A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.     

MicroRNA-22 Is a Master Regulator of Bone Morphogenetic Protein-7/6 Homeostasis in the Kidney

Accumulating evidence suggests that microRNAs (miRNAs) contribute to a myriad of kidney diseases. However, the regulatory role of miRNAs on the key molecules implicated in kidney fibrosis remains poorly understood. Bone morphogenetic protein-7 (BMP-7) and its related BMP-6 have recently emerged as key regulators of kidney fibrosis. Using the established unilateral ureteral obstruction (UUO) model of kidney fibrosis as our experimental model, we examined the regulatory role of miRNAs on BMP-7/6 signaling. By analyzing the potential miRNAs that target BMP-7/6 in silica, we identified miR-22 as a potent miRNA targeting BMP-7/6. We found that expression levels of BMP-7/6 were significantly elevated in the kidneys of the miR-22 null mouse. Importantly, mice with targeted deletion of miR-22 exhibited attenuated renal fibrosis in the UUO model. Consistent with these in vivo observations, primary renal fibroblast isolated from miR-22-deficient UUO mice demonstrated a significant increase in BMP-7/6 expression and their downstream targets. This phenotype could be rescued when cells were transfected with miR-22 mimics. Interestingly, we found that miR-22 and BMP-7/6 are in a regulatory feedback circuit, whereby not only miR-22 inhibits BMP-7/6, but miR-22 by itself is induced by BMP-7/6. Finally, we identified two BMP-responsive elements in the proximal region of miR-22 promoter. These findings identify miR-22 as a critical miRNA that contributes to renal fibrosis on the basis of its pivotal role on BMP signaling cascade.     

Effect of Interleukin 6 Deficiency on Renal Interstitial Fibrosis

Our recent studies have shown that bone marrow-derived fibroblast precursors contribute significantly to the pathogenesis of renal fibrosis. However, the molecular mechanisms underlying the recruitment and activation of bone marrow-derived fibroblast precursors are incompletely understood. We found that interleukin 6 was induced in the kidney in a murine model of renal fibrosis induced by unilateral ureteral obstruction. Therefore, we investigated if interleukin 6 play a role in the recruitment and maturation of bone marrow-derived fibroblast precursors in the kidney during the development of renal fibrosis. Wild-type and interleukin 6 knockout mice were subjected to unilateral obstructive injury for up to two weeks. Interleukin 6 knockout mice accumulated similar number of bone marrow-derived fibroblast precursors and myofibroblasts in the kidney in response to obstructive injury compared to wild-type mice. Furthermore, IL-6 knockout mice expressed comparable α-SMA in the obstructed kidney compared to wild-type mice. Moreover, targeted disruption of Interleukin 6 did not affect gene expression of profibrotic chemokine and cytokines in the obstructed kidney. Finally, there were no significant differences in renal interstitial fibrosis or expression of extracellular matrix proteins between wild-type and interleukin 6 knockout mice following obstructive injury. Our results indicate that interleukin 6 does not play a significant role in the recruitment of bone marrow-derived fibroblast precursors and the development of renal fibrosis.     

Bone morphogenetic protein-7 expression in human pyelonephritis

Bone morphogenetic protein 7 (BMP- 7) is a member of the transforming growth factor (TGF) beta superfamily and is involved in regeneration, repair, and development of specific tissues, for example kidney and skeleton. The experimental studies have shown its protective role against fibrotic processes. Tubulointerstitial changes are present in the pyelonephritic kidney which progresses to fibrosis. Renal fibrosis may lead to the loss of renal function. The aim of this study was to investigate BMP-7 expression in acute and chronic pyelonephritis in humans. Seven patients with acute pyelonephritis and 7 with chronic pyelonephritis were treated in Department of Nephrology Clinical Hospital, Rijeka. Tissue biopsy was taken and renal tissue was studied histopathologically by use of hematoxylin and eosin and scored for diagnosis of pyelonephritis. BMP-7 expression was studied by immunohistochemical staining. BMP-7 expression was observed in the tubular area of the pyelonephritic kidneys. The expression of BMP- 7 was stronger in the acute pyelonephritic group and less in the chronic pyelonephritic group of patients. The results imply that BMP-7 has a role in chronic pyelonephritis. Tubular BMP-7 expression had a negative correlation with fibrosis and tubular, atrophy. Our results are suggesting that BMP- 7 plays an important protective role in renal inflammatory diseases preventing greater damage and fibrosis.     

Mediators and mechanisms of heat shock protein 70 based cytoprotection in obstructive nephropathy

Urinary heat shock protein 70 (Hsp70) is rapidly increased in patients with clinical acute kidney injury, indicating that it constitutes a component of the endogenous stress response to renal injury. Moreover, experimental models have demonstrated that Hsp70 activation is associated with the cytoprotective actions of several drugs following obstruction, including nitric oxide (NO) donors, geranylgeranylacetone, vitamin D, and rosuvastatin. Discrete and synergistic effects of the biological activities of Hsp70 may explain its cytoprotective role in obstructive nephropathy. Basic studies point to a combination of effects including inhibition of apoptosis and inflammation, repair of damaged proteins, prevention of unfolded protein aggregation, targeting of damaged protein for degradation, and cytoskeletal stabilization as primary effectors of Hsp70 action. This review summarizes our understanding of how the biological actions of Hsp70 may affect renal cytoprotection in the context of obstructive injury. The potential of Hsp70 to be of central importance to the mechanism of action of various drugs that modify the genesis of experimental obstructive nephropathy is considered.     

BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury

Bone morphogenic protein (BMP)-7 is a 35-kDa homodimeric protein and a member of the transforming growth factor (TGF)-beta superfamily. BMP-7 expression is highest in the kidney, and its genetic deletion in mice leads to severe impairment of eye, skeletal and kidney development. Here we report that BMP-7 reverses TGF-beta1-induced epithelial-to-mesenchymal transition (EMT) by reinduction of E-cadherin, a key epithelial cell adhesion molecule. Additionally, we provide molecular evidence for Smad-dependent reversal of TGF-beta1-induced EMT by BMP-7 in renal tubular epithelial cells and mammary ductal epithelial cells. In the kidney, EMT-induced accumulation of myofibroblasts and subsequent tubular atrophy are considered key determinants of renal fibrosis during chronic renal injury. We therefore tested the potential of BMP-7 to reverse TGF-beta1-induced de novo EMT in a mouse model of chronic renal injury. Our results show that systemic administration of recombinant human BMP-7 leads to repair of severely damaged renal tubular epithelial cells, in association with reversal of chronic renal injury. Collectively, these results provide evidence of cross talk between BMP-7 and TGF-beta1 in the regulation of EMT in health and disease.     

Bone morphogenic protein-7 induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney

In the kidney, a unique plasticity exists between epithelial and mesenchymal cells. During kidney development, the metanephric mesenchyme contributes to emerging epithelium of the nephron via mesenchymal to epithelial transition (MET). In the injured adult kidney, renal epithelia contribute to the generation of fibroblasts via epithelial-mesenchymal transition, facilitating renal fibrosis. Recombinant human bone morphogenic protein (BMP)-7, a morphogen that is essential for the conversion of epithelia from condensing mesenchyme during kidney development, enhances the repair of tubular structures in the kidney. In this setting, BMP-7 inhibits epithelial-mesenchymal transition involving adult renal epithelial tubular cells and decreases secretion of type I collagen by adult renal fibroblasts. In search of a mechanism behind the ability of BMP-7 to repair damaged renal tubules, we hypothesized that systemic treatment with BMP-7 might induce MET involving adult renal fibroblasts in the injured kidney, generating functional epithelial cells. Here we report that BMP-7 induces formation of epithelial cell aggregates in adult renal fibroblasts associated with reacquisition of E-cadherin expression and decreased motility, mimicking the effect of BMP-7 on embryonic metanephric mesenchyme to generate epithelium. In addition, we provide evidence that BMP-7-mediated repair of renal injury is associated with MET involving adult renal interstitial fibroblasts in mouse models for renal fibrosis. Collectively, these findings suggest that adult renal fibroblasts might retain parts of their original embryonic imprint and plasticity, which can be re-engaged by systemic administration of BMP-7 to mediate repair of tubular injury in a fibrotic kidney.     

Exploring the connection between chronic renal fibrosis and bone morphogenic protein-7

Tubulointerstitial fibrosis is a hallmark feature of chronic renal injury. Specific therapies to control the progression of renal fibrosis towards end-stage renal failure are still limited. Transforming growth factor-beta1 (TGF-beta1) has been identified as a major mediator of renal fibrosis. Recent reports have suggested that Bone Morphogenic Protein-7 (BMP-7), another member of the TGF-beta superfamily, accelerates repair of acute renal injury and ameliorates progression of chronic renal fibrosis in a variety of animal models. Interestingly, BMP-7, an endogenous molecule which is present in the normal kidney, vastly decreases its expression during renal injury. Although, the mechanism of BMP-7 action in the kidney is not yet fully understood, the idea of an endogenous molecule with reno-protective function is intriguing.     

Bone morphogenetic proteins in development and homeostasis of kidney

Bone morphogenetic proteins play a key role in kidney development and postnatal function. The kidney has been identified as a major site of bone morphogenetic protein (BMP)-7 synthesis during embryonic and postnatal development, which mediates differentiation and maintenance of metanephric mesenchyme. Targeted disruption of BMP-7 gene expression in mice resulted in dysgenic kidneys with hydroureters, causing uremia within 24h after birth. Several experimental animal models of acute and chronic renal injury have all unequivocally shown beneficial effect of BMP-7 in ameliorating the severity of damage by preventing inflammation and fibrosis. Apart from the beneficial effect on kidney disease itself, BMP-7 improves important complications of chronic renal impairment such as renal osteodystrophy and vascular calcification.     

P2X7 receptor inhibition improves recovery after spinal cord injury

Secondary injury exacerbates the extent of spinal cord insults, yet the mechanistic basis of this phenomenon has largely been unexplored. Here we report that broad regions of the peritraumatic zone are characterized by a sustained process of pathologic, high ATP release. Spinal cord neurons expressed P2X7 purine receptors (P2X7R), and exposure to ATP led to high-frequency spiking, irreversible increases in cytosolic calcium and cell death. To assess the potential effect of P2X7R blockade in ameliorating acute spinal cord injury (SCI), we delivered P2X7R antagonists OxATP or PPADS to rats after acute impact injury. We found that both OxATP and PPADS significantly improved functional recovery and diminished cell death in the peritraumatic zone. These observations demonstrate that SCI is associated with prolonged purinergic receptor activation, which results in excitotoxicity-based neuronal degeneration. P2X7R antagonists inhibit this process, reducing both the histological extent and functional sequelae of acute SCI.     

Regression of Glomerular and Tubulointerstitial Injuries by Dietary Salt Reduction with Combination Therapy of Angiotensin II Receptor Blocker and Calcium Channel Blocker in Dahl Salt-Sensitive Rats

A growing body of evidence indicates that renal tissue injuries are reversible. We investigated whether dietary salt reduction with the combination therapy of angiotensin II type 1 receptor blocker (ARB) plus calcium channel blocker (CCB) reverses renal tissue injury in Dahl salt-sensitive (DSS) hypertensive rats. DSS rats were fed a high-salt diet (HS; 4% NaCl) for 4 weeks. Then, DSS rats were given one of the following for 10 weeks: HS diet; normal-salt diet (NS; 0.5% NaCl), NS + an ARB (olmesartan, 10 mg/kg/day), NS + a CCB (azelnidipine, 3 mg/kg/day), NS + olmesartan + azelnidipine or NS + hydralazine (50 mg/kg/day). Four weeks of treatment with HS diet induced hypertension, proteinuria, glomerular sclerosis and hypertrophy, glomerular podocyte injury, and tubulointerstitial fibrosis in DSS rats. A continued HS diet progressed hypertension, proteinuria and renal tissue injury, which was associated with inflammatory cell infiltration and increased proinflammatory cytokine mRNA levels, NADPH oxidase activity and NADPH oxidase-dependent superoxide production in the kidney. In contrast, switching to NS halted the progression of hypertension, renal glomerular and tubular injuries. Dietary salt reduction with ARB or with CCB treatment further reduced blood pressure and partially reversed renal tissues injury. Furthermore, dietary salt reduction with the combination of ARB plus CCB elicited a strong recovery from HS-induced renal tissue injury including the attenuation of inflammation and oxidative stress. These data support the hypothesis that dietary salt reduction with combination therapy of an ARB plus CCB restores glomerular and tubulointerstitial injury in DSS rats.     

The Multifaceted Effects of Agmatine on Functional Recovery after Spinal Cord Injury through Modulations of BMP-2/4/7 Expressions in Neurons and Glial Cells

Presently, few treatments for spinal cord injury (SCI) are available and none have facilitated neural regeneration and/or significant functional improvement. Agmatine (Agm), a guanidinium compound formed from decarboxylation of L-arginine by arginine decarboxylase, is a neurotransmitter/neuromodulator and been reported to exert neuroprotective effects in central nervous system injury models including SCI. The purpose of this study was to demonstrate the multifaceted effects of Agm on functional recovery and remyelinating events following SCI. Compression SCI in mice was produced by placing a 15 g/mm² weight for 1 min at thoracic vertebra (Th) 9 segment. Mice that received an intraperitoneal (i.p.) injection of Agm (100 mg/kg/day) within 1 hour after SCI until 35 days showed improvement in locomotor recovery and bladder function. Emphasis was made on the analysis of remyelination events, neuronal cell preservation and ablation of glial scar area following SCI. Agm treatment significantly inhibited the demyelination events, neuronal loss and glial scar around the lesion site. In light of recent findings that expressions of bone morphogenetic proteins (BMPs) are modulated in the neuronal and glial cell population after SCI, we hypothesized whether Agm could modulate BMP- 2/4/7 expressions in neurons, astrocytes, oligodendrocytes and play key role in promoting the neuronal and glial cell survival in the injured spinal cord. The results from computer assisted stereological toolbox analysis (CAST) demonstrate that Agm treatment dramatically increased BMP- 2/7 expressions in neurons and oligodendrocytes. On the other hand, BMP- 4 expressions were significantly decreased in astrocytes and oligodendrocytes around the lesion site. Together, our results reveal that Agm treatment improved neurological and histological outcomes, induced oligodendrogenesis, protected neurons, and decreased glial scar formation through modulating the BMP- 2/4/7 expressions following SCI.     

Negative Regulation of TGFβ Signaling by Stem Cell Antigen-1 Protects against Ischemic Acute Kidney Injury

Acute kidney injury, often caused by an ischemic insult, is associated with significant short-term morbidity and mortality, and increased risk of chronic kidney disease. The factors affecting the renal response to injury following ischemia and reperfusion remain to be clarified. We found that the Stem cell antigen-1 (Sca-1), commonly used as a stem cell marker, is heavily expressed in renal tubules of the adult mouse kidney. We evaluated its potential role in the kidney using Sca-1 knockout mice submitted to acute ischemia reperfusion injury (IRI), as well as cultured renal proximal tubular cells in which Sca-1 was stably silenced with shRNA. IRI induced more severe injury in Sca-1 null kidneys, as assessed by increased expression of Kim-1 and Ngal, rise in serum creatinine, abnormal pathology, and increased apoptosis of tubular epithelium, and persistent significant renal injury at day 7 post IRI, when recovery of renal function in control animals was nearly complete. Serum creatinine, Kim-1 and Ngal were slightly but significantly elevated even in uninjured Sca-1-/- kidneys. Sca-1 constitutively bound both TGFβ receptors I and II in cultured normal proximal tubular epithelial cells. Its genetic loss or silencing lead to constitutive TGFβ receptor—mediated activation of canonical Smad signaling even in the absence of ligand and to KIM-1 expression in the silenced cells. These studies demonstrate that by normally repressing TGFβ-mediated canonical Smad signaling, Sca-1 plays an important in renal epithelial cell homeostasis and in recovery of renal function following ischemic acute kidney injury.     

Lin28a attenuates TGF-β-induced renal fibrosis

Lin28a has diverse functions including regulation of cancer, reprogramming and regeneration, but whether it promotes injury or is a protective reaction to renal injury is unknown. We studied how Lin28a acts in unilateral ureteral obstruction (UUO)-induced renal fibrosis following unilateral ureteral obstruction, in a mouse model. We further defined the role of Lin28a in transforming growth factor (TGF)-signaling pathways in renal fibrosis through in vitro study using human tubular epithelium-like HK-2 cells. In the mouse unilateral ureteral obstruction model, obstruction markedly decreased the expression of Lin28a, increased the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin. In TGF-β-stimulated HK-2 cells, the expression of Lin28a was reduced and the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin was increased. Adenovirus-mediated overexpression of Lin28a inhibited the expression of TGF-β-stimulated type I collagen, α-SMA, vimentin and fibronectin. Lin28a inhibited TGF-β-stimulated SMAD3 activity, via inhibition of SMAD3 phos-phorylation, but not the MAPK pathway ERK, JNK or p38. Lin28a attenuates renal fibrosis in obstructive nephropathy, making its mechanism a possible therapeutic target for chronic kidney disease.     

Increased renal tubular synthesis of prostaglandins in the rabbit kidney in response to ureteral obstruction.

The hydronephrotic rabbit kidney exhibits elevated basal prostaglandin synthesis and supersensitivity to peptide stimulation of vascular prostaglandin and thromboxane formation. In this study the distribution of the prostaglandin-forming cyclooxygenase in hydronephrotic and contralateral rabbit kidneys following one and four day ureteral obstructions was compared using immunohistofluorescence. No alterasions were detected in the distribution or intensity of cyclooxygenase-positive fluorescence in the renal vasculature in response to ureteral obstructions. However, two significant differences were noted between hydronephrotic and contralateral kidneys in the staining of renal tubules: (a) the intensity of fluorescent staining in cortical and medullary collecting tubules of the hydronephrotic kidney was increased and (b) cyclooxygenase antigenicity appeared in the thin limbs of Henle's loop in the hydronephrotic organ. Although alterations in prostaglandin formation by the renal vasculature have been documented previously, our results indicate that ureteral obstruction also causes increased prostaglandin synthesis by renal tubules.