Harald S. Mackenzie

Suppression Subtractive Hybridization Identifies High Glucose Levels as a Stimulus for Expression of Connective Tissue Growth Factor and Other Genes in Human Mesangial Cells

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mm versus 5 mm) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor β1 (TGF-β1), and addition of TGF-β1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-β1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFβ1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFβ1-driven matrix production within a diabetic milieu.

Fewer nephrons at birth: A missing link in the etiology of essential hypertension?

In 1988, Brenner et al advanced the hypothesis that the nephron endowment at birth is inversely related to the risk of developing essential hypertension in later life (Am J Hypertens 1:335-347, 1988). This novel perspective on the origins of essential hypertension was taken from the viewpoint that the development and maintenance of hypertension must involve a renal factor favoring sodium retention, thereby preventing pressure-induced natriuresis from restoring blood pressure toward normal levels. Since nephron numbers in the normal population range from 300,000 to 1,100,000 or more, it was reasoned that a congenital deficit in nephron endowment itself could be the renal risk factor for hypertension: demographic groups in whom hypertension is unusually prevalent tend to have smaller kidneys, implying fewer nephrons, and some inbred hypertensive rat strains have, on average, fewer nephrons than their respective normotensive controls. We argue that recent independent observations in humans relating low birth weight to both increased risk of hypertension in later life and the formation of fewer nephrons at birth lend strong support to the nephron number hypothesis. Moreover, independent experimental studies in rodents suggest that maternal protein intake during gestation is directly related to he numbers of nephrons formed and that when protein intake is restricted, the offspring develop hypertension in maturity. The concept that nephron numbers may be programmed during gestation, as these observations imply, is discussed in relation to the potential advantages and disadvantages of such a mechanism for the next generation; parallels are drawn with the relationship of low birth weight to pancreatic beta cell development and maturity-onset diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of inflammatory mediators in rat renal isografts by donor brain death

BACKGROUND Brain death (BD) has been thought to influence the early course of transplanted organs by triggering a series of nonspecific inflammatory events that in turn may increase the kinetics and intensity of the immunological host responses. In this study early nonspecific, cellular, and molecular changes occurring in kidney isografts from BD donors are compared with those from normal anesthetized, ventilated controls. METHODS After induction of brain death, the animals were mechanically ventilated for 6 hr before organ removal. Only rats with stable blood pressure (mean arterial pressure >80 mmHg) were included. Serum creatinines were measured daily. Representative grafts were harvested 6 hr after brain death and between 1 hr and 5 days after engraftment for morphology, immunohistology, and reverse transcriptase-polymerase chain reaction. The presence of serum cytokines was assessed by enzyme linked immunoabsorbant assay. RESULTS Serum creatinine levels rose slightly in recipients from BD donors. Serum interleukin-1beta levels increased within 6 hr versus controls (P<0.05). mRNA levels of interleukin-1beta and macrophage inhibitory protein-1 in the kidneys were up-regulated transiently before engraftment (6 hr after BD) and 1 hr after revascularization (P<0.05). By immunohistology, numbers of infiltrating polymorphonuclear leukocytes peaked at 24 hr in parallel with intragraft induction of P- and E-selectin, complement, and other proinflammatory chemokines and cytokines. At 5 days, the isografts from BD donors were highly infiltrated by host leukocyte populations associated with intense up-regulation of their products. In contrast, those from control donors remained relatively normal through this initial follow-up period. CONCLUSIONS The intense nonimmune inflammation produced in isografts after donor BD may represent the initial stages of a continuum between an initial nonspecific and later immune reactivity, when placed in the context of allotransplantation.

High Glucose-altered Gene Expression in Mesangial Cells ACTIN-REGULATORY PROTEIN GENE EXPRESSION IS TRIGGERED BY OXIDATIVE STRESS AND CYTOSKELETAL DISASSEMBLY

High extracellular glucose plays a pivotal role in the pathophysiology of diabetic nephropathy. Here we report 200 genes, identified using suppression-subtractive hybridization, that are differentially expressed when human mesangial cells are propagated in high ambient glucose in vitro. The major functional classes of genes identified included modulators and products of extracellular matrix protein metabolism, regulators of cell growth and turnover, and a cohort of actin cytoskeleton regulatory proteins. Actin cytoskeletal disassembly is a prominent feature of diabetic nephropathy. The induction of actin cytoskeleton regulatory gene expression by high glucose was attenuated by the inhibitor of reactive oxygen species generation, carbonyl cyanide m-chlorophenylhydrazone but not by the protein kinase C inhibitor GF 109203X and was not mimicked by the addition of transforming growth factor β. Enhanced expression of actin cytoskeleton regulatory genes was also observed following disruption of the mesangial cell actin cytoskeleton by cytochalasin D. In aggregate, these results suggest that the induction of genes encoding actin cytoskeleton regulatory proteins (a) is a prominent component of the mesangial cell transcriptomic response in diabetic nephropathy and (b) is dependent on oxidative stress, is independent of protein kinase C and transforming growth factor-β, and represents an adaptive response to actin cytoskeleton disassembly.

Cellular and molecular mediators in common pathway mechanisms of chronic renal disease progression.

Injury mechanisms activated by the hemodynamic adaptations to nephron loss are considered to represent a final common pathway that underlies the progressive nature of chronic renal disease. In this article, we review experimental evidence that the induction of cell adhesion molecule, cytokine and profibrotic growth factor gene expression and the resultant renal infiltration by inflammatory cells, especially macrophages, are important components of these common pathway mechanisms. Interventions aimed at inhibiting these mechanisms may offer new treatments for slowing or arresting the progression of chronic renal disease.

Adrenomedullin and its receptor complexes in remnant kidneys of rats with renal mass ablation: decreased expression of calcitonin receptor-like receptor and receptor-activity modifying protein-3.

Adrenomedullin (AM) has vasodilator and diuretic actions, similarly to natriuretic peptides. AM receptor complexes are composed of calcitonin receptor-like receptor (CRLR) and receptor-activity modifying protein-2 (RAMP2), or CRLR and RAMP3. We aimed to know whether gene expression of AM and AM receptor complexes are regulated in kidneys under pathophysiological conditions. Expression of AM, RAMP2, RAMP3 and CRLR mRNA was studied in the remnant kidney of rats with renal mass ablation using competitive quantitative RT-PCR techniques. Partial cloning was performed to determine the rat RAMP3 nucleotide sequence. In normal rat kidneys, expression levels of RAMP2, RAMP3, CRLR and AM mRNAs were 26.5 +/- 1.9 mmol/mole of GAPDH, 7.7 +/- 0.9 mmol/mole of GAPDH, 3.6 +/- 0.2 mmol/mole of GAPDH and 0.57 +/- 0.03 mmol/mole of GAPDH (mean +/- SE, n = 6), respectively. RAMP3 mRNA levels decreased significantly to about 50% and about 70% of control (sham-operated rats) 4 days and 14 days after 5/6 nephrectomy, respectively. CRLR mRNA levels also decreased significantly to about 30% and about 43% of control. Sodium intake restriction had no significant effects on the RAMP3 and CRLR gene expression. On the other hand, RAMP2 mRNA expression in the kidney was suppressed by sodium intake restriction regardless of nephrectomy, while RAMP2 levels in the remnant kidney were not significantly changed by 5/6 nephrectomy. Neither 5/6 nephrectomy or sodium intake restriction had any significant effects on the AM gene expression in the kidney. The present study showed that expression of mRNAs encoding AM, RAMP2, RAMP3 and CRLR were differentially regulated in remnant kidneys of rats with renal mass ablation.

Renal Effects of High-Dose Natriuretic Peptide Receptor Blockade in Rats With Congestive Heart Failure

Previous studies suggest that elevated plasma atrial natriuretic peptide (ANP) levels participate in regulating renal excretory function in rats with congestive heart failure (CHF). To define the role of natriuretic peptides (NPs) in the regulation of renal function in CHF, the renal responses to HS-142-1 (HS), a potent NP receptor antagonist, were studied in anesthetized rats subjected to coronary ligation that developed left ventricular infarction and CHF or in sham-operated (SO) control rats. Plasma ANP levels averaged > 14-fold higher in rats with CHF than in SO rats. In response to HS (20 mg/kg IV bolus), both mean arterial pressure and renal vascular resistance increased in rats with CHF but not in SO rats; glomerular filtration rate (GFR, 1.26 +/- 0.04 versus 0.76 +/- 0.11 mL/min) and renal plasma flow rate (RPF, 3.52 +/- 0.27 versus 2.70 +/- 0.32 mL/min) were significantly reduced in rats with CHF; and in SO rats, GFR (1.26 +/- 0.06 versus 1.20 +/- 0.07 mL/min) and RPF (3.98 +/- 0.21 versus 3.99 +/- 0.18 mL/min) were not significantly affected by HS. The sodium excretion rate (0.18 +/- 0.04 to 0.06 +/- 0.01 muEq/min) and fractional sodium excretion (0.01 +/- 0.02% to 0.04 +/- 0.01%) also fell markedly after HS administration in rats with CHF, but these parameters were unchanged in SO rats. These data indicate that NPs play a critical role in maintaining renal hemodynamic function and inhibiting tubule sodium reabsorption in rats with CHF, thus opposing sodium retention and preserving sodium balance in this model.

Recipient hypertension potentiates chronic functional and structural injury of rat renal allografts

Background. Systemic hypertension affects many allograft recipients, is an important risk factor for chronic graft dysfunction, and is linked to reduced graft survival. The condition may up-regulate the expression of inflammatory host cells and their products. These, in turn, may significantly injure vascular endothelium and other components of allografted kidneys. Methods. Lewis rats received orthotopic F344 renal allografts, a standard model of chronic rejection. Renovascular hypertension was produced by placing a silver clip (0.25 mm) on the renal artery of the retained contralateral native kidney 4 weeks after transplantation. Sham-clipped rats served as normotensive controls. Four recipient groups (Gp) were studied: Gp 1, rats with an allograft plus a clipped native kidney; Gp 2, those with an allograft and a sham-clipped native kidney; Gp 3, isografted animals with a clipped native kidney; and Gp 4, those bearing an isograft and a sham-clipped native kidney. Systolic blood pressure and proteinuria were measured every 2 weeks for 24 weeks. Grafts were assessed serially for morphologic and immunohistologic changes. Results. Systemic blood pressure rose to hypertensive levels in Gps 1 and 3 within a week of clipping but never increased in Gps 2 and 4. Proteinuria developed in hypertensive animals but remained at baseline in normotensive controls. Intimal thickening of allograft arteries progressed to luminal obliteration with extensive perivascular and interstitial fibrosis by 24 weeks. In contrast, vascular changes in isografts of hypertensive hosts were restricted to medial hypertrophy. Tumor necrosis factor (TNF)-&agr;, transforming growth factor (TGF)-&bgr;, platelet derived growth factor (PDGF), endothelin, Il-6, major histocompatibility complex (MHC) class II, and B7 were up-regulated in allografts in hypertensive hosts. Vascular deposition of immunoglobulin (IgG) was increased. These changes were markedly less pronounced in Gp 3 isografts and minimal in the kidneys of the normotensive animals of Gps 2 and 4. Conclusions. An experimental model is presented that examines the influence of recipient hypertension in the pathogenesis of chronic dysfunction and injury developing in rat renal allografts over time.

Nephron endowment and the pathogenesis of chronic renal failure

When glomerular filtration rate (GFR) in humans falls below about half of normal, a relentless progressive loss of function ensues, even when the original disease becomes inactive [1], This clinical phenomenon is modelled by partial ablation of renal mass in rats which leads to progressive proteinuria, systemic hypertension and glomerulosclerosis [2]. Study of rats subjected to extensive renal mass ablation has provided major insights into the progressive nature of kidney disease. As a compensatory response to reduced renal mass, surviving nephrons undergo adaptations in structure and function, including nephron hypertrophy and increases in single-nephron GFR to meet excretory demands [3] Brenner and colleagues proposed that maladaptive glomerular hemodynamic changes associated with increased single-nephron GFR initiate and perpetuate glomerular injury following renal mass ablation and suggested that similar events occur when nephrons are lost through disease [3, 4]. In response to reduced total GFR, increased single-nephron GFR was observed, mediated by elevated glomerular flows and pressures. Raised glomerular hydraulic pressure (glomerular hypertension) appears to be the major effector of glomerular injury following renal mass reduction [5–8]. In addition to the detrimental effects of acquired nephron loss, severe inborn deficits in total nephron supply also lead to progressive glomerular injury.

Nephron endowment at birth and the pathogenesis of hypertension and chronic renal failure

Summary: It is well established that extensive ablation of renal mass initiates a cycle of accelerated, progressive glomerular injury in remnant kidneys. This process is associated with extreme nephron hypertrophy and hyperfiltration and systemic hypertension. Severe congenital deficiencies in nephron number are also associated with adverse effects on the kidney. In rodents, more subtle reductions in nephron supply are known to advance age‐related glomerular injury. Since intra‐uterine growth retardation is associated with formation of fewer nephrons, the recent observation that low birthweight is associated with increased risk of hypertension in later life raises the possibility that comparatively modest reductions in nephron complement may predispose to hypertension and renal injury over 4–5 decades. the finding of an association between short stature, a marker of low birthweight and nephropathy among type I diabetics also implies that a modest congenital nephron underendowment predisposes to glomerular injury. the numbers of viable nephrons supplied to renal allograft recipients may be critical determinants of late allograft success or failure, since unique circumstances combine to lower the nephron complement to levels akin to those seen in patients with surgical reduction of a solitary kidney, in whom predisposition to hypertension and glomerulosclerosis is evident. This article summarizes recent findings that suggest that congenital nephron endowment is a significant and overlooked factor in the pathogenesis of chronic renal disease and hypertension.