Experimental Models Of Renal Injury Research Articles

FGFR regulator Memo1 is dispensable for FGF23 expression by osteoblasts during folic acid-driven kidney injury.

Loss of the mediator Of cell motility 1 (Memo1) in mice caused kidney disease and a bone disease with diminished osteoblast and osteoclast biomarkers in serum, resembling alterations occurring in adynamic bone disease in humans with chronic kidney disease or in Klotho-deficient mice. Here, we investigated whether Memo1 expression in osteoblasts is required for normal bone structure and FGF23 expression. We deleted Memo1 in the osteoblast-osteocyte lineage in Memo fl/fl mice using a Cre under Col1a1 promotor to obtain osteoblast-specific knockout (obKO) mice. We studied organs by micro-computed tomography, qPCR, and western blot. We challenged mice with folic acid for acute kidney injury (AKI) and analyzed organs. Memo obKO were viable without changes in gross anatomy, serum electrolytes, or circulating FGF23 concentrations compared to controls. Memo1 expression was blunted in bones of Memo obKO, whereas it remained unchanged in other organs. Micro-CT revealed no differences between genotypes in bone structure of vertebrae, femur, and tibia. During AKI, Fgf23 expression in calvaria, and renal transcriptional changes were comparable between genotypes. However, renal injury marker expression, circulating FGF23, and parathyroid hormone revealed a sex difference with more severely affected females than males of either genotype. The present data imply that Memo1 in osteoblasts is dispensable for bone structure and expression of Fgf23. Moreover, we found evidence of potential sex differences in murine folic acid nephropathy similar to other experimental models of renal injury that are important to consider when using this experimental model of renal injury.

Understanding the opposite effects of sex hormones in mediating renal injury.

According to epidemiological studies, chronic kidney disease (CKD) affects more women than men, but the incidence of end-stage renal disease is higher in men than in women. However, most of these studies have not considered the incidence of CKD in women of reproductive or post-menopausal age, and even fewer with hormone replacement therapy. Some meta-analyses have reported an exacerbated progression of CKD in men compared with women. Consequently, in most of the experimental models of renal injury, men of reproductive age exhibit more abnormalities in renal function and structure that lead to greater progression to CKD than women, which suggests that these differences are mediated by sex hormones rather than by other factors. This review intends to show the mechanisms regulated by oestrogen or testosterone that may explain the different risks and evolution of renal diseases between men and women. Regardless of the initial cause of kidney disease, sex hormones have been implicated in modulating vascular tone, oxidative stress, inflammation and apoptosis. Finally, our previous study highlights the mechanisms by which the transition from acute kidney injury to CKD does not occur in female rats as commonly as it does in male rats. This review not only identifies sex differences in several kidney diseases but also supports potential therapeutic opportunities to reduce or prevent the progression of CKD and highlights the importance of considering sex differences in the design of any clinical study.

Adaptive immunity and IL-17A are not involved in the progression of chronic kidney disease after 5/6 nephrectomy in mice.

The adaptive immune response and IL-17A contribute to renal damage in several experimental models of renal injury. To evaluate the role of the adaptive immune response, 5/6 nephrectomy was performed in wildtype DBA/1J mice and in recombination-activating gene-1 (RAG-1) deficient mice that lack B and T-cells. To assess the role of IL-17A, we carried out 5/6 nephrectomy in IL-17A deficient mice. Flow cytometric analysis, immunohistochemistry and RT-PCR were used. Infiltration of CD3+ T-cells in the remnant kidney was increased after 5/6 nephrectomy in wildtype mice, along with a robust induction of IL-17A production in CD4+ T and γδ T-cells. After 5/6 nephrectomy, wildtype mice developed albuminuria in the nephrotic range over 10weeks. This was accompanied by severe glomerular sclerosis and tubulointerstitial injury, and as well as renal mRNA expression of markers of inflammation and fibrosis (the chemokine CCL2, plasminogen activator inhibitor-1; PAI-1 and neutrophil gelatinase-associated lipocalin; NGAL). Unexpectedly, RAG-1 deficient mice and IL-17A deficient mice developed renal injury, similar to that in wildtype mice. No differences were found for albuminuria, glomerular sclerosis, tubulointerstitial injury and mRNA expression of CCL2, PAI-1 and NGAL. Mortality did not differ between the three groups. Numbers of CD3+ T-cells and IL-17A+ lymphocytes infiltrating the kidney were increased after 5/6 nephrectomy. In contrast to other experimental models of renal injury, genetic deficiency of the adaptive immune system or of IL-17A did not attenuate induction or progression of chronic kidney disease after 5/6 nephrectomy. This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.

Gremlin activates the Notch pathway linked to renal inflammation.

Preclinical studies suggest that Gremlin participates in renal damage and could be a potential therapeutic target for human chronic kidney diseases. Inflammation is a common characteristic of progressive renal disease, and therefore novel anti-inflammatory therapeutic targets should be investigated. The Notch signaling pathway is involved in kidney development and is activated in human chronic kidney disease, but whether Gremlin regulates the Notch pathway has not been investigated. In cultured tubular cells, Gremlin up-regulated gene expression of several Notch pathway components, increased the production of the canonical ligand Jagged-1, and caused the nuclear translocation of active Notch-1 (N1ICD). In vivo administration of Gremlin into murine kidneys elicited Jagged-1 production, increased N1ICD nuclear levels, and up-regulated the gene expression of the Notch effectors hes-1 and hey-1 All these data clearly demonstrate that Gremlin activates the Notch pathway in the kidney. Notch inhibition using the γ-secretase inhibitor DAPT impaired renal inflammatory cell infiltration and proinflammatory cytokines overexpression in Gremlin-injected mice and in experimental models of renal injury. Moreover, Notch inhibition blocked Gremlin-induced activation of the canonical and noncanonical nuclear factor-κB (NF-κB) pathway, identifying an important mechanism involved in the anti-inflammatory actions of Notch inhibition. In conclusion, Gremlin activates the Notch pathway in the kidney and this is linked to NF-κB-mediated inflammation, supporting the hypothesis that Notch inhibition could be a potential anti-inflammatory strategy for renal diseases.

Ameliorating Effect of Gemigliptin on Renal Injury in Murine Adriamycin-Induced Nephropathy.

Background. Previous studies have shown the antiapoptotic and anti-inflammatory potential of DPP-IV inhibitor in experimental models of renal injury. We tested whether DPP-IV inhibitor (gemigliptin) ameliorates renal injury by suppressing apoptosis, inflammation, and oxidative stress in mice with adriamycin nephropathy. Methods. Mice were treated with normal saline (control), gemigliptin (GM), adriamycin (ADR), or adriamycin combined with gemigliptin (ADR+GM). Apoptosis, inflammation, and oxidative stress were analyzed via western blotting, real-time PCR, light microscopy, and immunofluorescence. Results. In the ADR+GM group, urine albumin creatinine ratio decreased significantly compared with that in the ADR group on day 15. Glomerulosclerosis index and tubulointerstitial injury index in mice with adriamycin-induced nephropathy decreased after gemigliptin treatment. ADR group showed higher levels of apoptosis, inflammation, and oxidative stress-related molecules compared with the control group. The upregulation of these molecules was significantly reduced by gemigliptin. In the ADR group, the staining intensities of WT-1 and nephrin reduced, but these changes were ameliorated in the ADR+GM group. Conclusion. We demonstrated that gemigliptin ameliorates nephropathy by suppressing apoptosis, inflammation, and oxidative stress in mice administered adriamycin. Our data demonstrate that gemigliptin has renoprotective effects on adriamycin-induced nephropathy.

Association of functional haem oxygenase-1 gene promoter polymorphism with polycystic kidney disease and IgA nephropathy

Haem oxygenase-1 (HO-1) is a cytoprotective molecule that is reported to have a protective role in a variety of experimental models of renal injury. A functional dinucleotide repeat (GT)(n) polymorphism, within the HO-1 promoter, regulates HO-1 gene expression; a short number of repeats (S-allele <25) increases transcription. We report the first assessment of the role of this HO-1 gene promoter polymorphism in chronic kidney disease due to autosomal dominant polycystic kidney disease (ADPKD) and IgA nephropathy (IgAN). The DNA from 160 patients (99% Caucasian) on renal replacement therapy (RRT) was genotyped. The primary renal disease was ADPKD in 100 patients and biopsy-proven IgAN in 60 patients. Overall, the mean age at commencement of RRT was not significantly different between patients with and without an S-allele (44.1 years versus 45.0 years, P = 0.64). In patients with ADPKD, the age at commencement of RRT was comparable regardless of the HO-1 genotype (47.7 years versus 46.7 years, P = 0.59). The same was true in patients with IgAN (38.3 years versus 42.2 years, P = 0.28). This suggests that the functional HO-1 promoter polymorphism does not influence renal survival in CKD due to ADPKD or IgAN.

NF-kappaB activation and overexpression of regulated genes in human diabetic nephropathy.

Nuclear factor-kappaB (NF-kappaB) regulates genes involved in renal disease progression, such as the chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES. NF-kappaB is activated in experimental models of renal injury, and in vitro studies also suggest that proteinuria and angiotensin II could be important NF-kappaB activators. It has been proposed that locally produced MCP-1 may be involved in the development of diabetic nephropathy (DN). We examined the hypothesis that NF-kappaB could be an indicator of renal damage progression in DN. Biopsy specimens from 11 patients with type 2 diabeties and overt nephropathy were studied by southwestern histochemistry for the in situ detection of activated NF-kappaB. In addition, by immunohistochemistry and/or in situ hybridization, we studied the expression of MCP-1 and RANTES, whose genes are regulated by NF-kappaB. NF-kappaB was detected mainly in cortical tubular epithelial cells and, to a lesser extent, in some glomerular and interstitial cells. A strong upregulation of MCP-1 and RANTES was observed in all the cases, mainly in tubular cells, and there was a strong correlation between the expression of these chemokines and NF-kappaB activation in the same cells, as observed in serial sections (r = 0.7; P = 0.01). In addition, the tubular expression of these chemokines was correlated mainly with the magnitude of the proteinuria (P = 0.002) and with interstitial cell infiltration (P<0.05). The activation of NF-kappaB and the transcription of certain pro-inflammatory chemokines in tubular epithelial cells are markers of progressive DN. Proteinuria might be one of the main factors inducing the observed pro-inflammatory phenotype.

Role of apoptosis in hypoxic/ischemic damage in the kidney

Cell death by hypoxia/ischemia may occur by apoptosis as well as necrosis in experimental models of renal injury both in vivo and in vitro. Necrosis can occur during hypoxia/ischemia as a result of widespread cellular degradation, and during reoxygenation/reperfusion as a consequence of the development of the mitochondrial permeability transition pore (PTP). In vitro models of hypoxia/reoxygenation suggest that apoptotic cell death may occur during reoxygenation as a consequence of mitochondrial release of cytochrome c (Cyt c) during hypoxia. In hypoxic renal cells, Bax and Bak, 2 pro-apoptotic proteins of the Bcl-2 family, collaborate to permeabilize the mitochondrial outer membrane to intermembrane proteins such as Cyt c, although Bax, per se, appears to play the dominant role. Cyt c then acts to trigger the downstream apoptotic cascade. Caspase inhibitors suppress these downstream events, but not Cyt c release. However, the anti-apoptotic Bcl-2 prevents mitochondrial permeabilization and maintains viability. Inflammation is known to play a major role in exacerbating parenchymal damage during reperfusion. Recent studies suggest that the apoptosis-related mechanisms contribute to the inflammatory process. By inhibiting tubular cell apoptosis, by suppressing an apoptotic chain reaction in accumulating inflammatory cells, and by inhibiting caspase-1 processing in injured tissue, caspase inhibitors may reduce inflammation, and thereby reduce the cascading parenchymal injury that is associated with inflammation.

Tubular NF-κB and AP-1 activation in human proteinuric renal disease

Nuclear factor-kappaB (NF-kappaB) and activated protein-1 (AP-1) are transcription factors that regulate many genes involved in the progression of renal disease. Recent data have shown that NF-kappaB is activated in tubules and glomeruli in various experimental models of renal injury. In vitro studies also suggest that proteinuria could be an important NF-kappaB activator. We therefore approached the idea that NF-kappaB may be an indicator of renal damage progression. Paraffin-embedded renal biopsy specimens from 34 patients with intense proteinuria [14 with minimal change disease (MCD) and 20 with idiopathic membranous nephropathy (MN)] and from 7 patients with minimal or no proteinuria (IgA nephropathy) were studied by Southwestern histochemistry for the in situ detection of activated transcription factors NF-kappaB and AP-1. In addition, by immunohistochemistry, we performed staining for the NF-kappaB subunits (p50 and p65) and AP-1 subunits (c-fos, c-jun). By immunohistochemistry and/or in situ hybridization, the expression of some chemokines [monocyte chemoattractant protein-1 (MCP-1), RANTES, osteopontin (OPN)] and profibrogenic cytokines [transforming growth factor-beta (TGF-beta)], whose genes are regulated by NF-kappaB and/or AP-1, were studied further. NF-kappaB was detected mainly in the tubules of proteinuric patients, but rarely in nonproteinuric IgA nephropathy (IgAN) patients. In addition, there was a significant relationship between the intensity of proteinuria and NF-kappaB activation in MCD (r = 0.64, P = 0.01) and MN patients (r = 0.64, P < 0.01). Unexpectedly, patients with MCD had a significantly higher NF-kappaB tubular activation than those with MN (P < 0.01). To assess whether there was a different composition of NF-kappaB protein components, immunostaining was performed for the NF-kappaB subunits p50 and p65. However, no differences were noted between MCD and MN patients. In those patients, there was a lower tubular activation of AP-1 compared with NF-kappaB. Moreover, a strong correlation in the expression of both transcription factors was observed only in MN (r = 0.7, P = 0.004). Patients with progressive MN had an overexpression of MCP-1, RANTES, OPN, and TGF-beta, mainly in the proximal tubules, while no significant expression was found in MCD patients. On the whole, our results show that a tubular overactivation of NF-kappaB and AP-1 and a simultaneous up-regulation of certain proinflammatory and profibrogenic genes are markers of progressive renal disease in humans. Increased activation of solely NF-kappaB and/or AP-1 may merely indicate the response of tubular renal cells to injury.

The role of transforming growth factor beta in chronic renal allograft nephropathy.

The role of transforming growth factor beta in chronic renal allograft nephropathy.

Effect of a specific endothelin receptor A antagonist and an angiotensin-converting enzyme inhibitor on glomerular mRNA levels for extracellular matrix components, metalloproteinases (MMP) and a tissue inhibitor of MMP in aminonucleoside nephrosis.

We previously reported that mRNA levels for extracellular matrix (ECM) components and endothelin (ET)-1 are upregulated in glomeruli of puromycin aminonucleoside (PAN) nephrosis. Angiotensin-converting enzyme (ACE) inhibitors are effective in experimental models of renal injury, including PAN nephrosis. This study was designed to assess whether the glomerular expression of mRNA for ECM components, ET-1, metalloproteinases (MMP), and a tissue inhibitor of metalloproteinases (TIMP) is modulated by a specific endothelin receptor A antagonist (FR139317) or angiotensin-converting enzyme inhibitor (enalapril) in PAN-injected rats. Animals were divided into six groups. Group 1 consisted of PAN-injected rats given no treatment. In group 2, PAN-injected rats were given enalapril 35 mg/l. In group 3, PAN-injected rats were given an intraperitoneal injection of FR139317. Group 4 consisted of saline-injected rats given no treatment. In group 5, saline-injected rats were given enalapril. In group 6, saline-injected rats were given FR139317. We prepared glomerular RNA and performed Northern blot analysis in all groups. In PAN nephrosis, glomerular mRNA levels for alpha 1 (IV) collagen chain, laminin B1 and B2 chains, ET-1, MMP-2 and TIMP-1 increased at the peak of proteinuria on day 8 and then decreased to the control level by day 20, whereas those for alpha 1 (I) and alpha 1 (III) collagen chains, MMP-1, MMP-3 and GAPDH showed little change in PAN nephrosis throughout the experimental periods. In contrast, mRNA levels for heparan sulphate proteoglycan (HSPG) decreased on day 8 and then increased to the control level by day 20. Both enalapril and FR139317 attenuated the increases in mRNA levels for alpha 1 (IV) collagen chain (P < 0.01), laminin chains (P < 0.01), and ET-1 (P < 0.01), and attenuated the decreases in mRNA levels for HSPG (P < 0.01) in glomeruli of PAN-injected rats. Enalapril had little effect on increased glomerular mRNA levels for MMP-2 and TIMP-1 in PAN nephrosis, whereas FR139317 attenuated the increases in glomerular mRNA levels for MMP-2 (P < 0.01) and TIMP-1 (P < 0.01). These data suggest that the beneficial effects of enalapril and FR139317 may be related to modulation of glomerular mRNA expression of ECM components and ET-1 and that these agents may follow a different mechanism in regulating the glomerular mRNA expression for MMP-2 and TIMP-1 in PAN nephrosis.

Quinapril decreases renal endothelin-1 expression and synthesis in a normotensive model of immune-complex nephritis.

Angiotensin-converting enzyme (ACE) inhibitors diminish proteinuria and the progression to renal failure in several experimental models of renal injury. Endothelin-1 (ET-1) possesses potent biological actions on renal vessels and has been considered as a potential mediator of renal damage. Because angiotensin II (Ang II) induces ET-1 synthesis in endothelial and mesangial cells, we hypothesized that some of the beneficial effects of the ACE inhibitors could result from the blockade of ET-1 synthesis. In a normotensive model of immune-complex nephritis, in which there exists an increase in renal ACE activity, the effect of the ACE inhibitor quinapril on preproET-1 and ETA receptor mRNA expression, as well as on ET-1 protein levels, was examined in this study. In relation to controls, nephritic rats showed an increase in preproET-1 and ETA receptor gene expression in renal cortex and medulla, coinciding with the maximal renal ACE activity. PreproET-1 mRNA (in situ hybridization) and ET-1 protein (immunohistochemistry) were localized in glomerular capillary walls, mesangial and glomerular epithelial cells, as well as in the brush border of some proximal tubules, and in small vessels. In nephritic rats, there was an increase in preproET-1 mRNA levels and ET-1 protein in all of these areas, without modification of their distribution. The administration of the ACE inhibitor quinapril decreased proteinuria and morphological lesions, preproET-1 gene transcription, and ET-1 protein levels, as well as the ETA receptor mRNA. The results from this study show that in a normotensive model of immune-complex nephritis, there was an overexpression of ET-1 in several structures of the kidney that was downregulated by quinapril administration. The beneficial effect of ACE inhibitors could be a result of the modulation of local production of Ang II and ET-1.

Acquired essential fatty acid depletion in the remnant kidney: Amelioration with U-63557A

Previous studies have demonstrated an association between renal cortical fatty acid composition and experimental models of renal injury. The present study was designed to extend these observations to the remnant kidney and to investigate the hypothesis that increased endogenous turnover of arachidonic acid metabolites results in the depletion of progenitor fatty acids. Remnant kidney cortex demonstrated a relative reduction of the essential fatty acids, linoleate and arachidonate (20 +/- 7.2% and 11 +/- 0.3%, respectively), nine weeks after subtotal nephrectomy. In addition, the monounsaturated fatty acid, oleate, was increased (48 +/- 10.6%) while its saturated progenitor, stearate, was decreased (13 +/- 4.3%). Serial evaluation of dienoic prostanoids revealed a significant increase in the renal excretion of TXB2 in rats with remnant kidneys (27 +/- 3.0, 29 +/- 1.1, and 34 +/- 3.3 ng/day vs. 21 +/- 0.8, 20 +/- 1.5, and 22 +/- 3.3 ng/day in control rats, at 3, 6, and 9 weeks, respectively). Moreover, TXB2 excretion inversely correlated with dienoic progenitor fatty acids [18:2(n-6), r2 = 0.76; 20:4(n-6), r2 = 0.79], suggesting that these events are biochemically coupled. Endogenous turnover of precursor fatty acids, confirmed by an increase in renal TXB2 excretion, preceded overt depletion of essential fatty acids by several weeks. Importantly, blockade of endogenous synthesis of TXA2 with the specific TXA2 synthase antagonist, U-63557A, restored the essential fatty acid composition to normal and ameliorated progressive glomerular destruction. Moreover, the ancillary fatty acid disturbances were attenuated by administration of U-63557A.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of angiotensin II and endothelin in matrix protein production and renal sclerosis

To review the evidence that links angiotensin II and endothelin as growth factors and as modifiers of extracellular matrix synthesis in renal cells, with particular reference to the effects of angiotensin converting enzyme (ACE) inhibition in models of renal injury. In cultured mesangial cells, both angiotensin II and endothelin promote contraction, proliferation/hypertrophy, signal transduction pathways, the activation of early growth genes, and the generation of inflammatory mediators, cytokines and growth factors. Both hormones have been shown to promote the synthesis of fibronectin and collagen in a dose-dependent manner. ACE inhibition attenuates the effect of endothelin-1, one of three isoforms of endothelin. In experimental models of renal injury, chiefly in those characterized by increased intraglomerular pressure, ACE inhibition has reduced proteinuria and glomerular and interstitial sclerosis. ACE inhibition has been shown to have major beneficial effects in patients with diabetic nephropathy, even in those with normal blood pressure. Although the renal-protective effects of ACE inhibitors in experimental and human renal injury may reflect systemic and/or local hemodynamic effects of these drugs, their modulatory actions on extracellular matrix synthesis and proteinuria may contribute to the benefit of ACE-inhibitor therapy.