated induction of mRNAs for HSP70 and betaine transporter in Madin-Darby canine kidney cells. The inhibition is dose-dependent and correlates with thein situ activity of native p38 kinase, determined as MAPKAPK-2 activity in cell extracts. As reported previously, the activities of ERK-1 and -2 were not affected by SB203580, but surprisingly, inhibition of native p38 kinase activity correlates with up-regulation of native JNK-1 activity in osmotically stressed cells. p38 mRNA is induced by hypertonic stress and is attenuated with p38 kinase inhibition. We also find that thermal induction of HSP70 mRNA is not affected by p38 kinase inhibition. Such findings suggest that p38 kinase activity is essential for the induction of genes involved in the adaptation of mammalian cells to osmotic stress and that the increased activity of JNK-1 during p38 kinase inhibition is consistent with regulation of JNK-1 by p38 kinase in osmotically stressed cells. In addition, the transduction pathways mediating HSP70 mRNA induction by different stresses appear to be divergent; osmotic induction of HSP70 is p38 kinase-dependent, while thermal induction is not.

Ultrasound-Microbubble-Mediated Gene Transfer of Inducible Smad7 Blocks Transforming Growth Factor-β Signaling and Fibrosis in Rat Remnant Kidney

Transforming growth factor (TGF)-beta1 has been shown to play a critical role in hypertensive nephropathy. We hypothesized that blocking TGF-beta1 signaling could attenuate renal fibrosis in a rat model of remnant kidney disease. Groups of six rats were subjected to 5/6 nephrectomy and received renal arterial injection of a doxycycline-regulated Smad7 gene or control empty vector using an ultrasound-microbubble-mediated system. Smad7 transgene expression within the kidney was tightly controlled by the addition of doxycycline in the daily drinking water. All animals were euthanized at week 4 for renal functional and histological examination. Hypertension of equivalent magnitude (190 to 200 mmHg) developed in both Smad7- and empty vector-treated rats. However, treatment with Smad7 substantially inhibited Smad2/3 activation and prevented progressive renal injury by inhibiting the rise of 24-hour proteinuria (P < 0.001) and serum creatinine (P < 0.001), preserving creatinine clearance (P < 0.05), and attenuating renal fibrosis and vascular sclerosis such as collagen I and III expression (P < 0.01) and myofibroblast accumulation (P < 0.001). In conclusion, TGF-beta/Smad signaling plays a critical role in renal fibrosis in a rat remnant kidney model. The ability of Smad7 to block Smad2/3 activation and attenuate renal and vascular sclerosis demonstrates that ultrasound-mediated Smad7 gene therapy may be a useful therapeutic strategy for the prevention of renal fibrosis in association with hypertension.

Osmotic Response Element Enhancer Activity REGULATION THROUGH p38 KINASE AND MITOGEN-ACTIVATED EXTRACELLULAR SIGNAL-REGULATED KINASE KINASE

Hypertonicity induces a group of genes that are responsible for the intracellular accumulation of protective organic osmolytes such as sorbitol and betaine. Two representative genes are the aldose reductase enzyme (AR, EC 1.1.1.21), which is responsible for the conversion of glucose to sorbitol, and the betaine transporter (BGT1), which mediates Na+-coupled betaine uptake in response to osmotic stress. We recently reported that the induction of BGT1 mRNA in the renal epithelial Madin-Darby canine kidney cell line is inhibited by SB203580, a specific p38 kinase inhibitor. In these studies we report that the hypertonic induction of aldose reductase mRNA in HepG2 cells as well as the osmotic response element (ORE)-driven reporter gene expression in transfected HepG2 cells are both inhibited by SB203580, suggesting that p38 kinase mediates the activation and/or binding of the transcription factor(s) to the ORE. Electrophoretic gel mobility shift assays with cell extracts prepared from SB203580-treated, hypertonically stressed HepG2 cells further show that the binding of trans-acting factors to the ORE is prevented and is thus also dependent on the activity of p38 kinase. Similarly, treatment of hypertonically stressed cells with PD098059, a mitogen-activated extracellular regulated kinase kinase (MEK1) inhibitor, results in inhibition of the hypertonic induction of aldose reductase mRNA, ORE-driven reporter gene expression, and the binding of trans-acting factors to the ORE. ORE-driven reporter gene expression was not affected by p38 kinase inhibition or MEK1 inhibition in cells incubated in iso-osmotic media. These data indicate that p38 kinase and MEK1 are involved in the regulation of the hyperosmotic stress response.

Mammalian stanniocalcin-1 activates mitochondrial antioxidant pathways: new paradigms for regulation of macrophages and endothelium

The mammalian homolog of the fish calcium regulatory hormone stanniocalcin-1 (STC1) is ubiquitously expressed and likely functions in an autocrine/paracrine fashion. Mammalian STC1 does not appear to exert significant effects on serum calcium, and its physiological role remains to be determined. In macrophages, STC1 decreases intracellular calcium and cell mobility; attenuates the response to chemoattractants; and diminishes superoxide generation through induction of uncoupling protein-2 (UCP2). In cytokine-treated endothelial cells, STC1 attenuates superoxide generation and the activation of inflammatory pathways [c-Jun NH(2)-terminal kinase (JNK) and NF-kappaB]; maintains the expression of tight junction proteins, preserving the endothelial monolayer seal; and decreases transendothelial migration of leukocytes. Combined, the effects of STC1 on endothelial cells and macrophages predict potent anti-inflammatory action. Indeed, application of the anti-glomerular basement membrane (GBM) glomerulonephritis model to STC1 transgenic mice that display increased expression of STC1 transgene in endothelial cells and macrophages yields renal protection. Our data suggest that STC1 activates antioxidant pathways in endothelial cells and macrophages and displays cytoprotective and anti-inflammatory actions.

Stanniocalcin-1 suppresses superoxide generation in macrophages through induction of mitochondrial UCP2.

Mammalian STC1 decreases the mobility of macrophages and diminishes their response to chemokines. In the current experiments, we sought to determine the impact of STC1 on energy metabolism and superoxide generation in mouse macrophages. STC1 decreases ATP level in macrophages but does not affect the activity of respiratory chain complexes I–IV. STC1 induces the expression of mitochondrial UCP2, diminishing mitochondrial membrane potential and superoxide generation; studies in UCP2 null and gp91phox null macrophages suggest that suppression of superoxide by STC1 is UCP2‐dependent yet is gp91phox‐independent. Furthermore, STC1 blunts the effects of LPS on superoxide generation in macrophages. Exogenous STC1 is internalized by macrophages within 10 min and localizes to the mitochondria, suggesting a role for circulating and/or tissue‐derived STC1 in regulating macrophage function. STC1 induces arrest of the cell cycle at the G1 phase and reduces cell necrosis and apoptosis in serum‐starved macrophages. Our data identify STC1 as a key regulator of superoxide generation in macrophages and suggest that STC1 may profoundly affect the immune/inflammatory response.

Angiotensin II directly increases rabbit renal brush-border membrane sodium transport: presence of local signal transduction system.

SummaryIn the present study, we have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10−11–10−7m) was found to stimulate22Na− uptake by the isolated BBM vesicles directly. AII did not affect the Na+-dependent BBM glucose uptake, and the effect of AII on BBM22Na+ uptake was inhibited by amiloride, suggesting the involvement of Na+/H+ exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na+/H+ antiport system.In search of the signal transduction mechanism, it was found that AII activated BBM phospholipase A2 (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-ßS or PTX abolished, the effects of AII on BBM PLA and22Na+ uptake, suggesting the involvement of G-protein in AIIs actions. On the other hand, inhibition of PLA by mepacrine prevented AIIs effect on BBM22Na+ uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBM22Na+ uptake, suggesting the role of PLA activation in mediating AIIs effect on BBM22Na+ uptake.In summary, results of the present study show a direct stimulatory effect of AII on BBM Na+/H+ antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation.

Acute effects of vasopressin V2-receptor antagonist on kidney AQP2 expression and subcellular distribution

The acute effect of treatment with the vasopressin V2-receptor antagonist OPC-31260 (OPC) on aquaporin-2 (AQP2) distribution and expression in rat kidney was examined. Immunofluorescence and semi-quantitative immunoelectron microscopy revealed that 15 and 30 min of OPC treatment resulted in significant reduction in apical plasma membrane labeling of AQP2, with a concomitant increase in labeling of vesicles and multivesicular bodies. In parallel, OPC treatment induced a large increase in urine output [0.6 ± 0.2 vs. 8.3 ± 1.0 ml/h ( n = 4)]. Northern blotting using a 32P-labeled AQP2 cDNA probe and a digoxigenin-labeled AQP2 RNA probe revealed a band of ∼1.6 kb corresponding to the predicted size of AQP2 mRNA. In control experiments, thirsting increased, whereas water loading decreased AQP2 mRNA levels. Treatment of rats with OPC caused a significant reduction in AQP2 mRNA within 30 min (52 ± 21%, n = 8, P < 0.025) and 60 min (56 ± 7%, n = 4, P < 0.001) of treatment compared with intravenous saline-injected controls. Thus a very rapid reduction in AQP2 mRNA was observed in response to vasopressin-receptor antagonist treatment. The reduction in AQP2 mRNA persisted after 24 h (40 ± 17%, n = 5, P < 0.05) of OPC treatment. There was a parallel increase in diuresis and reduction in urine osmolality. In conclusion, V2-receptor blockade produced a rapid internalization of AQP2 parallel with a rapid increase in urine output. Furthermore, OPC treatment caused a rapid and significant reduction in AQP2 mRNA expression, demonstrating that for rapid regulation of AQP2 expression, modulation of AQP2 mRNA levels is regulated via vasopressin-receptor signaling pathways.

Anti-Inflammatory and Renal Protective Actions of Stanniocalcin-1 in a Model of Anti-Glomerular Basement Membrane Glomerulonephritis

We have previously shown that stanniocalcin-1 (STC1) inhibits the transendothelial migration of macrophages and T cells, suppresses superoxide generation in macrophages, and attenuates macrophage responses to chemoattractants. To study the effects of STC1 on inflammation, in this study we induced a macrophage- and T-cell-mediated model of anti-glomerular basement membrane disease in STC1 transgenic mice, which display elevated serum STC1 levels and preferentially express STC1 in both endothelial cells and macrophages. We examined the following parameters both at baseline and after anti-glomerular basement membrane antibody treatment: blood pressure; C(3a) levels; urine output; proteinuria; blood urea nitrogen; and kidney C(3) deposition, fibrosis, histological changes, cytokine expression, and number of T cells and macrophages. Compared with wild-type mice, after anti-glomerular basement membrane treatment STC1 transgenic mice exhibited: i) diminished infiltration of inflammatory macrophages in the glomeruli; ii) marked reduction in crescent formation and sclerotic glomeruli; iii) decreased interstitial fibrosis; iv) preservation of kidney function and lower blood pressure; v) diminished C(3) deposition in the glomeruli; and vi) reduced expression of macrophage inhibitory protein-2 and transforming growth factor-beta2 in the kidney. Compared with baseline, wild-type mice, but not STC1 transgenic mice, had higher proteinuria and a marked reduction in urine output. STC1 had minimal effects, however, on both T-cell number in the glomeruli and interstitium and on cytokine expression characteristic of either TH1 or TH2 activation. These data suggest that STC1 is a potent anti-inflammatory and renal protective protein.

Climate Change and the Emergent Epidemic of CKD from Heat Stress in Rural Communities: The Case for Heat Stress Nephropathy

Climate change has led to significant rise of 0.8°C-0.9°C in global mean temperature over the last century and has been linked with significant increases in the frequency and severity of heat waves (extreme heat events). Climate change has also been increasingly connected to detrimental human health. One of the consequences of climate-related extreme heat exposure is dehydration and volume loss, leading to acute mortality from exacerbations of pre-existing chronic disease, as well as from outright heat exhaustion and heat stroke. Recent studies have also shown that recurrent heat exposure with physical exertion and inadequate hydration can lead to CKD that is distinct from that caused by diabetes, hypertension, or GN. Epidemics of CKD consistent with heat stress nephropathy are now occurring across the world. Here, we describe this disease, discuss the locations where it appears to be manifesting, link it with increasing temperatures, and discuss ongoing attempts to prevent the disease. Heat stress nephropathy may represent one of the first epidemics due to global warming. Government, industry, and health policy makers in the impacted regions should place greater emphasis on occupational and community interventions.