The swan-neck lesion: proximal tubular adaptation to oxidative stress in nephropathic cystinosis.

Cystinosis is an inherited disorder resulting from a mutation in the CTNS gene, causing progressive proximal tubular cell flattening, the so-called swan-neck lesion (SNL), and eventual renal failure. To determine the role of oxidative stress in cystinosis, histologic sections of kidneys from C57BL/6 Ctns(-/-) and wild-type mice were examined by immunohistochemistry and morphometry from 1 wk to 20 mo of age. Additional mice were treated from 1 to 6 mo with vehicle or mitoquinone (MitoQ), an antioxidant targeted to mitochondria. The leading edge of the SNL lost mitochondria and superoxide production, and became surrounded by a thickened tubular basement membrane. Progression of the SNL as determined by staining with lectin from Lotus tetragonolobus accelerated after 3 mo, but was delayed by treatment with MitoQ (38 ± 4% vs. 28 ± 1%, P < 0.01). Through 9 mo, glomeruli had retained renin staining and intact macula densa, whereas SNL expressed transgelin, an actin-binding protein, but neither kidney injury molecule-1 (KIM-1) nor cell death was observed. After 9 mo, clusters of proximal tubules exhibited localized oxidative stress (4-hydroxynonenal binding), expressed KIM-1, and underwent apoptosis, leading to the formation of atubular glomeruli and accumulation of interstitial collagen. We conclude that nephron integrity is initially maintained in the Ctns(-/-) mouse by adaptive flattening of cells of the SNL through loss of mitochondria, upregulation of transgelin, and thickened basement membrane. This adaptation ultimately fails in adulthood, with proximal tubular disruption, formation of atubular glomeruli, and renal failure. Antioxidant treatment targeted to mitochondria delays initiation of the SNL, and may provide therapeutic benefit in children with cystinosis.

Responses of proximal tubular cells to injury in congenital renal disease: fight or flight.

Most chronic kidney disease in children results from congenital or inherited disorders, which can be studied in mouse models. Following 2 weeks of unilateral ureteral obstruction (UUO) in the adult mouse, nephron loss is due to proximal tubular mitochondrial injury and cell death. In neonatal mice, proximal tubular cell death is delayed beyond 2 weeks of complete UUO, and release of partial UUO allows remodeling of remaining nephrons. Progressive cyst expansion develops in polycystic kidney disease (PKD), a common inherited renal disorder. The polycystic kidney and fibrosis (pcy)-mutant mouse (which develops late-onset PKD) develops thinning of the glomerulotubular junction in parallel with growth of cysts in adulthood. Renal insufficiency in nephropathic cystinosis, a rare inherited renal disorder, results from progressive tubular cystine accumulation. In the Ctns knockout mouse (a model of cystinosis), proximal tubular cells become flattened, with loss of mitochondria and thickening of tubular basement membrane. In each model, persistent obstructive or metabolic stress leads ultimately to the formation of atubular glomeruli. The initial "fight" response (proximal tubular survival) switches to a "flight" response (proximal tubular cell death) with ongoing oxidative injury and mitochondrial damage. Therapies should be directed at reducing proximal tubular mitochondrial oxidative injury to enhance repair and regeneration.

Formation of atubular glomeruli in the developing kidney following chronic urinary tract obstruction.

Congenital urinary tract obstruction is a major cause of progressive renal disease in children. We developed a model of partial unilateral ureteral obstruction (UUO) in the neonatal mouse, in which nephrogenesis at birth is similar to that of the midtrimester human fetus. The proximal tubule responds to UUO by undergoing apoptosis and necrosis, likely due to mitochondrial sensitivity to hypoxia and reactive oxygen species in the face of reduced endogenous antiapoptotic factors such as eNOS. Damage to the glomerulotubular junction is followed by scission and formation of atubular glomeruli and aglomerular tubules. This is an orchestrated process, with atubular glomeruli surrounded by a continuous layer of regenerated parietal epithelial cells. Relief of UUO at 7 days of age results in remodeling of the renal parenchyma by adulthood. In contrast to proximal tubular destruction, collecting ducts remain dilated and patent, with remodeling due to apoptosis and proliferation (a process associated with recruitment of intercalated cells as progenitor cells following UUO in the fetal monkey). Formation of atubular glomeruli occurs in other renal disorders (congenital nephrotic syndrome and cystinosis), and may represent a maladaptive response to proximal tubular injury reflecting an evolutionary adaptation by an ancestor we share with aglomerular marine fish.

Inducible nitric oxide synthase modulates hydronephrosis following partial or complete unilateral ureteral obstruction in the neonatal mouse.

To investigate the role of endogenous inducible nitric oxide synthase (iNOS) in the response of the developing kidney to unilateral ureteral obstruction (UUO), neonatal iNOS null mutant (-/-) and wild-type (WT) mice were subjected to partial or complete UUO. At 7 and 21 days of age, apoptosis, renin, vascular endothelial growth factor (VEGF), fibroblasts (anti-fibroblast-specific peptide 1), myofibroblasts (alpha-smooth muscle actin), macrophages (F4/80), and collagen were measured in kidney tissue. Compared with WT, renal parenchymal thickness was increased, with preservation of the papilla, in -/- mice with partial UUO, but decreased in -/- mice with complete UUO. Ureteral peristalsis increased with severity of pelvic dilatation in WT, and increased further in -/- mice with partial UUO. Apoptosis, fibroblasts, and macrophages were increased in -/- mice with complete UUO, but there was no effect of iNOS on other histological parameters following complete UUO. Renin was decreased in -/- mice with partial UUO. There was no effect of iNOS genotype on renal collagen accumulation at either 7 or 21 days of age. These results are consistent with an injurious role for endogenous iNOS following partial UUO by inhibiting ureteral peristalsis and increasing renal renin although renal fibrosis is not affected. In contrast, in mice with complete UUO, iNOS attenuates apoptosis and enhances renal parenchymal thickness. Alterations in the severity of ureteral obstruction may therefore influence the effect of iNOS on long-term renal injury.

Angiotensin AT1-receptor inhibition exacerbates renal injury resulting from partial unilateral ureteral obstruction in the neonatal rat.

The renin-angiotensin system is activated in the developing kidney and is necessary for normal renal development, but is further activated by unilateral ureteral obstruction (UUO). During nephrogenesis, there is a switch from a preponderance of angiotensin AT(2) to AT(1) receptors in the rat. We examined the renal cellular response to angiotensin II receptor inhibition in the neonatal rat subjected to partial UUO under anesthesia within 48 h of birth. Group I ("early") received saline vehicle, losartan (AT(1) inhibitor), or PD-123319 (AT(2) inhibitor) during the completion of nephrogenesis in the first 10 days of life. Group II ("late") received each of the three treatments throughout the subsequent 10 days of life. Kidneys were harvested at 21 days, and the distribution of renin, apoptosis, macrophages, alpha-smooth muscle actin, and collagen was determined. Losartan and PD-123319 each increased vascular renin distribution in both kidneys. Partial UUO reduced growth and increased apoptosis, macrophages, alpha-smooth muscle actin, and collagen in the obstructed kidney. Early losartan treatment further increased alpha-smooth muscle actin and collagen in the obstructed kidney and induced apoptosis, macrophages, and collagen in the contralateral kidney. Late losartan treatment had no effect on any of the parameters in either kidney, and PD-123319 had no effect on either kidney. We conclude that selective inhibition of AT(1) receptors during nephrogenesis (but not during subsequent renal maturation) exacerbates injury to the obstructed kidney and also injures the contralateral kidney. These results suggest that angiotensin II receptor blockers should be avoided in the developing hydronephrotic kidney.

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.

Ablation of renin-expressing juxtaglomerular cells results in a distinct kidney phenotype.

Renin-expressing cells are peculiar in that they act as differentiated cells, producing the hormone renin, while they also seem to act as progenitors for other renal cell types. As such, they may have functions independent of their ability to generate renin/angiotensin. To test this hypothesis, we ablated renin-expressing cells during development by placing diphtheria toxin A chain (DTA) under control of the Ren1d mouse renin promoter by homologous recombination in a two-renin gene strain (Ren2 and Ren1d). Renin-expressing cells are essentially absent from kidneys in homozygotes (DTA/DTA) which, unlike wild-type mice, are unable to recruit renin-expressing cells when homeostasis is threatened. In contrast, renin staining in the submandibular gland (SMG), which expresses mainly Ren2, is normal. Homozygous mice survive normally, but the kidneys are small and have morphological abnormalities: 25% of the glomeruli are hyperplastic or atrophic, tubules are dilated and atrophic, and areas of undifferentiated cells exist near the atrophic glomeruli and tubules. However, in contrast to the very abnormal renal vessels found when renin-angiotensin system genes are deleted, the kidney vessels in homozygotes have normal wall thickness and no decrease in lumen size. Homozygotes have severely reduced kidney and plasma renin concentrations and females have reduced blood pressure. Homozygotes have elevated blood urea nitrogen and potassium levels, which are suggestive of altered renal function. We conclude that renin cells per se are necessary for the morphological integrity of the kidney and may have a role in maintenance of normal kidney function.

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.

Selectins mediate macrophage infiltration in obstructive nephropathy in newborn mice.

BACKGROUND: Urinary tract obstruction during development leads to tubular atrophy and causes interstitial fibrosis. Macrophage infiltration into the interstitium plays a central role in this process. Selectins, a family of three adhesion molecules, are involved in leukocyte recruitment to sites of inflammation and immune activity. We investigated the role of selectins in obstructive nephropathy in newborn mice. METHODS: Triple selectin-deficient mice (EPL-/-), L-selectin deficient mice (L-/-) and wild type mice (WT) were subjected to complete unilateral ureteral obstruction (UUO) or sham operation within the first 48 hours of life, and were sacrificed 5 and 12 days later. Kidneys were removed, and sections were stained for macrophage infiltration (mAb F4/80), apoptosis (TUNEL), tubular atrophy (periodic acid-Schiff) and interstitial fibrosis (Masson trichrome). RESULTS: Selectin deficient mice showed a marked reduction in macrophage infiltration into the obstructed kidney compared to WT at day 5 and day 12 after UUO. Tubular apoptosis was strongly reduced in EPL-/- at day 5 after UUO, and in EPL-/- and L-/- at day 12 after UUO when compared to WT. The number of apoptotic tubular cells was correlated with macrophage infiltration, suggesting that macrophages stimulate tubular apoptosis in obstructive nephropathy. In addition, tubular atrophy and interstitial fibrosis were significantly diminished in EPL-/- and L-/- compared to WT at day 12 after UUO. CONCLUSION: Following UUO, selectins mediate macrophage infiltration into the obstructed kidney, which in turn may induce tubular apoptosis, tubular atrophy and interstitial fibrosis.