Background/Aims

The mammalian target of rapamycin (mTOR) is a serine kinase that regulates phosphorylation (p) of its target ribosomal S6 kinase (S6K1), whose activation can lead to glomerular and proximal tubular cell (PTC) injury and associated proteinuria. Increased mTOR/S6K1 signaling regulates signaling pathways that target fibrosis through adherens junctions. Recent data indicate aldosterone signaling through the mineralocorticoid receptor (MR) can activate the mTOR pathway. Further, antagonism of the MR has beneficial effects on proteinuria that occur independent of hemodynamics.

Methods

Accordingly, hypertensive transgenic TG(mRen2)27 (Ren2) rats, with elevated serum aldosterone and proteinuria, and age-matched Sprague-Dawley rats were treated with either a low dose (1 mg/kg/day) or a conventional dose (30 mg/kg/day) of spironolactone (MR antagonist) or placebo for 3 weeks.

Results

Ren2 rats displayed increases in urine levels of the PTC brush border lysosomal enzyme N-acetyl-β-aminoglycosidase (β-NAG) in conjunction with reductions in PTC megalin, the apical membrane adherens protein T-cadherin and basolateral α-(E)-catenin, and fibrosis. In concert with these abnormalities, Ren2 renal cortical tissue also displayed increased Ser2448 (p)/activation of mTOR and Thr389 (p)-S6K1 and increased 3-nitrotyrosine (3-NT) content, a marker for peroxynitrite. Low-dose spironolactone had no effect on blood pressure but decreased proteinuria and β-NAG comparable to a conventional dose of this MR antagonist. Both doses of spironolactone attenuated ultrastructural maladaptive alterations and led to comparable reductions in (p)-mTOR/(p)-S6K1, 3-NT, fibrosis, and increased expression of α-(E)-catenin, T- and N-cadherin.

Conclusions

Thereby, MR antagonism improves proximal tubule integrity by targeting mTOR/S6K1 signaling and redox status independent of changes in blood pressure.

Background/Aims

Angiotensin (Ang) II contributes to tubulointerstitial fibrosis. Recent data highlight mammalian target of rapamycin (mTOR)/S6 kinase 1 (S6K1) signaling in tubulointerstitial fibrosis; however, the mechanisms remain unclear. Thereby, we investigated the role of Ang II on mTOR/S6K1-dependent proximal tubule (PT) injury, remodeling, and fibrosis.

Methods

We utilized young transgenic Ren2 rats (R2-T) and Sprague-Dawley rats (SD-T) treated with the Ang type 1 receptor (AT1R) blocker telmisartan (2 mg · kg−1 · day−1) or vehicle (R2-C; SD-C) for 3 weeks to examine PT structure and function.

Results

Ren2 rats displayed increased systolic blood pressure, proteinuria and increased PT oxidant stress and remodeling. There were parallel increases in kidney injury molecule-1 and reductions in neprilysin and megalin with associated ultrastructural findings of decreased clathrin-coated pits, endosomes, and vacuoles. Ren2 rats displayed increased Serine2448 phosphorylation of mTOR and downstream S6K1, in concert with ultrastructural basement membrane thickening, tubulointerstitial fibrosis and loss of the adhesion molecule N-cadherin. Telmisartan treatment attenuated proteinuria as well as the biochemical and tubulointerstitial structural abnormalities seen in the Ren2 rats.

Conclusions

Our observations suggest that Ang II activation of the AT1R contributes to PT brush border injury and remodeling, in part, due to enhanced mTOR/S6K1 signaling which promotes tubulointerstitial fibrosis through loss of N-cadherin.

The risk of developing type 2 diabetes and cardiovascular disease in women who had previously been diagnosed with gestational diabetes (GDM) is well established. There is increasing evidence that the offspring of women with GDM are at increased risk for the development of all components of the cardiorenal metabolic syndrome. Overall, it appears that these offspring have an increased risk for overweight/obesity, insulin resistance, higher blood pressure, renal disease, and type 2 diabetes. However, distinct differences in regional populations, lack of routine screening and treatment of GDM worldwide, and long follow-up periods for offspring represent a challenge in assessing the risk for development of these abnormalities in the offspring of women who have had GDM.

The presence of a group of interacting maladaptive factors, including hypertension, insulin resistance, metabolic dyslipidemia, obesity, and microalbuminuria and/or reduced renal function, collectively constitutes the cardiorenal metabolic syndrome (CRS). Nutritional and other environmental cues during fetal development can permanently affect the composition, homeostatic systems, and functions of multiple organs and systems; this process has been referred to as ‘programming’. Since the original formulation of the notion that low birth weight is a proxy for ‘prenatal programming’ of adult hypertension and cardiovascular disease, evidence has also emerged for programming of kidney disease, insulin resistance, obesity, metabolic dyslipidemia, and other chronic diseases. The programming concept was initially predicated on the notion that in utero growth restriction due to famine was responsible for increased hypertension, and cardiovascular and renal diseases. On the other hand, we are now more commonly exposed to increasing rates of maternal obesity. The current review will discuss the overarching role of maternal overnutrition, as well as fetal undernutrition, in epigenetic programming in relation to the pathogenesis of the CRS in children and adults.

The renin-angiotensin-aldosterone-system (RAAS) is central to the pathogenesis of hypertension, cardiovascular and kidney disease. Emerging evidence support various pathways through which a local renal RAAS can affect kidney function, hypertension, and cardiovascular disease. A prominent mechanism appears to be loss of redox homeostasis and formation of excessive free radicals. Free radicals such as reactive oxygen species (ROS) are necessary in normal physiologic processes including development of nephrons, erythropoeisis and tubular sodium transport. However, loss of redox homeostasis contributes to pro-inflammatory and pro-fibrotic pathways in the kidney that in turn lead to reduced vascular compliance, podocyte pathology and proteinuria. Both blockade of the RAAS and oxidative stress produces salutary effects on hypertension and glomerular filtration barrier injury. Thus, the focus of current research is on understanding the pathophysiology of chronic kidney disease in the context of an elevated RAAS and unbalanced redox mechanisms.

Abstract

Loss of redox homeostasis and formation of excessive free radicals play an important role in the pathogenesis of kidney disease and hypertension. Free radicals such as reactive oxygen species (ROS) are necessary in physiologic processes. However, loss of redox homeostasis contributes to proinflammatory and profibrotic pathways in the kidney, which in turn lead to reduced vascular compliance and proteinuria. The kidney is susceptible to the influence of various extracellular and intracellular cues, including the renin–angiotensin–aldosterone system (RAAS), hyperglycemia, lipid peroxidation, inflammatory cytokines, and growth factors. Redox control of kidney function is a dynamic process with reversible pro– and anti-free radical processes. The imbalance of redox homeostasis within the kidney is integral in hypertension and the progression of kidney disease. An emerging paradigm exists for renal redox contribution to hypertension. Antioxid. Redox Signal. 11, 2047–2089.

Introduction

Redox Control of Cellular Function: How Is It Achieved?

Free radical contribution to redox control of hypertension

Clinical contribution to redox control of hypertension

Prooxidant enzymes and pathways

NAD(P)H oxidase

Xanthine oxidase (XO)

Lipooxygenases (LOX) and cyclooxygenases (COX)

P450 monooxygenase and mitochondrial respiratory chain enzymes (I–IV)

Antioxidant enzymes and pathways

Role of ROS in physiologic processes

Pathologic Role of ROS in Hypertension

Non–RAAS-mediated oxidative stress in hypertension

High intravascular pressure

Shear stress

Lipids

Eicosanoids

High salt

Cigarette smoke

Insulin resistance/hyperinsulinemia

eNOS uncoupling

Dopaminergic system (DS)/sympathetic nervous system

Role of the RAAS in oxidative stress and hypertension

Ang II, ROS, and systemic hypertension

Ang II stimulation of NAD(P)H oxidase and hypertension

p22phox and hypertension

gp91phox (Nox2) and hypertension

p47phox and hypertension

p67phox and hypertension

p40phox and hypertension

Kidney Redox Function and Hypertension

ROS in normal kidney physiology

RAAS in the kidney

RAAS expression in developing and adult kidneys

RAAS-mediated redox mechanisms

Methods for detecting ROS in the laboratory and clinic

Nephron handling of ROS and hypertension: redox control of renal function

Redox control of kidney function

Tubuloglomerular feedback and role of ROS in macula densa

Medullary perfusion and renal hemodynamics

Pressure natriuresis

Tubular sodium transport

Renal sympathetic nerves

Nephron components and their contribution to ROS and hypertension

ROS in the glomeruli/podocytes

ROS and the glomerular basement membrane (GBM)

ROS and the mesangium

ROS and the tubule

NAD(P)H Oxidase Inhibition for the Treatment of Hypertension: Promises and Limitations

NAD(P)H oxidase–specific inhibitors

Apocynin

DPI

Neopterin/phenylarsine oxide

Phycobilins

gp91ds/gp91ds-tat

PR-39

VAS2870, SI7834, and AEBSF

siRNAs

Monoclonal antibodies

Nonspecific NAD(P)H oxidase inhibitors

PKC inhibitors

Antioxidants

Statins, ACE inhibitors/ARBs, and aldosterone antagonists

Future Perspectives/Conclusions

Diabetic kidney disease is characterized by persistent albuminuria (>300 mg/dl or >200 μg/min) that is confirmed on at least 2 occasions 3 to 6 months apart, with a progressive decline in the glomerular filtration rate (GFR), elevated arterial blood pressure, and an increased risk for cardiovascular morbidity and mortality. Diabetic kidney disease is the leading cause of end stage renal disease (ESRD) prompting investigators to evaluate mechanisms by which to slow disease progression. One such mechanism is to block the activity of angiotensin II at the receptor site and agents that follow this mechanism are referred to as angiotensin receptor blockers (ARB). There is sufficient clinical evidence to support that ARB have protective effects on kidney function in patients with diabetes and hypertension. However, in the past decade there have been few investigations comparing individual ARBs on renal outcomes. Telmisartan, a lipophilic ARB with a long half-life, has been hypothesized to have a greater anti-proteinuric effect when compared to the shorter acting losartan. Therefore, the A comparison of telMisartan versus losArtan in hypertensive type 2 DiabEtic patients with Overt nephropathy (AMADEO) trial sought to investigate renal and cardiovascular endpoints. In this review, we discuss the pathophysiology of diabetic kidney disease and implications of the AMADEO trial in the context of current understanding from recent outcome trials.

Activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase by angiotensin II is integral to the formation of oxidative stress in the vasculature and the kidney. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition is associated with reductions of oxidative stress in the vasculature and kidney and associated decreases in albuminuria. Effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition on oxidative stress in the kidney and filtration barrier integrity are poorly understood. To investigate, we used transgenic TG(mRen2)27 (Ren2) rats, which harbor the mouse renin transgene and renin-angiotensin system activation, and an immortalized murine podocyte cell line. We treated young, male Ren2 and Sprague-Dawley rats with rosuvastatin (20 mg/kg IP) or placebo for 21 days. Compared with controls, we observed increases in systolic blood pressure, albuminuria, renal NADPH oxidase activity, and 3-nitrotryosine staining, with reductions in the rosuvastatin-treated Ren2. Structural changes on light and transmission electron microscopy, consistent with periarteriolar fibrosis and podocyte foot-process effacement, were attenuated with statin treatment. Nephrin expression was diminished in the Ren2 kidney and trended to normalize with statin treatment. Angiotensin II–dependent increases in podocyte NADPH oxidase activity and subunit expression (NOX2, NOX4, Rac, and p22phox) and reactive oxygen species generation were decreased after in vitro statin treatment. These data support a role for increased NADPH oxidase activity and subunit expression with resultant reactive oxygen species formation in the kidney and podocyte. Furthermore, statin attenuation of NADPH oxidase activation and reactive oxygen species formation in the kidney/podocyte seems to play roles in the abrogation of oxidative stress-induced filtration barrier injury and consequent albuminuria.

P1-derived artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) have become very useful as tools to study gene expression and regulation in cells and in transgenic mice. They carry large fragments of genomic DNA (≥100 kb) and therefore may contain all of the cis-regulatory elements required for expression of a gene. Because of this, even when inserted randomly in the genome, they can emulate the native environment of a gene resulting in a tightly regulated pattern of expression. Because these large genomic clones often contain DNA sequences which can manipulate chromatin at the local level, they become immune to position effects which affect expression of smaller transgenes, and thus their expression is proportional to copy number. Transgenic mice containing large BACs and PACs have become excellent models to examine the regulation of gene expression. Their usefulness would certainly be increased if easy and efficient methods are developed to manipulate them. We describe herein a method to make deletion mutations reliably and efficiently using a novel modification of the Chi-stimulated homologous recombination method. Specifically, we generated and employed a Lox511 ‘floxed’ CAM resistance marker that first affords selection for homologous recombination in Escherichia coli, and then can be easily deleted leaving only a single Lox511 site as the footprint.