Evolution of the urinary proteome during human renal development and maturation: variations with gestational and postnatal age.

BACKGROUND: Low birth weight is associated with deficits in nephron number in the infant kidney and increased risk of adulthood hypertension and renal dysfunction. Urinary biomarkers may be potential indicators of renal reserve, but little is known about the influence of gestational and postnatal age on the expression of urinary proteins. The aims of this study were to determine the relationships between selected urinary proteins and renal maturation. We hypothesized that urinary protein patterns would change over time during late nephrogenesis and renal maturation. METHODS: Urine samples were collected at birth and over 12 mo from preterm (33-35 wk) and term (38-40 wk) infants. Candidate urinary proteins were identified by antibody array and quantified with enzyme-linked immunosorbent assay. RESULTS: Preterm infants at birth were found to have relatively elevated levels of insulin-like growth factor binding protein-1, -2, and -6, monocyte chemotactic protein-1, CD14, and sialic acid-binding Ig-like lectin 5. These markers gradually decline to levels similar to those of full-term infants by 2-6 mo of life. In contrast, many urinary markers in healthy full-term infants remain stable over the first year of life. CONCLUSION: Gestational and postnatal age must be considered when evaluating the utility of urinary biomarkers.

Mechanisms of renal injury and progression of renal disease in congenital obstructive nephropathy.

Congenital obstructive nephropathy accounts for the greatest fraction of chronic kidney disease in children. Genetic and nongenetic factors responsible for the lesions are largely unidentified, and attention has been focused on minimizing obstructive renal injury and optimizing long-term outcomes. The cellular and molecular events responsible for obstructive injury to the developing kidney have been elucidated from animal models. These have revealed nephron loss through cellular phenotypic transition and cell death, leading to the formation of atubular glomeruli and tubular atrophy. Altered renal expression of growth factors and cytokines, including angiotensin, transforming growth factor-beta, and adhesion molecules, modulate cell death by apoptosis or phenotypic transition of glomerular, tubular, and vascular cells. Mediators of cellular injury include hypoxia, ischemia, and reactive oxygen species, while fibroblasts undergo myofibroblast transformation with increased deposition of extracellular matrix. Progression of the lesions involves interstitial inflammation and interstitial fibrosis, both of which impair growth of the obstructed kidney and result in compensatory growth of the contralateral kidney. The long-term outcome depends on timing and severity of the obstruction and its relief, minimizing ongoing injury, and enhancing remodeling. Advances will depend on new biomarkers to evaluate the severity of obstruction, to determine therapy, and to follow the evolution of lesions.

Urinary biomarkers: the future looks promising.

Soler-García and collaborators show that changing levels of urinary biomarkers associated with the pathology of HIV-associated nephropathy may identify HIV-infected children at greatest risk for this complication. These changes appear relatively early in disease progression and remain responsive to antiviral therapy. Will these trends be borne out in a multicenter study?

Species differences in renal Src activity direct EGF receptor regulation in life or death response to EGF.

In rodent models of obstructive nephropathy, exogenous epidermal growth factor (EGF) attenuates tubule cell death in rats and exacerbates cell death in mice. To identify species differences in EGF receptor (EGFR) regulation and signaling, cell lysates were prepared from rat, mouse, and human proximal tubule cells (PTC) and compared by immunoblot analysis for expression and phosphorylation of Src and EGFR. Frozen kidney tissue was also analyzed. Results indicate mouse PTC have constitutive Src- and EGFR-kinase activities not detected in rat or human PTC. Immunoblots of rat, mouse, and human kidney homogenates confirmed this finding in vivo. Src-specific inhibitor PP2 and EGFR kinase inhibitor AG1478 decreased EGF-induced apoptosis in mouse PTC by 74% (P < 0.001) and 70% (P < 0.001), respectively. Expression of a constitutive Src mutant cDNA in rat PTC rendered cells susceptible to EGF-induced death. EGF decreased stretch-induced apoptosis by 66% (P < 0.001) relative to vehicle control in human PTC, similar to rat PTC response. We conclude that elevated Src activity in mouse tubular cells alters downstream EGFR signaling and increases susceptibility to EGF-induced cell death. The unexpected finding that a therapeutic agent (EGF) in rats is detrimental in mice underscores the importance of determining which animal best represents the response of human kidneys to a given agent.

Renal vascular endothelial growth factor in neonatal obstructive nephropathy. II. Exogenous VEGF.

Chronic unilateral ureteral obstruction (UUO) in the neonatal rat causes delayed renal maturation, tubular apoptosis, and interstitial inflammation. Vascular endothelial growth factor (VEGF) acts as a survival factor for tubular cells and reduces renal injury in several models of renal disease. To determine whether exogenous VEGF attenuates renal injury from UUO, rats were subjected within the first 48 h of life to sham operation, partial UUO, or complete UUO. Saline vehicle or VEGF(121) (50 mg/kg) was injected twice daily for 7 days, after which kidneys were harvested for histological study. The density of peritubular capillaries was measured with platelet-endothelial cell adhesion molecule-1 immunostaining, proliferating nuclei were detected by proliferating-cell nuclear antigen staining, apoptosis by the transferase-mediated dUTP nick end-labeling technique, macrophages by ED-1 immunostaining, and collagen by Sirius red staining. Glomerular number and maturation index were also determined in each group. Following chronic complete UUO in the neonatal rat, peritubular capillary density was significantly decreased. Cortical capillary density was further reduced by exogenous VEGF in the partially obstructed kidney. While UUO also decreased glomerular number and delayed glomerular maturation, exogenous VEGF exerted no additional effects. Cellular proliferation and tubular apoptosis increased in proportion to the severity of obstruction, but exogenous VEGF had no additional effects on proliferation, tubular apoptosis, or macrophage infiltration. However, VEGF reduced interstitial apoptosis in the kidney with partial UUO. We conclude that VEGF does not have salutary effects on the renal lesions caused by chronic UUO in the neonatal rat and may actually worsen obstructive nephropathy by aggravating the interstitial lesions.

Renal vascular endothelial growth factor in neonatal obstructive nephropathy. I. Endogenous VEGF.

Obstructive nephropathy constitutes a major cause of renal impairment in children. Chronic unilateral ureteral obstruction (UUO) impairs maturation of the developing kidney and leads to tubular apoptosis and interstitial inflammation. Vascular endothelial growth factor (VEGF) is involved in recovery from various forms of renal injury. We questioned whether the renal expression of endogenous VEGF and its receptor (VEGFR2/Flk-1) is modified by UUO in early development. Neonatal rats were subjected to partial or complete UUO or sham operation. The distribution of immunoreactive VEGF in each kidney was examined after 7, 14, or 28 days. Adult rats were also subjected to sham operation or complete UUO. Tubular VEGF increased between 14 and 28 days in sham-operated rats and in some partially obstructed neonatal rats but decreased with complete UUO. Parallel changes were found by Western blotting, but not by RT-PCR. Immunoreactive VEGF colocalized with mitochondria in proximal and distal tubules and also appeared in type A intercalated cells, glomerular vascular endothelium, and podocytes. While neonatal microvascular renal VEGFR2 receptor staining was strongly positive regardless of UUO, staining was weak in sham-operated adults but increased following UUO. Parallel changes in VEGFR2 expression were verified by RT-PCR and Western blotting. We conclude that endogenous renal VEGF is developmentally regulated in the neonatal rat and is differentially regulated by partial and complete UUO. Following UUO in the adult, the VEGF receptor is upregulated. Endogenous VEGF may serve an adaptive role in responding to tubular injury caused by UUO and may modulate adaptation by the contralateral kidney.

Angiotensin-converting enzyme inhibition aggravates renal interstitial injury resulting from partial unilateral ureteral obstruction in the neonatal rat.

Congenital urinary tract obstruction is the most important cause of renal insufficiency in infants and children, and angiotensin-converting enzyme (ACE) inhibitors attenuate the progression of renal disease in adults. ACE inhibitors are increasingly utilized in children with progressive renal disease. Because angiotensin is necessary for normal renal development, we examined the effects of ACE inhibition both during and immediately following the period of postnatal nephrogenesis in the neonatal rat subjected to sham operation or partial unilateral ureteral obstruction (UUO) under general anesthesia within the first 48 h of life. Rats in group I received enalapril 30 mg/kg body wt (or vehicle) daily for the first 10 days, while in group II, the 10 days of treatment began 10 days after surgery. Kidneys were harvested at day 21 and analyzed for apoptosis (TUNEL), interstitial macrophages (ED-1 immunohistochemistry), myofibroblasts (alpha-smooth muscle actin), and collagen (Sirius red). Partial UUO delayed glomerular maturation and increased ipsilateral renal macrophage infiltration, alpha-smooth muscle actin and Sirius red staining. In group I, enalapril increased myofibroblast accumulation in sham-operated kidneys, but not in obstructed kidneys. In contrast, in group II, enalapril further increased macrophage, myofibroblast, and collagen accumulation following partial UUO. The relative abundance of components of the kallikrein-kinin system, measured by Western blot, was not altered by partial UUO in the 14- and 28-day-old rat. Thus, in contrast to its salutary effects at later ages, ACE inhibition can worsen injury to the partially obstructed kidney during renal maturation even after the completion of nephrogenesis.

Epidermal growth factor potentiates renal cell death in hydronephrotic neonatal mice, but cell survival in rats.

BACKGROUND: Epidermal growth factor (EGF) markedly attenuates tubular apoptosis induced by unilateral ureteral obstruction (UUO) in the neonatal rat, and reduces apoptosis induced by mechanical stretch of cultured rat tubular cells. METHODS: To investigate the role of EGF in modulating apoptosis resulting from UUO, neonatal wild type and mutant mice lacking EGF (knockout), or with diminished EGF receptor activity (waved-2 mutant) were compared to control mice for tubular apoptosis and atrophy. Rat and mouse kidneys were compared for localization of the EGF receptor. Apoptosis was also measured in cultured mouse tubular cells subjected to stretch and exposed to EGF. RESULTS: UUO reduced endogenous renal EGF expression in wild-type mice. Unlike the rat, exogenous EGF did not decrease tubular apoptosis or atrophy in the obstructed kidney, and significantly increased stretch-induced apoptosis of cultured mouse tubular cells. Tubular apoptosis was 50% lower in the obstructed kidney of EGF knockout and waved-2 mice relative to wild type and heterozygous animals. Exogenous EGF increased tubular apoptosis and doubled atrophy in the obstructed kidney of waved-2 mice. Species differences in EGF receptor localization were detected in 3-day-old kidneys. CONCLUSION: EGF acts as a survival factor in the neonatal rat, but potentiates tubular cell death in the neonatal mouse. Species differences are maintained in cultured cells, suggesting that differences in EGF receptor signaling underlie these opposing effects.

Ureteral obstruction in neonatal mice elicits segment-specific tubular cell responses leading to nephron loss.

BACKGROUND: To elucidate the sequence of renal responses leading to nephron loss in obstructive nephropathy, we examined the evolution of segmental nephron cellular changes consequent to chronic unilateral ureteral obstruction (UUO) in the neonatal mouse. METHODS: Neonatal mice were subjected to UUO or sham-operation, and kidneys were harvested 5, 12 or 19 days after surgery. Proximal tubules (PT), distal tubules (DT) and collecting ducts (CD) were identified with lectins. Histomorphometric quantitation was made for cellular necrosis, apoptosis, proliferation, tubular dilatation, tubular basement membrane (TBM) thickening, interstitial collagen, and glomerular maturation. The distribution of hypoxic tissue was determined using pimonidazole as a marker. Additional studies were performed by mechanically stretching monolayer cultures of mouse proximal tubular and collecting duct cells, and measuring apoptosis. RESULTS: Neonatal UUO induced an arrest of glomerular maturation throughout the period of study. Chronic UUO induced hypoxia, tubular necrosis, proliferation, and TBM thickening in the PT, but stimulated apoptosis in the DT and CD. Tubular dilation in the obstructed kidney was most severe in CD and least severe in PT. Tubular cell apoptosis closely paralleled tubular dilation (P < 0.05), and fibrosis surrounding individual tubules also correlated with tubular dilation (P < 0.001). Mechanical stretching of cultured mouse tubular cells induced apoptosis directly proportional to the magnitude of axial strain: apoptosis was consistently greater in CD than in PT cells (P < 0.05). CONCLUSIONS: Following UUO, the co-localization of hypoxia with cellular proliferation, necrosis, and TBM thickening of the PT is consistent with ischemic injury resulting from vasoconstriction. In contrast, a selective dilation of the distal portion of the nephron (DT and CD), which results from the greater tubular compliance there, leads to stretch-induced epithelial cell apoptosis, along with a progressive peritubular fibrosis. Nephron loss in the obstructed developing kidney likely results from complex, segment-specific cellular responses.

Growth factor-mediated phosphorylation of proapoptotic BAD reduces tubule cell death in vitro and in vivo.

BACKGROUND: Exogenous growth factors administered during unilateral ureteral obstruction (UUO) in neonatal rats significantly reduce apoptosis and tubular atrophy. Because the mechanism underlying these salutary effects is largely unknown, we investigated signaling pathways potentially activated by growth factors to determine their roles in therapeutic action. METHODS: Mechanical strain was applied to confluent cultures of immortalized rat proximal tubule cells to simulate obstruction-induced stretch injury in vivo. Growth factors, inhibitory antibodies or pharmacological inhibitors were added to cultures that were subsequently processed for TUNEL analysis or immunoblots to identify signaling pathways that could be modulating cell survival. For in vivo studies, kidneys harvested from rats +/- UUO +/- epidermal growth factor (EGF) were fixed or frozen for immunohistochemistry or immunoblot analysis. RESULTS: Treatment with EGF or insulin-like growth factor-1 (IGF-1) during stretch decreased apoptosis by 50% (P < 0.001). Neutralizing antibodies (Abs) directed against either growth factor or its receptor blocked the reduction in apoptosis. Stretch decreased BAD phosphorylation by approximately 50% (P < 0.001) relative to unstretched cells and each growth factor restored phosphorylation to basal levels. Kinase-specific inhibitors that blocked growth factor-mediated BAD phosphorylation promoted apoptosis in vitro. BAD phosphorylation decreased by approximately 50% (P < 0.001) in the tubules of obstructed hydronephrotic rat kidneys and administration of EGF restored BAD phosphorylation to basal levels. CONCLUSIONS: Signaling pathways converging at BAD phosphorylation are key to growth factor-mediated attenuation of stretch-induced apoptosis in vitro and in vivo.