Peroxisome proliferator-activated receptor gamma activators downregulate angiotensin II type 1 receptor in vascular smooth muscle cells.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARgamma) activators, such as troglitazone (Tro), not only improve insulin resistance but also suppress the neointimal formation after balloon injury. However, the precise mechanisms have not been determined. Angiotensin II (Ang II) plays crucial roles in the pathogenesis of atherosclerosis, hypertension, and neointimal formation after angioplasty. We examined the effect of PPARgamma activators on the expression of Ang II type 1 receptor (AT(1)-R) in cultured vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: AT(1)-R mRNA and AT(1)-R protein levels were determined by Northern blot analysis and radioligand binding assay, respectively. Natural PPARgamma ligand 15-deoxy-Delta(12,14)-prostaglandin J(2), as well as Tro, reduced the AT(1)-R mRNA expression and the AT(1)-R protein level. The PPARgamma activators also reduced the calcium response of VSMCs to Ang II. PPARgamma activators suppressed the AT(1)-R promoter activity measured by luciferase assay but did not affect the AT(1)-R mRNA stability, suggesting that the suppression occurs at the transcriptional level. CONCLUSIONS: PPARgamma activators reduced the AT(1)-R expression and calcium response to Ang II in VSMCs. Downregulation of AT(1)-R may contribute to the inhibition of neointimal formation by PPARgamma activators.

Critical role of Rho-kinase and MEK/ERK pathways for angiotensin II-induced plasminogen activator inhibitor type-1 gene expression.

Plasminogen activator inhibitor type-1 (PAI-1) plays an integral role not only in the regulation of fibrinolytic activity but also in the pathogenesis of atherosclerosis and hypertension. We investigated the signaling pathways of angiotensin II (Ang II) leading to PAI-1 gene expression. Ang II increased the PAI-1 mRNA and protein levels in a time- and dose-dependent manner through the Ang II type 1 receptor in vascular smooth muscle cells. PAI-1 gene promoter activity measured by luciferase assay was significantly increased by Ang II. PAI-1 mRNA stability was also increased by Ang II. Ang II-induced PAI-1 mRNA upregulation was inhibited by BAPTA-AM, genistein, and AG1478, suggesting that intracellular calcium, tyrosine kinase, and epidermal growth factor receptor transactivation are involved. Furthermore, PD98059, an inhibitor of extracellular signal-regulated kinase (ERK) kinase (MEK), almost completely suppressed Ang II-induced PAI-1 upregulation. Adenovirus-mediated overexpression of the dominant-negative form of Rho-kinase or Y27632, a Rho-kinase inhibitor, also completely prevented PAI-1 induction by Ang II without affecting Ang II-induced ERK activation. These data suggest that activation of MEK/ERK and Rho-kinase pathways plays a pivotal role in PAI-1 gene upregulation by Ang II. The Rho-kinase pathway may be a novel target to inhibit Ang II signaling, and its inhibition may be useful in the treatment of hypertension as well as atherosclerosis.

Thrombin induces interleukin-6 expression through the cAMP response element in vascular smooth muscle cells.

The plasma level of interleukin-6 (IL-6) is elevated in patients with acute coronary syndromes and has prognostic value. Thrombin is a potent mitogen for vascular smooth muscle cells (VSMCs) and plays an important role in the progression of atherosclerosis. We examined the mechanism of thrombin-induced IL-6 expression in VSMCs. Thrombin induced IL-6 mRNA and protein expression in a dose-dependent manner. Pharmacological inhibition of extracellular signal-regulated protein kinase (ERK), p38 mitogen-activated protein kinase (MAPK), or epidermal growth factor receptor (EGF-R) suppressed the thrombin-induced IL-6 expression. Deletion and mutation analysis of the promoter region of the IL-6 gene by using luciferase as a reporter showed that the DNA segment between -228 and -150 bp containing the cAMP response element (CRE) site played a critical role. Thrombin also induced phosphorylation of CRE binding protein (CREB) in an ERK- and a p38 MAPK-dependent manner. Overexpression of the dominant-negative form of CREB inhibited thrombin-induced IL-6 mRNA expression. These results suggest that the CRE site and CREB play an important role in thrombin-induced IL-6 gene expression in VSMCs. Transactivation of EGF-R and activation of ERK and p38 MAPK are involved in this process. CREB may be a novel transcription factor that regulates thrombin-induced gene expression.

cAMP response element-binding protein mediates thrombin-induced proliferation of vascular smooth muscle cells.

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMCs) and plays an important role in the progression of atherosclerosis. Although recent reports have suggested that cAMP response element-binding protein (CREB) is necessary for the survival of neuronal cells, the role of CREB in VSMC proliferation is not determined. We examined the role of CREB in thrombin-induced VSMC proliferation and the effect of thrombin on phosphorylation of CREB at Ser133, which is a critical marker for activation by Western blot analysis. Thrombin induced phosphorylation of CREB in a dose-dependent manner. An oligopeptide, SFLLRN, which activates the thrombin receptor, also induced the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase or inhibition of p38 mitogen-activated protein kinase suppressed the thrombin-induced CREB phosphorylation. Inhibition of the epidermal growth factor receptor by AG1478 also inhibited the thrombin-induced CREB phosphorylation. Overexpression of the dominant-negative form of CREB inhibited thrombin-induced c-fos mRNA expression and incorporation of [(3)H]thymidine and [(3)H]leucine. These results suggest that CREB-dependent gene transcription plays a critical role in thrombin-induced proliferation and hypertrophy of VSMCs. Transactivation of the epidermal growth factor receptor and 2 mitogen-activated protein kinase pathways are involved in this process. CREB may be a novel transcription factor mediating the vascular remodeling process induced by thrombin.

Downregulation of angiotensin II type 1 receptor by hydrophobic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in vascular smooth muscle cells.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, so-called statins, reduce the relative risk of a major coronary event by lowering the serum cholesterol level. In addition, statins may confer beneficial effects by cholesterol-lowering independent mechanisms, which are incompletely characterized. Because angiotensin II (Ang II) plays crucial roles in the pathogenesis of cardiovascular diseases, we examined the effect of statins on the expression of the Ang II type 1 receptor (AT(1)-R) in cultured vascular smooth muscle cells (VSMCs). Cerivastatin and fluvastatin reduced the AT(1)-R mRNA and the AT(1)-R protein levels; however, pravastatin lacked this effect. Cerivastatin and fluvastatin suppressed the AT(1)-R promoter activity measured by luciferase assay but did not affect AT(1)-R mRNA stability, suggesting that the suppression occurs at the transcriptional level. Coincubation of VSMCs with mevalonate or geranylgeranyl pyrophosphate but not with farnesyl pyrophosphate reversed the cerivastatin-induced AT(1)-R downregulation. Overexpression of dominant-negative Rho A also suppressed AT(1)-R mRNA expression. Treatment with cerivastatin for 24 hours reduced the calcium response of VSMCs to Ang II. Taken together, statins downregulate AT(1)-R expression through a mevalonate-dependent, geranylgeranyl pyrophosphate-dependent, and Rho A-dependent manner and attenuate the biological function of Ang II. Downregulation of AT(1)-R may contribute to the cholesterol-independent beneficial effect of statins on the cardiovascular system.

Determination of optimal blood pressure for patients with IgA nephropathy based on renal histology.

To evaluate the optimal BP control for patients with IgA nephropathy (IgAN) based on the histologic severity of the nephropathy and the degree of renal dysfunction. We analyzed 332 consecutive renal biopsy specimens and clinical data from patients with IgAN. Patients were divided into three groups based on their BP at the time of biopsy: an optimal BP (SBP<120 mmHg and DBP<80 mmHg), a hypertensive BP (SBP > or = 140 mmHg and/or DBP > or = 90 mmHg), and an intermediate BP group. Each biopsy specimen was evaluated for mesangial proliferation, degree of sclerosis and/or hyalinosis of the arterioles and the interlobular artery using a semiquantitative method. Creatinine clearance and the percentage of sclerosed glomeruli were also determined. Both the degree of renal dysfunction and the histologic changes correlated significantly with BP, even in patients with a BP <140/90 mmHg. The patients with an optimal BP at the time of biopsy had significantly less histologic damage with respect to mesangial proliferation and vessel changes than those with an intermediate or hypertensive BP. In the patients with a hypertensive BP, the percentage of sclerotic glomeruli was significantly higher and the creatinine clearance was significantly lower. The optimal BP proposed by the WHO in 1999 prevents histologic evidence of renal damage for patients with IgAN.

alpha-Amylase expressed in human liver is encoded by the AMY-2B gene identified in tumorous tissues.

BACKGROUND: An alpha-Amylase in human liver is detected with an anti-human salivary amylase antibody, but the enzyme activity is very low. We previously found that the rat liver contained an amylase which differed from the enzyme of mice. In this study, we characterized the human liver amylases biochemically and immunohistochermically. METHODS AND RESULTS: Although the amylase activity of human liver was much lower than that of rat, protein moiety and sugar chains of the human amylase were identified as similar to the rat liver enzyme with an anti-human salivary amylase antibody and by concanavalin A (Con A) affinity chromatography. Liver amylases from human and rat were the same size, 50 kDa, on Western blot analysis and had the same isoelectric points. The cytoplasm of hepatocytes was moderately stained immunohistochemically with the anti-human salivary amylase antibody. Intrahepatic bile ducts were also stained weak-to-moderately. RT-PCR, with a specific primer for the consensus sequence of human amylases, amplified a single 474-bp product from the human liver total RNA. The PCR product was sequenced and referred to the homology. Thirteen bases in the 434-bp fragment of the human liver amylase differed from the corresponding region of the AMY-1 gene transcript and the deduced amino acid sequence differed at five residues. The human liver amylase cDNA sequence was identical to the corresponding cDNA of the AMY-2B, which was known to expressed in tumorous tissues. In situ hybridization revealed the expression of AMY-2B mRNA in non-tumorous human liver. CONCLUSIONS: The present results suggest the possibility that a novel amylase detected in tumorous tissues and encoded by the AMY-2B gene is a liver-specific amylase expressed in the human liver.

Successful perioperative blood purification therapy in patients with maintenance hemodialysis therapy who underwent living donor liver transplantation.

Living donor liver transplantation (LDLT) is a treatment for end-stage liver failure, and was developed to overcome the distinct insufficiency of cadaveric donors. Case 1 is a 56-year-old man who had undergone maintenance hemodialysis therapy for 4 years. An LDLT was performed for the treatment of advanced liver cirrhosis and hepatocellular carcinoma. Continuous hemodiafiltration (CHDF) was performed from the 2nd to 5th days after the operation. Case 2 is a 55-year-old man with primary amyloidosis and chronic renal failure. An LDLT was performed for the treatment of severe abdominal distention caused by a large liver volume. Although CHDF was started at the 3rd day after the operation, it was discontinued within 24 hours because of an increased urinary volume. CHDF was required again from the 6th-8th days, after which the blood purification mode was switched to regular intermittent hemodialysis. Meanwhile, no major problems occurred in either case. In conclusion, CHDF was required for about 5 days from the 2nd day after the operation. The application of careful and aggressive blood purification therapy during the perioperative period is a key to successful LDLT in dialysis patients.

An improved method for the syntheses of p-aminophenyl 1-thio-beta-D-glycosides.

The condensation of the appropriate acetylglycosyl bromides with p-aminobenzenethiol in the presence of sodium methoxide afforded p-aminophenyl 1-thio-beta-D-glucopyranoside, 1-thio-beta-D-galactopyranoside, 1-thio-beta-D-xylopyranoside, and 2-acetamido-2-deoxy-1-thio-beta-D-glucopyranoside. p-Aminophenyl 1-thio-beta-D-glucopyranosiduronic acid was synthesized by condensation of methyl (2,3,4-tri-O-acetyl-alpha-D-glucopyranosyl bromide)uronate with p-aminobenzenethiol, followed by saponification with sodium hydroxide.

Treatment of membranoproliferative glomerulonephritis associated with hepatitis C virus infection. Niigata Research Group of Glomerulonephritis and Nephrotic Syndrome.

OBJECTIVE: Interferon has been used as a new therapeutic agent for glomerulonephritis since a manifest relationship between membranoproliferative glomerulonephritis (MPGN) and hepatitis C virus (HCV) infection was documented. However, several side effects and rebound phenomenon have been significant problems. We retrospectively evaluated the therapeutic effect and safety of the standard treatment with steroids and/or immunosuppressive agents for MPGN patients with an HCV infection. METHODS: Remission and renal survival rates as well as clinical and histological data were compared between MPGN groups with or without an HCV infection. In addition, the hepatic function was followed-up after the treatment. PATIENTS: The subjects were 42 biopsy proven MPGN patients. Seven were positive for an HCV infection. Secondary causes of MPGN excluding an HCV infection were omitted. Most patients were treated with steroids and/or immunosuppressive agents. RESULTS: The mean age of the MPGN patients with an HCV infection was significantly higher than that of those without an HCV infection. The renal function and the interstitial change of the former group were significantly worse than those of the latter. Nevertheless, remission and renal survival rates were not significantly different between the two groups. None in the HCV positive MPGN group showed an impairment of hepatic function during the clinical course. However, 2 subjects died from severe pneumonia during the treatment. CONCLUSION: The standard treatment with steroids and/or immunosuppressive agents did not reveal a statistical difference in the therapeutic efficacy between MPGN patients with or without an HCV infection. However, some in the former group showed a poor prognosis.

Unilateral agenesis and hypoplasia of the internal carotid artery: a report of three cases.

We report one patient with agenesis and two with hypoplasia of the internal carotid artery (ICA) from an angiographic series of 1275 consecutive patients. In all three patients, MRI and MR angiography were used to establish the anatomical situation; however, they may not clearly distinguish between hypoplasia and agenesis or acquired stenosis or occlusion of the ICA. In such cases, CT of the skull base may be obtained to clarify matters. In all patients, retrograde flow via a large posterior communicating artery was demonstrated. Single-photon emission computed tomography confirmed both the adequacy of cerebral perfusion and the preservation of vasomotor reactivity.

Chromatography of beta-glucuronidase from bovine liver. A study of the enzyme binding sites of prepared adsorbents.

beta-Glucuronidase from bovine liver was adsorbed to the adsorbents prepared with CH-Sepharose 4B and either the competitive inhibitor or its analogs such as p-aminophenyl 1-thio-beta-D-glucuronic acid, -glucoside, -galactoside, and N-acetyl glucosaminide. The adsorbed enzyme was eluted at 0.1 or 0.5 M NaCl by a stepwise gradient. Chromatography of the enzyme was also performed by using the adsorbents prepared with Epoxy-activated Sepharose 6B and amine compounds or other compounds. In order to see whether the hydroxyl groups of the sugar parts in the ligand are necessary for the adsorption of the enzyme, chromatography was performed by using the adsorbents prepared with sugar derivatives as the ligand. As a result, it was found that beta-glucuronidase had an affinity for adsorbents prepared with either acetyl derivatives or methoxy derivatives of glycosides and CH-Sepharose 4B. From the results of elution of the enzyme with NaCl from adsorbents having amide bonding, it was clarified that the affinity of the enzyme for adsorbents without glycosides in the ligands correlated with acidity of the amide in the adsorbents. Hydrogen bond chromatography was performed with the prepared adsorbents. The enzyme was adsorbed under a high concentration of ammonium sulfate, and the elution of the adsorbed enzyme from adsorbents was examined by the degradation of salt. The enzyme was most easily eluted from aminoethyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B at 0.9 M ammonium sulfate and at 0.5 M concentration of the salt with p-aminophenyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-dimensional analysis of nephrogenesis in the neonatal rat kidney: light and scanning electron microscopic studies.

In order to clarify the process of renal development more precisely than previously, the present study observed the rat neonatal kidney by scanning electron microscopy (SEM) of KOH digested tissue as well as by light microscopy of plastic sections. In the subcapsular region, aggregation of the mesenchymal cells was closely associated with the upper side of the ureteric duct ampulla. These mesenchymal cells projected a number of fine irregular processes at the basal portion facing the ureteric duct. A spherical cluster transformed from the mesenchymal cell aggregation was found on the lower side of the terminal ampulla, and was differentiated into the renal vesicle. Some cells at the top of the renal vesicle formed a cone-shaped projection and invaded the ureteric duct ampulla, forming a connection with it. In the advanced stage, a shallow transverse cleft appeared on the outer lateral side of the renal vesicle, and a second cleft was formed on the opposite side close to the junction between the renal vesicle and the ampulla. As the two clefts deepened, the vesicle assumed the well-known S-shaped body. In the advanced S-shaped body, the lower limb became cup-shaped, while the segment between the middle and lower limbs of the "S" elongated to form a tubular structure (i.e., the prospective proximal tubule and Henle's loop). The upper limb of the "S" also increased its length to form a distal tubule. The middle limb of the "S", however, was attached firmly to the cup-shaped lower limb (i.e., the prospective renal corpuscle) and was considered to become the macula densa of the mature nephron. In the maturing renal corpuscle, irregularly shaped cells were observed as a sheet-like aggregation at its vascular pole and were continuous with the vascular smooth muscle cells. These findings will help toward a better understanding of the morphological complexities of nephrogenesis.

The three-dimensional structure of the neonatal mouse kidney as revealed by scanning electron microscopy after KOH treatment.

The shape and arrangement of the developing nephrons were studied three-dimensionally by scanning electron microscopy (SEM) of the neonatal mouse kidney. The specimens were treated with the KOH digestion method in order to remove extracellular connective tissue components, thus enabling the direct observation of the developing nephrons at various stages. At the subcapsular region of the renal cortex, the ureteric ducts were observed as branched tubules with terminal swellings or ampullae. Newly formed blood vessels were often associated with terminals of these ureteric ducts. The cup-shaped renal corpuscles had aggregations of mesangial cells with blood vessels in the groove. At the vascular pole of mature nephrons, extraglomerular mesangial cells were observed as a cellular sheet, which was continuous with the smooth muscle layer of afferent and efferent blood vessels. The present study also demonstrated the shape of the immature podocytes in relation to the endothelial morphology of glomerular capillaries.

Reactive oxygen species-mediated homologous downregulation of angiotensin II type 1 receptor mRNA by angiotensin II.

Recent studies suggest a crucial role of reactive oxygen species (ROS) for the signaling of angiotensin (Ang) II through Ang II type 1 receptor (AT(1)-R). However, the role of ROS in the regulation of AT(1)-R expression has not been explored. In this study, we examined the effect of an antioxidant on the homologous downregulation of AT(1)-R by Ang II. Ang II (10(-6) mol/L) decreased AT(1)-R mRNA with a peak suppression at 6 hours of stimulation in rat aortic vascular smooth muscle cells. Preincubation of vascular smooth muscle cells with N:-acetylcysteine (NAC), a potent antioxidant, almost completely inhibited the Ang II-induced downregulation of AT(1)-R mRNA. The effect of NAC was due to stabilization of the AT(1)-R mRNA that was destabilized by Ang II. The Ang II-induced AT(1)-R mRNA downregulation was also blocked by PD98059, an extracellular signal-regulated protein kinase (ERK) kinase inhibitor. Ang II-induced ERK activation was inhibited by NAC as well as by PD98059. Exogenous H(2)O(2) also suppressed AT(1)-R mRNA. These results suggest that the production of ROS and the activation of ERK are critical for the downregulation of AT(1)-R mRNA. The generation of ROS through stimulation of AT(1)-R not only mediates signaling of Ang II but also may play a crucial role in the adaptation process of AT(1)-R to the sustained stimulation of Ang II.

15-Deoxy-delta 12,14-prostaglandin J2 and thiazolidinediones activate the MEK/ERK pathway through phosphatidylinositol 3-kinase in vascular smooth muscle cells.

Peroxisome proliferator-activated receptor (PPAR) gamma belongs to the nuclear receptor superfamily of ligand-dependent transcription factors. Recent results have shown that the ligands for nuclear receptors have rapid effects so called "nongenomic" effects, which are observed within minutes after stimulation. We examined whether 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-d-PGJ2) had rapid effects on cultured vascular smooth muscle cells. Phosphorylation of ERK and c-fos mRNA expression were determined by Western and Northern blot analyses, respectively. PPAR gamma agonists 15-d-PGJ2 and thiazolidinediones such as pioglitazone and troglitazone elicited rapid activation of ERK within 15 min and induced c-fos mRNA expression within 30 min, whereas the PPAR alpha agonist bezafibrate failed to activate ERK. 15-d-PGJ2-induced expression of c-fos mRNA was blocked by PD98059 or U0126, two ERK kinase inhibitors, suggesting that the MEK/ERK pathway mediates 15-d-PGJ2-induced c-fos gene expression. Furthermore, pretreatment with wortmannin, an inhibitor of phosphatidylinositol 3 (PI3)-kinase, inhibited 15-d-PGJ2-induced ERK activation and c-fos mRNA expression, suggesting that PI3-kinase is involved in the process. An electrophoretic mobility shift assay showed that 15-d-PGJ2 enhanced AP-1 binding activity to AP-1 consensus sequence in a time-dependent manner. 15-d-PGJ2 increased thymidine incorporation in a PI3-kinase-dependent manner. Taken together, our findings show that 15-d-PGJ2 and thiazolidinediones activate the MEK/ERK pathway through PI3-kinase and lead to c-fos mRNA expression and DNA synthesis. These findings indicate a novel regulatory mechanism of gene expression by 15-d-PGJ2 and thiazolidinediones.

Kidney-targeted naked DNA transfer by retrograde injection into the renal vein in mice.

We recently developed a novel kidney-targeted gene transfer technique in rats, using the retrograde renal vein injection of naked plasmid DNA. Many animal disease models are created in mice by transgenic or knockout technologies. However, it is much harder to perform renal vein injection in mice than in rats because they have a thin and short vein. Here we transferred the mouse interleukin (IL)-10 gene into mice by retrograde renal vein injection, using an IL-10 and immunoglobulin fusion protein (IL-10/Fc) (96-kDa) expression plasmid, pCAGGS-IL10/Fc. We observed a dose-response relationship between serum IL-10 levels and the amount of injected DNA. The serum IL-10 levels peaked at day 1 and then were sustained for at least 2 weeks. These results demonstrate that the kidney-targeted naked plasmid DNA transfer of mice by retrograde renal vein injection can be achieved, and the kidney serves as a depot organ for the production of large proteins.

Hydrodynamics-based transfer of PCR-amplified DNA fragments into rat liver.

A high level of plasmid DNA expression in rat liver can be achieved by the rapid injection of a large volume of a naked DNA solution into the tail vein, called the 'hydrodynamics-based procedure.' The preparation of PCR-amplified DNA fragments is easier than that of naked DNA. In this paper we evaluated the effects of expressing the erythropoietin (Epo) gene in the rat liver by injecting fCAGGS-Epo, an Epo-expressing PCR-amplified DNA fragment, via the tail vein. After injection of 5 pmol fCAGGS-Epo (10 microg) or pCAGGS-Epo (18.4 microg), plasmid DNA, the serum Epo levels peaked at week 1, then persisted for at least 12 weeks. Transgene-derived Epo secretion resulted in significant erythropoiesis. These results demonstrated that transfer of PCR-amplified DNA fragments into the rat liver via rapid tail vein injection can be achieved. This method may provide a useful means for studying the physiologic function of a putative gene.

Ultrastructural characteristics of diabetic nephropathy.

Diabetic nephropathy is a major cause of chronic renal failure in Japan, and the prevalence rate has markedly increased during the past decade. Diabetic nephropathy shows various specific histological changes not only in glomeruli but also in the interstitial region. Nodular, diffuse, and exudative lesions, so-called diabetic glomerulosclerosis, are well known as glomerular lesions. At first, they were historically evaluated only by light microscopy, and thus which components of the glomeruli were modified was not sufficiently clear. Subsequent electron microscopic studies clarified that the expansion of the mesangial matrix was the true form of nodular and diffuse lesions, and that insudated serum substance was the real appearance of an exudative lesion. Interstitial lesions also exhibit specific features in diabetic nephropathy. In electron microscopic studies, it was proved that the size of mitochondria and thickness of the tubular basement membrane were increased in diabetic nephropathy. In this review, we introduce typical electron microscopic findings in diabetic nephropathy and recent opinions on the progression of diabetic nephropathy.