Original Research: Potential of urinary nephrin as a biomarker reflecting podocyte dysfunction in various kidney disease models.

Urinary nephrin is a potential non-invasive biomarker of disease. To date, however, most studies of urinary nephrin have been conducted in animal models of diabetic nephropathy, and correlations between urinary nephrin-to-creatinine ratio and other parameters have yet to be evaluated in animal models or patients of kidney disease with podocyte dysfunction. We hypothesized that urinary nephrin-to-creatinine ratio can be up-regulated and is negatively correlated with renal nephrin mRNA levels in animal models of kidney disease, and that increased urinary nephrin-to-creatinine ratio levels are attenuated following administration of glucocorticoids. In the present study, renal nephrin mRNA, urinary nephrin-to-creatinine ratio, urinary protein-to-creatinine ratio, and creatinine clearance ratio were measured in animal models of adriamycin nephropathy, puromycin aminonucleoside nephropathy, anti-glomerular basement membrane glomerulonephritis, and 5/6 nephrectomy. The effects of prednisolone on urinary nephrin-to-creatinine ratio and other parameters in puromycin aminonucleoside (single injection) nephropathy rats were also investigated. In all models tested, urinary nephrin-to-creatinine ratio and urinary protein-to-creatinine ratio increased, while renal nephrin mRNA and creatinine clearance ratio decreased. Urinary nephrin-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA in almost all models, as well as a significant positive correlation with urinary protein-to-creatinine ratio and a significant negative correlation with creatinine clearance ratio. Urinary protein-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA. Following the administration of prednisolone to puromycin aminonucleoside (single injection) nephropathy rats, urinary nephrin-to-creatinine ratio was significantly suppressed and exhibited a significant positive correlation with urinary protein-to-creatinine ratio. In addition, the decrease in number of glomerular Wilms tumor antigen-1-positive cells was attenuated, and urinary nephrin-to-creatinine ratio exhibited a significant negative correlation in these cells. In conclusion, these results suggest that urinary nephrin-to-creatinine ratio level is a useful and reliable biomarker for predicting the amelioration of podocyte dysfunction by candidate drugs in various kidney disease models with podocyte dysfunction. This suggestion will also be validated in a clinical setting in future studies.

Antifibrotic effects of pirfenidone in rat proximal tubular epithelial cells.

OBJECTIVE: Renal fibrosis is a common cause of renal dysfunction with chronic kidney disease. We previously investigated the renoprotective effects of the antifibrotic agent pirfenidone in a rat model of subtotal nephrectomy. Here, we further evaluated the antifibrotic effects of pirfenidone in rat proximal tubular epithelial cells. METHODS: NRK52E cells were incubated in a medium containing either transforming growth factor (TGF)-ß1 (3 ng/mL) or platelet-derived growth factor (PDGF)-BB (5 Ang/mL) or both, with or without pirfenidone (0.1-1 mmol/L), for 24 h to assess mRNA expression, for 48 h to assess protein production, and for 1 h or various time (5-120 min) to assess phosphorylation of signal kinase. RESULTS: TGF-ß1, a key mediator in renal fibrosis, induced increases in the mRNA expression of various profibrotic factors and extracellular matrix, including plasminogen activator inhibitor type 1 (PAI-1), fibronectin, type 1 collagen, and connective tissue growth factor (CTGF)-increases which pirfenidone significantly inhibited. Specifically, pirfenidone potently inhibited TGF-ß1-induced increases in the mRNA expression and protein secretion of PAI-1, an effect mediated, at least in part, via the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. Further, PDGF-BB, which has been implicated in renal interstitial fibrosis, potently activated PAI-1 expression under TGF-ß1 stimulation, and pirfenidone significantly inhibited TGF-ß1- and PDGF-BB-induced increases in PAI-1 expression. CONCLUSIONS: Taken together, these results suggest that TGF-ß1 closely correlates with renal fibrosis in cooperation with several fibrosis-promoting molecules, such as PAI-1 and PDGF, in rat proximal tubular epithelial cells, and pirfenidone inhibits TGF-ß1-induced fibrosis cascade and will therefore likely exert antifibrotic effects under pathological conditions.

Vasopressin induces human mesangial cell growth via induction of vascular endothelial growth factor secretion.

Vasoactive hormones, growth factors, and cytokines are important in promoting mesangial cell growth, a characteristic feature of many glomerular diseases. Vascular endothelial growth factor (VEGF) is an endothelial mitogen and promoter of vascular permeability that is constitutively expressed in human glomeruli, but its role in the kidney is still unclear. In the present study, we investigated the ability of vasopressin (AVP) to stimulate VEGF secretion by and correlation with AVP-induced cell growth in human mesangial cells. AVP caused time- and concentration-dependent increases in VEGF secretion from human mesangial cells, which was in turn potently inhibited by a V(1A) receptor-selective antagonist, confirming that this secretion is a V(1A) receptor-mediated event. VEGF also induced mesangial cell growth which was completely inhibited on administration of an anti-VEGF neutralizing antibody. Further, AVP-induced mesangial cell growth was completely abolished by the V(1A) receptor-selective antagonist and partially inhibited by an anti-VEGF neutralizing antibody. These results suggest that AVP stimulates VEGF secretion by human mesangial cells via V(1A) receptors. This secreted VEGF may function as an autocrine hormone to regulate mesangial cell growth, a mechanism by which AVP might contribute to progressive glomerular diseases such as diabetic nephropathy.

Vasopressin regulates rat mesangial cell growth by inducing autocrine secretion of vascular endothelial growth factor.

Mesangial cell growth is a key feature of several glomerular diseases. Vascular endothelial growth factor (VEGF) is a potent mitogen of vascular endothelial cells and promoter of vascular permeability. Here, we examined the ability of vasopressin (AVP), which causes mesangial cell proliferation and hypertrophy, to stimulate VEGF secretion from cultured rat mesangial cells. AVP potently induced a time- and concentration-dependent increase in VEGF secretion in these cells, which was then inhibited by a V(1A) receptor-selective antagonist, confirming this is a V(1A) receptor-mediated event. VEGF also induced hyperplasia and hypertrophy in mesangial cells, which was completely abolished by an anti-VEGF antibody. In addition, AVP-induced hyperplasia and hypertrophy were completely inhibited by the V(1A) receptor-selective antagonist and partially abolished by the anti-VEGF antibody. These results indicate that AVP increases VEGF secretion in rat mesangial cells via V(1A) receptors and modulates mesangial cell growth not only by direct action but also through stimulation of VEGF secretion. This autocrine mechanism might contribute to glomerulosclerosis in renal diseases such as diabetic nephropathy.

Synthesis and biological activity of novel 4,4-difluorobenzazepine derivatives as non-peptide antagonists of the arginine vasopressin V1A receptor.

To find potent and selective antagonists of the arginine vasopressin (AVP) V1A receptor, optimization studies of compounds structurally related to (Z)-N-{4'-[(4,4-difluoro-5-carbamoylmethylidene-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl)carbonyl]phenyl}carboxamide were performed. The synthesis and pharmacological properties of these compounds are described. We first investigated the effect of the carboxamide moiety, and found that a 2-methylfuran-3-carbonyl group at this position increased V1A binding affinity and selectivity for the V1A receptor versus the V2 receptor. The amino group of the 5-carbamoylmethylidene moiety was also examined, and a 4-piperidinopiperidino group was found to be optimal at this position. The hemifumarate of compound 12l (YM218) was shown to exhibit potent binding affinity, V1A receptor selectivity, and in vivo antagonist activity.

Progression of renal failure with anaemia and multiple effects of angiotensin-converting enzyme inhibitor in rats with renal mass reduction.

Several factors such as proteinuria and renal fibrosis may be important in the progression of many forms of chronic renal diseases. The purposes of the current study were to investigate the progressive renal failure of the rats with surgical renal mass reduction (RMR) and the effect of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, and to document correlation of several factors associated with progressive renal failure. Rats were subtotal (5/6) nephrectomized by resection of the renal poles and sham-operated. The functional, histological and haematological changes of the rats were studied for up to 10 weeks. After 2 weeks of RMR, oral administration of lisinopril (10 mg kg(-1) per day) was performed for 8 weeks. RMR resulted in progressive renal failure with proteinuria, monocyte/macrophage (ED1+) infiltration, anaemia as assessed by haemoglobin and haematocrit (Htc), renal hypertrophy as assessed by left kidney to body weight ratio (BKW/BW), and renal fibrosis as assessed by glomerular lesions and tubulointerstitial changes. Lisinopril exhibited renoprotection with antiproteinuric effect and inhibition of monocyte/macrophage (ED1+) infiltration. However, beneficial effect of lisinopril on anaemia was not observed. At 10 weeks after surgery, severity of proteinuria positively correlated with plasma creatinine (Pcr), BKW/BW, histological damage, and systolic blood pressure, and negatively correlated with haemoglobin. Severity of tubulointerstitial changes positively correlated with Pcr and blood urea nitrogen, and negatively correlated with haemoglobin and Htc. Moreover, monocyte/macrophage (ED1+) infiltration positively correlated with severity of proteinuria and tubulointerstitial changes. These findings strongly support that proteinuria, monocyte/macrophage infiltration and renal fibrosis appear to play principal roles in the progressive renal failure with anaemia and renoprotection of ACE inhibition may be mediated by multiple actions of ACE inhibitor. The present study confirms that rats with RMR is useful to explore target molecules for renoprotective drugs and evaluate renoprotective effect of new molecular entities.