Pedro Cortes

Connective tissue growth factor and its regulation: a new element in diabetic glomerulosclerosis.

Connective tissue growth factor (CTGF), a member of the closely related CCN family of cytokines appears to be fibrotic in skin. To determine whether CTGF is implicated in diabetic glomerulosclerosis we studied cultured rat mesangial cells (MC) as well as kidney cortex and microdissected glomeruli from obese, diabetic db/db mice and their normal counterparts. Exposure of MC to rhCTGF significantly increased fibronectin and collagen type I secretion. Further, unstimulated MC expressed low levels of CTGF message and secreted minimal amounts of CTGF protein (36–38 kDa). However, exposure to TGF-β, increased glucose concentrations, or cyclic mechanical strain, all causal factors in glomerulosclerosis, markedly induced the expression of CTGF transcripts. With all but mechanical strain there was a concomitant stimulation of CTGF protein secretion. TGF-β also induced abundant quantities of a small molecular weight form of CTGF (18kDa). The induction of CTGF protein by a high glucose concentration was mediated by TGF-β, since a TGF-β neutralizing antibody blocked this stimulation. In vivo studies using quantitative RT-PCR demonstrated that while CTGF transcripts were low in the glomeruli of control mice, expression was increased 27-fold after approximately 3.5 months of diabetes. These changes occurred early in diabetic nephropathy when mesangial expansion was mild, and interstitial disease and proteinuria were absent. A substantially reduced elevation of CTGF mRNA (2-fold) observed in whole kidney cortices indicted that the primary alteration of CTGF expression was in the glomerulus. These results suggest that CTGF upregulation is an important factor in the pathogenesis of mesangial matrix accumulation in both diabetic and non-diabetic glomerulosclerosis, acting downstream of TGF-β.

Relationship Between Renal Function and Metabolic Alterations in Early Streptozocin-Induced Diabetes in Rats

To investigate the temporal relationship of diabetes-induced renal growth and its associated metabolic alterations to the early development of renal hyperfunction, parallel functional and metabolic studies were performed shortly after the onset of diabetes in rats. Hyperglycemia and hypoinsulinemia were evident 18 h after streptozocin injection, and significant hyperglucagonemia and acidosis were present at 36-48 h. Glomerular filtration rate (GFR), expressed per unit of body weight, first increased at 3 days of diabetes [1.35 ± 0.07 (SE) (N = 14)] and was 18% greater than in controls [1.14 · ± 0.03 ml · min−1 100 g−1 (SE) (N = 38)] (P < .005). Renal enlargement preceded GFR changes, so that GFR per unit of kidney weight was lower at 48 h in diabetics [1.31 ± 0.06 (SE) (N = 16)] than in controls [1.54 ± 0.04 ml · min−1 · g−1 (SE) (N = 38)] (P < .01). Nucleotide and RNA metabolism was studied in the renal cortex after infusion of radiolabeled orotate or adenine. Rate of RNA synthesis, total cellular RNA, and the pools of ATP, UTP, and uridine 5′-diphospho-N-acetyl glucosamine were significantly increased 13–51% in 48-h diabetics. Nucleotide precursor incorporation was significantly increased only in uracil ribonucleotides. The increase in uracil ribonucleotide pool exceeded the degree of cell hypertrophy. Our studies indicate that renal hypertrophy and specific increases in uracil ribonucleotide synthesis precede functional changes in early diabetes. Renal metabolic changes may be the critical primary factors in diabetic nephropathy.

Cyclic stretching of mesangial cells up-regulates intercellular adhesion molecule-1 and leukocyte adherence: a possible new mechanism for glomerulosclerosis.

Intraglomerular hypertension is a primary causal factor in the progressive glomerulosclerosis that characterizes diabetic nephropathy or severe renal ablation. However, inflammation of the glomerular mesangium also participates in at least the early phase of these diseases. In glomerulonephritis, where inflammation is thought to be the predominant causal factor, intraglomerular hypertension is also often present. Mesangial cells (MCs) are critical in orchestrating key functions of the glomerulus including extracellular matrix metabolism, cytokine production, and interaction with leukocytes. Because MCs are subject to increased stretching when intraglomerular hypertension is present, and in glomerulonephritis MC/leukocyte interactions seem to be mediated primarily via the up-regulation of intercellular adhesion molecule-1 (ICAM-1), we examine the possibility that cyclic stretching is a stimulus for increased MC ICAM-1 activity. We demonstrate that the normal low levels of MC ICAM-1 mRNA and protein are dramatically up-regulated by even short intervals of cyclic stretch. This effect is dose- and time-dependent, and requires little amplitude and a brief period of elongation for significant induction. Stretch-induced MC ICAM-1 also leads to a marked elevation in phagocytic leukocyte adherence. This stimulated adherence is equal or greater than that induced by the inflammatory cytokine tumor necrosis factor-alpha, whereas an additive effect occurs when both are applied in combination. Our results indicate that stretch-induced ICAM-1 may provide a direct link between hypertension and inflammation in the progression of injury and glomerulosclerosis in diabetes, renal ablation, and other forms of glomerulonephritis.

The role of dietary protein restriction in progressive azotemia

In rats, renal diseases progress to the end stage, but the rate of progression can be slowed dramatically by dietary protein restriction1. In humans, most renal diseases also progress to death due to uremia, unless transplantation or dialysis is provided2. Despite several studies to the contrary, the results of the Modification of Diet in Renal Diseases (MDRD) study, reported in this issue of the Journal by Klahr et al., suggest that the progression of renal diseases in humans is only minimally slowed by dietary protein restriction3. What accounts for these differences? In the studies in rats the .xa0.xa0.

Effects of Orotate Administration on the Normal Rat Kidney: Similarity to Changes Observed in Diabetes Mellitus

Glomerular filtration rate, tubular reabsorptive capacity, and renal size are increased early in the course of human insulin-dependent diabetes mellitus. In short-term experimental diabetes in rats, renal weight, total protein, and RNA content are greater than in control rats. These changes are associated with a 12% increase in the cortical uridine 5-triphosphate (UTP)/DNA ratio. We measured the UTP content in the renal cortex of long-term diabetic rats and studied the effects of long-term orotate feeding (0.5%) as a means of UTP pool expansion. Rats with streptozotocin-induced diabetes for six months had a 42% increase in serum creatinine concentration and an 18% increase in width of the glomerular basement membrane (GBM). Renal cortex UTP/DNA/kg was 54% higher than in control rats. Long-term orotate feeding of diabetic rats produced further deterioration of function and increase in basement membrane width. All diabetic animals developed cataracts bilaterally. Chronic orotate feeding in nondiabetic animals produced thickening of the GBM when compared with age-matched controls (2330 ± 24 vs. 2056 ± 16 Å; P < 0.01) that were morphologically undistinguishable from diabetic GBM. UTP/DNA/kg body weight were no different from control values. Chronic orotate feeding for 15 mo was associated with a higher renal cortex glycogen content and an increase in creatinine clearance. No animal in this group had cataracts. Orotate feeding did not change plasma concentrations of glucose or insulin and was associated with increased glucagon concentrations in both diabetic and nondiabetic rats. We postulate that the renal changes induced by orotate, which resemble those of diabetes, are mediated through alterations in pyrimidine metabolism.

Potentiation Of Glucose‐Mediated Glomerular Injury By Mechanical Strain

1. The glomerular injury of diabetes is characterized by the progressive accumulation of extracellular matrix in the mesangial regions, ultimately resulting in glomerulosclerosis.

The Nature of the Diabetic Glomerulus: Pressure-Induced and Metabolic Aberrations

Early clinical and experimental studies on the pathogenesis of diabetic glomerulosclerosis identified specific mechanisms believed to be unique and crucial in the development and progression of the glomerular lesion. The relative importance of these mechanisms, broadly classified as hemodynamic or metabolic, has been long debated The hemodynamic factors included systemic arterial hypertension [1–3], glomerular hyperfunction and increased glomerular capillary hydrostatic pressure [4–6]. The metabolic factors related to changes associated with glomerular hypertrophy [7–9], hyperlipidemia [10, 11] and the effects of hyperglycemia exerted either directly [12–14] or through the formation of advanced glycosylation end products [15–17] and increased polyol pathway activity [18]. More recent studies have demonstrated that stimulated cytokine expression is also an important pathogenetic component [19–21]. These cytokines, specially transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) have been shown to induce extracellular matrix (ECM) deposition in glomeruli and to stimulate the mesangial cell synthesis of ECM components [22–25].

The Nature of the Glomerulus: Pressure-Induced and Metabolic Aberrations

The experimental search for mechanisms responsible for the development and progression of glomerulosclerosis has resulted in the identification of factors, broadly classified as hemodynamic and metabolic, which may be involved in both the glomerular injury of diabetes and in that occurring in the kidney remnant following subtotal nephrectomy. The hemodynamic factors include systemic arterial hypertension [1–3], glomerular hyperfunction and increased glomerular capillary hydrostatic pressure [4–6]. The metabolic factors include changes associated with glomerular hypertrophy [7–9], hyperlipidaemia [10,11] and, in the case of diabetes, the effects of hyperglycaemia exerted either directly [12–14] or through the formation of advanced glycosylation end products [15–17] and increased polyol pathway activity [18]. In addition, stimulated cytokine expression is also an important pathogenetic component [19–21]. These cytokines, specially transforming growth factor-s (TGF-s) and platelet-derived growth factor (PDGF) are known to induce extracellular matrix deposition in glomeruli and to stimulate the mesangial cell synthesis of extracellular matrix components [22–25].

Pressure Induced and Metabolic Alterations in the Glomerulus: Role in Cytokine Activity and Progressive Sclerosis

The experimental search for mechanisms responsible for the development and progression of glomerulosclerosis has resulted in the identification of factors, broadly classified as hemodynamic and metabolic, which may be involved in both the glomerular injury of diabetes and in that occurring in the kidney remnant following subtotal nephrectomy. The hemodynamic factors include systemic arterial hypertension [1–3], glomerular hyperfunction and increased glomerular capillary hydrostatic pressure [4–6]. The metabolic factors relate to changes associated with glomerular hypertrophy [7–9], hyperlipidemia [10, 11] and, in the case of diabetes, the effects of hyperglycemia exerted either directly [12–14] or through the formation of advanced glycosylation end products and increased oxidative stress [15–19] or the increased activity of the polyol pathway [20].