Robert L. Chevalier

Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy

Renal fibrosis is the hallmark of progressive renal disease of virtually any etiology. The model of unilateral ureteral obstruction (UUO) in the rodent generates progressive renal fibrosis. Surgically created UUO can be experimentally manipulated with respect to timing, severity, and duration, while reversal of the obstruction permits the study of recovery. The use of genetically engineered mice has greatly expanded the utility of the model in studying molecular mechanisms underlying the renal response to UUO. Ureteral obstruction results in marked renal hemodynamic and metabolic changes, followed by tubular injury and cell death by apoptosis or necrosis, with interstitial macrophage infiltration. Proliferation of interstitial fibroblasts with myofibroblast transformation leads to excess deposition of the extracellular matrix and renal fibrosis. Phenotypic transition of resident renal tubular cells, endothelial cells, and pericytes has also been implicated in this process. Technical aspects of the UUO model are discussed in this review, including the importance of rodent species or strain, the age of the animal, surgical procedures, and histological methods. The UUO model is likely to reveal useful biomarkers of progression of renal disease, as well as new therapies, which are desperately needed to allow intervention before the establishment of irreversible renal injury.

Reduced angiotensinogen expression attenuates renal interstitial fibrosis in obstructive nephropathy in mice

A novel approach was employed to assess the contribution of the renin-angiotensin system (RAS) to obstructive nephropathy in neonatal mice having zero to four functional copies of the angiotensinogen gene (Agt). Two-day-old mice underwent unilateral ureteral obstruction (UUO) or sham operation; 28 days later, renal interstitial fibrosis and tubular atrophy were quantitated. In all Agt genotypes, UUO reduced ipsilateral renal mass and increased that of the opposite kidney. Renal interstitial collagen increased after UUO linearly with Agt expression, from a fractional area of 25% in zero-copy mice to 54% in two-copy mice. Renal expression of transforming growth factor-beta1 was increased by ipsilateral UUO in mice expressing Agt, but not in zero-copy mice. However, the prevalence of atrophic tubules due to UUO did not vary with Agt expression. Blood pressure was not different in all groups, except for a reduction in sham zero-copy mice. We conclude that a functional RAS is not necessary for compensatory renal growth. This study demonstrates conclusively that angiotensin regulates at least 50% of the renal interstitial fibrotic response in obstructive nephropathy, an effect independent of systemic hemodynamic changes. Angiotensin-induced fibrosis likely is a mechanism common to the progression of many forms of renal disease.

Obstructive nephropathy: towards biomarker discovery and gene therapy

Obstructive nephropathy is a major cause of renal failure, particularly in infants and children. Cellular and molecular mechanisms responsible for the progression of the tubular atrophy and interstitial fibrosis—processes that lead to nephron loss—have been elucidated in the past 5 years. Following urinary tract obstruction and tubular dilatation, a cascade of events results in upregulation of the intrarenal renin–angiotensin system, tubular apoptosis and macrophage infiltration of the interstitium. This is followed by accumulation of interstitial fibroblasts through proliferation of resident fibroblasts and epithelial–mesenchymal transformation of renal tubular cells. Under the influence of cytokines, chemokines and other signaling molecules produced by tubular and interstitial cells, fibroblasts undergo transformation to myofibroblasts that induce expansion of the extracellular matrix. The cellular interactions that regulate development of interstitial inflammation, tubular apoptosis and interstitial fibrosis are complex. Changes in renal gene expression and protein production afford many potential biomarkers of disease progression and targets for therapeutic manipulation. These include signaling molecules and receptors involved in macrophage recruitment and proliferation, tubular death signals and survival factors, and modulators of epithelial–mesenchymal transformation. Targeted gene deletion and various forms of gene therapy have been used in experimental obstructive nephropathy, mostly rodent models of unilateral ureteral obstruction or cell culture techniques. Further refinement of these models is needed to develop a matrix of biomarkers with clinical predictive value, as well as molecular therapies that will prevent or reverse the renal structural and functional consequences of obstructive nephropathy.

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-β, 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.

Molecular and cellular pathophysiology of obstructive nephropathy

Abstract Congenital obstructive nephropathy remains one of the most-important causes of renal insufficiency in children. This review focuses on the unique interactions that result from urinary tract obstruction during the period of renal development in the neonatal rodent. Following unilateral ureteral obstruction (UUO), growth of the obstructed kidney is impaired and compensatory growth by the intact opposite kidney is related directly to the duration of obstruction. Development of the renal vasculature is delayed by UUO, and the activity of the intrarenal renin-angiotensin system is enhanced throughout the period of obstruction. Glomerular maturation is also delayed by UUO, and nephrogenesis is permanently impaired. The effects of UUO on the developing tubule are also profound, with a suppression of proliferation, stimulation of apoptosis, and the maintenance of an immature phenotype by tubular epithelial cells. Expression of tubular epidermal growth factor is suppressed and transforming growth factor-β1 and clusterin are increased. Maturation of interstitial fibroblasts is delayed, with progression of tubular atrophy and interstitial fibrosis resulting in part from continued activation of the renin-angiotensin system and oxygen radicals. Future efforts to prevent the consequences of congenital urinary tract obstruction must account for the dual effects of obstruction: interference with normal renal development and progression of irreversible tubulointerstitial injury.

ARRESTED DEVELOPMENT OF THE NEONATAL KIDNEY FOLLOWING CHRONIC URETERAL OBSTRUCTION

PURPOSE The purpose of this study was to investigate the role of growth-related peptides in the impairment of renal growth and development resulting from unilateral ureteral obstruction (UUO) in the neonatal rats. MATERIALS AND METHODS Sprague-Dawley rats underwent UUO or sham-operation in the first 48-hours of life, and kidneys were harvested 1 to 28 days later. Renal messenger RNA (mRNA) was quantitated for renin, transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF). Renal interstitial volume was measured in Masson-trichrome-stained sections, and renin and alpha-smooth muscle actin (alpha-SM actin) distribution were determined by immunocytochemistry. RESULTS The normal developmental increase in renal mass and DNA content were suppressed in ipsilateral UUO and increased in the intact opposite kidney. Renal interstitial volume was increased more than 10-fold by ipsilateral UUO. Unilateral ureteral obstruction resulted in a sustained increased in ipsilateral renal renin mRNA and persistence of fetal renin distribution. Renin in the contralateral kidney was suppressed. Transforming growth factor-beta 1 expression increased progressively in the obstructed kidney, but decreased after 7 days in sham-operated kidneys. While renal EGF expression was undetectable in the normal sham kidney during the first 3 days of life, it increased steadily with maturation. However, EGF expression remained suppressed in the obstructed kidney. Whereas alpha-SM actin disappeared from the interstitium of normal rat kidneys by 15 days of age, it persisted in the obstructed neonatal kidney. CONCLUSIONS As revealed by changes in expression of growth-related peptides, neonatal UUO delays ipsilateral renal development, which may contribute to impaired renal growth.

Developmental Renal Physiology of the Low Birth Weight Pre-Term Newborn

PURPOSE The remarkable growth of the kidney and the rapid changes in renal function in the second half of gestation and early postnatal period are discussed. MATERIALS AND METHODS Adaptation to the extrauterine environment involves immediate postnatal natriuresis that is prolonged in the pre-term neonate, followed by the sodium retention necessary for growth. Glomerular filtration rate increases throughout the postnatal period, and it is modulated by the renin-angiotensin system and prostaglandins. Because of this, the fetus and neonate are particularly susceptible to renal injury following the administration of angiotensin converting enzyme inhibitors or nonsteroidal anti-inflammatory drugs. Renal tubular function in the neonate is characterized by reduced renal concentration and acidification ability, which can be further compromised by obstructive uropathy. Urine calcium excretion is high in the neonate, which can be aggravated by calciuric drugs, such as furosemide and glucocorticoids. RESULTS Reduced renal mass results in compensatory renal growth even in the fetus, a response that could prove maladaptive later in life through excessive glomerular hyperfiltration and progressive interstitial fibrosis. CONCLUSIONS These factors underscore the importance of attempting to maximize functional renal mass in the neonate or infant with renal impairment of any etiology.

Pathogenesis of renal injury in obstructive uropathy

Purpose of review This review focuses on recent advances in understanding the factors contributing to obstructive nephropathy, the most important cause of renal failure in children. The major focus is on renal cellular and molecular events, with emphasis on those affecting the developing kidney. Recent findings Experiments in the fetal sheep or neonatal rat, mouse, or pig reveal dramatic effects of urinary tract obstruction on renal growth and development. Surgical relief of obstruction can reverse some of the structural and functional deficits, but cannot restore normalcy. Renal tubular apoptosis is a major factor leading to tubular atrophy following unilateral ureteral obstruction. Increased reactive oxygen species, and a renal environment favoring pro-apoptotic, over survival, signals, contribute to cell death. A variety of intrarenal factors lead to progressive interstitial fibrosis, including the newly described process of epithelial–mesenchymal transition, whereby tubular epithelial cells are transformed into activated fibroblasts. A number of endogenous antifibrotic counter-regulatory molecules have been identified, opening the possibility of enhancing the kidneys own defenses against progressive fibrosis. Summary The renal response to urinary tract obstruction is complex and involves a wide array of interacting molecules. Elucidation of these interactions will lead to the identification of biomarkers that will allow a more precise prediction to the response to surgical intervention and, hopefully, to novel therapies to prevent renal deterioration.

UNILATERAL URETERAL OBSTRUCTION IN EARLY DEVELOPMENT ALTERS RENAL GROWTH: DEPENDENCE ON THE DURATION OF OBSTRUCTION

PURPOSE Over 90% of nephrogenesis in the rat takes place postnatally in the first 10 days, analogous to the midtrimester human fetus. We wished to determine the relationship between the duration of unilateral ureteral obstruction and growth and morphology of both kidneys following relief of the obstruction in the neonatal rat. MATERIALS AND METHODS One ureter of 1 day-old rats was sham-operated or occluded and released 1, 2, 3, or 5 days later, or not released. Fourteen or 28 days later, renal mass, tubular atrophy, and interstitial fibrosis were determined in the obstructed and contralateral kidney of each group. RESULTS At 28 days, there was a linear relationship between kidney/body weight ratio and duration of obstruction, such that the decrement in renal mass resulting from ipsilateral obstruction was precisely compensated by an equal increment in the mass of the contralateral kidney (both, p <0.0001). Tubular atrophy was increased 100-fold in kidneys of rats with 28 days continuous ipsilateral obstruction, while relief of obstruction after 2 to 5 days reduced tubular atrophy by 90% (p <0.01). Interstitial fibrosis was also markedly reduced by relief of obstruction, with the severity of fibrosis being proportional to the duration of obstruction. CONCLUSIONS We conclude that ureteral obstruction during the critical period of nephrogenesis impairs growth of the obstructed kidney and stimulates growth of the contralateral kidney in direct proportion to the duration of obstruction. Moreover, counterbalance between the two kidneys is finely regulated. Even 2 days of ureteral obstruction (with subsequent relief) induces contralateral renal growth, and induces ipsilateral tubular atrophy. However, the time dependence of renal injury on duration of obstruction suggests that earlier relief of obstruction in the developing kidney may allow greater ultimate preservation of functional renal mass.

Proximal tubular injury and rapid formation of atubular glomeruli in mice with unilateral ureteral obstruction: a new look at an old model

Unilateral ureteral obstruction (UUO), employed extensively as a model of progressive renal interstitial fibrosis, results in rapid parenchymal deterioration. Atubular glomeruli are formed in many renal disorders, but their identification has been limited by labor-intensive available techniques. The formation of atubular glomeruli was therefore investigated in adult male mice subjected to complete UUO under general anesthesia. In this species, the urinary pole of Bowmans capsule is normally lined by tall parietal epithelial cells similar to those of the proximal tubule, and both avidly bind Lotus tetragonolobus lectin. Following UUO, these cells became flattened, lost their affinity for Lotus lectin, and no longer generated superoxide (revealed by nitroblue tetrazolium infusion). Based on Lotus lectin staining, stereological measurements, and serial section analysis, over 80% of glomeruli underwent marked transformation after 14 days of UUO. The glomerulotubular junction became stenotic and atrophic due to cell death by apoptosis and autophagy, with concomitant remodeling of Bowmans capsule to form atubular glomeruli. In this degenerative process, transformed epithelial cells sealing the urinary pole expressed α-smooth muscle actin, vimentin, and nestin. Although atubular glomeruli remained perfused, renin immunostaining was markedly increased along afferent arterioles, and associated maculae densae disappeared. Numerous progressive kidney disorders, including diabetic nephropathy, are characterized by the formation of atubular glomeruli. The rapidity with which glomerulotubular junctions degenerate, coupled with Lotus lectin as a marker of glomerular integrity, points to new investigative uses for the model of murine UUO focusing on mechanisms of epithelial cell injury and remodeling in addition to fibrogenesis.