Axonal projection of the medullary expiratory neurons in the feline thoracic spinal cord.

Expiratory neurons in the caudal ventral respiratory group extend descending axons to the lumbar and sacral spinal cord, and they possess axon collaterals, the distribution of which has been well-documented. Likewise, these expiratory neurons extend axons to the thoracic spinal cord and innervate thoracic expiratory motoneurons. These axons also give rise to collaterals, and their distribution may influence the strength of synaptic connectivity between the axons and the thoracic expiratory motoneurons. We investigated the distribution of axon collaterals in the thoracic spinal cord using a microstimulation technique. This study was performed on cats; one cat was used to make an anatomical atlas and six were used in the experiment. Extracellular spikes of expiratory neurons were recorded in artificially ventilated cats. The thoracic spinal gray matter was microstimulated from dorsal to ventral sites at 100-µm intervals using a glass-insulated tungsten microelectrode with a current of 150-250 µA. The stimulation tracks were made at 1 mm intervals along the spinal cord in segments Th9 to Th13, and the effective stimulating sites of antidromic activation in axon collaterals were systematically mapped. The effective stimulating sites in the contralateral thoracic spinal cord with expiratory neurons in the caudal ventral respiratory group (cVRG) occupied 14.4% of the total length of the thoracic spinal cord examined. The mean percentage of effective stimulating tracks per unit was 18.6 ± 4.4%. The distribution of axon collaterals of expiratory neurons in the feline thoracic spinal cord indeed resembled that reported in the upper lumbar spinal cord. We propose that a single medullary expiratory neuron exerts excitatory effects across multiple segments of the thoracic spinal cord via its collaterals.

Morphological analysis of neck muscle nerves and neurons in cats.

Previous studies have failed to show morphological differences between neck muscle alpha and gamma motor fibers or alpha and gamma motoneurons. The present study aimed to investigate the morphological features of neck muscle motor nerves and motoneurons in cats. To determine the morphological features of peripheral motor fibers, the value of the outer contours of each fiber was converted into a perfect circle after ganglionectomy to remove sensory fibers, and the fiber diameters were calculated based on their circumferences. The sizes of neck motor fibers in the peripheral nerves had an evident bimodal distribution into small and large fiber groups, as depicted in histograms. The sizes of small and large motor fibers ranged from 2 to 12 µm and from 12 to 40 µm, respectively. The small fiber group is likely to correspond to gamma motor fibers and the large fiber group to alpha motor fibers. The morphological features of neck muscle motoneurons sectioned in the horizontal plane were examined using the horseradish peroxidase (HRP) retrograde labeling technique. The diameters of the biventer cervicis and complexus motoneurons had bimodal distributions. The inflection point between the small and large diameter population was 28 µm for the biventer cervicis and 26 µm for the complexus. We also observed that larger neurons displayed more dendrites. In conclusion, we could identify morphological differences likely to correlate with alpha and gamma motoneurons in both neck muscle peripheral nerves and neck motoneurons.

Impaired Bile Acid Synthesis in a Taurine-Deficient Cat Model.

Deficiency of the functional amino acid-like compound taurine induced in cats by taurine-depleted food was previously shown to significantly decrease the levels of taurine-conjugated bile acids (BAs) and significantly increase the levels of unconjugated BAs, with a significant decrease in total BA concentration in the bile. Because the ratios of primary BAs (cholic acid [CA] and chenodeoxycholic acids [CDCA]) have also been shown to be altered in the bile, taurine has been suggested to play an important role in BA synthesis in the liver. The present study showed that in the liver of taurine-deficient cats, CYP7A1 protein expression and its metabolites (7α-hydroxycholesterol and α-hydroxy-4-cholesten-3-one) were significantly increased and, therefore, the ratio of the CA product in this pathway was decreased. On the other hand, the expression of the mitochondrial CYP27A1 protein and its metabolite 27-hydroxycholesterol (27HC) were significantly decreased in the taurine-deficient liver. Thus, a significantly decreased ratio of CDCA, which is the main product of 27HC, was found. The decreased activity of the CDCA-producing pathway might be related to mitochondrial dysfunction induced by taurine deficiency. In addition, a significant decrease in cholesterol levels in the liver was induced by a decrease in intestinal cholesterol absorption because of decreased hepatic-intestinal circulation of taurine-conjugated BAs. The results of this study showed that taurine deficiency alters both the quality and quantity of BAs through inactivity of the mitochondrial CDCA production pathway caused by impaired mitochondrial function and inhibited the absorption of cholesterol in the intestine.

LRBA is essential for urinary concentration and body water homeostasis.

Protein kinase A (PKA) directly phosphorylates aquaporin-2 (AQP2) water channels in renal collecting ducts to reabsorb water from urine for the maintenance of systemic water homeostasis. More than 50 functionally distinct PKA-anchoring proteins (AKAPs) respectively create compartmentalized PKA signaling to determine the substrate specificity of PKA. Identification of an AKAP responsible for AQP2 phosphorylation is an essential step toward elucidating the molecular mechanisms of urinary concentration. PKA activation by several compounds is a novel screening strategy to uncover PKA substrates whose phosphorylation levels were nearly perfectly correlated with that of AQP2. The leading candidate in this assay proved to be an AKAP termed lipopolysaccharide-responsive and beige-like anchor protein (LRBA). We found that LRBA colocalized with AQP2 in vivo, and Lrba knockout mice displayed a polyuric phenotype with severely impaired AQP2 phosphorylation. Most of the PKA substrates other than AQP2 were adequately phosphorylated by PKA in the absence of LRBA, demonstrating that LRBA-anchored PKA preferentially phosphorylated AQP2 in renal collecting ducts. Furthermore, the LRBA-PKA interaction, rather than other AKAP-PKA interactions, was robustly dissociated by PKA activation. AKAP-PKA interaction inhibitors have attracted attention for their ability to directly phosphorylate AQP2. Therefore, the LRBA-PKA interaction is a promising drug target for the development of anti-aquaretics.

Updates and Perspectives on Aquaporin-2 and Water Balance Disorders.

Ensuring the proper amount of water inside the body is essential for survival. One of the key factors in the maintenance of body water balance is water reabsorption in the collecting ducts of the kidney, a process that is regulated by aquaporin-2 (AQP2). AQP2 is a channel that is exclusively selective for water molecules and impermeable to ions or other small molecules. Impairments of AQP2 result in various water balance disorders, including nephrogenic diabetes insipidus (NDI), which is a disease characterized by a massive loss of water through the kidney and consequent severe dehydration. Dysregulation of AQP2 is also a cause of water retention with hyponatremia in heart failure, hepatic cirrhosis, and syndrome of inappropriate antidiuretic hormone secretion (SIADH). Antidiuretic hormone vasopressin is an upstream regulator of AQP2. Its binding to the vasopressin V2 receptor promotes AQP2 targeting to the apical membrane and thus enables water reabsorption. Tolvaptan, a vasopressin V2 receptor antagonist, is effective and widely used for water retention with hyponatremia. However, there are no studies showing improvement in hard outcomes or long-term prognosis. A possible reason is that vasopressin receptors have many downstream effects other than AQP2 function. It is expected that the development of drugs that directly target AQP2 may result in increased treatment specificity and effectiveness for water balance disorders. This review summarizes recent progress in studies of AQP2 and drug development challenges for water balance disorders.

Minimal change disease concurrent with acute interstitial nephritis after long-term use of sorafenib in a patient with renal cell carcinoma.

Sorafenib is one of the multi-targeted tyrosine kinase inhibitors (TKI), mainly used for treating advanced renal cell carcinoma. Accumulated evidence indicates a minority of patients develop nephrotic syndrome (NS) as a high-grade nephrotoxic injury; however, evidence of NS after long-term use of sorafenib remains unclear. A 64-year-old man developed NS following 2-year use of sorafenib and his NS persisted even after sorafenib use was discontinued. Renal biopsy disclosed minimal change disease (MCD) concurrent with acute tubulointerstitial nephritis, indicating secondary MCD with which sorafenib may be involved. To prevent permanent renal insufficiency, we administered glucocorticoid and succeeded in achieving complete remission from NS. Nephrotoxic injuries could occur at any time with variable onset after sorafenib. Renal biopsy should be pursued in the case of NS associated with TKI therapy. To facilitate recovery of renal dysfunction, administration of prednisolone should be considered, particularly when NS does not disappear after cessation of TKIs.

Properties of Renshaw-like cells excited by recurrent collaterals of pudendal motoneurons in the cat.

Although anatomical studies have indicated pudendal motoneurons to give off recurrent collaterals, they are not considered to make synapses onto interneurons, such as Renshaw cells, and rather terminate their own signals. No study till date has examined interneurons being driven by recurrent collaterals of pudendal motoneurons. Here, we aimed to investigate the existence of Renshaw cells driven by pudendal motoneurons along with the recurrent inhibition of the latter. Extracellular recordings were obtained from the ventral horn of the sacral spinal cord of anesthetized cats. Dorsal roots were sectioned, and motor axons were electrically stimulated. Renshaw-like cells driven by recurrent collaterals, with high-frequency firings at short latency discharge, were observed around Onuf's nucleus. However, the recurrent inhibitory post-synaptic potentials were not recorded by intracellular recordings from the pudendal motoneurons. In summary, we found Renshaw-like cells driven by pudendal motoneurons, but we could not identify the synaptic connection of these neurons.

Phosphorylation profile of human AQP2 in urinary exosomes by LC-MS/MS phosphoproteomic analysis.

BACKGROUND: Aquaporin-2 (AQP2) is a key water channel protein which determines the water permeability of the collecting duct. Multiple phosphorylation sites are present at the C-terminal of AQP2 including S256 (serine at 256 residue), S261, S264 and S/T269, which are regulated by vasopressin (VP) to modulate AQP2 trafficking. As the dynamics of these phosphorylations have been studied mostly in rodents, little is known about the phosphorylation of human AQP2 which has unique T269 in the place of S269 of rodent AQP2. Because AQP2 is excreted in urinary exosomes, the phosphoprotein profile of human AQP2 can be easily examined through urinary exosomes without any intervention. METHODS: Human urinary exosomes digested with trypsin or glutamyl endopeptidase (Glu-C) were examined by the liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) phosphoproteomic analysis. RESULTS: The most dominant phosphorylated AQP2 peptide identified was S256 phosphorylated form (pS256), followed by pS261 with less pS264 and far less pT269, which was confirmed by the western blot analyses using phosphorylated AQP2-specific antibodies. In a patient lacking circulating VP, administration of a VP analogue showed a transient increase (peak at 30-60 min) in excretion of exosomes with pS261 AQP2. CONCLUSION: These data suggest that all phosphorylation sites of human AQP2 including T269 are phosphorylated and phosphorylations at S256 and S261 may play a dominant role in the urinary exosomal excretion of AQP2.

Impaired bile acid metabolism with defectives of mitochondrial-tRNA taurine modification and bile acid taurine conjugation in the taurine depleted cats.

Taurine that conjugates with bile acid (BA) and mitochondrial-tRNA (mt-tRNA) is a conditional essential amino acid in humans, similarly to cats. To better understand the influence of acquired depletion of taurine on BA metabolism, the profiling of BAs and its intermediates, BA metabolism-enzyme expression, and taurine modified mt-tRNAs were evaluated in the taurine deficient diet-supplemented cats. In the taurine depleted cats, taurine-conjugated bile acids in bile and taurine-modified mt-tRNA in liver were significantly decreased, whereas unconjugated BA in serum was markedly increased. Impaired bile acid metabolism in the liver was induced accompanied with the decreases of mitochondrial cholesterol 27-hydroxylase expression and mitochondrial activity. Consequently, total bile acid concentration in bile was significantly decreased by the low activity of mitochondrial bile acid synthesis. These results implied that the insufficient dietary taurine intake causes impaired bile acid metabolism, and in turn, a risk for the various diseases similar to the mitochondrial diseases would be enhanced.

Firing properties of medullary expiratory neurons during fictive straining in cats.

Expiratory (E) neurons in the caudal nucleus retroambigualis extend descending spinal axons to the lumbar and sacral spinal cord. Discharge rates of single E neurons were recorded to examine differences in activity of E neurons projecting to the lumbar or sacral spinal cord during fictive straining induced by distention of the colon with a balloon. Firing frequencies of E neurons with descending axons in the thoracic and lumbar spinal cord increased during the repetitive rise of rectum pressure, whereas those of E neurons with descending axons in the sacral spinal cord decreased. E neurons with descending axons in the thoracic/lumbar and sacral spinal cord exhibit different firing characteristics during the repetitive rise of rectum pressure when straining during defecation. The activity of abdominal nerves during fictive straining is in phase with changes in rectum pressure, but out of phase with the activity of E neurons.

Influences of Taurine Deficiency on Bile Acids of the Bile in the Cat Model.

Taurine content in the body is maintained by both biosynthesis from sulfur-contained amino acids in the liver and ingestion from usual foods, mainly seafoods and meat. Contrary to the rodents, the maintenance of taurine content in the body depends on the oral taurine ingestion in cats as well as humans because of the low ability of the biosynthesis. Therefore, insufficient of dietary taurine intake increases the risks of various diseases such as blind and expanded cardiomyopathy in the cats. One of the most established physiological roles of taurine is the conjugation with bile acid in the liver. In addition, taurine has effect to increase the expression and activity of bile acid synthesis rate-limiting enzyme CYP7A1. Present study purposed to evaluate the influence of taurine deficiency on bile acids in the cats fed taurine-lacking diet. Adult cats were fed the soybean protein-based diet with 0.15% taurine or without taurine for 30 weeks. Taurine concentration in serum and liver was undetectable, and bile acids in the bile were significantly decreased in the taurine-deficient cats. Taurine-conjugated bile acids in the bile were significantly decreased, and instead, unconjugated bile acids were significantly increased in the taurine-deficient cats. Present results suggested that the taurine may play an important role in the synthesis of bile acids in the liver.

Electrophysiological properties of Ia excitation and recurrent inhibition in cat abdominal motoneurons.

Ia excitation and recurrent inhibition are basic neuronal circuits in motor control in hind limb. Renshaw cells receive synaptic inputs from axon collaterals of motoneurons and inhibit motoneurons and Ia inhibitory interneurons. It is important to know properties of Ia excitation and recurrent inhibition of trunk muscle such as abdominal muscles. The abdominal muscles have many roles and change those roles for different kind of functions. Intracellular recordings were obtained from the abdominal motoneurons of the upper lumbar segments in cats anesthetized. First, dorsal roots were left intact, and sensory and motor axons were electrically stimulated. Ia excitatory post-synaptic potentials were elicited in five of eight motoneurons at same segment stimulated. Second, dorsal roots were sectioned, and motor axons were electrically stimulated. Recurrent inhibitory post-synaptic potentials were elicited in one of 11 abdominal motoneurons. Renshaw cells extracellularly fired high-frequency bursts at short latency and at same segment stimulated.

AKAPs-PKA disruptors increase AQP2 activity independently of vasopressin in a model of nephrogenic diabetes insipidus.

Congenital nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine. Congenital NDI is mainly caused by loss-of-function mutations in the vasopressin type 2 receptor (V2R), leading to impaired aquaporin-2 (AQP2) water channel activity. So far, treatment options of congenital NDI either by rescuing mutant V2R with chemical chaperones or by elevating cyclic adenosine monophosphate (cAMP) levels have failed to yield effective therapies. Here we show that inhibition of A-kinase anchoring proteins (AKAPs) binding to PKA increases PKA activity and activates AQP2 channels in cortical collecting duct cells. In vivo, the low molecular weight compound 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives increase AQP2 activity to the same extent as vasopressin, and increase urine osmolality in the context of V2R inhibition. We therefore suggest that FMP-API-1 may constitute a promising lead compound for the treatment of congenital NDI caused by V2R mutations.

AQP2 in human urine is predominantly localized to exosomes with preserved water channel activities.

BACKGROUND: AQP2 water channel is critical for urinary concentration in the kidney. Interestingly, AQP2 is abundantly excreted in the urine as extracellular vesicles (EVs), which is known to be a useful biomarker for water-balance disorders although the character of AQP2-enriched EVs is poorly understood including water channel function. METHODS: Human urine EVs were obtained by a differential centrifugation method. AQP2-bearing EVs were isolated by immunoprecipitation with an AQP2-specific antibody, and the proteins in the EVs were analyzed by LC-MS/MS proteomic analysis. Osmotic water permeability (Pf) of the AQP2-rich EVs was measured by a stopped-flow method monitoring scattered light intensity in response to outwardly directed osmotic gradient. RESULTS: Sequential centrifugation of human urine showed that AQP2 was present predominantly (80%) in low-density EVs (160,000 g), whereas negligible amount in high-density EVs (17,000 g). Proteomic analysis of the AQP2-bearing EVs identified 137 proteins, mostly in the endosome pathway, including the components of ESCRT (endosomal sorting complex required transporter)-I, II, III. Pf value of the 160,000 g EVs was 4.75 ± 0.38 × 10-4 cm s-1 (mean ± SE) with the activation energy of 3.51 kcal mol-1 which was inhibited with 0.3 mM HgCl2 by 63%, suggesting a channel-mediated water transport. Moreover, Pf value showed a significant correlation with the abundance of AQP2 protein in EVs. CONCLUSION: Taken together, AQP2 is localized predominantly to urinary exosomes with preserved water channel activities.

Prognosis of chronic kidney disease with normal-range proteinuria: The CKD-ROUTE study.

BACKGROUND: Although lower estimated glomerular filtration rate (eGFR) and higher proteinuria are high risks for mortality and kidney outcomes, the prognosis of chronic kidney disease (CKD) in patients with normal-range proteinuria remains unclear. METHODS: In this prospective cohort study, 1138 newly visiting stage G2-G5 CKD patients were stratified into normal-range and abnormal-range proteinuria groups. Study endpoints were CKD progression (>50% eGFR loss or initiation of dialysis), cardiovascular events, and all-cause death. RESULTS: In total, 927 patients who were followed for >6 months were included in the analysis. The mean age was 67 years, and 70.2% were male. During a median follow-up of 35 months, CKD progression, cardiovascular events, and mortality were observed in 223, 110, and 55 patients, respectively. Patients with normal-range proteinuria had a significantly lower risk for CKD progression (hazard ratio, 0.20; 95% confidence interval, 0.10-0.38) than those with abnormal-proteinuria by multivariate Cox proportional hazard analysis. We also analyzed patients with normal-range proteinuria (n = 351). Nephrosclerosis was the most frequent cause of CKD among all patients with normal-range proteinuria (59.7%). During a median follow-up of 36 months, CKD progression, cardiovascular events, and mortality were observed in 10, 28, and 18 patients, respectively. The Kaplan-Meyer analysis demonstrated that the risks of CKD progression and cardiovascular events were not significantly different among CKD stages, whereas the risk of death was significantly higher in patients with advanced-stage CKD. Multivariate Cox proportional hazard analysis showed that the risk of three endpoints did not significantly differ among CKD stages. CONCLUSION: Newly visiting CKD patients with normal-range proteinuria, who tend to be overlooked during health checkups did not exhibit a decrease in kidney function even in advanced CKD stages under specialized nephrology care.

Low white blood cell count is independently associated with chronic kidney disease progression in the elderly: the CKD-ROUTE study.

BACKGROUND: Elevated white blood cell (WBC) count is a well-known predictor of chronic kidney disease (CKD) progression. However, elderly patients commonly fail to develop a high WBC count in response to several diseased states and may instead present a low WBC count. Therefore, we hypothesized that low WBC count, in addition to high WBC count, is associated with CKD progression in the elderly. METHODS: We conducted a prospective cohort study using 3-year follow-up data from the CKD Research of Outcomes in Treatment and Epidemiology study. In the present study, participants aged over 60 years with pre-dialysis CKD stages G2-G5 were eligible. Patients were stratified into three groups according to WBC count using tertiles (T). The primary outcome was a composite of end-stage renal disease and a 50% reduction in estimated glomerular filtration rate. Data were analyzed using Cox proportional hazard models with adjustments for covariates. RESULTS: We enrolled 697 patients (males, 69%). The median WBC count was 6100 cells/µl (T1, <5400, n =  222; T2, 5400-6900, n =  235; T3, ≥6900, n = 240). During a median follow-up of 868 days, the primary outcome was observed in 170 patients, whereas 54 patients died. T1 and T3 had significantly higher hazard ratios (HR) than T2 (T1, HR 1.69, 95% confidence interval 1.14-2.51; T3, HR 1.63, 95% confidence interval 1.10-2.41). Moreover, T1 had a significantly higher adjusted HR (1.54, 95% confidence interval 1.00-2.37). CONCLUSION: Low WBC count is independently associated with CKD progression in the elderly.

Infective endocarditis in a patient with lupus nephritis who was undergoing immunosuppressive therapy: A case of survival.

Systemic lupus erythematosus is an autoimmune disease associated with mild valvular regurgitation. However, there have been no detailed reports of infective endocarditis in patients with systemic lupus erythematosus. Here, we describe a case of a 55-year-old woman without any cardiac abnormalities who was diagnosed with lupus nephritis by renal biopsy; she contracted infective endocarditis while receiving immunosuppressive therapy. Our case emphasizes that special consideration of the occurrence of infective endocarditis, and its early diagnosis and treatment are mandatory for patient survival. We propose that echocardiography should be performed before treating patients with systemic lupus erythematosus who have an uncertain cardiac status.

Ser-261 phospho-regulation is involved in pS256 and pS269-mediated aquaporin-2 apical translocation.

Vasopressin catalyzes aquaporin-2 phosphorylation at several serine sites in the C-terminal region. Compared with Ser-256 and Ser-269 phosphorylation, the role of Ser-261 phospho-regulation on vasopressin-regulated AQP2 apical translocation is largely unknown. In addition, recent discovery of transcytotic apical delivery of AQP2 made the concept of its intracellular trafficking even more complicated. In this study, we evaluated how intact phospho-AQP2 signals fit with the transcytosis trafficking model in Madin-Darby canine kidney cells. PS256 and pS269 signals were intracellularly detectable in wild-type AQP2 at the beginning of forskolin stimulation (1 min). These phospho-signals were detectable in basolateral membranes even after 10 min of stimulation. AQP2 stably inserted in the apical membrane increased pS269 and decreased pS261 signals. In an NDI-causing mutant P262L-AQP2, in which Ser-261 phospho-regulation is impaired, the pS256 and pS269 signals were detectable in the basolateral membranes with increased pS261 signals after forskolin stimulation. These results suggest that Ser-261 phospho-regulation is involved in pS256- and pS269-mediated AQP2 apical translocation.