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Severe AKI is often associated with multiorgan dysfunction, but the mechanisms of this remote tissue injury are unknown. We hypothesized that renal necroinflammation releases cytotoxic molecules that may cause remote organ damage. In hypoxia-induced tubular epithelial cell necrosis in vitro , histone secretion from ischemic tubular cells primed neutrophils to form neutrophil extracellular traps. These traps induced tubular epithelial cell death and stimulated neutrophil extracellular trap formation in fresh neutrophils. In vivo , ischemia-reperfusion injury in the mouse kidney induced tubular necrosis, which preceded the expansion of localized and circulating neutrophil extracellular traps and the increased expression of inflammatory and injury-related genes. Pretreatment with inhibitors of neutrophil extracellular trap formation reduced kidney injury. Dual inhibition of neutrophil trap formation and tubular cell necrosis had an additive protective effect. Moreover, pretreatment with antihistone IgG suppressed ischemia-induced neutrophil extracellular trap formation and renal injury. Renal ischemic injury also increased the levels of circulating histones, and we detected neutrophil infiltration and TUNEL-positive cells in the lungs, liver, brain, and heart along with neutrophil extracellular trap accumulation in the lungs. Inhibition of neutrophil extracellular trap formation or of circulating histones reduced these effects as well. These data suggest that tubular necrosis and neutrophil extracellular trap formation accelerate kidney damage and remote organ dysfunction through cytokine and histone release and identify novel molecular targets to limit renal necroinflammation and multiorgan failure., (Copyright © 2017 by the American Society of Nephrology.)

The Banff working group on preimplantation biopsy was established to develop consensus criteria (best practice guidelines) for the interpretation of preimplantation kidney biopsies. Digitally scanned slides were used (i) to evaluate interobserver variability of histopathologic findings, comparing frozen sections with formalin-fixed, paraffin-embedded tissue of wedge and needle core biopsies, and (ii) to correlate consensus histopathologic findings with graft outcome in a cohort of biopsies from international medical centers. Intraclass correlations (ICCs) and univariable and multivariable statistical analyses were performed. Good to fair reproducibility was observed in semiquantitative scores for percentage of glomerulosclerosis, arterial intimal fibrosis and interstitial fibrosis on frozen wedge biopsies. Evaluation of frozen wedge and core biopsies was comparable for number of glomeruli, but needle biopsies showed worse ICCs for glomerulosclerosis, interstitial fibrosis and tubular atrophy. A consensus evaluation form is provided to help standardize the reporting of histopathologic lesions in donor biopsies. It should be recognized that histologic parameters may not correlate with graft outcome in studies based on organs deemed to be acceptable after careful clinical assessment. Significant limitations remain in the assessment of implantation biopsies., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Acute kidney injury (AKI) involves nephron injury leading to irreversible nephron loss, ie, chronic kidney disease (CKD). Both AKI and CKD are associated with distinct histological patterns of tissue injury, but kidney atrophy in CKD involves tissue remodeling with interstitial inflammation and scarring. No doubt, nephron atrophy, inflammation, fibrosis, and renal dysfunction are associated with each other, but their hierarchical relationships remain speculative. To better understand the pathophysiology, we provide an overview of the fundamental danger response programs that assure host survival upon traumatic injury from as early as the first multicellular organisms, ie, bleeding control by coagulation, infection control by inflammation, epithelial barrier restoration by re-epithelialization, and tissue stabilization by mesenchymal repair. Although these processes assure survival in the majority of the populations, their dysregulation causes kidney disease in a minority. We discuss how, in genetically heterogeneous population, genetic variants shift balances and modulate danger responses toward kidney disease. We further discuss how classic kidney disease entities develop from an insufficient or overshooting activation of these danger response programs. Finally, we discuss molecular pathways linking, for example, inflammation and regeneration or inflammation and fibrosis. Understanding the causative and hierarchical relationships and the molecular links between the danger response programs should help to identify molecular targets to modulate kidney injury and to improve outcomes for kidney disease patients.

A 13-year-old girl presented with proteinuria and acute kidney failure. She was born at full term via cesarean delivery (due to nuchal cord), but there were no other prenatal or perinatal complications. In early childhood the patient had two hospitalizations at ages 4.5 and 9 years, respectively, the latter for pneumonia. She had no history of symptoms of kidney disease. She came to the hospital at age 12 years for routine bilateral molar extractions. She was treated with oral antibiotics and discharged after the procedure without complications. At age 13 years, 10 months after the molar extraction, she was seen by a pediatrician because of puffiness and increased BP. She had had respiratory symptoms 2 weeks before presentation. The pediatrician prescribed furosemide and amlodipine. A few days later, the patient returned to the pediatrician's office because of hand, ankle, and facial swelling and malaise. The pediatrician recommended hospitalization and the patient was admitted at this time.

Background/aims: The search for new therapies providing cardiorenal protection in chronic kidney disease (CKD) has led to treatments that combine conventional renin-angiotensin-aldosterone-system inhibitors with other drugs that exhibit potential in disease management., Methods: In rats made uremic by renal ablation, we examined the effects of addition of the endothelin-A receptor antagonist atrasentan to a previously examined combination of enalapril (angiotensin converting enzyme inhibitor) and paricalcitol (vitamin D receptor activator) on cardiac and renal parameters. The effects of the individual and combined drugs were examined after a 3-month treatment., Results: A decrease in systolic blood pressure, serum creatinine and proteinuria, and improvement of renal histology in uremic rats were attributed to enalapril and/or paricalcitol treatment; atrasentan alone had no effect. In heart tissue, individual treatment with the drugs blunted the increase in cardiomyocyte size, and combined treatment additively decreased cardiomyocyte size to normal levels. Perivascular fibrosis was blunted in uremic control rats with atrasentan or enalapril treatment., Conclusions: We found distinct cardiac and renal effects of atrasentan. Combination treatment with atrasentan, enalapril and paricalcitol provided positive effects on cardiac remodeling in uremic rats, whereas combination treatment did not offer further protective effects on blood pressure, proteinuria or renal histology.

The role of hyperphosphatemia in the pathogenesis of secondary hyperparathyroidism, cardiovascular disease, and progression of renal failure is widely known. Here we studied effects of dietary phosphate restriction on mortality and vascular calcification in uremic rats. Control and uremic rats were fed a high-phosphate diet and at 3 months a portion of rats of each group were killed. Serum phosphate and the calcium phosphate product increased in uremic rats, as did aortic calcium. Of the rats, 56% had positive aortic staining for calcium (von Kossa), RUNX2, and osteopontin. The remaining uremic rats were continued on diets containing high phosphate without and with sevelamer, or low phosphate, and after 3 more months they were killed. Serum phosphate was highest in uremic rats on high phosphate. Serum PTH and FGF-23 were markedly lower in rats on low phosphate. Mortality on high phosphate was 71.4%, with sevelamer reducing this to 37.5% and phosphate restriction to 5.9%. Positive aortic staining for von Kossa, RUNX2, and osteopontin was increased, but phosphate restriction inhibited this. Kidneys from low-phosphate and sevelamer-treated uremic rats had less interstitial fibrosis, glomerulosclerosis, and inflammation than those of uremic rats on high phosphate. Importantly, kidneys from rats on low phosphate showed improvement over kidneys from high-phosphate rats at 3 months. Left ventricles from rats on low phosphate had less perivascular fibrosis and smaller cardiomyocyte size compared to rats on high phosphate. Thus, intensive phosphate restriction significantly reduces mortality in uremic rats with severe vascular calcification.

Electron microscopy (EM) has been an indispensable tool for kidney research since its inception more than half a century ago. Much of the substantial advances were propelled by the need to find methods to best visualize and analyze the kidney's structure deduced from the fundamental principle that has structure and function intimately related. The result of 3 decades of experimental kidney work between 1950 and 1980 coincided with remarkable advances in nephrology that marked a renaissance era for renal pathology and resulted in the morphologic classification of medical kidney diseases. In the era of genetics and molecular medicine TEM continues to contribute significant clinical and pathogenetic insights in kidney disease. The basic principles as applied to kidney disease experimental models are discussed with emphasis on crescent formation in Col4A3-deficient mice and a mouse model of experimental oxalosis (CaOx).

Renal biopsy was introduced in the 1950s. By 1980 the pathologic diagnostic criteria for the majority of medical kidney diseases known today, including pediatric diseases, were established using light, electron microscopy and immunohistochemistry. However, it has become clear that there are limitations in the morphologic evaluation, mainly because a given pattern of injury can be caused by different aetiologies and, conversely, a single aetiology may present with more than one histological pattern. An explosion in kidney disease research in the last 20-30 years has brought new knowledge from bench to bedside rapidly and resulted in new molecular and genetic tools that enhance the diagnostic and prognostic power of the renal biopsy. Genomic technologies such as polymerase chain reaction (PCR), in situ hybridization and oligonucleotide microarrays, collectively known as genomics, detect single or multiple genes underscoring the pathologic changes and revealing specific causes of injury that may require different treatment. The aims of this review are to (1) summarize current recommendations for diagnostic renal biopsies encompassing light microscopy, immunofluorescence or immunohistochemistry and electron microscopy; (2) address the limitations of morphology; (3) show current contributions of genomic technologies adjunct to the renal biopsy, and provide examples of how these may transform pathologic interpretation into molecular disease phenotypes.

Congenital renal dysplasia (RD) is a severe form of congenital renal malformation characterized by disruption of normal renal development with cyst formation, reduced or absent nephrons, and impaired renal growth. The authors previously identified that matrilysin (matrix metalloproteinase-7) was overexpressed in a microarray gene expression analysis of human RD compared to normal control kidneys. They now find that active matrilysin gene transcription and protein synthesis occur within dysplastic tubules and epithelial cells lining cysts in human RD by RT-PCR and immunohistochemistry. Similar staining patterns were seen in obstructed kidneys of pouch opossums that show histological features similar to that of human RD. In vitro, matrilysin inhibits formation of branching structures in mIMCD-3 cells stimulated by bone morphogenetic protein-7 (BMP-7) but does not inhibit hepatocyte growth factor-stimulated branching. BMP-7 signaling is essential for normal kidney development, and overexpression of catalytically active matrilysin in human embryonic kidney 293 cells reduces endogenous BMP-7 protein levels and inhibits phosphorylation of BMP-7 SMAD signaling intermediates. These findings suggest that matrilysin expression in RD may be an injury response that disrupts normal nephrogenesis by impairing BMP-7 signaling.

The role of axial structures, especially the notochord, in metanephric kidney development has not been directly examined. Here, we showed that disruption of the notochord and floor plate by diphtheria toxin (DTA)-mediated cell ablation did not disrupt nephrogenesis, but resulted in kidney fusions, resembling horseshoe kidneys in humans. Axial disruptions led to more medially positioned metanephric mesenchyme (MM) in midgestation. However, neither axial disruption nor the ensuing positional shift of the MM affected the formation of nephrons and other structures within the kidney. Response to Shh signaling was greatly reduced in midline cell populations in the mutants. To further ascertain the molecular mechanism underlying these abnormalities, we specifically inactivated Shh in the notochord and floor plate. We found that depleting the axial source of Shh was sufficient to cause kidney fusion, even in the presence of the notochord. These results suggested that the notochord is dispensable for nephrogenesis but required for the correct positioning of the metanephric kidney. Axial Shh signal appears to be critical in conferring the effects of axial structures on kidney positioning along the mediolateral axis. These studies also provide insights into the pathogenesis of horseshoe kidneys and how congenital kidney defects can be caused by signals outside the renal primordia., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Congenital renal dysplasia (RD) is a major cause of renal failure in the pediatric population. Although molecular and genetic aspects of RD have been studied in animal models, limited studies have been done in human RD primarily due to lack of available material. To identify novel genes that are associated with RD and normal kidney development, we performed microarray analysis on total RNA extracted from age-matched fetal kidneys of normal and RD patients. In midgestational RD kidneys, we found 180 upregulated and 104 downregulated transcripts compared with normal kidneys. Among the increased transcripts in the dysplastic kidneys were matrix-degrading enzymes (MMP7, MMP19, TIMP1), inflammation- and immunity-related genes, and growth factors. Expression of genes known to be essential for normal kidney development, such as WT1, BMP7, renin, angiotensin receptor 2 (AGTR2), SAL-like 1 (SALL1) and glypican 3 (GPC3), were decreased in dysplastic kidneys. Expression of selected gene products (BMP7, renin, and MMP7) was further confirmed in parallel sections and in several normal and human dysplastic kidneys, supporting the role of these genes as putative RD biomarkers. These results are among the first to reveal disrupted expression profiles during gestation in human RD patients.

Many molecules have been implicated in kidney development, often based on experimental animal studies with organ cultures and cell lines. There are very few studies, however, that have directly addressed equivalent living human embryonic tissues. We generated renal mesenchymal cell lines from normal human metanephroi and used a microarray strategy to define changes in gene expression after stimulation with growth factors which enhance nephrogenesis in rodents. Changes were observed in 1) genes modulating diverse general cellular processes, such as matrix metalloproteinase 1 and stanniocalcin 1; 2) genes previously implicated in organogenesis e.g., sprouty 4 and midline 1; and 3) genes involved in blood vessel growth, including angiopoietin 1 and 4. Expression of these same genes was subsequently confirmed in vivo. Our novel data have identified several previously unhighlighted genes that may be implicated in differentiation programs within early human nephrogenesis.

Background: In chronic renal failure (CRF), hyperphosphatemia and an elevated calcium-phosphate product are associated with vascular calcification and increased cardiovascular morbidity and mortality. Previous data have demonstrated that 3-month treatment of uremic rats with sevelamer was associated with less nephrocalcinosis compared to calcium carbonate (CaCO3), despite similar control of serum phosphorus, calcium-phosphorus product (Ca x P product), and secondary hyperparathyroidism. There was no evidence of aortic calcification after 3 months of uremia (J Am Soc Nephrol 13:2299-2308, 2002). The present studies explore the influence of sevelamer and CaCO3 on cardiovascular and kidney calcifications in long-term experimental uremia over 6 months., Methods: Normal and 5/6 nephrectomized rats (U) were fed a high phosphorus (HP) diet for 6 months. Two phosphate binders, CaCO3 and sevelamer, were administered and their influence on hyperphosphatemia, secondary hyperparathyroidism, kidney/myocardial/aortic calcification, and renal function was compared., Results: All uremic rats began the study with the same degree of renal failure. Sevelamer was as effective as CaCO3 in reducing serum phosphorus, Ca x P product, and attenuating secondary hyperparathyroidism. Despite similar serum cholesterol levels, rats in the U-HP + sevelamer group had markedly lower calcium deposition in the myocardium and aorta (myocardium, 72 +/- 4 microg/g wet tissue; aorta, 736 +/- 156 microg/g wet tissue) compared to rats in either the U-HP + CaCO3 group (myocardium, 179 +/- 48, P < 0.05; aorta, 1308 +/- 343, P < 0.05) or the U-HP group (myocardium, 98 +/- 10, NS; aorta, 2150 +/- 447, P < 0.05). Dual immunohistochemical analysis for calcium and endothelial cell markers demonstrated that myocardial calcium deposition was intravascular within capillaries. Furthermore, calcium deposition in the kidney of uremic rats treated with sevelamer (582 +/- 111 microg/g wet tissue) was lower than that found in uremic rats treated with CaCO3 (1196 +/- 180 microg/g wet tissue). Sevelamer-treated rats had less deterioration in renal function with an associated lower serum creatinine, higher creatinine clearance, and less proteinuria. There was no difference in overall mortality between the three experimental groups., Conclusion: In long-term experimental CRF, in addition to controlling serum phosphorus and secondary hyperparathyroidism as efficiently as CaCO3, treatment with the phosphate-binder sevelamer attenuates vascular and kidney calcification.

Background: Quantitative blood polymerase chain reaction (PCR) for cytomegalovirus (CMV) is used to direct therapy in kidney transplant patients, but cytomegalic inclusions are rarely found in allograft renal biopsies even with an elevated serum creatinine and apparent CMV disease. The relationship between quantitative blood CMV and renal allograft pathology is unknown., Methods: Thirteen biopsy samples were available for analysis from patients suspected of CMV disease, who had a buffy coat CMV-PCR drawn within 2-5 days of a renal allograft biopsy for an elevated creatinine. All were evaluated for CMV pathologically, by light microscopy, immunohistochemistry, in situ hybridization and tissue PCR., Results: Qualitative and quantitative buffy coat CMV-PCR were positive in 10/13 (77%) patients. Tissue CMV-PCR was positive in five (50%) biopsies, including two with CMV inclusions and three with no inclusions. Quantitative buffy coat CMV-PCR levels did not correlate with detection of CMV inclusions in renal tissue. Paradoxically, quantitative buffy coat CMV-PCR was low (239 and 538 copies/microg of DNA) when CMV inclusions were detected. All five biopsies with acute rejection were associated with CMV viraemia and two of the five with allograft CMV inclusions. A quantitative buffy coat CMV-PCR of <100 copies/microg of DNA ruled out disease with CMV inclusions., Conclusions: CMV nephropathy is much more common than previously reported when sensitive techniques are used for detection in tissue. Acute rejection and CMV viraemia occur commonly together in patients at risk for CMV. Quantitative buffy coat CMV-PCR does not correlate with the presence of CMV inclusions. These findings have implications for management of patients who have elevated serum creatinine and are at risk for CMV disease.

Congenital obstructive nephropathy is a common disease affecting fetuses and young children. The kidney shows profound morphologic and functional changes. The physiologic developmental kidney program is disturbed in the most advanced cases, arguing for altered temporal/spatial expression of genes which control normal nephrogenesis. Major regulators of mesenchymal-epithelial transformation and collecting duct and tubular development such as WT1 and Sall1 are decreased with obstruction. Additional candidate genes include GDNF/cRET, LIM1 and Pax2. Excessive apoptosis is an undisputed mechanism in these processes, mediated by decreased expression of apoptosis inhibiting genes (Bcl-2, HGF, IGF, BMP7), and overexpression of pro-apoptotic genes like Bax and TGF-beta. Renin and AT2R implicated in renal vascular development are decreased. Numerous extracellular matrix genes including Matrilysin are altered. The emerging theories of the biology of congenital obstructive nephropathy suggest new targets for therapeutic interventions with profound implications for these children., (Copyright 2003 S. Karger AG, Basel)

Purpose: We studied the long-term effects of complete unilateral ureteral obstruction on the extracellular matrix composition of the fetal kidney interstitium in neonatal opossums., Materials and Methods: We created complete left ureteral obstruction in 2-week-old opossum pups. Sham operated animals served as controls. A total of 12 animals were sacrificed 2, 3 and 5 weeks after obstruction, and the kidneys were harvested. Resultant tissue sections were prepared for light and electron microscopy, and immunohistochemical testing for alpha-smooth muscle action (SMA), fibronectin, laminin and type IV collagen. In situ hybridization was done to determine interstitial collagen type I. Western blot analysis was performed on single kidney protein extracts for alpha-SMA, fibronectin and laminin., Results: Light microscopy revealed established interstitial fibrosis and tubular atrophy 2 weeks after obstruction which increased with the duration of obstruction. Electron microscopy showed an increased number of interstitial fibroblasts, increased extracellular lucent substance and thin collagen fibrils. The tubular basement membranes were irregular and thickened compared with the continuous and even contours in controls. The deposition of alpha-SMA, fibronectin, collagen types I and type IV were markedly increased in the interstitium compared with controls and were highest at 5 weeks. Type IV collagen was associated with aberrant peritubular capillaries within the scarred interstitium. There were no appreciable differences in laminin expression in obstructed and sham operated kidneys., Conclusions: Experimental obstruction in opossums alters the cell phenotype, extracellular matrix and microcapillary composition in the fetal kidney interstitium. Biosynthetic properties of the interstitial fibroblasts in obstructed kidneys appear similar to those of undifferentiated mesenchyma before the induction of nephrons. We propose that these aberrant extracellular matrices coincident with cytoarchitectural disturbance may form a faulty scaffold that hampers normal kidney development secondary to complete unilateral ureteral obstruction.

Angiotensin II upregulates tumor necrosis factor-alpha (TNF-alpha) in the rat kidney with unilateral ureteral obstruction (UUO). In a mouse model of UUO, we found that tubulointerstitial fibrosis is blunted when the TNF-alpha receptor, TNFR1, is functionally knocked out. In this study, we used mutant mice with UUO in which the angiotensin II receptor AT(1a) or the TNF-alpha receptors TNFR1 and TNFR2 were knocked out to elucidate interactions between the two systems. The contribution of both systems to renal fibrosis was assessed by treating TNFR1/TNFR2-double knockout (KO) mice with an angiotensin-converting enzyme inhibitor, enalapril. The increased interstitial volume (Vv(int)) in the C57BI/6 wild-type mouse was decreased in the AT(1a) KO from 32.8 +/- 4.0 to 21.0 +/- 3.7% (P < 0.005) or in the TNFR1/TNFR2 KO to 22.3 +/- 2.1% (P < 0.005). The Vv(int) of the TNFR1/TNFR2 KO was further decreased to 15.2 +/- 3.7% (P < 0.01) by enalapril compared with no treatment. The induction of TNF-alpha mRNA and transforming growth factor-beta1 (TGF-beta1) mRNA in the kidney with UUO was significantly blunted in the AT(1a) or TNFR1/TNFR2 KO mice compared with the wild-type mice. Treatment of the TNFR1/TNFR2 KO mouse with enalapril reduced both TNF-alpha and TGF-beta1 mRNA and their proteins to near normal levels. Also, alpha-smooth muscle actin expression and myofibroblast proliferation were significantly inhibited in the AT(1a) or TNFR1/TNFR2 KO mice, and they were further inhibited in enalapril-treated TNFR1/TNFR2 KO mice. Incapacitating the angiotensin II or the TNF-alpha systems individually leads to partial blunting of fibrosis. Incapacitating both systems, by using a combination of genetic and pharmacological means, further inhibited interstitial fibrosis and tubule atrophy in obstructive nephropathy.

To determine whether transplanted metanephroi grow, differentiate, and function in hosts that differ in major histocompatibility complex loci (RT1 loci in rats) from donors in a defined way, we implanted metanephroi from embryonic day (E) 15 PVG (RT1(c)) rat embryos into the omentum of nonimmunosupressed uninephrectomized PVG-RT1(avl) (host) rats. By 4 wk posttransplantation, metanephroi had grown and differentiated such that glomeruli, proximal and distal tubules, and collecting ducts had normal structure and ultrastructure. At 12 wk posttransplantation, weights of metanephroi were 54 +/- 8 mg. Inulin clearances were 0.9 +/- 0.3 microl. min(-1). 100 g rat wt(-1). In vitro, splenocytes from PVG rats stimulated the proliferation of cells originating from both PVG-RT1(avl) rats in which a transplant had been performed and PVG-RT1(avl) rats with no transplant. Full-thickness PVG-RT1(avl) skin engrafted normally on PVG-RT1(avl) rats in which PVG metanephroi had been previously implanted and metanephroi retained a normal appearance. In contrast, skin from PVG rats sloughed, and the tubular architecture of metanephroi was obliterated by a mononuclear cell infiltrate consistent with acute rejection. Here we show for the first time that functional chimeric kidneys develop from metanephroi transplanted across the MHC into nonimmunosupressed hosts and provide evidence that a state of peripheral immune tolerance secondary to T cell "ignorance" permits their survival.

Purpose: Complete ureteral obstruction (CUTO) in the fetal kidney induces tubular and glomerular cysts, interstitial fibrosis, and halts renal development. Previous studies have shown that apoptosis is a predominant mechanism in the chronically injured kidney following obstruction, but the precise cellular and molecular mechanisms are poorly understood., Materials and Methods: We have used CUTO in opossum pups with early metanephric kidneys, sacrificed at two weeks, to evaluate the role of cell proliferation, apoptosis and apoptosis regulating genes, Bcl-2 and Bax., Results: Obstructed fetal kidneys demonstrate high apoptosis in the renal pelvis and tubulointerstitium, compared with sham operated animals. Apoptosis is accompanied by statistically significant increased cell proliferation in the interstitium but not in tubules. Apoptosis in the tubules is accompanied by increased Bax and decreased Bcl-2 staining. In the nephrogenic zone apoptosis is increased, even though it is not statistically significant. Bcl-2 and Bax in the nephrogenic zone are unchanged compared with sham, but cell proliferation is increased., Conclusions: We suggest that abnormal patterns of cell kinetics may contribute to disease pathogenesis in the obstructed fetal kidney.

Unilateral ureteral obstruction (UUO) is a model of renal injury characterized by progressive tubulointerstitial fibrosis and renal damage, while relatively sparing the glomerulus and not producing hypertension or abnormalities in lipid metabolism. Tubulointerstitial fibrosis is a major component of several kidney diseases associated with the progression to end-stage renal failure. Here we report that when a critical renal developmental morphogen, osteogenic protein-1 (OP-1; 100 or 300 microg/kg body wt), is administered at the time of UUO and every other day thereafter, interstitial inflammation and fibrogenesis are prevented, leading to preservation of renal function during the first 5 days after obstruction. Compared with angiotensin-converting enzyme inhibition with enalapril treatment, OP-1 was more effective in preventing tubulointerstitial fibrosis and in preserving renal function. The mechanism of OP-1- induced renal protection was associated with prevention of tubular atrophy, an effect not shared with enalapril, and was related to preservation of tubular epithelial integrity. OP-1 blocked the stimulation of epithelial cell apoptosis produced by UUO, which promoted maintenance of tubular epithelial integrity. OP-1 preserved renal blood flow (RBF) during UUO, but enalapril also stimulated RBF. Thus OP-1 treatment inhibited tubular epithelial disruption stimulated by the renal injury of UUO, preventing tubular atrophy and diminishing the activation of tubulointerstitial inflammation and fibrosis and preserving renal function.

The K-Cl cotransporter protein KCC1 is a membrane transport protein that mediates the coupled, electroneutral transport of K and Cl across plasma membranes. The precise cell type(s) in the kidney that express the K-Cl cotransporter have remained unknown. The aim of the present investigation was to define the distribution of KCC1 mRNA in the human kidney. We used in situ hybridization with a nonradioactive digoxigenin-labeled riboprobe. We identified abundant KCC1 mRNA expression in the epithelial cells throughout the distal and proximal renal tubular epithelium. The transporter was also expressed in glomerular mesangial cells and endothelial cells of the renal vessels. These findings suggest that the K-Cl cotransporter may have an important role in transepithelial K and Cl reabsorption.

Members of the forkhead/winged-helix transcription factor family play crucial roles during vertebrate development. A human hepatocyte nuclear factor/forkhead homolog (HFH)-4 cDNA encoding a 421-amino acid protein was isolated from a human fetal lung cDNA library. By Southern blot analysis of human-rodent somatic cell hybrid genomic DNA, the human HFH-4 gene localizes to chromosome 17q23-qter. This is the locus of another forkhead/winged-helix gene, the interleukin enhancer binding factor gene. RNA blot analysis revealed a 2.5-kilobase human HFH-4 transcript in fetal lung, kidney, and brain as well as in adult reproductive tissues, lung, and brain. By in situ hybridization, HFH-4 expression is associated with differentiation of the proximal pulmonary epithelium, starting during the pseudoglandular stage of human lung development. During human renal morphogenesis, HFH-4 is expressed in the developing epithelial cells of the ureteric duct, glomerulus, and epithelial vesicles. The unique pattern of HFH-4 expression during human fetal development suggests a role for this forkhead/winged-helix factor during pulmonary and renal epithelial development.

We have used our opossum model of fetal nephrotoxicity to investigate uranyl nitrate-induced morphologic changes in the developing kidney. The present study establishes a renal dose response curve for the uranyl nitrate (UN). We find that pups treated with nonlethal doses of UN do not demonstrate growth retardation compared to saline-treated controls. The kidneys of UN-treated pups are heavier than the control animals, an effect less apparent the longer the pups are followed. A low dose of 60 mg/kg of UN administered to small pups causes slight histologic derangement but nevertheless more change than the same dose administered to larger more mature pups. Using a dose of 100 mg/kg of UN that effectively causes nonfatal renal disruption, we examined the kidneys from 4 to 42 days following injection. We find that tubular dilation and epithelial necrosis starts soon after treatment (day 4) and reaches its maximum during the second and third week (11 and 22 days). Architectural restoration appears complete by the end of the third week. By electron microscopy, UN induces sequential structural damage with loss of proximal tubule brush border, epithelial necrosis with intact basement membranes and regeneration at 4, 11, and 22 days. Residual tubular mitochondrial damage is present at 42 days in spite of histologically normal tubules. No apparent lesions are seen in glomeruli. Fibroblastic interstitial proliferation in UN-treated kidneys at 11 days is not followed by appreciable fibrosis when assessed at 22 and 42 days. As the structural changes caused by 100 mg/ml UN administration in fetal opossum kidneys are reversible, this is a useful model to study the molecular mediators responsible for this form of renal damage and repair.

The expression of platelet-derived growth factor A (PDGF-A), and its spatial and temporal relationship to interstitial collagens in kidneys with congenital multicystic dysplasia using in situ hybridization, have been examined. Seventeen dysplastic kidneys (16 weeks to 7 months) and 20 normal age-matched controls were used in the study. Increased PDGF-A mRNA was detected in dysplastic compared to normal kidneys in all age groups including extensively fibrotic postnatal kidneys. An abundant PDGF-A mRNA signal was seen within the epithelial cells of cystically dilated or dysplastic tubules and within interstitial fibroblasts and disorganized primitive mesenchyme. A comparable amount of PDGF-A protein was detected by Western blotting. Procollagen I and III mRNA were increased in fibroblasts surrounding cystic and dysplastic tubules. We conclude that tubular epithelial production of PDGF-A may induce collagenous matrix production by adjacent fibroblasts, while marked up-regulation of PDGF-A by interstitial cells may be responsible for sustainable fibrogenic effects in the fetal kidney contributing to renal maldevelopment.

Due to the transient transcription of the gene of erythropoietin (EPO) in response to hypoxic stimuli, localization within the cell of origin in humans has not been possible. Under acutely induced transcription of the EPO gene, through severe bleeding (hematocrit < 10%), EPO transcripts were detected within renal interstitial cells in rodents. However, by immunohistochemistry or tissue culture, human tubular epithelial cells or glomerular mesangium have been shown to produce EPO, suggesting a species difference. Aiming to investigate the precise location of EPO in intact human kidneys, 38 fetal, infant, and adult kidneys were examined by in situ hybridization. Kidneys from patients with conditions predisposing to severe hypoxia and/or induced polycythemia were utilized, because under these conditions active EPO mRNA synthesis is expected. We found specific EPO mRNA transcripts within a small population of cortical interstitial cells of near-term or postnatal kidneys.

Complete ureteral obstruction in fetal opossum kidneys has been used as an experimental method to induce tubulointerstitial damage and interstitial fibrosis. However, the molecular events underlying extracellular matrix deposition are currently unknown. Cytokines such as platelet-derived growth factor (PDGF) are considered possible participants in these processes. In this study we used a marsupial model of ureteral obstruction to examine the expression of PDGF-A and type I procollagen mRNAs by in situ hybridization. Complete unilateral ureteral obstruction was performed in six animals at midtrimester human equivalent. Obstructed kidneys, as well as the contralateral and age-matched sham kidneys, were harvested at 1, 3, 5, 10, and 20 days post obstruction. Morphological assessment of the obstructed kidneys harvested between 1 and 5 days post obstruction showed mild tubulocystic changes, interstitial fibrosis, and inflammation compared with controls. Kidneys harvested at days 10 and 20 showed moderate tubulointerstitial damage compared with kidneys harvested after 1 and 5 days. PDGF-A mRNA signal of low abundance was detected within renal interstitial cells, urothelial cells of the pelvis, and focally within epithelial cells of immature distal convoluted tubules in non-obstructed kidneys. Type I procollagen mRNA expression was spatially co-distributed with PDGF-A-expressing interstitial cells. PDGF-A and type I procollagen signal intensities in obstructed kidneys harvested 10 and 20 days post obstruction were increased several fold compared with controls and kidneys harvested 1-5 days post obstruction. Both PDGF-A and type I procollagen mRNA increases correlated with morphological features of tubulointerstitial damage. Our results suggest that PDGF-A may participate in this form of fetal kidney damage.