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Fibrosis is the end point of most renal diseases, and several glycosaminoglycans have been shown to attenuate this process. Marine invertebrate glycosaminoglycans with unique structures have opened the possibility to test these new compounds on renal fibrosis. The effect of a fucosylated chondroitin sulfate from an echinoderm marine species is reported with the use of a model of renal fibrosis in rats, termed unilateral ureteral obstruction. Animals were given 4 mg/kg body wt of fucosylated chondroitin sulfate intraperitoneally, once a day. After 14 days, their kidneys were examined by histological, immunohistochemical, and biochemical methods. Compared with control mice, collagen deposition decreased in the course of renal fibrosis in the animals receiving fucosylated chondroitin sulfate, as revealed by Sirius red staining and hydroxyproline content. The cellularity related to myofibroblasts and macrophages was also reduced, as was the production of transforming growth factor (TGF)-[beta]. The glycosaminoglycan content increased in the renal interstitium of animals submitted to unilateral ureteral obstruction compared with the control contralateral kidney, mostly due to an increase of chondroitin sulfate content. Interestingly, no change in the pattern of glycosaminoglycan deposition was observed after administration of fucosylated chondroitin sulfate. Fibrosis induced by unilateral ureteral obstruction is attenuated in P-selectin-deficient mice, which also do not respond to the invertebrate glycosaminoglycan. In conclusion, fucosylated chondroitin sulfate attenuates renal fibrosis on a ureteral obstruction model in mice preponderantly through a P-selectin-mediated mechanism. glycosaminoglycans; inflammatory cells; anti-selectin activity; cytokine expression; collagen accumulation doi: 10.1152/ajprenal.00217.2010

Kidney fibrosis, a typical characteristic of chronic renal disease, is associated with tubular epithelial cell apoptosis. The results of our recent studies have shown that Omi/HtrA2 (Omi), a proapoptotic mitochondrial serine protease, performs a crucial function in renal tubular epithelial apoptotic cell death in animal models of acute kidney injury, including cisplatin toxicity and ischemia-reperfusion insult. However, the role of Omi in tubulointerstitial disease-associated fibrosis in the kidney remains to be clearly defined. We evaluated the potential function and molecular mechanism of Omi in ureteral obstruction-induced kidney epithelial cell apoptosis and fibrosis. The mice were subjected to unilateral ureteral obstruction (UUO) via the ligation of the left ureter near the renal pelvis. UUO increased the protein level of Omi in the cytosolic fraction of the kidney, with a concomitant reduction in the mitochondrial fraction. UUO reduced the X-linked inhibitor of apoptosis protein (XIAP), a substrate of Omi, and pro-caspase-3, whereas it increased cleaved poly(ADP-ribose) polymerase (cleaved PARP) and the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells. When mice were treated with ucf-101, an inhibitor of the proteolytic activity of Omi (6.19 [micro]g/day ip), on a daily basis beginning 2 days before UUO and continuing until the end of the experiment, the Omi inhibitor protected XIAP cleavage after UUO and reduced the increment of PARP cleavage and the numbers of TUNEL-positive cells. Furthermore, the Omi inhibitor significantly attenuated UUO-induced increases in fibrotic characteristics in the kidney, including the atrophy and dilation of tubules, expansion of the interstitium, and increases in the expression of collagens, [alpha]-smooth muscle actin, and fibronectin. In conclusion, Omi/HtrA2 is associated with apoptotic signaling pathways in tubular epithelial cells activated by unilateral ureteral obstruction, thereby resulting in kidney fibrosis. XIAP; kidney fibrosis; ucf-101; [alpha]-SMA doi: 10.1152/ajprenal.00737.2009.

Norregaard R, Jensen BL, Topcu SO, Wang G, Schweer H, Nielsen S, Frokiaer J. Urinary tract obstruction induces transient accumulation of COX-2-derived prostanoids in kidney tissue. Am J Physiol Regul Integr Comp Physiol 298: R1017-R1025, 2010. First published February 10, 2010; doi:10.11 152/ajpregu.00336.2009.--Inhibitors of cyclooxygenase (COX)-2 prevent suppression of aquaporin-2 and reduce polyuria in the acute phase after release of bilateral ureteral obstruction (BUO). We hypothesized that BUO leads to COX-2-mediated local accumulation of prostanoids in inner medulla (IM) tissue. To test this, rats were subjected to BUO and treated with selective COX-1 or COX-2 inhibitors. Tissue was examined at 2, 6, 12, and 24 h after BUO. COX-2 protein abundance increased in IM 12 and 24 h after onset of BUO but did not change in cortex. COX-I did not change at any time points in any region. A full profile of all five primary prostanoids was obtained by mass spectrometric determination of PG[E.sub.2], PG[F.sub.2[alpha]], 6-keto-PG[F.sub.1[alpha]], PG[D.sub.2], and thromboxane (Tx) [B.sub.2] concentrations in kidney cortex/outer medulla and IM fractions. IM concentration of PG[E.sub.2], 6-keto-PG[F.sub.1[alpha]], and PG[F.sub.2[alpha]] was increased at 6 h BUO, and PG[E.sub.2] and PG[F.sub.2[alpha]] increased further at 12 h BUO. Tx[B.sub.2] increased after 12 h BUO. 6-keto-PG[F.sub.1[alha]] remained significantly increased after 24 h BUO. The COX-2 inhibitor parecoxib lowered IM PG[E.sub.2]. Tx[B.sub.2], 6-keto-PG[F.sub.1[alpha]], and PG[F.sub.2[alpha]] below vehicle-treated BUO and sham rats at 6, 12 and, 24 h BUO. The COX-1 inhibitor SC-560 lowered PG[E.sub.2], PG[F.sub.2[alpha]], and PG[D.sub.2] in IM compared with untreated 12 h BUO, but levels remained significantly above sham. In cortex tissue, PG[E.sub.2] and 6-keto-PG[F1[alpha]] concentrations were elevated at 6 h only. In conclusion, COX-2 activity contributes to the transient increase in prostacyclin metabolite 6-keto-PG[F.sub.1[alpha]] and Tx[B.sub.2] concentration in the kidney IM, and COX-2 is the predominant isoform that is responsible for accumulation of PGE2 and PG[F.sub.2[alpha]] with minor, but significant, contributions from COX-1. PG[D.sub.2] synthesis is mediated exclusively by COX-1. In BUO, therapeutic interventions aimed at the COXprostanoid pathway should target primarily COX-2. bilateral ureteral obstruction; cyclooxygenase-2; mass spectrometric doi: 10.1152/Npregu.00336.2009.

Unilateral ureteral obstruction (UUO), a model of tubulointerstitial scarring (TIS), has a propensity toward regeneration of renal parenchyma after release of obstruction (RUUO). No information exists on the contribution of stem cells to this process. We performed UUO in FVB/N mice, reversed it after 10 days, and examined kidneys 3 wk after RUUO. UUO resulted in attenuation of renal parenchyma. FACS analysis of endothelial progenitor (EPC), mesenchymal stem (MSC) and hematopoietic stem (HSC) cells obtained from UUO kidneys by collagenase-dispersed single-cell suspension showed significant increase in EPC, MSC, and HSC compared with control. After RUUO cortical parenchyma was nearly restored, and TIS score improved by 3 wk. This reversal process was associated with return of stem cells toward baseline level. When animals were chronically treated with nitric oxide synthase (NOS) inhibitor at a dose that did not induce hypertension but resulted in endothelial dysfunction, TIS scores were not different from control UUO, but EPC number in the kidney decreased significantly; however, parenchymal regeneration in these mice was similar to control. Blockade of CXCR4-mediated engraftment resulted in dramatic worsening of UUO and RUUO. Similar results were obtained in caveolin-1-deficient but not -overexpressing mice, reflecting the fact that activation of CXCR4 occurs in caveolae. The present data show increase in EPC, HSC, and MSC population during UUO and a tendency for these cells to decrease to control level during RUUO. These processes are minimally affected by chronic NOS inhibition. Blockade of CXCR4-stromal cell-derived factor-1 (SDF-1) interaction by AMD3100 or caveolin-1 deficiency significantly reduced the UUO-associated surge in stem cells and prevented parenchymal regeneration after RUUO. We conclude that the surge in stem cell accumulation during UUO is a prerequisite for regeneration of renal parenchyma. fibrosis; caveolin; AMD3100; mesenchymal stem cells doi: 10.1152/ajprenal.00542.2009

Am J Physiol Renal Physiol 298: F133-F141, 2010. First published November 11, 2009; doi: 10.1152/ajprenal.00400.2009.--Histone deacetylase (HDAC) regulates gene expression by modifying chromatin structure. Although changes in the expression and activities of HDAC may affect the course of kidney disease, the role of HDAC in tubulointerstitial injury has not been explored. We therefore investigated the alterations in HDAC expression and determined the effects of HDAC inhibition on the tubulointerstitial injury induced by unilateral ureteral obstruction. The induction of HDAC1 and HDAC2, accompanied by a decrease in histone acetylation was observed in kidneys injured by ureteral obstruction. Immunohistochemical analysis revealed that HDAC1 and HDAC2 were induced in renal tubular cells. Treatment with an HDAC inhibitor, trichostatin A (TSA), attenuated macrophage infiltration and fibrotic changes in tubulointerstitial injury induced by ureteral obstruction. The induction of colony-stimulating factor-1 (CSF-1), a chemokine known to be involved in macrophage infiltration in tubulointerstitial injury, was reduced in injured kidneys from mice treated with TSA. TSA, valproate, and the knockdown of HDAC1 or HDAC2 significantly reduced CSF-1 induced by TNF-[alpha] in renal tubular cells. These results suggest that tubular HDAC1 and HDAC2, induced in response to injury, may contribute to the induction of CSF-1 and the initiation of macrophage infiltration and profibrotic responses. These findings suggest a potential of HDAC inhibition therapy aimed at reducing inflammation and fibrosis in tubulointerstitial injury. epigenetic; chemokine; ureteral obstruction

Am J Physiol Renal Physiol 298: F62-F71, 2010. First published November 4, 2009; doi: 10.1152/ajprenal.00234.2009.--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. apoptosis; collagen; fibrosis; macrophages; ureteral peristalsis

Li L, Zepeda-Orozco D, Patel V, Truong P, Karner CM, Carroll T J, Lin F. Aberrant planar cell polarity induced by urinary tract obstruction. Am J Physiol Renal Physiol 297: F1526-F1533, 2009. First published September 30, 2009; doi: 10.1152/ajprenal.00318.2009.--Flow sensing by primary cilia of the epithelial cells is involved in cystogenesis in polycystic kidney disease. We investigate whether a similar mechanism applies to the pathogenesis of cyst-like tubular dilatation induced by ureteral obstruction in mice. Robust proliferation occurs in the obstructed tubules when urine flow is interrupted as well as in the repairing tubules when urine flow is reestablished after relief of the obstruction, suggesting a urine flow-independent mechanism of proliferation. In the urothelium, proliferation is only detected above the obstruction, although urine flow ceased both above and below the obstruction. Our results support mechanical strain- rather than flow-mediated proliferation in obstructive uropathy. To understand the mechanism of cell proliferation leading to increased tubular diameter in cyst-like tubular dilatation, we examine planar cell polarity (PCP), which is necessary for oriented cell division and maintenance of tubular diameter. In dilated tubules, the orientation of cell division is randomized, atypical PKC (aPKC) is mislocalized, and the pattern of the expression of a core PCP protein, Frizzled3 (Fz3), is altered. In addition, the level of Fz3 expression is increased. These results indicate that aberrant PCP may contribute to cyst-like tubular dilatation in obstructive uropathy. Interestingly, the orientation of cell division, localization of aPKC, and Fz3 expression return to normal when obstruction is relieved, which suggest a role of normal PCP signaling in tubular repair. obstructive injury; orientation of cell division; mechanical strain; urine flow doi: 10.1152/ajprenal.00318.2009

Activation of renal interstitial fibroblasts is critically involved in the development of tubulointerstitial fibrosis in chronic kidney diseases. In this study, we investigated the effect of trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor, on the activation of renal interstitial fibroblasts in a rat renal interstitial fibroblast line (NRK49F) and the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO). [alpha]-Smooth muscle actin ([alpha]-SMA) and fibronectin, two hallmarks of fibroblast activation, were highly expressed in cultured NRK-49F cells, and their expression was inhibited in the presence of TSA. Similarly, administration of TSA suppressed the expression of [alpha]-SMA and fibronectin and attenuated the accumulation of renal interstitial fibroblasts in the kidney after the obstructive injury. Activation of renal interstitial fibroblasts was accompanied by phosphorylation of signal transducer and activator of transcription 3 (STAT3), and TSA treatment also abolished these responses. Furthermore, inhibition of the STAT3 pathway with AG490 inhibited expression of [alpha]-SMA and fibronectin in NRK-49F cells. Finally, TSA treatment inhibited tubular cell apoptosis and caspase-3 activation in the obstructive kidney. Collectively, we suggest that pharmacological HDAC inhibition may induce antifibrotic activity by inactivation of renal interstitial fibroblasts and inhibition of renal tubular cell death. STAT3 may mediate those actions of HDACs. trichostatin A; histone deacetylase; renal interstitial fibroblasts; unilateral ureteral obstruction; STAT3 doi: 10.1152/ajprenal.00282.2009.

Ureteral obstruction is characterized by decreased renal blood flow that is associated with hypoxia within the kidney. Adrenomedullin (AM) is a peptide hormone with tissue-protective capacity that is stimulated through hypoxia. We tested the hypothesis that ureteral obstruction stimulates expression of AM and hypoxia-inducible factor-1 (HIF-a[alpha]) in kidneys. Rats were exposed to bilateral ureteral obstruction (BUO) for 2, 6, 12, and 24 h or sham operation and compared with unilateral obstruction (UUO). AM mRNA expression was measured by quantitative PCR in cortex and outer medulla (C+OM) and inner medulla (IM). AM and HIF-1[alpha] protein abundance and localization were determined in rats subjected to 24-h BUO. AM mRNA expression in C+OM increased significantly after 12-h BUO and further increased after 24 h. In IM, AM mRNA expression increased significantly in response to BUO for 6 h and further increased after 24 h. AM peptide abundance was enhanced in C+OM and IM after 24-h BUO. Immunohistochemical labeling of kidneys showed a wider distribution and more intense AM signal in 24-h BUO compared with Sham. In UUO rats, AM mRNA expression increased significantly in IM of the obstructed kidney compared with nonobstructed and Sham kidney whereas AM peptide increased in IM compared with Sham. HIF-1[alpha] protein abundance increased significantly in IM after 24-h BUO compared with Sham and HIF-1[alpha] immunoreactive protein colocalized with AM. In summary, AM and HIF-1[alpha] expression increases in response to ureteral obstruction in agreement with expected oxygen gradients. Hypoxia acting through HIF-1[apha] accumulation may be an important pathway for the renal response to ureteral obstruction. hypoxia-inducible factor-1[alpha]; bilateral ureteral obstruction; medullary hypoxia; inflammation; tumor neurosis factor [alpha]

The sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase2 (SERCA2) is down-regulated in cardiac hypertrophy with decompensation. We sought to determine whether mice heterozygous for the SERCA2 allele would develop greater bladder hypertrophy and decompensation than their wild-type littermates following partial bladder outlet obstruction (pBOO). We found that following 4 wk of surgically created pBOO, SERCA2 heterozygous murine bladders showed significantly less hypertrophy, improved in vitro cystometry performance, diminished expression of the slow myosin isoform A analyzed by RT-PCR, a significant drop in nuclear translocation of nuclear factor of activated T cells by EMSA, and decreased cell proliferation within the smooth muscle layer following 5-bromo-2'-deoxyuridine labeling compared with their wild-type littermates. Thus, in contrast to cardiac muscle, deletion of a SERCA2 allele confers protection against bladder hypertrophy in a murine model of pBOO. Compensatory mechanisms in heterozygous mice seem to be related to the calcineurin pathway. Further studies are underway to better define the molecular basis of this observation, which has potential clinical applications. urinary bladder; obstruction; sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase2; nuclear factor of activated T cells

Interstitial fibrosis is a universal feature of progressive kidney disease. Urokinase-type plasminogen activator (uPA) is thought to participate for several reasons: 1) uPA is produced predominantly in kidney, 2) its inhibitor plasminogen activator inhibitor-1 (PAI-1) is a strong promoter of interstitial fibrosis, whereas its receptor (uPAR) attenuates renal fibrosis, 3) uPA reduces fibrosis in liver and lung, and 4) uPA can activate hepatocyte growth factor (HGF), a potent antifibrotic growth factor. The present study tested the hypothesis that endogenous uPA reduces fibrosis severity by investigating the unilateral ureteral obstruction (UUO) model in wild-type (WT) and uPA-/-mice. Several outcomes were measured: renal collagen 3-21 days after UUO, macrophage accumulation (F4/80 Western blotting), interstitial myofibroblast density ([alpha]-smooth muscle actin immunostaining), and tubular injury (E-cadherin and Ksp-cadherin Western blotting). None of these measures differed significantly between WT and uPA-/- mice. uPA genetic deficiency was not associated with compensatory changes in renal uPAR mRNA levels, PAI-1 protein levels, or tissue plasminogen activator activity levels after UUO. Despite the known ability of uPA to activate latent HGF, immunoblotting failed to detect significant differences in levels of the active HGF [alpha]-chain and phosphorylated cMET (the activated HGF receptor) between the WT and uPA-/- groups. These findings suggest that the profibrotic actions of PAI-I are uPA independent and that an alternative pathway must activate HGF in kidney. Finally, these results highlight a significant organ-specific difference in basic fibrogenic pathways, as enhanced uPA activity has been reported to attenuate pulmonary and hepatic fibrosis. renal interstitial fibrosis: unilateral ureteral obstruction; serine protease doi: 10.1152/ajpregu.00380.2006

Release of bilateral ureteral obstruction (BUO) is associated with reduced expression of renal aquaporins (AQPs), polyuria, and impairment of urine-concentrating capacity. Recently, we demonstrated that 24 h of BUO is associated with increased cyclooxygenase (COX)-2 expression in the inner medulla (IM) and that selective COX-2 inhibition prevents downregulation of AQP2. In the present study, we tested the hypothesis that COX-2 activity increases in the postobstructive phase and that this increase in COX-2 activity contributes to polyuria and impaired urine-concentrating capacity. We examined the effect of the selective COX-2 inhibitor parecoxib (5 mg x [kg.sup.-1] x [day.sup.-1] via osmotic minipumps) on renal functions and protein abundance of AQP2, AQP3, Na-K-2Cl cotransporter type 2 (NKCC2), and Na-K-ATPase 3 days after release of BUO. At 3 days after release of BUO, rats exhibited polyuria, dehydration and urine and IM tissue osmolality were decreased. There were inverse changes of COX-1 and COX-2 in the IM: COX-2 mRNA, protein, and activity increased, while COX-1 mRNA and protein decreased. Parecoxib reduced urine output 1 day after release of BUO, but sodium excretion and glomerular filtration rate were unchanged. Parecoxib normalized urinary PG[E.sub.2] and PG[I.sub.2] excretion and attenuated downregulation of AQP2 and AQP3, while phosphorylated AQP2 and NKCC2 remained suppressed. Parecoxib did not improve urine-concentrating capacity in response to 24 h of water deprivation. We conclude that decreased NKCC2 and collapse of the IM osmotic gradient, together with suppressed phosphorylated AQP2, are likely causes for the impaired urine-concentrating capacity and that COX-2 activity is not likely to mediate these changes in the chronic postobstructive phase after ureteral obstruction. cyclooxygenase; ureteral obstruction; PGE2; parecoxib; AQP2; NKCC2; urine-concentrating capacity doi:10.1152/ajprenal.00394.2006

Angiotensin II (ANG II) plays an important role in the development of obstructive nephropathy. Here, we examined the effects of the ANG II receptor type 1 (AT1R) blockade using candesartan on long-term renal molecular and functional changes in response to partial unilateral ureteral obstruction (PUUO). Newborn rats were subjected to severe PUUO or sham operation (Sham) within the first 48 h of life. Candesartan was provided in the drinking water (10 mg*[kg.sup.-1]*[day.sup.-1]) from day 21 of life until 10 wk of age. Renal blood flow (RBF) was evaluated by MRI, glomerular filtration rate (GFR) was measured using the renal clearance of [.sup.51]Cr-EDTA, and the renal expression of Na-K-ATPase and the collecting duct water channel aquaporin-2 (AQP2) was examined by immunoblotting and immunocytochemistry. At 10 wk of age, PUUO significantly reduced RBF (0.8 [+ or -] 0.1 vs. 1.6 [+ or -] 0.1 ml*[min.sup.-1]*100 g body [wt.sup.-1]; p < 0.05) and GFR (37 [+ or -] 16 vs. 448 [+ or -] 111 [micro]l*[min.sup.-1]*100 g body [wt.sup.-1]; P < 0.05) compared with Sham. Candesartan prevented the RBF reduction (PUUO+CAN: 1.6 [+ or -] 0.2 vs. PUUO: 0.8 [+ or -] 0.1 ml*[min.sup.-1]*100 g body [wt.sup.-1]; p < 0.05) and attenuated the GFR reduction (PUUO+CAN: 265 [+ or -] 68 vs. PUUO: 37 [+ or -] 16 [micro]l*[min.sup.-1]*100 g body [wt.sup.-1]; P < 0.05). PUUO was also associated with a significant downregulation in the expression of Na-K-ATPase (75 [+ or -] 12 vs. 100 [+ or -] 5%, P < 0.05) and AQP2 (52 [+ or -] 15 vs. 100 [+ or -] 4%, P < 0.05), which were also prevented by candesartan (Na-K-ATPase: 103 [+ or -] 8 vs. 100 [+ or -] 5% and AQP2: 74 [+ or -] 13 vs. 100 [+ or -] 4%). These findings were confirmed by immunocytochemistry. Consistent with this, candesartan treatment partly prevented the reduction in solute free water reabsorption and attenuated fractional sodium excretion in rats with PUUO. In conclusion, candesartan prevents or attenuates the reduction in RBF, GFR and dysregulation of AQP2 and Na-K-ATPase in response to congenital PUUO in rats, suggesting that AT1R blockade may protect the neonatally obstructed kidney against development of obstructive nephropathy. congenital ureteral obstruction; newborn rat; aquaporin water channels; sodium transporter; A[T.sub.1] receptor blockage

Although the mammalian urinary tract is generally held to be solely a transit and storage vehicle for urine made by the kidney, in vivo data suggest reabsorption of urea and other urine constituents across urinary tract epithelia. To determine whether urinary tract tissue concentrations are increased as a result of such reabsorption, we measured urea nitrogen and creatinine concentrations and determined whether urea transporter B (UT-B) was present in bladder, ureter, and other tissues from dogs and rats. Mean urea nitrogen and creatinine concentrations in dogs and rats were three- to sevenfold higher in urinary tract tissues than in serum and were comparable to those in renal cortex. In waterrestricted or water-loaded rats, urea nitrogen concentrations in bladder tissues fell inversely with the state of hydration, were proportional to urine urea nitrogen concentrations, and were greater than the corresponding serum urea nitrogen concentration in every animal. Immunoblots of rat and dog urinary tract tissues demonstrated the presence of UT-B in homogenates of bladder and ureter, and immunocytochemical analysis localized UT-B to epithelial cell membranes. These findings are consistent with the notion that urea and creatinine are continuously reabsorbed from the urine across the urothelium, urea in part via UT-B, and that urine is thus altered in its passage through the urinary tract. Urea reabsorption across urinary tract epithelia may be important during conditions requiring nitrogen conservation and may contribute to pathophysiological states characterized by high blood urea nitrogen, such as prerenal azotemia and obstructive uropathy. urothelial transport; bladder; ureter

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 rots 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 VEGP-R2 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. mitochondria; renal maturation; VEGFR2

Discs-large homolog 1 (DLGH1) is a mouse ortholog of the Drosophila discs-large (DLG) tumor suppressor protein, a founding member of the PDZ and MAGUK protein families. DLG proteins play important roles in regulating cell proliferation, epithelial cell polarity, and synapse formation and function. Here, we generated a null allele of Dlgh1 and studied its role in urogenital development. [Dlgh1.sup.-/-] mice developed severe urinary tract abnormalities, including congenital hydronephrosis, which is the leading cause of renal failure in infants and children. DLGH1 is expressed in the developing ureter; in its absence, the stromal cells that normally lie between the urothelial and smooth muscle layers were missing. Moreover, in ureteric smooth muscle, the circular smooth muscle cells were misaligned in a longitudinal orientation. These abnormalities in the ureter led to severely impaired ureteric peristalsis. Similar smooth muscle defects are observed frequently in patients with ureteropelvic junction obstruction, a common form of hydronephrosis. Our results suggest that (i) besides its well documented role in regulating epithelial polarity, Dlgh1 also regulates smooth muscle orientation, and (ii) human DLG1 mutations may contribute to hereditary forms of hydronephrosis. SAP97 | kidney | postsynaptic density-95/discs-large/zonula occludens-1 urogenital | sonic hedgehog

Role of Hox11 paralogous genes in the development of kidney is studied. An extensive microarray analysis of the development of Hoxa11/Hoxd 11 mutant kidney is presented.

Ureteric obstruction is frequently encountered in primary care urology and can lead to damage to the ipsilateral kidney. Relief of all types of obstruction generally leads to the normalization of any deterioration in renal function noted at diagnosis. However, some evidence from animal models suggests that obstruction can cause progressive deleterious effects on renal function and blood pressure control, especially in the presence of preexisting pathologies such as essential hypertension. The last 10 years have seen a proliferation of studies in rodents wherein complete unilateral ureteric obstruction has been used as a model of renal fibrosis. However, the relevance of the findings to human obstructive uropathy has, in many cases, not been the primary aim. In this review, we outline the major events linking damage to the renal parenchyma and cell death to the evolution of fibrosis following obstruction. Special focus is given to the role of apoptosis as a major cause of cell death during and post-complete ureteric obstruction. Several interventions that reduce tubular apoptosis are discussed in terms of their ability to prevent subsequent progression to end-organ damage and fibrosis. tubular epithelium; oxidative stress; growth factors; protection

Bilateral ureteral obstruction (BUO) is associated with marked changes in the expression of renal aquaporins (AQPs) and sodium transport proteins. To examine the role of prostaglandin in this response, we investigated whether 24-h BUO changed the expression of cyclooxygenases (COX-1 and -2) in the kidney and tested the effect of the selective COX-2 inhibitor parecoxib (5 mg*[kg.sup.-1]*day J via osmotic minipumps) on AQPs and sodium transport. Sham and BUO kidneys were analyzed by semiquantitative immunoblotting, and a subset of kidneys was perfusion fixed for immunocytochemistry. BUO caused a significant 14-fold induction of inner medullary COX-2 (14.40 [+ or -] 1.8 vs. 1.0 [+ or -] 0.4, n = 6; P < 0.0001) and a reduction in medullary tissue osmolality, whereas COX-1 did not change. Immunohistochemistry confirmed increased COX-2 labeling associated with medullary interstitial cells. COX isoforms did not change in cortex/outer medulla after 24-h BUO. In BUO kidneys, inner medullary AQP2 expression was reduced, and this decrease was prevented by parecoxib. In the inner stripe of outer medulla, the type 3 [Na.sup.+]/[H.sup.+] exchanger (NHE3) and apical [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (BSC-1) were significantly reduced by BUO, and this decrease was significantly attenuated by parecoxib. Immunohistochemistry for AQP2, NHE3, and BSC-1 confirmed the effect of parecoxib. Parecoxib had no significant effect on the Na-K-ATPase [[alpha].sub.1]-subunit, type II Na-[P.sub.i] cotransporter, or AQP3. In conclusion, acute BUO leads to marked upregulation of COX-2 in inner medulla and selective COX-2 inhibition prevents dysregulation of AQP2, BSC-1, and NHE3 in response to BUO. These data indicate that COX-2 may be an important factor contributing to the impaired renal water and sodium handling in response to BUO. cyclooxygenase; PG[E.sub.2]; parecoxib; AQP2

Byline: S. K. Chowdhary (1), A. Lander (1), K. Parashar (1), J. J. Corkery (1) Keywords: Keywords Ectopic ureters; Solitary kidney Abstract: Ectopic ureters present in childhood with symptoms related to an abnormal site or structure (refluxing, obstructed) of the ureteric orifice. The majority drain duplex kidneys. The diagnosis is relatively easy if the poles are functioning or hydronephrotic. Associated malformations are rarely seen and the results of surgery are gratifying. If an ectopic ureter drains a single kidney, it is called a single-system ectopic ureter (SSEU). We reviewed a 15-year experience (1980--1995) with 127 ectopic ureters from our hospital:11 SSEUs in ten consecutive children were managed during this period. Our data lead us to believe that SSEUs are a special subset of ectopic ureters. Diagnosis is often delayed because the ectopic ureter may be associated with a single small, dysplastic, poorly-functioning, non-visualised kidney and the child may be thought to have a contralateral normal 'solitary kidney'. Associated systemic malformations are common. Residual symptoms of wetting may persist in the early postoperative period. A high degree of suspicion must be maintained for this entity when a child presents with urinary symptoms of wetting or recurrent infection and a 'solitary kidney'. Early endoscopic examination of the genitourinary tract will clinch the diagnosis in the majority of cases. Residual symptoms of wetting in the postoperative period generally resolve with passage of time. Author Affiliation: (1) Diana, Princess Of Wales Children's Hospital, Steelhouse Lane, Birmingham B4 6NH, UK, GB (2) Advanced Paediatric Centre, PGIMER, Chandigarh 160012, India, IN Article note: Accepted: 20 November 2000

Byline: K. Hohenfellner (1), Tracy E. Hunley (2), Carde Schloemer (3), Walburgis Brenner (3), Elizabeth Yerkes (4), Fred Zepp (1), John W. Brock III. (4), Valentina Kon (2) Keywords: Key wordsaAngiotensin II; Angiotensin 2 receptor; Obstructive megaureter; Posterior urethral valves Abstract: aIn humans, the actions of angiotensin II are transduced through the AT1 and AT2 receptors which have recently been implicated in renal organogenesis. Polymorphisms in the human angiotensin II receptor genes have been linked to cardiovascular and nephrological disorders. In this study we evaluated 35 patients with either primary obstructive megaureter or posterior urethral valves. Each was genotyped for the A1166 AT1 polymorphism and the recently described A-1332G AT2 transition. The incidence of these genetic variants was also evaluated in normal controls without any ultrasonographic urological abnormalities. Similar to our previous findings in congenital urological abnormalities, the AT1 receptor genotype distribution did not differ between patients with either primary obstructive megaureter or posterior urethral valves versus controls. In contrast, compared with normal controls, there was a dramatic increase in the occurrence of the AT2 A-1332G transition in patients with primary obstructive megaureter (75.0% vs. 41.9% in controls, P&lt 0.025). In patients with posterior urethral valves, there was no difference in the occurrence of the transition versus controls (36.9%, P=NS). Thus, there is no correlation between the AT1 receptor gene polymorphism and urological abnormalities. However there is an increased incidence in the AT2 genetic variant in patients with primary obstructive megaureter. Author Affiliation: (1) Universitats-Kinderklinik, Langenbeckstrasse 1, D-55131 Mainz Tel.: +49-6131-177183, Fax: +49-06131-173918 (2) Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA, US (3) Department of Urology, Universitats-Kinderklinik, Mainz, Germany, DE (4) Department of Urology, Vanderbilt University Medical Center, Nashville, Tennessee, USA, US Article note: Received: 2 March 1998 / Received: 1 July 1998 / Accepted: 6 July 1998

Byline: LuAnn Klemme (1), Alfred J. Fish (1), Stephen Rich (2), Beryl Greenberg (4), Beverly Senske (4), Miriam Segall (2) Keywords: Key words: Linkage analysis; Hydronephrosis; Hydroureter; Vesicoureteral reflux; Inherited ureteral defects; Chromosome 6p Abstract: Abnormal development of the ureter during embryogenesis, when occurring in multiple family members, appears to be a genetically determined defect with autosomal dominant inheritance and high penetrance, which can lead to significant kidney damage, renal failure, and death. We have studied 48 individuals within a large kindred in which ureteral-related abnormalities (including vesicoureteral reflux, ureteropelvic junction obstruction, duplicated ureters, and medullary sponge kidney) were segregated. Family members who had not had previous diagnostic studies were evaluated for presence or absence of ureteral abnormalities and we attempted to map the locus for this familial ureteral abnormalities syndrome (FUAS). These studies identified 11 asymptomatic individuals, previously assumed to be unaffected, with minor abnormalities. When linkage analysis between the inheritance of ureteral abnormalities and six marker loci glyoxalase I (GLO-1), major histocompatibility antigens (HLA-A, B, and DR/DQ), D6S288, and factor XIII antigen (F13A1) on the short arm of chromosome 6 was performed, the lod scores significantly rejected linkage over a 77.1-cM distance. These findings are in contrast to previous data suggesting linkage between the presence of ureteral abnormalities and HLA, and indicate the possibility of genetic heterogeneity of FUAS. Author Affiliation: (1) Department of Pediatrics, University of Minnesota School of Medicine, Minneapolis, Minnesota, USA, US (2) Department of Laboratory Medicine and Pathology, University of Minnesota School of Medicine, Minneapolis, Minnesota, USA, US (3) The Institute of Human Genetics, University of Minnesota School of Medicine, Minneapolis, Minnesota, USA, US (4) Clinical Research Center, University of Minnesota School of Medicine, Minneapolis, Minnesota, USA, US Article note: Received April 29, 1997 received in revised form September 15, 1997 accepted September 22, 1997

Patch-clamp and single-cell reverse transcription-polymerase chain reaction (RT-PCR) methods were employed on embryonic rat ureteric buds (UBs) microdissected from the outer cortex to characterize their membrane conductive proteins. Results revealed the functional characteristics of the cells at the tip of the UB (tUB). Also, the tUB cell represents a Ca-activated-like Cl conductance, which is phenotypical for embryonic nonpolarized cells.

Byline: S. V. S. Soundappan (1,2), A. P. Barker (1) Keywords: Hydronephrosis; Retrocaval ureter Abstract: Retrocaval ureter is a relatively rare anomaly that usually manifests in the third or fourth decades. Symptoms are due to ureteric obstruction, either extrinsic by the abnormal inferior vena cava (IVC), or intrinsic ureteric hypoplasia. Surgery is needed for symptomatic cases and involves transection and relocation of the ureter anterior to the IVC. We report our experience with two such children who needed surgery because of increasing hydronephrosis and who have done well since. Author Affiliation: (1) Department of Paediatric Surgery, Princess Margaret Hospital, Perth, Western Australia (2) 1 First Cross Street, Lambert Nagar, 600-087, Chennai, Tamil Nadu, India Article History: Registration Date: 01/01/2003 Online Date: 10/02/2004

Urothelium synthesizes a group of integral membrane proteins called uroplakins, which form two-dimensional crystals (urothelial plaques) covering [is greater than] 90% of the apical urothelial surface. We show that the ablation of the mouse uroplakin III (UPIII) gene leads to overexpression, defective glycosylation, and abnormal targeting of uroplakin Ib, the presumed partner of UPIII. The UPIII-depleted urothelium features small plaques, becomes leaky, and has enlarged ureteral orifices resulting in the back flow of urine, hydronephrosis, and altered renal function indicators. Thus, UPIII is an integral subunit of the urothelial plaque and contributes to the permeability barrier function of the urothelium, and UPIII deficiency can lead to global anomalies in the urinary tract. The ablation of a single urothelial-specific gene can therefore cause primary vesicoureteral reflux (VUR), a hereditary disease affecting ~1% of pregnancies and representing a leading cause of renal failure in infants. The fact that VUR caused by UPIII deletion seems distinct from that caused by the deletion of angiotensin receptor II gene suggests the existence of VUR subtypes. Mutations in multiple gene, including some that are urothelial specific, may therefore cause different subtypes of primary reflux. Studies of VUR in animal models caused by well-defined genetic defects should lead to improved molecular classification, prenatal diagnosis, and therapy of this important hereditary problem. Key words: urothelium * permeability * knockout mice * vesicoureteral reflux * hydronephrosis

Byline: V. Sripathi (1) Keywords: Horseshoe kidney Multicystic dysplasia Ureteric atresia Abstract: During evaluation of chronic abdominal pain, a 9-year-old male was found to have a horseshoe kidney (HSK) with multicystic dysplasia (MCD) of the left-sided component. Attached to the MCD was a very large, tortuous ureter occupying almost the whole left side of the abdomen. This ureter on dissection was found to end blindly adjacent to the bladder. MCD of one-half of a HSK is an unusual lesion. Its association with a large megaureter with juxtavesical atresia is a unique event. In HSKs, controversy exists about the need to remove a small dysplastic segment. If this segment is associated with a large ureter, as in our case, removal is mandatory in order to avoid pain and infection. Author Affiliation: (1) Department of Pediatric Urology, CHILDS Trust Hospital, Sundaram Medical Foundation Community Hospital, Chennai, South India (2) 1, Damodara Mudali Street, Chennai -- 600 031, India Article History: Accepted Date: 15/04/2001 Article note: Electronic Publication

Endostatin (ES) inhibits endothelial cell migration and has been found to bind to glypicans (Gpcs) on both endothelial cells and renal epithelial cells. We examined the possibility that ES might regulate epithelial cell morphogenesis. The addition of ES to cultured epithelial cells causes an inhibition of both hepatocyte growth factor- and epidermal growth factor-dependent process formation and migration. In contrast, ES does not inhibit epidermal growth factor-dependent morphogenesis in renal epithelial cells derived from Gpc-3 -/mice, whereas expression of Gpc-1 in these cells reconstitutes ES responsiveness. Gpc-3 -/mice have been shown to display enhanced ureteric bud (UB) branching early in development, and cultured UB cells release ES into the media, suggesting that ES binding to Gpcs may regulate UB branching. The addition of ES inhibits branching of the explanted UB, whereas a neutralizing Ab to ES enhances UB outgrowth and branching. Thus, local expression of ES at the tips of the UB may play a role in the regulation of UB arborization.