Evaluating the State of Glomerular Disease by Analyzing Urinary Sediments: mRNA Levels and Immunofluorescence Staining for Various Markers.

Renal biopsy is the gold standard for making the final diagnosis and for predicting the progression of renal disease, but monitoring disease status by performing biopsies repeatedly is impossible because it is an invasive procedure. Urine tests are non-invasive and may reflect the general condition of the whole kidney better than renal biopsy results. We therefore investigated the diagnostic value of extensive urinary sediment analysis by immunofluorescence staining for markers expressed on kidney-derived cells (cytokeratin: marker for tubular epithelial cells, synaptopodin: marker for podocytes, claudin1: marker for parietal epithelial cells, CD68: marker for macrophages (MΦ), neutrophil elastase: marker for neutrophils). We further examined the expression levels of the mRNAs for these markers by real-time reverse transcription polymerase chain reaction. We also examined the levels of mRNAs associated with the M1 (iNOS, IL-6) and M2 (CD163, CD204, CD206, IL-10) MΦ phenotypes. Evaluated markers were compared with clinical and histological findings for the assessment of renal diseases. Claudin1- and CD68-positive cell counts in urinary sediments were higher in patients with glomerular crescents (especially cellular crescents) than in patients without crescents. The relative levels of mRNA for CD68 and the M2 MΦ markers (CD163, CD204, CD206, and IL-10) in urinary sediments were also higher in patients with glomerular crescents. These data suggest that immunofluorescence staining for claudin1 and CD68 in urinary sediments and the relative levels of mRNA for CD68 and M2 MΦ markers in urinary sediments are useful for evaluating the state of glomerular diseases.

Renoprotective effects of a dipeptidyl peptidase 4 inhibitor in a mouse model of progressive renal fibrosis.

Although the effects of dipeptidyl peptidase 4 (DPP-4) inhibitors beyond their hypoglycemic action have been reported, whether these inhibitors have renoprotective effects in nondiabetic chronic kidney disease (CKD) is unclear. We examined the therapeutic effects of DPP-4 inhibition in mice with unilateral ureteral obstruction (UUO), a nondiabetic model of progressive renal fibrosis. After UUO surgery, mice were administered either the DPP-4 inhibitor alogliptin or a vehicle by oral gavage once a day for 10 days. Physiological parameters, degrees of renal fibrosis and inflammation, and molecules related to renal fibrosis and inflammation were then evaluated using sham-operated mice as controls. Positive area of α-smooth muscle actin was significantly smaller and expression of transforming growth factor ß messenger RNA was significantly lower in the alogliptin-treated group than in the vehicle-treated group. Renal total collagen content was also significantly lower in the alogliptin-treated group than in the vehicle-treated group. These results suggest that alogliptin exerted renoprotective antifibrotic effects. The positive area of F4/80 was significantly smaller and expression of CD68 messenger RNA was significantly lower in the alogliptin-treated group than in the vehicle-treated group, suggesting an anti-inflammatory action by the DPP-4 inhibitor. Compared to the results for the vehicle-treated group, expression of markers for M1 macrophages tended to be lower in the alogliptin-treated group, and the relative expression of M2 macrophages tended to be higher. These data indicate the various protective effects of DPP-4 inhibition in nondiabetic mice with UUO. DPP-4 inhibitors may therefore be promising therapeutic choices even for nondiabetic CKD patients.

Role of tubulointerstitial plasmin in the progression of IgA nephropathy.

BACKGROUND: Plasmin has recently been reported to be associated with renal fibrosis in experimental models, but its role in human renal diseases is unclear. METHODS: Fifty-seven patients with IgA nephropathy (IgAN) were evaluated retrospectively. Plasmin in their renal biopsy tissues was assessed by in situ zymography using a plasmin-sensitive synthetic peptide, and the relationships between patients' histologic or clinical parameters and their renal plasmin activity [assessed semiquantitatively by calculating the positively stained percentage of the total tubulointerstitial (TI) area] were evaluated. RESULTS: Plasmin activity was observed almost exclusively in the TI space (mainly in the interstitium and partly in the tubular epithelial cells) and was significantly stronger in patients with TI lesion (tubular atrophy/interstitial fibrosis and tubulointerstitial inflammation) than in those without TI lesion. It was significantly and positively correlated with the global glomerulosclerosis rate and significantly and negatively correlated with estimated glomerular filtration rate not only at the time of renal biopsy but also at the end of the follow-up period. Double stainings for plasmin activity and inflammatory cells, cytokeratin, or α-smooth muscle actin (α-SMA) in selected patients revealed TI infiltration of inflammatory cells, attenuated tubular epithelial expression of cytokeratin, and augmented interstitial expression of α-SMA close to upregulated plasmin activity in the TI space. CONCLUSIONS: These data suggest that TI plasmin is associated with TI inflammation leading to renal fibrosis, and can cause the decline in renal function seen in patients with IgAN. Reducing plasmin in situ may therefore be a promising therapeutic approach slowing renal fibrogenesis and improving renal function.

Observation of Angiolymphoid Hyperplasia with Eosinophilia (ALHE) at Three Arterial Sites and Its Association with Membranous Nephropathy.

We herein report a case involving the systemic presentation of angiolymphoid hyperplasia with eosinophilia (ALHE) in association with membranous nephropathy (MN). A 34-year-old Japanese man presented with leg edema and bilateral temporal nodules. He had a history of Buerger's disease and recurrent coronary stenosis. A renal biopsy performed to assess nephrotic syndrome revealed MN. Furthermore, a temporal nodule was excised, and ALHE was diagnosed. We reevaluated the coronary and posterior tibial artery specimens obtained in his twenties and presumed that these lesions were also vascular tumors arising from ALHE. The association of ALHE and MN is quite rare.

Expression and functions of ß1- and ß2-adrenergic receptors on the bulbospinal neurons in the rostral ventrolateral medulla.

The expression and effects of ß-adrenergic receptors (ß-ARs) on the neurons of the bulbospinal rostral ventrolateral medulla (RVLM) have been limitedly examined to date. The objective of this study was to examine the expression of ß1- and ß2-ARs on the bulbospinal RVLM neurons electrophysiologically and histologically. To directly investigate whether RVLM neurons display sensitivity to metoprolol (a ß1-AR antagonist), dobutamine (a ß1-AR agonist), butoxamine (a ß2-AR antagonist), and salbutamol (a ß2-AR agonist), we examined changes in the membrane potentials of the bulbospinal RVLM neurons using the whole-cell patch-clamp technique during superfusion of these drugs. During metoprolol superfusion, 16 of the 20 RVLM neurons were hyperpolarized, and 5 of the 6 RVLM neurons were depolarized during dobutamine superfusion. During butoxamine superfusion, 11 of the 16 RVLM neurons were depolarized, and all of the 8 RVLM neurons were hyperpolarized during salbutamol superfusion. These results suggest the expression of ß1- and ß2-ARs on the RVLM neurons. To determine the presence of ß1- and ß2-ARs histologically, immunofluorescence examination was performed. Five metoprolol-hyperpolarized neurons were examined for ß1-AR and tyrosine hydroxylase (TH) immunoreactivity. All of the neurons displayed ß1-AR immunoreactivity, whereas three of the neurons displayed TH immunoreactivity. All of the five RVLM neurons that became depolarized during metoprolol superfusion and hyperpolarized during butoxamine superfusion displayed ß1- and ß2-AR immunoreactivity. Our findings suggest that ß1-AR antagonists or ß2-AR agonists may decrease blood pressure through decreasing the activity of the bulbospinal RVLM neurons.

Clinical and immunological implications of increase in CD208+ dendritic cells in tonsils of patients with immunoglobulin A nephropathy.

BACKGROUND: The therapeutic effect of tonsillectomy for immunoglobulin A nephropathy (IgAN) has been widely recognized, but the mechanism by which tonsillar immunity leads to glomerulonephritis has been unclear. We investigated subtypes and localization of dendritic cells (DCs) in tonsils and looked for relationships between the tonsillar DCs and the clinical features and renal histological changes of patients with IgAN. METHODS: We examined tonsils from 33 IgAN patients, using as control tonsillar specimens from subjects without glomerulonephritis. Five distinct markers of DCs (CD303, CD1c, CD209, CD208 and CD1a) were analyzed by immunohistochemistry and flow cytometry. The mRNA levels of these DC markers were evaluated using real-time polymerase chain reaction. The clinical data and histological results obtained evaluating renal biopsy tissues were statistically compared with immunological data. RESULTS: Of the five subtypes of DCs, CD208(+) DCs were significantly increased in the tonsils of IgAN patients compared with that of controls. Furthermore, the number of CD208(+) DCs in the tonsils was positively and linearly correlated with the proportion of crescentic glomeruli in renal biopsy tissues and with the urinary protein level. Only few CD208(+) cells, however, were found in the kidney biopsy specimens of IgAN patients. CONCLUSIONS: These observations suggest that increased CD208(+) DCs in tonsils may play a directive role in the pathogenesis of IgAN. The present results support the therapeutic significance of tonsillectomy for IgAN patients.

Protective effects of Rho kinase inhibitor fasudil on rats with chronic kidney disease.

The protective effects of Rho kinase inhibitor fasudil against renal diseases have recently been reported. We compared the therapeutic effects of fasudil on the spontaneously hypercholesterolemic (SHC) rat, a model of chronic kidney disease (CKD) with proteinuria, with those of the angiotensin receptor blocker olmesartan (OL) by paying attention to the proteinuria and the macrophage phenotype. SHC rats were allocated to six treatment groups: a vehicle (Ve) group, a low-dose fasudil (FL) group, a high-dose fasudil (FH) group, an OL group, a combination of low-dose fasudil and OL (CL) group, and a combination of high-dose fasudil and OL (CH) group. Sprague-Dawley rats treated with vehicle served as a control (n = 7/each). The rats were treated for 24 wk. Compared with the Ve group, proteinuria was significantly decreased in the FH, OL, and CL groups, and it completely disappeared in the CH group. Glomerular stainings of nephrin and F-actin were focally impaired in the Ve group but were restored in the CH group. Western blotting showed that the CH group had significantly increased renal nephrin expression compared with the Ve group. Interstitial infiltration of macrophages was significantly increased in the Ve group, which was significantly attenuated in all treatment groups. The ratio of CD206 (M2 macrophage marker) to CD68 mRNA was significantly greater in the CH group than in the Ve group. These results indicate that fasudil with OL reduces proteinuria by protecting podocyte integrity and alters the interstitial macrophage density/phenotype, thereby exerting renoprotective effects against CKD.

Aldosterone is synthesized in and activates bulbospinal neurons through mineralocorticoid receptors and ENaCs in the RVLM.

The effects of aldosterone and mineralocorticoid receptor (MR) blockers on presympathetic neurons in the rostral ventrolateral medulla (RVLM) are well studied. To directly investigate whether aldosterone, eplerenone (an MR blocker), FAD286 (an aldosterone synthase inhibitor) and benzamil (an epithelial sodium channel (ENaC) blocker) affect RVLM neurons, we examined changes in the membrane potentials (MPs) of bulbospinal RVLM neurons using the whole-cell patch-clamp technique during superfusion with these drugs to brainstem-spinal cord preparations. Aldosterone superfusion (0.1 µmol/l) depolarized the RVLM neurons. In contrast, eplerenone superfusion (1 µmol/l) hyperpolarized them. To evaluate the existence of aldosterone, FAD286 superfusion (10 µmol/l) was performed, and the RVLM neurons became hyperpolarized during FAD superfusion. These data suggest that MRs exist and that aldosterone is synthesized in the brainstem. Benzamil superfusion (1 µmol/l) hyperpolarized the RVLM neurons. To clarify whether aldosterone, eplerenone, FAD286 and benzamil acted directly on the RVLM neurons, a low-Ca(2+), high-Mg(2+) solution was used to block the synaptic input to the RVLM neurons, and the above-mentioned drugs were added during the low-Ca(2+) superfusion. During the aldosterone superfusion, the RVLM neurons became depolarized, and they became hyperpolarized during eplerenone, FAD286 or benzamil superfusion. Importantly, when aldosterone was superfused after the benzamil solution, the MPs of the RVLM neurons did not depolarize. These results suggest that MRs are present in the RVLM neurons and that aldosterone is synthesized in the RVLM. The RVLM neurons themselves possess ENaCs, and ENaCs are the underlying mechanism by which aldosterone activates RVLM neurons.

The role of nephritis-associated plasmin receptor (NAPlr) in glomerulonephritis associated with streptococcal infection.

It is well known that glomerulonephritis can occur after streptococcal infection, which is classically referred to as acute poststreptococcal glomerulonephritis (APSGN). The pathogenic mechanism of APSGN has been described by so-called immune complex theory, which involves glomerular deposition of nephritogenic streptococcal antigen and subsequent formation of immune complexes in situ and/or the deposition of circulating antigen-antibody complexes. However, the exact entity of the causative antigen has remained a matter of debate. We isolated a nephritogenic antigen for APSGN from the cytoplasmic fractions of group A streptococcus (GAS) depending on the affinity for IgG of APSGN patients. The amino acid and the nucleotide sequences of the isolated protein revealed to be highly identical to those of reported plasmin(ogen) receptor of GAS. Thus, we termed this antigen nephritis-associated plasmin receptor (NAPlr). Immunofluorescence staining of the renal biopsy tissues with anti-NAPlr antibody revealed glomerular NAPlr deposition in essentially all patients with early-phase APSGN. Furthermore, glomerular plasmin activity was detected by in situ zymography in the distribution almost identical to NAPlr deposition in renal biopsy tissues of APSGN patients. These data suggest that NAPlr has a direct, nonimmunologic function as a plasmin receptor and may contribute to the pathogenesis of APSGN by maintaining plasmin activity.

[Syndrome of inappropriate antidiuretic hormone secretion following adjuvant CDDP and 5-FU administration in a patient with esophageal carcinoma].

A case of hyponatremia following the first course of systemic adjuvant chemotherapy with cisplatin (CDDP) and 5-FU in a previously treated patient with esophageal cancer is reported. A 61-year-old man was admitted to our hospital for adjuvant chemotherapy after transthoracic esophagectomy and 3-field lymphadenectomy for esophageal cancer. Six days following chemotherapy, his serum sodium concentration was found to be 118 mEq/L, without edema or dehydration. This hyponatremic state was diagnosed as the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) induced by CDDP, based on the hypo-osmolality of his serum and urine, and an inappropriate level of plasma vasopressin.