Neonatal Unilateral Ureteral Obstruction Research Articles

Renal developmental genes are differentially regulated after unilateral ureteral obstruction in neonatal and adult mice

Congenital obstructive nephropathy hinders normal kidney development. The severity and the duration of obstruction determine the compensatory growth of the contralateral, intact opposite kidney. We investigated the regulation of renal developmental genes, that are relevant in congenital anomalies of the kidney and urinary tract (CAKUT) in obstructed and contralateral (intact opposite) kidneys after unilateral ureteral obstruction (UUO) in neonatal and adult mice. Newborn and adult mice were subjected to complete UUO or sham-operation, and were sacrificed 1, 5, 12 and 19 days later. Quantitative RT-PCR was performed in obstructed, intact opposite kidneys and sham controls for Gdnf, Pax2, Six4, Six2, Dach1, Eya1, Bmp4, and Hnf-1β. Neonatal UUO induced an early and strong upregulation of all genes. In contrast, adult UUO kidneys showed a delayed and less pronounced upregulation. Intact opposite kidneys of neonatal mice revealed a strong upregulation of all developmental genes, whereas intact opposite kidneys of adult mice demonstrated only a weak response. Only neonatal mice exhibited an increase in BMP4 protein expression whereas adult kidneys strongly upregulated phosphatidylinositol 3 kinase class III, essential for compensatory hypertrophy. In conclusion, gene regulation differs in neonatal and adult mice with UUO. Repair and compensatory hypertrophy involve different genetic programs in developing and adult obstructed kidneys.

DAMPs in Unilateral Ureteral Obstruction.

Damage-associated molecular patterns (DAMPs) are released from tubular and interstitial cells in the kidney after unilateral ureteral obstruction (UUO). DAMPs are recognized by pattern recognition receptors (PRRs), which mediate the initiation of an immune response and the release of inflammatory cytokines. The animal model of UUO is used for various purposes. UUO in adult mice serves as a model for accelerated renal fibrosis, which is a hallmark of progressive renal disease. UUO in adult mice enables to study cell death, inflammation, and extracellular matrix deposition in the kidney. Neonatal UUO is a model for congenital obstructive nephropathies. It studies inflammation, apoptosis, and interstitial fibrosis in the neonatal kidney, when nephrogenesis is still ongoing. Following UUO, several DAMPs as well as DAMP receptors are upregulated. In adult UUO, soluble uric acid is upregulated and activates the NOD-like receptor family, pyrin domain containing-3 (NLRP3) inflammasome, which promotes fibrosis, apoptosis, and reactive oxygen species (ROS) injury. Further DAMPs associated with UUO are uromodulin, members of the IL-1 family, and necrotic cell DNA, all of which promote sterile inflammation. In neonatal UUO, the receptor for advanced glycation endproducts (RAGE) is highly upregulated. RAGE is a ligand for several DAMPs, including high mobility group box 1 (HMGB1) and S100 proteins, which play an important role in renal fibrosis. Additionally, necroptosis is an important mechanism of cell death, besides apoptosis, in neonatal UUO. It is highly inflammatory due to release of cytokines and specific DAMPs. The release and recognition of DAMPs initiate sterile inflammation, which makes them good candidates to develop and improve diagnostic and therapeutic strategies in renal fibrosis and congenital obstructive nephropathies.

Heat shock protein 70/nitric oxide effect on stretched tubular epithelial cells linked to WT-1 cytoprotection during neonatal obstructive nephropathy.

Mechanical stress is a key pathogenic driver of apoptosis in the tubular epithelium in obstructive nephropathy. Heat shock protein 70 (Hsp70) and Wilms' tumor (WT-1) have been proposed to represent linked downstream effectors of the cytoprotective properties of NO. In the present study, we sought to evaluate whether the cytoprotective effects of L-arginine in neonatal obstructive nephropathy may be associated with NO-dependent increases in WT-1 and Hsp70 expression. Neonatal Wistar-Kyoto rats were submitted to complete unilateral ureteral obstruction (UUO) and treated thereafter with vehicle, L-NAME or L-arginine by daily gavage for 14days to block or augment NO levels, respectively. Normal rat kidney epithelial cells by NRK-52E were exposed to mechanical stress in vitro in the presence or absence of L-NAME, L-arginine, sodium nitroprusside (SNP), L-arginine+SNP or L-arginine/L-NAME. Induction of apoptosis and the mRNA expression of WT-1 and Hsp70 genes were assessed. WT-1 and Hsp70 genes expression decreased in the presence of L-NAME and following UUO coincident with increased tubular apoptosis. L-arginine treatment increased NO levels, reduced apoptosis and restored expression levels of WT-1 and Hsp70 to control levels. L-arginine treatment in vitro reduced basal apoptotic rates and prevented apoptosis in response to mechanical strain, an effect enhanced by SNP co-incubation. L-NAME increased apoptosis and prevented the anti-apoptotic action of L-arginine. L-arginine treatment in experimental neonatal UUO reduces apoptosis coincident with restoration of WT-1 and Hsp70 expression levels and directly inhibits mechanical strain-induced apoptosis in an NO-dependent manner in vitro. This potentially implicates an NO-Hsp70-WT-1 axis in the cytoprotective effects of L-arginine.

Systems biology combining human- and animal-data miRNA and mRNA data identifies new targets in ureteropelvic junction obstruction

BackgroundAlthough renal fibrosis and inflammation have shown to be involved in the pathophysiology of obstructive nephropathies, molecular mechanisms underlying evolution of these processes remain undetermined. In an attempt towards improved understanding of obstructive nephropathy and improved translatability of the results to clinical practice we have developed a systems biology approach combining omics data of both human and mouse obstructive nephropathy.ResultsWe have studied in parallel the urinary miRNome of infants with ureteropelvic junction obstruction and the kidney tissue miRNome and transcriptome of the corresponding neonatal partial unilateral ureteral obstruction (UUO) mouse model. Several hundreds of miRNAs and mRNAs displayed changed abundance during disease. Combination of miRNAs in both species and associated mRNAs let to the prioritization of five miRNAs and 35 mRNAs associated to disease. In vitro and in vivo validation identified consistent dysregulation of let-7a-5p and miR-29-3p and new potential targets, E3 ubiquitin-protein ligase (DTX4) and neuron navigator 1 (NAV1), potentially involved in fibrotic processes, in obstructive nephropathy in both human and mice that would not be identified otherwise.ConclusionsOur study is the first to correlate a mouse model of neonatal partial UUO with human UPJ obstruction in a comprehensive systems biology analysis. Our data revealed let-7a and miR-29b as molecules potentially involved in the development of fibrosis in UPJ obstruction via the control of DTX4 in both man and mice that would not be identified otherwise.

Growing evidence suggests WT1 effects in the kidney development are modulated by Hsp70/NO interaction.

The study of kidney development at the cellular and molecular levels remains an active area of nephrology research. The functional integrity of the kidney depends on normal development as well as on physiological cell turnover. Apoptosis induction is essential for these mechanisms. A route to cell death revealed in the past decade shows that heat shock proteins (HSPs) and their cofactors are responsible for regulating the apoptotic pathway. Specifically, heat shock protein 70 (Hsp70), the most ubiquitous and highly conserved HSP, helps proteins adopt native conformation or regain function after misfolding. Hsp70 is an important cofactor for the function of Wilms' tumour 1 (WT1) and suggests a potential role for this chaperone during kidney differentiation. In addition, we have demonstrated that WT1 expression is modulated by nitric oxide (NO) availability and Hsp70 interaction after neonatal unilateral ureteral obstruction. NO has been identified as playing an important role in the developing kidney. These findings suggest that Hsp70 and NO may play a critical and fundamental role in the capacity to modulate both apoptotic pathway and oxidative stress during kidney development. Furthermore, the design of experimental protocols that assess renal epithelial functionality in this context, could contribute to the understanding of renal development and alterations.

Chronic unilateral ureteral obstruction in the neonatal mouse delays maturation of both kidneys and leads to late formation of atubular glomeruli

Unilateral ureteral obstruction (UUO) in the adult mouse is the most widely used model of progressive renal disease: the proximal tubule is the nephron segment most severely affected and atubular glomeruli are formed after only 7 days of UUO. To determine the proximal nephron response to UUO in the maturing kidney, neonatal mice were examined 7 to 28 days following complete UUO under general anesthesia. Proximal tubular mass and maturation were determined by staining with Lotus tetragolonobus lectin. Superoxide was localized by nitroblue tetrazolium and collagen by Sirius red. Cell proliferation, cell death, PAX-2, megalin, α-smooth muscle actin (α-SMA), renin, and fibronectin were identified by immunohistochemistry. During the first 14 days of ipsilateral UUO, despite oxidative stress (4-hydroxynonenal staining), glomerulotubular continuity was maintained and mitochondrial superoxide production persisted. However, from 14 to 28 days, papillary growth was impaired and proximal tubules collapsed with increased apoptosis, autophagy, mitochondrial loss, and formation of atubular glomeruli. Fibronectin, α-SMA, and collagen increased in the obstructed kidney. Oxidative stress was present also in the contralateral kidney: renin was decreased, glomerulotubular maturation and papillary growth were delayed, followed by increased cortical and medullary growth. We conclude that neonatal UUO initially delays renal maturation and results in oxidative stress in both kidneys. In contrast to the adult, proximal tubular injury in the neonatal obstructed kidney is delayed at 14 days, followed only later by the formation of atubular glomeruli. Antioxidant therapies directed at proximal tubular mitochondria during early renal maturation may slow progression of congenital obstructive nephropathy.

Wt-1 Expression Linked to Nitric Oxide Availability during Neonatal Obstructive Nephropathy

The wt-1 gene encodes a zinc finger DNA-binding protein that acts as a transcriptional activator or repressor depending on the cellular or chromosomal context. The wt-1 regulates the expression of a large number of genes that have a critical role in kidney development. Congenital obstructive nephropathy disrupts normal renal development and causes chronic progressive interstitial fibrosis, which contributes to renal growth arrest, ultimately leading to chronic renal failure. Wt-1 is downregulated during congenital obstructive nephropathy, leading to apoptosis. Of great interest, nitric oxide bioavailability associated with heat shock protein 70 (Hsp70) interaction may modulate wt-1 mRNA expression, preventing obstruction-induced cell death during neonatal unilateral ureteral obstruction. Moreover, recent genetic researches have allowed characterization of many of the complex interactions among the individual components cited, but the realization of new biochemical, molecular, and functional experiments as proposed in our and other research labs allows us to establish a deeper level of commitment among proteins involved and the potential pathogenic consequences of their imbalance.

Angiotensin AT(1) receptor inhibition-induced apoptosis by RhoA GTPase activation and pERK1/2 signaling pathways in neonatal obstructive nephropathy.

Intrarenal renin-Angiotensin system (RAS) activity is increased during early development and is further enhanced by unilateral ureteral obstruction (UUO). We studied the involvement of mitogen-activated protein (MAP) kinase members and the RhoA GTPase signaling pathways on the regulation of renal cell response after AT1 Angiotensin II receptor inhibition in obstruction. Neonatal rats subjected to sham operation or complete UUO within the first 48 hours of life received saline vehicle, Losartan (AT1 inhibitor), or PD-123319 (AT2 inhibitor) during the first 14 days of life. Cortex tubular epithelial cell apoptotic response was shown by TUNEL and confirmed by electron microscopy associated with mitochondrial signaling pathway through the increased proapoptotic ratio Bax/BcL-2, and consequently increased caspase 3 expression and activity in obstructed kidney before and after Type 1 (AT1) receptor blockade. Non injury of contralateral kidney was shown. The convergence of two independent signal pathways, the RhoA GTPase and pERK and concurrent inhibition of JNK MAP kinase, were required for the apoptotic response in 14 day kidney obstructed tubular cells either with or without Losartan treatment. Absence of increased AT2 protein expression after AT1 receptor inhibition on day 14 of obstruction was shown. Selective AngiotensinAT2-receptor inhibition with PD-123319 had no protective effect on the renal response to complete 14 day UUO. We suggest a role of both RhoA GTPase activation and the opposing actions of the ERK and JNK-MAP kinase signaling pathways as events involved in tubular cell apoptosis regulation in neonatal UUO. The selective AT1-receptor inhibition had no effect on the renal cellular response in the kidney subjected to UUO for 14 days.

Caveolin-1–eNOS/Hsp70 interactions mediate rosuvastatin antifibrotic effects in neonatal obstructive nephropathy

Evidence suggesting that statins may contribute to renoprotection has been provided in experimental and clinical studies. Statins restore endothelial nitric oxide (NO) levels by mechanisms including up-regulation of endothelial NO synthase (eNOS) expression. Caveolin-1/eNOS interaction is essential preventing inadequate NO levels. Here, we evaluated whether caveolin-1 associated with eNOS/Hsp70 expression may be involved in the mechanism by which rosuvastatin exerts tubulointerstitial fibrosis protection in neonatal unilateral ureteral obstruction (UUO). Neonatal rats subjected to UUO within 2 days of birth and controls were treated daily with vehicle or rosuvastatin (10 mg/kg/day) by oral gavage for 14 days. After UUO, morphometric evaluation of interstitial fibrosis showed increased interstitial volume (Vv) associated with reduced NO availability, increased mRNA and protein caveolin-1 expression as well as downregulation eNOS and heat shock protein 70 (Hsp70) expression. Conversely, rosuvastatin treatment attenuated the fibrotic response linked to high NO availability, decreased mRNA and protein caveolin-1 expression, and marked upregulation of eNOS and Hsp70 expression at transcriptional and posttranscriptional levels. Moreover, protein-protein interactions determined by immunoprecipitation and by immunofluorescence co-localization have shown decreased caveolin-1/eNOS as well as increased Hsp70/eNOS interaction, after rosuvastatin treatment. A dose dependent effect of rosuvastatin on decreased caveolin-1 expression was shown in control cortex. In conclusion, our data suggest that statins contribute to the protection against tubulointerstitial fibrosis injury in neonatal early kidney obstruction by increased NO availability, involving interaction of up-regulated eNOS/Hsp70 and down-regulated caveolin-1.

The Nrf2–Keap1 cellular defense pathway and heat shock protein 70 (Hsp70) response. Role in protection against oxidative stress in early neonatal unilateral ureteral obstruction (UUO)

Perturbation of renal tubular antioxidants and overproduction of reactive oxygen species may amplify the proinflammatory state of renal obstruction, culminating in oxidative stress and tubular loss. Here, we analyzed the heat shock protein 70 (Hsp70) response and the function and signal transduction of NF-E2-related protein 2 (Nrf2) transcription factor on oxidative stress modulation in obstruction. Rats were subjected to unilateral ureteral obstruction or sham operation and kidneys harvested at 5, 7, 10, and 14days after obstruction. Hsp70 expression and Nrf2 activity and its downstream target gene products were assessed. After 10 and 14days of obstruction, enhanced lipid peroxidation through higher thiobarbituric acid reactive substances levels and increased oxidative stress resulted in reduced total antioxidant activity and enhanced nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity were demonstrated. This was accompanied by decreased inducible Hsp70 expression and a progressive reduction of nuclear Nrf2 and its target gene products glutathione S-transferase A2 (GSTA2) and NADPH/quinone oxidoreductase 1 (NQO1), whereas the Nrf2 repressor Kelch-like ECH-associated protein-1 (Keap1) was upregulated. By contrast, on early obstruction for 7days, lack of increased oxidative markers associated with higher inducible Hsp70 protein levels and a rapid nuclear accumulation of Nrf2, Keap1 downregulation, and mRNA induction of the identified Nrf2-dependent genes, NQO1 and GSTA2, were shown. For these results, we suggest that the magnitude of cytoprotection in early obstruction depends on the combined contribution of induced activation of Nrf2 upregulating its downstream gene products and Hsp70 response. Impaired ability to mount the biological response to the prevailing oxidative stress leading to renal injury was shown in prolonged obstruction.

Cytoprotective role of nitric oxide associated with Hsp70 expression in neonatal obstructive nephropathy

Nitric oxide (NO) has emerged as an important endogenous inhibitor of apoptosis. In this study, we postulated that the mechanism of apoptosis inhibition by NO would include stimulation of heat shock protein 70 (Hsp70) expression. Rats were subjected to unilateral ureteral obstruction (UUO) or sham operation, and kidneys were harvested 5 and 14 days after obstruction. After 14 days of obstruction, decreased endogenous NO and lower inducible nitric oxide synthase (iNOS) expression at mRNA and protein levels associated with downregulation of Hsp70 protein expression were shown in apoptosis induction, regulated by mitochondrial signal pathway, through the increased pro-apoptotic ratio Bax/BcL 2 and consequently caspase 3 activity. Conversely, 5 days after kidney obstruction, increased Hsp70 expression linked to increase NO and iNOS expression at transcriptional and post-transcriptional levels with absence of apoptotic response, were demonstrated. In obstructed neonatal rats, in vivo administration of l-Arginine induced heat shock protein 70 (Hsp70) expression, which was associated with cytoprotection from apoptosis and transiently decreased nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activity. Opposite effects were obtained after nitro l-Arginine methyl ester ( l-NAME) treatment. The interaction between B-cell lymphoma 2 anti-apoptotic members (BcL 2) and Hsp70 in the presence of l-Arginine and l-NAME, was determined by coimmunoprecipitation. Binding of BcL 2 and Hsp70 increased after l-Arginine administration. These findings suggest that NO can produce resistance to obstruction-induced cell death by mitochondrial apoptotic pathway, through the induction of Hsp70 expression, in neonatal unilateral ureteral obstruction.

Leukocytes Induce Epithelial to Mesenchymal Transition after Unilateral Ureteral Obstruction in Neonatal Mice

Urinary tract obstruction during renal development leads to tubular apoptosis, tubular atrophy, and interstitial fibrosis. Epithelial to mesenchymal transition (EMT) has been proposed as a key mechanism of myofibroblast accumulation in renal fibrosis. We studied the interplay of leukocyte infiltration, tubular apoptosis, and EMT in renal fibrosis induced by unilateral ureteral obstruction (UUO) in neonatal mice. We show that leukocytes mediate tubular apoptosis and EMT in the developing kidney with obstructive nephropathy. Blocking leukocyte recruitment by using the chemokine receptor-1 antagonist BX471 protected against tubular apoptosis and interstitial fibrosis, as evidenced by reduced monocyte influx, a decrease in EMT, and attenuated collagen deposition. EMT was rapidly induced within 24 hours after UUO along with up-regulation of the transcription factors Snail1 and Snail2/Slug, preceding the induction of alpha-smooth muscle actin and vimentin. In the presence of BX471, the expression of chemokines, as well as of Snail1 and Snail2/Slug, in the obstructed kidney was completely attenuated. This was associated with reduced macrophage and T-cell infiltration, tubular apoptosis, and interstitial fibrosis in the developing kidney. Our findings provide evidence that leukocytes induce EMT and renal fibrosis after UUO and suggest that chemokine receptor-1 antagonism may prove beneficial in obstructive nephropathy.

Review : Biochemical-molecular markers in unilateral ureteral obstruction

Congenital obstructive nephropathy is the primary cause of end-stage renal disease in children. Rapid diagnosis and initiation of the treatment are vital to preserve function and/or to slow down renal injury. Obstructive uropathy effects -decline in the plasmatic renal flow and glomerular filtration rate, interstitial infiltrate of leukocytes, significant decrease of the urine concentration, loss of the capacity to concentrate urine as well as fibrosis and apoptosis- are a consequence of a variety of factors that work in complex ways and are still not fully understood. Mediators as angiotensin II, transforming growth factor-beta (TGF-beta) and nitric oxide (NO) have been implicated in congenital obstructive nephropathy. The renin-angiotensin system is regulated in different ways, affecting both renal structure and function, and that it in turn depends upon the duration of the obstruction. On the other hand, the role of nitric oxide in renal injury remains somewhat controversial due to the fact that it can exert opposite effects such as cytoprotective and prooxidant / proapoptotic efects as well as proinflammatory and anti-inflammatory effects. In addition, reactive oxidative species (ROS) might contribute to the progression of renal disease. During unilateral ureteral obstruction induced uncoordinated and aberrant growth may lead to the loss of cellular phenotype and apoptosis. Promoting inflammatory responses, the oxidizers can regulate the adherence of certain molecules and proinflammatory mediators, transcription factors and fibrogenic cytokines, that are clearly involved in the progression of renal disease. The congenital obstructive nephropathy is characterized by tubular atrophy, cellular proliferation, apoptosis and fibrosis; immature kidney is more susceptible than adult kidney to showing the above mentioned alterations. Apoptosis seems to be the principal mechanism that leads to tubular atrophy during the neonatal unilateral ureteral obstruction (UUO). Considering the significant role of the apoptosis in UUO, we believe of big interest the study of the regulatory factors of apoptosis in the renal obstruction neonatal. The complex biochemical and molecular events during the development, maintenance and progression of the renal injury in unilateral ureteral obstruction require further major studies to better understand the alterations mentioned above.

Altered expression of immune modulator and structural genes in neonatal unilateral ureteral obstruction

Congenital obstructive nephropathy is a condition characterized by hydronephrosis, tubular dilatation, apoptosis, and atrophy, as well as interstitial cellular infiltration and progressive interstitial fibrosis. The renal consequences of chronic unilateral ureteral obstruction (UUO) in the neonatal rat are similar to those of clinical congenital obstructive nephropathy. To define alterations in renal gene expression induced by chronic neonatal UUO, Sprague-Dawley rats were subjected to UUO or sham operation within the first 2 days of life, and kidneys were harvested after 12 days. Microarray analysis revealed that the mRNA expression of multiple immune modulators, including krox24, interferon-gamma regulating factor-1 (IRF-1), monocyte chemoattractant protein-1 (MCP-1), interleukin-1beta (IL-1beta), CCAAT/enhancer binding protein (C/EBP), p21, c-fos, c-jun, and pJunB, was significantly increased in obstructed compared to sham-operated kidneys (all P < 0.05). Western blot analysis revealed significant changes in immune modulator protein abundance in the obstructed versus sham-operated kidney for krox24 (P = 0.0004), IRF-1 (P = 0.005), MCP-1 (P = 0.01), and JunD (P = 0.0008). Alternatively, the abundance of all of the immune modulator proteins was similar in sham-operated and obstructed kidneys in rats subjected to acute (4 days) neonatal UUO. Microarray analysis studies also reveal that structural genes that comprise the cytoskeleton and cell matrix are significantly up-regulated by chronic neonatal UUO, including calponin, desmin, dynamin, and lumican (all P < 0.05). Multiple genes are aberrantly expressed in the kidney of rats subjected to chronic neonatal UUO. Elucidation of these genes involved in neonatal UUO may lead to new insight about congenital obstructive nephropathy.

Expression of connexins in the normal and obstructed developing kidney.

Connections between cells are achieved by proteins called connexins that comprise the gap junction. Connexins play a major role in organ development. Our reverse transcription-polymerase chain reaction (RT-PCR) studies demonstrate that Cx30, Cx36, Cx37, Cx40, Cx45, Cx46, and Cx50 are expressed in the kidney. Quantitative RT-PCR indicates that Cx37, Cx45, and Cx46 are preferentially expressed during early renal development. We also explored the expression of connexins in neonatal unilateral ureteral obstruction (UUO). After 12 days of neonatal UUO, the renal mRNA expression of Cx30, Cx37, and Cx40 was significantly elevated. In contrast, there was no change in connexin renal mRNA levels in adult UUO. We conclude that multiple connexins are expressed in the rat kidney and several are aberrantly expressed in neonatal UUO.

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

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. 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. 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). 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.

Interstitial Fibrosis in Chronic Hydronephrosis is Modulated by Angiotensinogen Expression • 1797

The renin-angiotensin system (RAS) may serve pathogenic as well as salutary roles in the development of renal disease, and angiotensin II has been implicated to regulate cellular mechanisms of fibrosis. To advance the understanding of the contribution of the RAS to the progression of renal disease, chronic obstructive nephropathy was studied in neonatal mice having 0, 1, 2 or 4 functional copies of the angiotensinogen gene (Agt) since: 1) all components of RAS are stimulated in obstructive nephropathy; and 2) these mice have genetically determined gradations in their steady-state plasma angiotensinogen levels. Two day-old mice underwent complete unilateral ureteral obstruction (UUO) or a sham operation (n=27 each). Obstructed (UUO), intact opposite (IO), and sham (S) kidneys were examined 28 d later for tubular atrophy and interstitial fibrosis, both hallmarks of chronic obstructive nephropathy. Sham and obstructed zero-copy mice exhibited a similar 20-30% reduction in body weight (P<0.05). In all genotypes, UUO induced a 40-80% reduction in renal mass compared to S, and IO demonstrated compensatory growth of 127-155% (both P<0.05). Compared to S, UUO increased tubular atrophy 100-fold in 1-4 copy mice. The elevated tubular atrophy in S of zero-copy mice was reduced in IO (both P<0.05). Interstitial collagen deposition, an index of fibrosis, increased in UUO linearly with Agt copy number, from 25.4 ± 9.6% in 0-copy mice to 54.2 ± 14.2% in 2-copy mice (R=0.723, slope=14.3 ± 3.3%, P<0.001), but fibrosis was not further elevated by UUO in 4-copy mice (58.5 ± 9.6%). Thus, in chronic neonatal UUO, less than normal levels of angiotensinogen decrease interstitial fibrosis. In contrast, tubular atrophy in the absence of Agt is prominent and not synergistic with UUO. We conclude: 1) Compensatory renal growth is independent of RAS activity; 2) Tubular integrity (measured by severity of tubular atrophy) is impaired in the absence of Atg; 3) Most importantly, this study demonstrates conclusively that the renal fibrotic response to chronic UUO is attenuated by decreasing angiotensinogen expression. It is likely that angiotensin-dependent fibrosis is a mechanism common to the progression of many forms of renal disease or 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 rat. 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 by 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 increase 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.

Arrested development of the neonatal kidney following chronic ureteral obstruction.

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. 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. 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. As revealed by changes in expression of growth-related peptides, neonatal UUO delays ipsilateral renal development, which may contribute to impaired renal growth.

Neonatal ureteral obstruction stimulates recruitment of renin-secreting renal cortical cells

Unilateral ureteral obstruction (UUO) in the neonate increases ipsilateral renal renin gene expression, an effect which is mediated by renal nerves. To determine whether neonatal UUO alters the number of renal cortical cells secreting renin and whether this change is modulated by renal nerve activity, newborn Sprague-Dawley rats were subjected to left UUO, right uninephrectomy, or sham operation and studied four weeks thereafter. To evaluate the importance of renal nerves in this response, an additional group of animals underwent chemical sympathectomy with guanethidine. Ureteral obstruction was associated with marked reduction in renal mass in the obstructed kidney and contralateral compensatory hypertrophy, changes which were not altered by sympathectomy. Renin messenger RNA and renal renin content were elevated in the obstructed kidney. The number of cells secreting renin, measured by the reverse hemolytic plaque assay, was markedly increased in the obstructed kidney (45 +/- 18 plaques/slide vs. 11 +/- 1 plaques/slide in sham animals), but not in the opposite kidney or following uninephrectomy. This effect was not significantly altered by sympathectomy. There was no change in the amount of renin secreted per cell or in the secretory response to Ca++. These results show that UUO results in recruitment of cells not previously secreting renin by a mechanism independent of renal nerve activity. This recruitment occurs without alteration of the quantity of renin secreted per cell or in the normal regulatory effect of Ca++ on renin secretion. An increase in the number of renin-secreting cells may contribute to the activation of the renin-angiotensin system, and thus to the vasoconstriction observed following ureteral obstruction.