A population of mitochondrion-rich cells in the pars recta of mouse kidney.

Following perfusion of adult mouse kidney with a solution of nitroblue tetrazolium (NBT), certain epithelial cells in the pars recta (S3) segments of proximal tubules react to form cytoplasmic deposits of blue diformazan particles. Such cells are characterized by dark cytoplasm, small and often elliptical nuclei, elaborate, process-bearing profiles, and abundant mitochondria. The atypical epithelial cells display the additional characteristic of immunoreactivity for a wide spectrum of antigens, including mesenchymal proteins such as vimentin. Though present in kidneys of untreated or sham-operated animals, they are particularly evident under experimental conditions such as unilateral ureteral obstruction (UUO), appearing in both contralateral and obstructed kidneys over the course of a week's duration, but disappearing from the obstructed kidney as it undergoes the profound atrophy attributable to deterioration of the population of its proximal tubules. The cells do not appear in neonatal kidneys, even those undergoing UUO, but begin to be recognizable soon after weaning (28 days). It is possible that diformazan-positive cells in the mouse S3 tubular segment constitute a resident population of cells that can replenish or augment the tubule. Although somewhat similar cells, with dark cytoplasm and vimentin expression, have been described in human, rat, and transgenic mouse kidney (Smeets et al. in J Pathol 229: 645-659, 2013; Berger et al. in Proc Natl Acad Sci U S A 111: 1533-1538, 2014), those cells-known as "scattered tubule cells" or "proximal tubule rare cells"- differ from the S3-specific cells in that they are present throughout the entire proximal tubule, often lack a brush border, and have only a few mitochondria.

Glomerulotubular disconnection in neonatal mice after relief of partial ureteral obstruction.

Ureteropelvic junction obstruction is a common cause of congenital obstructive nephropathy. To study the pathogenesis of nephropathy, a variable-partial, complete or a sham unilateral ureteral obstruction (UUO) was produced in mice within 2 days of birth. The obstruction was released in some animals at 7 days and kidneys harvested at 7-42 days of age for histologic and morphometric study. Renal parenchymal growth was stunted by partial UUO with the impairment proportional to the duration and severity of obstruction. Proximal tubule apoptosis and glomerulotubular disconnection led to nephron loss. Relief of partial UUO arrested glomerulotubular disconnection, resolved tubule atrophy, and interstitial fibrosis with remodeling of the renal architecture. Relief of severe UUO did not result in recovery. Compensatory growth of the contralateral kidney depended on the severity of obstruction. Our studies indicate that relief of moderate UUO will minimize nephron loss. Application of this technique to mutant mice will help develop future therapies to enhance nephron recovery.

Osteopontin regulates renal apoptosis and interstitial fibrosis in neonatal chronic unilateral ureteral obstruction.

Congenital obstructive nephropathy is a major cause of renal insufficiency in children. Osteopontin (OPN) is a phosphoprotein produced by the kidney that mediates cell adhesion and migration. We investigated the role of OPN in the renal response to unilateral ureteral obstruction (UUO) in neonatal mice. OPN null mutant (-/-) and wild-type (+/+) mice were subjected to sham operation or UUO within the first 2 days of life. At 7 and 21 days of age, fibroblasts (fibroblast-specific protein (FSP)-1), myofibroblasts (alpha-smooth muscle actin (SMA)), and macrophages (F4/80) were identified by immunohistochemical staining. Apoptotic cells were detected by terminal deoxy transferase uridine triphosphate nick end-labeling technique and interstitial collagen by Masson trichrome or picrosirius red stain. Compared to sham-operated or contralateral kidneys, obstructed kidneys showed increases in all parameters by 7 days, with further increases by 21 days. After 21 days UUO, there was an increase in tubular and interstitial apoptosis in OPN -/- mice as compared to +/+ animals (P<0.05). However, FSP-1- and alpha-SMA-positive cells and collagen in the obstructed kidney were decreased in OPN -/- compared to +/+ mice (P<0.05), whereas the interstitial macrophage population did not differ between groups. We conclude that OPN plays a significant role in the recruitment and activation of interstitial fibroblasts to myofibroblasts in the progression of interstitial fibrosis in the developing hydronephrotic kidney. However, OPN also suppresses apoptosis. Future approaches to limit the progression of obstructive nephropathy in the developing kidney will require targeting of specific renal compartments.

Distinct roles of Mac-1 and its counter-receptors in neonatal obstructive nephropathy.

Urinary tract obstruction during renal development leads to tubular atrophy and interstitial fibrosis. Inflammatory macrophages are crucial in this process, and beta2-integrins play a major role in leukocyte recruitment. We investigated the role of beta2-integrins and their major counter-receptors (intercellular adhesion molecule-1 (ICAM-1), receptor for advanced glycation endproducts (RAGE), junctional adhesion molecule (JAM)-C) in obstructive nephropathy in neonatal mice. Two-day-old beta2-integrin-deficient mice (Mac-1-/- and LFA-1-/-(deficient for leukocyte function-associated antigen-1)) and wild-type mice (C57BL/6) underwent unilateral ureteral obstruction (UUO) or sham operation. After 1, 5 or 12 days of obstruction, renal macrophage infiltration and tubulointerstitial damage were quantitated. Tissue abundance of Mac-1 and its ligands ICAM-1, RAGE and JAM-C was examined by Western blot and immunoprecipitation. Deficiency of either integrin was associated with reduced early macrophage invasion into the obstructed kidney. After 12 days of UUO, macrophage infiltration and tubulointerstitial injury were reduced only in Mac-1-/- but not in LFA-1-/- mice. Besides ICAM-1, an upregulation of two novel Mac-1 ligands, RAGE and JAM-C were observed, however, with distinct time courses. We conclude that beta2-integrins mediate macrophage infiltration in UUO. Mac-1 is the predominant leukocyte integrin involved in leukocyte recruitment after obstruction. ICAM-1 and its new ligands RAGE and JAM-C are sequentially activated in UUO. Blocking of Mac-1 and its ligands may confer synergistic renoprotective effects in neonatal obstructive nephropathy.

Renal apoptosis parallels ceramide content after prolonged ureteral obstruction in the neonatal rat.

Obstructive nephropathy, the primary cause of renal insufficiency in infants, is characterized by progressive renal apoptosis. Ceramide is a sphingolipid known to stimulate apoptosis in the kidney. We investigated the effects of unilateral ureteral obstruction (UUO) on endogenous renal ceramide content and apoptosis in neonatal and adult rats. Animals were subjected to UUO or sham operation on the first day of life and were studied 3-28 days later. Adult rats were similarly treated and then studied 3 or 14 days later. In additional neonatal rats, the obstruction was removed after 5 days, with study at 14 or 28 days. Renal ceramide content was measured by diacylglycerol kinase assay, and apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end-labeling technique. Renal ceramide content was 50-fold higher in the 3-day neonatal compared with the adult kidney and 10-fold higher in the 7-day neonatal compared with the adult kidney, but there was no additional effect of UUO on ceramide content at these ages. However, after 14 or 28 days UUO in the neonate, renal ceramide was elevated compared with sham or intact opposite kidneys, and renal apoptosis was directly related to ceramide content (r = 0.99, P < 0.001). Moreover, renal ceramide was reduced by relief of obstruction (P < 0.05). There was less apoptosis in the obstructed kidney of the adult than the neonate, and UUO had no effect on ceramide content at 14 days in the adult. We conclude that prolonged UUO (at least 14 days duration) increases endogenous renal ceramide in the neonatal but not the adult rat. It is likely that this contributes to the prolonged renal apoptotic response of the neonatal obstructed kidney.

The C sign: more specific for flatfoot deformity than subtalar coalition.

OBJECTIVE: To assess the sensitivity and specificity of the C sign, a C-shaped line created by the outline of the talar dome and the inferior margin of the sustentaculum tali on lateral ankle radiographs, for patients with both flatfoot deformity and specifically talocalcaneal (TC) coalition. DESIGN AND PATIENTS: All patients in this retrospective study were under 35 years of age and had a lateral ankle radiograph and follow-up CT scan for a non-traumatic indication. Forty-eight cases over the past 5 years fulfilled these criteria. Two masked musculoskeletal radiologists determined the presence or absence of the C sign for each lateral radiograph by consensus. Each CT study was then assessed by a third musculoskeletal radiologist for the presence of tarsal coalition. Observations were correlated with clinical history regarding presence or absence of flatfoot deformity. RESULTS: Ten cases of TC coalition were diagnosed, four of which demonstrated a C sign (40%). Eight cases with a C sign were encountered, four of which had TC coalition (50%) and four did not. All patients with a positive C sign had a flatfoot clinically (100%), while only eight of 24 flatfooted patients had a C sign (33%). CONCLUSION: The C sign is specific, but not sensitive, for flatfoot deformity, and is neither sensitive nor specific for subtalar coalition.

Unilateral ureteral obstruction in neonatal rats leads to renal insufficiency in adulthood.

BACKGROUND: Although unilateral ureteropelvic junction obstruction is the most common cause of congenital obstructive nephropathy in infants and children, management remains controversial, and follow-up after pyeloplasty is generally limited to the pediatric ages. We have developed a model of temporary unilateral ureteral obstruction (UUO) in the neonatal rat: One month following the relief of five-day UUO, the glomerular filtration rate (GFR) of the postobstructed kidney was normal despite a 40% reduction in the number of glomeruli and residual vascular, glomerular, tubular, and interstitial injury. METHODS: To determine whether hyperfiltration and residual injury of remaining nephrons leads to progression of renal insufficiency in later life, 31 rats were sham operated or subjected to left UUO at one day of age, with relief of UUO five days later, and were studied at one year of age. GFR was measured by inulin clearance, and the number of glomeruli, tubular atrophy, glomerular sclerosis, and interstitial fibrosis were measured by histomorphometry in sham, obstructed (UUO), and intact opposite kidneys. Intrarenal macrophages and alpha-smooth muscle actin were identified by immunohistochemistry. RESULTS: Despite relief of UUO, ultimate growth of the postobstructed kidney was impaired. The number of glomeruli was reduced by 40%, and GFR was decreased by 80%. However, despite significant compensatory growth of the opposite kidney, there was no compensatory increase in GFR, and proteinuria was increased. Moreover, glomerular sclerosis, tubular atrophy, macrophage infiltration, and interstitial fibrosis were significantly increased not only in the postobstructed kidney, but also in the opposite kidney. CONCLUSIONS: Although GFR is initially maintained following relief of five-day UUO in the neonatal rat, there is eventual profound loss of function of the postobstructed and opposite kidneys because of progressive tubulointerstitial and glomerular damage. These findings suggest that despite normal postoperative GFR in infancy, children undergoing pyeloplasty for ureteropelvic junction obstruction should be followed into adulthood. Elucidation of the cellular response to temporary UUO may lead to improved methods to assess renal growth, injury, and functional reserve in patients with congenital obstructive nephropathy.

Angiotensin stimulates TGF-beta1 and clusterin in the hydronephrotic neonatal rat kidney.

Unilateral ureteral obstruction (UUO) induces activation of the renin-angiotensin system and upregulation of transforming growth factor-beta1 (TGF-beta1; a cytokine modulating cellular adhesion and fibrogenesis) and clusterin (a glycoprotein produced in response to cellular injury). This study was designed to examine the regulation of renal TGF-beta1 and clusterin by ANG II in the neonatal rat. Animals were subjected to UUO in the first 2 days of life, and renal TGF-beta1 and clusterin mRNA were measured 3 days later. Rats were divided into treatment groups receiving saline vehicle, ANG, losartan (AT(1) receptor inhibitor), or PD-123319 (AT(2) receptor inhibitor). ANG stimulated renal TGF-beta1 expression via AT(1) receptors, a response similar to that in the adult. In contrast, clusterin expression was stimulated via AT(2) receptors, a response differing from that in the adult, in which ANG inhibits clusterin expression via AT(1) receptors. We speculate that the unique response of the neonatal hydronephrotic kidney to ANG II is due to the preponderance of AT(2) receptors in the developing kidney.

Apoptosis parallels ceramide content in the developing rat kidney.

Ceramide is emerging as an important hydrophobic sphingolipid involved in cell differentiation and apoptosis. Since apoptosis plays a significant role in cellular remodeling during renal morphogenesis, we measured ceramide content and apoptosis in the fetal (18 days gestation), neonatal (3, 7, and 14 days postnatal), and adult rat kidney. In addition, to determine whether developmental changes in ceramide content are tissue-specific, we compared renal ceramide content with that in lung and liver. Ceramide was measured by the diacylglycerol kinase assay, and apoptosis was determined by the TUNEL technique. Renal ceramide content fell over 100-fold from the fetus to the 7th postnatal day. Renal apoptosis paralleled ceramide content, with a greater than 300-fold decrease in apoptosis from fetal to adult life. Ceramide content of the lung and liver was significantly less than that of the kidney, and changed less with maturation. We conclude that maturational changes in ceramide content are tissue-specific, and that the high rate of apoptosis in the developing kidney may be related to the elevated ceramide content.

EGF improves recovery following relief of unilateral ureteral obstruction in the neonatal rat.

PURPOSE: Renal epidermal growth factor (EGF) is suppressed by unilateral ureteral obstruction (UUO), and we reported previously that exogenous EGF attenuates renal injury due to UUO in the neonatal rat. In this study, we wished to determine whether administration of epidermal growth factor (EGF) improves long-term renal cellular recovery after relief of obstruction. MATERIALS AND METHODS: One ureter of 1 day-old rats was occluded or sham-operated, and rats received daily injections of EGF, 0.1 mg./kg., or saline for the following 7 days. Five days following UUO, the obstruction was removed. Kidneys were removed 28 days following release of UUO or sham operation, and processed for histomorphometry and immunohistochemistry. RESULTS: Kidney weight and the number of glomeruli were reduced in the postobstructed kidney regardless of administration of EGF. However, EGF reduced tubular vimentin by 36% and clusterin expression by 70% (markers of tubular injury), and decreased tubular atrophy by 50% in the postobstructed kidney compared with saline-treated rats. EGF also reduced interstitial alpha-smooth muscle actin and interstitial collagen deposition by 50% in the postobstructed kidney. CONCLUSIONS: Short-term administration of EGF markedly attenuates both tubular and interstitial injury one month following the release of UUO in the neonatal rat. This suggests therapeutic potential for targeted delivery of growth factors to optimize recovery after release of urinary tract obstruction.

Renal cellular response to ureteral obstruction: role of maturation and angiotensin II.

Renal angiotensin II (ANG II) is increased as a result of unilateral ureteral obstruction (UUO), and angiotensin AT(2) receptors predominate over AT(1) receptors in the early postnatal period. To examine the renal cellular response to 3-day UUO in the neonatal and adult rat, AT(1) and AT(2) receptors were inhibited by losartan and PD-123319, respectively. Additional rats received exogenous ANG II, 0.5 mg. kg(-1). day(-1). Renal cellular proliferation and apoptosis were quantitated by proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling technique, respectively. In the neonate, UUO reduced proliferation and increased tubular apoptosis. Losartan had no detectable cellular effect, whereas PD-123319 increased cellular proliferation and suppressed apoptosis, and exogenous ANG II stimulated apoptosis. In the adult, UUO increased cellular proliferation as well as apoptosis, whereas losartan, PD-123319, and exogenous ANG II did not alter the cellular response. In conclusion, UUO impairs renal growth in the neonate by reducing proliferation and stimulating apoptosis, at least in part through angiotensin AT(2) receptors. UUO stimulates both renal cellular proliferation and apoptosis in the adult, but these effects are independent of ANG II. We speculate that the unique early responses of the developing kidney to urinary tract obstruction are mediated by a highly activated renin-angiotensin system and preponderance of AT(2) receptors.

Effect of peroxisome proliferator-activated receptor alpha activators on tumor necrosis factor expression in mice during endotoxemia.

Inflammatory mediators orchestrate the host immune and metabolic response to acute bacterial infections and mediate the events leading to septic shock. Tumor necrosis factor (TNF) has long been identified as one of the proximal mediators of endotoxin action. Recent studies have implicated peroxisome proliferator-activated receptor alpha (PPARalpha) as a potential target to modulate regulation of the immune response. Since PPARalpha activators, which are hypolipidemic drugs, are being prescribed for a significant population of older patients, it is important to determine the impact of these drugs on the host response to acute inflammation. Therefore, we examined the role of PPARalpha activators on the regulation of TNF expression in a mouse model of endotoxemia. CD-1 mice treated with dietary fenofibrate or Wy-14,643 had fivefold-higher lipopolysaccharide (LPS)-induced TNF plasma levels than LPS-treated control-fed animals. Higher LPS-induced TNF levels in drug-fed animals were reflected physiologically in significantly lower glucose levels in plasma and a significantly lower 50% lethal dose than those in LPS-treated control-fed animals. Utilizing PPARalpha wild-type (WT) and knockout (KO) mice, we showed that the effect of fenofibrate on LPS-induced TNF expression was indeed mediated by PPARalpha. PPARalpha WT mice fed fenofibrate also had a fivefold increase in LPS-induced TNF levels in plasma compared to control-fed animals. However, LPS-induced TNF levels were significantly decreased and glucose levels in plasma were significantly increased in PPARalpha KO mice fed fenofibrate compared to those in control-fed animals. Data from peritoneal macrophage studies indicate that Wy-14,643 modestly decreased TNF expression in vitro. Similarly, overexpression of PPARalpha in 293T cells decreased activity of a human TNF promoter-luciferase construct. The results from these studies suggest that any anti-inflammatory activity of PPARalpha in vivo can be masked by other systemic effects of PPARalpha activators.

Unilateral ureteral obstruction impairs renal antioxidant enzyme activation during sodium depletion.

BACKGROUND: Obstructive nephropathy leads to progressive renal tubular atrophy and interstitial fibrosis and is associated with sodium wasting and sodium depletion. Renal damage resulting from unilateral ureteral obstruction (UUO) may be aggravated by reactive oxygen species (ROS), which are produced by a variety of processes. Ideally, deleterious effects of ROS are attenuated by antioxidant enzymes, including the superoxide dismutases, glutathione peroxidases, catalase, and glutathione-S-transferases. The general paradigm is that tissue damage occurs when ROS production is greater than the protective capacity of the antioxidant enzymes. METHODS: This study was designed to investigate the response of renal antioxidant enzymes to UUO and sodium depletion. Adult, male Sprague-Dawley rats received normal-sodium or sodium-depleted siets and were subjected to UUO or sham operation. Obstructed (UUO), intact opposite, or sham-operated kidneys were harvested after 14 days, and antioxidant enzyme activities were measured in kidney homogenates. Thiobarbituric acid reactive substances were measured in these homogenates at 3 and 14 days after UUO or sham operation as an index of ROS production. RESULTS: Renal interstitial area, a measure of fibrosis, was increased by UUO and was doubled in sodium-depleted animals. Sodium depletion increased manganese superoxide dismutase, glutathione peroxidases, and glutathione-S-transferase activities in sham-operated kidneys but not in UUO kidneys. Relative to intact opposite kidneys, UUO kidneys had reduced activities of catalase, manganese superoxide dismutase, and glutathione-S-transferase in normal-sodium animals and all antioxidant enzymes tested in sodium-depleted animals. Renal thiobarbituric acid reactive substances were increased by three days of UUO and were increased further by 14 days of sodium depletion. CONCLUSION: In summary, sodium depletion increased several renal antioxidant enzymes, consistent with a stress response to increased ROS production. Further, UUO not only reduced antioxidant enzyme activities but also inhibited increases seen with sodium depletion. We conclude that suppression of renal antioxidant enzyme activities by UUO contributes to the progression of renal injury in obstructive nephropathy, a process exacerbated by sodium depletion.

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.

Recovery following relief of unilateral ureteral obstruction in the neonatal rat.

BACKGROUND: Obstructive nephropathy is a primary cause of renal insufficiency in infants and children. This study was designed to distinguish the reversible and irreversible cellular consequences of temporary unilateral ureteral obstruction (UUO) on the developing kidney. METHODS: Rats were subjected to UUO or sham operation in the first 48 hours of life, and the obstruction was removed five days later (or was left in place). Kidneys were removed for study 14 or 28 days later. In additional groups, kidneys were removed at the end of five days of obstruction. Immunoreactive distribution of renin was determined in arterioles, and the distribution of epidermal growth factor, transforming growth factor-beta1, clusterin, vimentin, and alpha-smooth muscle actin was determined in tubules and/or interstitium. The number of glomeruli, glomerular maturation, tubular atrophy, and interstitial collagen deposition was determined by morphometry. Renal cellular proliferation and apoptosis were measured by proliferating cell nuclear antigen and the TdT uridine-nick-end-label technique, respectively. The glomerular filtration rate was measured by inulin clearance. RESULTS: Renal microvascular renin maintained a fetal distribution with persistent UUO; this was partially reversed by the relief of obstruction. Although glomerular maturation was also delayed and glomerular volume was reduced by UUO, the relief of obstruction prevented the reduction in glomerular volume. Although relief of obstruction did not reverse a 40% reduction in the number of nephrons, the glomerular filtration rate of the postobstructed kidney was normal. The relief of obstruction did not improve tubular cell proliferation and only partially reduced apoptosis induced by UUO. This was associated with a persistent reduction in the tubular epidermal growth factor. In addition, the relief of obstruction reduced but did not normalize tubular expression of transforming growth factor-beta1, clusterin, and vimentin, all of which are evidence of persistent tubular injury. The relief of obstruction significantly reduced interstitial fibrosis and expression of alpha-smooth muscle actin by interstitial fibroblasts, but not to normal levels. CONCLUSIONS: The relief of obstruction in the neonatal rat attenuates, but does not reverse, renal vascular, glomerular, tubular, and interstitial injury resulting from five days of UUO. Hyperfiltration by remaining nephrons and residual tubulointerstitial injury in the postobstructed kidney are likely to lead to deterioration of renal function later in life.

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.

Tissue-specific regulation of growth factors and clusterin by angiotensin II.

Angiotensin II (ANG II) has been implicated in the hypertrophic and fibrotic responses of the heart and kidney to systemic hypertension. To determine whether these actions of ANG II are related to tissue-specific stimulation of growth factors, we infused adult Sprague-Dawley rats with ANG II at 50 ng/min (low dose), 100 ng/min (high dose), or vehicle for 1 week. Rats receiving vehicle or low-dose ANG II were normotensive with normal plasma aldosterone concentration, whereas rats receiving high-dose ANG II were hypertensive with increased plasma aldosterone. Tissue fibrosis was quantified morphometrically, and messenger RNA (mRNA) for transforming growth factor-beta1 (TGF-beta1) and prepro-epidermal growth factor (EGF) was measured in liver, heart, and renal glomeruli and tubules. In addition, mRNA was determined for clusterin, a glycoprotein expressed in response to tissue injury. Compared to vehicle, low-dose ANG II increased TGF-beta1 expression in glomeruli, tubules, and heart, but not in liver, and increased EGF expression in renal tubules only. High-dose ANG II decreased clusterin expression in liver only. Fibrosis was induced by low- and high-dose ANG II in kidney and heart, but not in liver. We conclude that ANG II selectively stimulates TGF-beta1 mRNA in the heart and kidney, which may contribute to cardiac and renal interstitial fibrosis resulting from activation of the renin-angiotensin system independent of hypertension. By stimulating cellular proliferation, selective stimulation by ANG II of EGF in renal tubules may amplify the effects of TGF-beta1. Suppression of clusterin expression in the liver of hypertensive rats may represent a specific response to high levels of circulating ANG II or a response to hypertensive injury.

Obstructive nephropathy in the neonatal rat is attenuated by epidermal growth factor.

BACKGROUND: Obstructive nephropathy is a primary cause of renal failure in infancy. Chronic unilateral ureteral obstruction (UUO) in the neonatal rat results in reduced renal expression of epidermal growth factor (EGF), renal tubular epithelial (RTE) cell apoptosis and interstitial fibrosis. We wished to determine whether these changes could be prevented by exogenous administration of EGF. METHODS: Thirty-three Sprague-Dawley rats underwent UUO within the first 48 hours of life, and received daily injections of either EGF (0.1 mg/kg/day) or saline (control) for the following seven days, after which obstructed and intact opposite kidneys were removed for study. These were compared to 11 sham-operated rats that received either no injections, EGF injections, or saline injections. Renal cell proliferation was determined by proliferating cell nuclear antigen, apoptosis was measured by the TUNEL technique, and the distribution of vimentin, clusterin, transforming growth factor-beta 1 (TGF-beta 1), and alpha-smooth muscle actin were determined by immunohistochemistry. Tubular dilation, tubular atrophy, and interstitial collagen deposition were quantitated by histomorphometry. RESULTS: Compared to controls, EGF treatment increased RTE cell proliferation in the obstructed kidney by 76%, decreased apoptosis by 80%, and reduced vimentin, clusterin and TGF-beta 1 immunostaining (all P < 0.05). EGF treatment reduced tubular dilation by 50%, atrophic tubules by 30%, and interstitial fibrosis by 50% (all P < 0.05). There was no significant effect of EGF on renal alpha smooth muscle actin distribution. There was no effect of saline or EGF injections on kidneys from sham-operated rats for any of the parameters studied. CONCLUSIONS: We conclude that EGF stimulates RTE cell proliferation and maturation and reduces apoptosis in the neonatal rat kidney subjected to chronic UUO. These effects may contribute to the reduction in tubular dilation, tubular atrophy, and interstitial fibrosis. By preserving renal development, administration of EGF attenuates the renal injury resulting from chronic UUO.

K+ channel subunit isoforms with divergent carboxy-terminal sequences carry distinct membrane targeting signals.

Kv3 K+ channel genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K+ channel proteins that differ only in their C-termini. This divergence does not affect the electrophysiological properties of the channels expressed by these proteins. A similar alternative splicing with unknown function is seen in K+ channel genes of other families. We have investigated the possibility that the alternative splicing serves to generate channel subunits with different membrane targeting signals by examining the sorting behavior of three alternatively-spliced Kv3.2 isoforms when expressed in polarized MDCK cells. Two Kv3.2 proteins, Kv3.2b and Kv3.2c were expressed predominantly in the apical membrane, while Kv3.2a was localized mainly to the basolateral side (thought to be equivalent to the axonal and somatodendritic compartments in neurons, respectively). The Kv3.2 mRNA transcripts used in these studies are identical except for their 3' sequence, encoding the extreme C-terminal domain of the protein and the 3'UTR of the mRNA. However, the proteins achieve the same localizations in MDCK cells when expressed from constructs containing or lacking the 3'UTR, indicating that the differential localization is due to targeting signals present in the C' terminal domain of the protein. These results suggest that the alternative splicing of Kv3 genes is involved in channel localization. Since the precise localization of any given ion channel on the neuronal surface has significant functional implications, the results shown here suggest an important function for the alternative splicing of 3' ends seen in many K+ channel genes.