Urine exosomal ceruloplasmin: a potential early biomarker of underlying kidney disease.

BACKGROUND: Previously we found that kidney tissue and urinary exosomes from patients of diabetic kidney disease showed high levels of ceruloplasmin (CP). Because CP is an acute-phase protein of kidney origin, it could be an early marker of many other kidney diseases. To investigate this hypothesis, we first measured urine exosomal and kidney expression of CP in non-diabetic chronic kidney disease (CKD) patients (membranous nephropathy, focal segmental glomerulosclerosis, lupus nephritis and IgA nephropathy) followed by a longitudinal study in rat passive Heymann nephritis (PHN), a model of human membranous nephropathy. METHODS: Urinary exosomes were isolated from urine of patients (and rats) by differential centrifugation. The exosomal extracts were used for measuring CP using ELISA. Kidney expression of CP was evaluated by immune-staining biopsy tissues. Similar techniques were applied in rat PHN model (produced by injection of anti-gp600 antiserum) to analyze urine exosomal and kidney CP. RESULTS: Urine exosomal CP levels were 10-20 times higher in CKD patients than in controls; consistent with this we found high immune-reactive CP localized in tubules and collecting ducts of biopsies of CKD patients. In the PHN model urinary exosomal CP level was significantly higher prior to the onset of proteinuria. Early rise of urine exosomal CP, which preceded proteinuria, correlated with high immunoreactive CP found in rat kidneys at this time. CONCLUSION: We propose that urine exosomal CP, observed to increase prior to proteinuria, makes it a potential urinary biomarker to diagnose early kidney disease.

Peptiduria: a potential early predictor of diabetic kidney disease.

BACKGROUND: To protect the kidney effectively with medication in type 2 diabetics, it is crucial to identify such at-risk patients early for treatment. We investigated whether peptiduria precedes proteinuria (the earliest urinary marker in our model), and thereby serve as an early predictor of diabetic nephropathy. METHODS: A longitudinal study was performed in a rat model of diabetic nephropathy. Peptides, defined as degradation products of proteins of < 13 kD size, were quantified by a previously validated method using a combination of Lowry and Biorad protein assays. Peptides in urine were also confirmed by chromatographically separating low molecular weight fractions from urine and quantifying albumin fragments in these fractions by enzyme immunoassay. Also, the mechanism of peptiduria was addressed by measuring acid phosphatase, a marker of lysosomal activity, in urine and on kidney sections (histochemically). RESULTS: In rats with diabetic nephropathy, proteinuria occurred after 12 weeks of diabetes, while peptiduria occurred as early as 2 weeks after diabetes. Peptiduria was confirmed by showing that the chromatographically separated low molecular weight fractions of urine containing albumin fragments is in proportion to the level of peptiduria. The time course of peptiduria paralleled the increase in urinary acid phosphatase suggesting that the mechanism of early peptiduria could be due to upregulation of lysosomal enzyme activity in the tubules. CONCLUSIONS: Our results showing that peptiduria precedes proteinuria in diabetic nephropathy provide a compelling rationale to perform a prospective human clinical trial to investigate whether peptiduria can serve as an early predictor of diabetic nephropathy.

In Diabetic Kidney Disease Urinary Exosomes Better Represent Kidney Specific Protein Alterations Than Whole Urine.

BACKGROUND: Predicting or diagnosing underlying kidney disease by analyzing whole urine remains the mainstay of nephrology practice. However, whole urine is a poor compartment to assess many structural changes in the kidney because whole urine contains only a few proteins derived from the kidney itself. Urinary exosomes, on the other hand, which are derived from the kidney, contain proteins secreted by the kidney. We experimentally tested the hypothesis that 'urinary exosomes more faithfully represent changes in the kidney tissue than whole urine'. A direct comparison between whole urine and urine exosomal levels of two chosen kidney disease markers, gelatinase and ceruloplasmin, was carried out on diabetic kidney disease patients. METHODS: Urinary exosomes were separated from whole urine by sequential centrifugation including ultra-centrifugation. Gelatinase activity was measured using fluorosceinated gelatin as the substrate, and ceruloplasmin was measured by sandwich ELISA. A few kidney specimens from patients biopsied for atypical features were histochemically stained for validation of the biochemical results. RESULTS: We found that changes in both, gelatinase (decreased activity) and ceruloplasmin (increased levels), in the urinary exosomes of diabetic kidney patients were in agreement with the alterations of these two proteins in the kidney tissue. In contrast, the levels of these two proteins in whole urine were highly variable and did not correlate with levels in the diabetic kidney tissue. CONCLUSION: In conclusion, these results confirmed our hypothesis that protein markers in urinary exosomes better reflected the underlying protein changes in the kidney than in whole urine samples.

Activated omentum slows progression of CKD.

Stem cells show promise in the treatment of AKI but do not survive long term after injection. However, organ repair has been achieved by extending and attaching the omentum, a fatty tissue lying above the stomach containing stem cells, to various organs. To examine whether fusing the omentum to a subtotally nephrectomized kidney could slow the progression of CKD, we used two groups of rats: an experimental group undergoing 5/6 nephrectomy only and a control group undergoing 5/6 nephrectomy and complete omentectomy. Polydextran gel particles were administered intraperitoneally before suture only in the experimental group to facilitate the fusion of the omentum to the injured kidney. After 12 weeks, experimental rats exhibited omentum fused to the remnant kidney and had lower plasma creatinine and urea nitrogen levels; less glomerulosclerosis, tubulointerstitial injury, and extracellular matrix; and reduced thickening of basement membranes compared with controls. A fusion zone formed between the injured kidney and the omentum contained abundant stem cells expressing stem cell antigen-1, Wilms' tumor 1 (WT-1), and CD34, suggesting active, healing tissue. Furthermore, kidney extracts from experimental rats showed increases in expression levels of growth factors involved in renal repair, the number of proliferating cells, especially at the injured edge, the number of WT-1-positive cells in the glomeruli, and WT-1 gene expression. These results suggest that contact between the omentum and injured kidney slows the progression of CKD in the remnant organ, and this effect appears to be mediated by the presence of omental stem cells and their secretory products.

A novel model of surgical injury in adult rat kidney: a "pouch model".

Regenerative mechanisms after surgical injury have been studied in many organs but not in the kidney. Studying surgical injury may provide new insights into mechanisms of kidney regeneration. In rodent models, extrarenal tissues adhere to surgical kidney wound and interfere with healing. We hypothesized that this can be prevented by wrapping injured kidney in a plastic pouch. Adult rats tolerated 5/6 nephrectomy with pouch application well. Histological analysis demonstrates that application of the pouch effectively prevented formation of adhesions and induced characteristic wound healing manifested by formation of granulation tissue. Additionally, selected tubules of the wounded kidney extended into the granulation tissue forming branching tubular epithelial outgrowths (TEOs) without terminal differentiation. Tubular regeneration outside of renal parenchyma was not previously observed, and suggests previously unrecognized capacity for regeneration. Our model provides a novel approach to study kidney wound healing.

Acute Epstein-Barr virus infection-associated collapsing glomerulopathy.

A 21-year-old woman presenting with acute Epstein-Barr virus (EBV) infection (infectious mononucleosis) was noted to have renal involvement. She had proteinuria, leukocyturia and microscopic hematuria, and 10 days after admission became nephrotic (23 g of protein per g of creatinine). Renal biopsy revealed glomerular tuft collapse, visceral epithelial cell proliferation and vacuolization consistent with collapsing glomerulopathy. She had only transient deterioration in renal function, attributed to contrast nephropathy, but after recovery remained proteinuric. Renal disease is well described in EBV infection, but collapsing glomerulopathy has not been reported previously.

Stem cells from foreign body granulation tissue accelerate recovery from acute kidney injury.

BACKGROUND: In previous studies, we obtained mesenchymal stem cells called granulation tissue stem cells (GTSC) from a regenerating granulation tissue created by placing a foreign body in the subcutaneous tissue of rats. Here, we used GTSC to ameliorate ischemia/reperfusion-induced acute kidney injury (AKI) in rats. METHODS: In two groups of Fischer rats, we induced ischemia/reperfusion injury. Group 1 (treated rats) received one intravenous injection of GTSC 3 h after injury; Group 2 (control rats) received vehicle. Both groups were subsequently studied by renal function tests, kidney histology and immunohistochemistry. RESULTS: At 24 and 48 h after injury, plasma creatinine and blood urea nitrogen were significantly lower in the treated rats as compared to control rats. The levels remained low and declined to near baseline levels by Day 4 in the treated group. At the cortico-medullary region, the treated rats showed significantly higher renal tubular cell proliferation and less tubular cell apoptosis. Histological analysis of the kidney for tubular dilatation, necrosis, congestion and casts was not significantly different in the two groups. To understand the mechanism of the GTSC effect, messenger RNA levels of several growth and immune modulatory factors were quantified in cultured GTSC and compared with those in cultured glomerular epithelial cell (GEC; a non-stem cell line). GTSC had 2- to 8-fold higher expression of FGF2, HGF, IGF-1, vascular endothelial growth factor (growth factors) and IL-4, IL-6 (anti-inflammatory factors) than GEC. CONCLUSIONS: Administration of GTSC accelerates recovery in rats with ischemia/reperfusion-induced AKI. This effect may be mediated by the paracrine action of growth and immune-suppressive factors secreted by these cells.

Culture of omentum-induced regenerating liver yielded hepatocyte-committed stem cells.

Earlier we showed that when omentum, activated by inert particles, is allowed to fuse to a wedge cut in the liver, it induces stem cell proliferation in the liver resulting in massive liver regeneration. Here, we attempt to culture stem cells from the omentum-induced regenerating liver tissue. Cells from regenerating liver tissue were harvested and cultured. Cultured cells were characterized by immune staining, fluorescence activated cell sorting analysis, growth factor assay, in vitro differentiation, and their ability to engraft to injured sites in vivo. Culture yielded cells with a mesenchymal stem cell phenotype that could be maintained in culture indefinitely. These cells, called regenerating liver stem cells, expressed both adult and embryonic stem cell markers, secreted high levels of vascular endothelial growth factor, and expressed albumin. When grown on matrigel in the presence of hepatocyte growth factor, these cells differentiated into hepatocyte-like cells in culture, but they did not differentiate to adipogenic and osteogenic lineages when grown in specific differentiation medium. The differentiated cells expressed α-fetoprotein and secreted high levels of albumin and urea. After systemic injection, the undifferentiated cells engrafted only to the injured sites in the liver and not to the normal areas of the liver. In conclusion, omentum-induced regenerating liver yields hepatocyte-committed stem cells in culture. Such cells could prove to be useful in cell transplantation therapies.

Foreign body-induced granulation tissue is a source of adult stem cells.

In the current study, we have cultured and propagated the cells obtained from the granulation tissue that forms around perforated polyvinyl tubes placed in the subcutaneous space of normal rats. We found that these cells (called granulation tissue-derived stem cells [GTSCs]) expressed markers of embryonic pluripotent cells (Oct-4 and Nanog) and of adult stem cells (CXCR4 and Thy1.1) as well as produced high levels of vascular endothelial growth factor (VEGF) for up to 10 passages. By fluorescence-activated cell-sorting (FACS) analysis, GTSCs were positive for stem-cell surface markers CD90, CD59, and CD44 and were negative for CD45, which suggests that they were of mesenchymal origin and not of hematopoietic lineage. When incubated in specific differentiation medium, these cells transformed into adipogenic, osteogenic, and chondrogenic lineages, which shows that they were multipotent. Furthermore, after systemic injection, these cells were found in the vicinity of an injured site created in the liver but not in normal areas of the liver, which indicates their propensity to seek and engraft to an injured area in the body. We conclude that granulation tissue induced by a large foreign body is a convenient source of adult stem cells that can be maintained in culture and can be used to repair and regenerate injured tissue.

Omentum facilitates liver regeneration.

AIM: To investigate the mechanism of liver regeneration induced by fusing the omentum to a small traumatic injury created in the liver. We studied three groups of rats. In one group the rats were omentectomized; in another group the omentum was left in situ and was not activated, and in the third group the omentum was activated by polydextran particles. METHODS: We pre-activated the omentum by injecting polydextran particles and then made a small wedge wound in the rat liver to allow the omentum to fuse to the wound. We monitored the regeneration of the liver by determining the ratio of liver weight/body weight, by histological evaluation (including immune staining for cytokeratin-19, an oval cell marker), and by testing for developmental gene activation using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: There was no liver regeneration in the omentectomized rats, nor was there significant regeneration when the omentum was not activated, even though in this instance the omentum had fused with the liver. In contrast, the liver in the rats with the activated omentum expanded to a size 50% greater than the original, and there was histologically an interlying tissue between the wounded liver and the activated omentum in which bile ducts, containing cytokeratin-19 positive oval cells, extended from the wound edge. In this interlying tissue, oval cells were abundant and appeared to proliferate to form new liver tissue. In rats pre-treated with drugs that inhibited hepatocyte growth, liver proliferation was ongoing, indicating that regeneration of the liver was the result of oval cell expansion. CONCLUSION: Activated omentum facilitates liver regeneration following injury by a mechanism that depends largely on oval cell proliferation.

A case of lymphangioleiomyomatosis with membranous nephropathy and likely systemic lupus.

Membranous glomerulonephritis is most often idiopathic, but it can be secondary to systemic lupus erythematosus, viral hepatitis, and drugs. A number of malignancies have also been associated with membranous glomerulonephritis, although a causal link has not been established yet. A young patient with lymphangioleiomyomatosis is described who developed massive proteinuria and was found to have membranous glomerulonephritis on renal biopsy. There were many pathological features and some clinical features that suggested the patient also suffered from systemic lupus erythematosus.

Stromal cells cultured from omentum express pluripotent markers, produce high amounts of VEGF, and engraft to injured sites.

When rat omentum becomes activated by intraperitoneal injection of inert polydextran particles, these particles are rapidly surrounded by cells that express markers of adult stem cells (SDF-1alpha, CXCR4, WT-1) and of embryonic pluripotent cells (Oct-4, Nanog, SSEA-1). We have cultured such cells, because they may offer a convenient source of adult stem cells, and have found that they retain stem cell markers and produce high levels of vascular endothelial growth factor for up to ten passages. After systemic or local injection of these cultured cells into rats with acute injury of various organs, the cells specifically engraft at the injured sites. Thus, our experiments show that omental stromal cells can be cultured from activated omentum, and that these cells exhibit stem cell properties enabling them to be used for repair and possibly for the regeneration of damaged tissues.

Impaired integration of endothelial progenitor cells in capillaries of diabetic wounds is reversible with vascular endothelial growth factor infusion.

To understand impaired angiogenesis in diabetic wounds, polyvinyl tubes were implanted subcutaneously in rats to form a granulation tissue for 2 weeks and the granulation tissue was studied after inducing diabetes with streptozotocin. By 1 week of diabetes, the granulation tissue was bloody and thinner than controls, its medial layer was depleted of microvessels, and the surviving vessels appeared dehisced. Vascular endothelial growth factor (VEGF) in the diabetic granulation tissue was reduced by 50% compared with control granulation tissue. After 3 days of diabetes, the diabetic tissue showed a greater degree of apoptosis in the microvessels. Chemotactic factors [stromal cell-derived factor-1alpha and chemokine receptor-4 (CXCR-4)], responsible for attracting bone marrow cells, showed equal intensity in control and diabetic tissues. As expected, progenitor endothelial CD-34 cells were observed in abundance in both the control and the diabetic granulation tissues. However, although the CD-34-positive cells appeared mostly to be integrated in the blood vessels of the control tissue, fewer such cells were present in the blood vessels of the diabetic tissues, suggesting that CD-34 failed to integrate into new blood vessels. Infusion of VEGF in the granulation tissue of diabetic rats for 1 week resulted in complete prevention of the microvascular defect compared with the contralateral granulation tissue that showed the typical diabetic changes. It was concluded that diabetes causes reduction of VEGF in the wound, resulting in loss of blood vessels by apoptosis and possible failure of CD-34 cells to integrate into the vessel structure.

Activated omentum becomes rich in factors that promote healing and tissue regeneration.

In order to study the mechanism by which an omental pedicle promotes healing when applied to an injured site, we injected a foreign body into the abdominal cavity to activate the omentum. One week after the injection, we isolated the omentum and measured blood vessel density, blood content, growth and angiogenesis factors (VEGF and others), chemotactic factors (SDF-1 alpha), and progenitor cells (CXCR-4, WT-1). We found that the native omentum, which consisted mostly of adipose tissue, expanded the mass of its non-adipose part (milky spots) 15- to 20-fold. VEGF and other growth factors increased by two- to four-fold, blood vessel density by three-fold, and blood content by two-fold. The activated omentum also showed increases in SDF-1 alpha, CXCR-4, and WT-1 cells (factors and cells positively associated with tissue regeneration). Thus, we propose that an omentum activated by a foreign body (or by injury) greatly expands its milky-spot tissue and becomes rich in growth factors and progenitor cells that facilitate the healing and regeneration of injured tissue.

Bilateral emphysematous pyelonephritis in a patient with no known risk factors.

Emphysematous pyelonephritis (EPN) is a rare life-threatening necrotizing infection of the kidney and perirenal space with gas formation. It is usually unilateral and affects patients with a risk factor such as diabetes or urinary obstruction. In the past, most patients required nephrectomy, and in bilateral cases long-term dialysis was inevitable. We present here the unusual case of a patient who developed bilateral EPN in the absence of any known risk factor. He was managed conservatively, required dialysis and bilateral nephrostomies, but eventually recovered completely.

Transplanting fragments of diabetic pancreas into activated omentum gives rise to new insulin producing cells.

To determine if pancreatic progenitor cells can be induced to form insulin producing cells in vivo, we auto-transplanted fragments of streptozotocin-induced diabetic pancreas into omentum pre-injected with a foreign material. As shown previously, omentum pre-activated in this manner becomes rich in growth factors and progenitor cells. After auto-transplanting diabetic pancreas in the activated omentum, new insulin secreting cells appeared in the omentum in niches surrounding the foreign particles--a site previously shown to harbor progenitor cells. Extracts of these omenta contained measurable insulin. Four of eight diabetic animals treated in this manner became normoglycemic. This shows that new insulin producing cells can be regenerated from diabetic pancreas by auto-transplanting pancreatic fragments into the activated omentum, an environment rich in growth factors and progenitor cells.

Idiopathic hypocomplementemic immune-complex-mediated tubulointerstitial nephritis.

BACKGROUND: A 42-year-old man presenting with flank pain was found to have renal failure with severe hypocomplementemia and eosinophilia. INVESTIGATIONS: Physical examination, laboratory testing, renal ultrasonography, and renal biopsies. DIAGNOSIS: Acute immune-complex-mediated tubulointerstitial nephritis. MANAGEMENT: Immunosuppressive therapy with 1 mg/kg/day prednisone.