Regulation of renal NaDC1 expression and citrate excretion by NBCe1-A

Citrate is critical for acid-base homeostasis and to prevent calcium nephrolithiasis. Both metabolic acidosis and hypokalemia decrease citrate excretion and increase expression of Na+-dicarboxylate cotransporter 1 (NaDC1; SLC13A2), the primary protein involved in citrate reabsorption. However, the mechanisms transducing extracellular signals and mediating these responses are incompletely understood. The purpose of the present study was to determine the role of the Na+-coupled electrogenic bicarbonate cotransporter (NBCe1) A variant (NBCe1-A) in citrate metabolism under basal conditions and in response to acid loading and hypokalemia. NBCe1-A deletion increased citrate excretion and decreased NaDC1 expression in the proximal convoluted tubules (PCT) and proximal straight tubules (PST) in the medullary ray (PST-MR) but not in the PST in the outer medulla (PST-OM). Acid loading wild-type (WT) mice decreased citrate excretion. NaDC1 expression increased only in the PCT and PST-MR and not in the PST-MR. In NBCe1-A knockout (KO) mice, the acid loading change in citrate excretion was unaffected, changes in PCT NaDC1 expression were blocked, and there was an adaptive increase in PST-MR. Hypokalemia in WT mice decreased citrate excretion; NaDC1 expression increased only in the PCT and PST-MR. NBCe1-A KO blocked both the citrate and NaDC1 changes. We conclude that 1) adaptive changes in NaDC1 expression in response to metabolic acidosis and hypokalemia occur specifically in the PCT and PST-MR, i.e., in cortical proximal tubule segments; 2) NBCe1-A is necessary for normal basal, metabolic acidosis and hypokalemia-stimulated citrate metabolism and does so by regulating NaDC1 expression in cortical proximal tubule segments; and 3) adaptive increases in PST-OM NaDC1 expression occur in NBCe1-A KO mice in response to acid loading that do not occur in WT mice.

Seasonal and Morphological Variations of Brown Trout (Salmo truttaf.fario) Kidney Peroxisomes: A Stereological Study

Literature about fish kidney peroxisomes is scarce. To tackle this caveat, a stereological approach on renal peroxisome morphological parameters was performed for the first time in a fish, establishing correlations with maturation stages as it was previously done in brown trout liver. Three-year-old brown trout males and females were collected at the major seasons of their reproductive cycle. Trunk kidney was fixed and processed for catalase cytochemistry. Classical stereological methods were applied to electromicrographs to quantitate morphological parameters. Different seasonal variation patterns were observed between genders, and between renal proximal tubule segments I and II. In males, peroxisomes from proximal tubule segment II had a relatively higher volume and number in May, being individually bigger in February. Females presented similar trends, though with less marked variations. Overall, males and females did not show exactly the same seasonal patterns for most peroxisomal parameters, and no correlations were found between the latter and the gonado-somatic index (GSI). Hence, and despite the variations, the morphology of renal peroxisomes is not strictly correlated with gonad maturation kinetics, therefore suggesting that kidney peroxisome morphology is not seasonally modulated by sex steroids, like estradiol, as it seems to happen in liver peroxisomes.

Abstract 402: Specified Proximal Tubule Segments Augmented Angiotensinogen mRNA and Protein Expressions in Diabetic Kidneys in Rats and Humans

Others and we showed the increased levels of intrarenal angiotensinogen (AGT) in diabetic humans, mice, and rats. However, the precise localization of the augmented AGT mRNA and protein in proximal tubule segments of kidneys in diabetes remains to be established. To investigate the detailed localization of AGT in 3 proximal tubule segments of the kidneys in diabetes, the Otsuka Long-Evans Tokushima fatty (OLETF) rat (15 weeks) and the age-matched genetic control, the Long-Evans Tokushima Otsuka (LETO) rat were used. Kidney tissues were also prepared from OLETF rats that were administered with angiotensin II type 1 receptor blocker, olmesartan (0.02% in chow) or a combination of vasodilator agents (0.03% hydralazine, 0.006% reserpine, and 0.012% hydrochlorothiazide in chow; HRH). Moreover, biopsied samples of human kidney cortex were obtained by fine-needle aspiration to confirm the below-mentioned results of the rat localization study. Using 3-μm sections of zinc-saturated formalin-fixed paraffin-embedded slides, we examined the co-localization of AGT mRNA or protein with segment-specific protein markers (Segment (S)1-specific anti-sodium glucose cotransporter 2 antibody, S2-specific anti-carbonic anhydrase IV antibody, and S3-specific anti-ecto-adenosine triphosphatase antibody) by double staining using fluorescence in situ hybridization and/or immunofluorescence. AGT mRNA expression was not recognized much in S1 segments of either OLETF or LETO rat kidneys. In S3 segments, the AGT mRNA expression area was augmented in the OLETF rats than in the LETO rats (2.31 ± 0.02-fold; P = 0.0029). AGT protein expression areas in S1 and S3 segments were also increased (8.50 ± 2.78-fold; P = 0.0476, and 3.71 ± 0.83-fold; P = 0.0317, respectively). Chronic treatment with olmesartan, but not with HRH, ameliorated the augmented AGT mRNA and protein expression areas. AGT mRNA and protein expression areas in S2 segments were unchanged in OLETF rats compared with LETO rats. Biopsied samples of human kidney cortex showed the same results as for rat AGT mRNA and protein expression in the S1 and S2 segments. These data suggest that the augmented AGT mRNA in S3 and AGT protein in S1 and S3 play an important role in the development and the progression of diabetic nephropathy.

The transcription factor HNF1α regulates expression of chloride-proton exchanger ClC-5 in the renal proximal tubule

The Cl−/H+ exchanger ClC-5 is essential for the endocytic activity of the proximal tubule cells and the tubular clearance of proteins filtered in the glomeruli. The mechanisms that regulate the expression of ClC-5 in general and its specific expression in the proximal tubule are unknown. In this study, we investigated the hypothesis that the hepatocyte nuclear transcription factor HNF1α, which is predominantly expressed in proximal tubule segments, may directly regulate the expression of ClC-5. In situ hybridization demonstrated that the expression of Clcn5 overlaps with that of Hnf1α in the developing kidney as well as in absorptive epithelia, including the digestive tract and yolk sac. Multiple binding sites for HNF1 were mapped in the 5′-regulatory sequences of the mouse and human Clcn5/CLCN5 genes. The transactivation of the Clcn5/CLCN5 promoter by HNF1α was verified in vitro, and the binding of HNF1α to the Clcn5 promoter in vivo was confirmed by chromatin immunoprecipitation in mouse kidney. The expression of Clcn5 was reduced in the proximal tubule segments of HNF1α-null kidneys, and it was rescued upon transfection of HNF1α-null cells with wild-type but not with mutant HNF1α. These data demonstrate that HNF1α directly regulates the expression of ClC-5 in the renal proximal tubule and yield insights into the mechanisms governing epithelial differentiation and specialized transport activities in the kidney.

Role of angiotensin II in the enhancement of ammonia production and secretion by the proximal tubule in metabolic acidosis

Acidosis and angiotensin II stimulate ammonia production and transport by the proximal tubule. We examined the modulatory effect of the type 1 angiotensin II receptor blocker losartan on the ability of metabolic acidosis to stimulate ammonia production and secretion by mouse S2 proximal tubule segments. Mice given NH4Cl for 7 days developed metabolic acidosis (low serum bicarbonate concentration) and increased urinary excretion of ammonia. S2 tubule segments from acidotic mice displayed higher rates of ammonia production and secretion compared with those from control mice. However, when losartan was coadministered in vivo with NH4Cl, both the acidosis-induced increase in urinary ammonia excretion and the adaptive increase in ammonia production and secretion of microperfused S2 segments were largely blocked. In renal cortical tissue, losartan blocked the acid-induced increase in brush-border membrane NHE3 expression but had no effect on the acid-induced upregulation of phosphate-dependent glutaminase or phosphoenolpyruvate carboxykinase 1 in cortical homogenates. Addition of angiotensin II to the microperfusion solution enhanced ammonia secretion and production rates in tubules from NH4Cl-treated and control mice in a losartan-inhibitable manner. These results demonstrate that a 7-day acid challenge induces an adaptive increase in ammonia production and secretion by the proximal tubule and suggest that during metabolic acidosis, angiotensin II signaling is necessary for adaptive enhancements of ammonia excretion by the kidney and ammonia production and secretion by S2 proximal tubule segments, as mediated, in part, by angiotensin receptor-dependent enhancement of NHE3 expression.

Na+-d-glucose cotransporter in the kidney of Leucoraja erinacea: molecular identification and intrarenal distribution

Studies on membrane vesicles from the kidney of Leucoraja erinacea suggested the sole presence of a sodium-d-glucose cotransporter type 1 involved in renal d-glucose reabsorption. For molecular characterization of this transport system, an mRNA library was screened with primers directed against conserved regions of human sglt1. A cDNA was cloned whose nucleotide and derived amino acid sequence revealed high homology to sodium glucose cotransporter 1 (SGLT1). Xenopus laevis oocytes injected with the respective cRNA showed sodium-dependent high-affinity uptake of d-glucose. Many positions considered functionally essential for sodium glucose cotransporter 1 (SGLT1) are also found in the skate protein. High conservation preferentially in transmembrane helices and small linking loops suggests early appearance and continued preservation of these regions. Larger loops, especially loop 13, which is associated with phlorizin binding, were more variable, as is the interaction with the specific inhibitor in various species. To study the intrarenal distribution of the transporter, a skate SGLT1-specific antibody was generated. In cryosections of skate kidney, various nephron segments could be differentiated by lectin staining. Immunoreaction with the antibody was observed in the proximal tubule segments PIa and PIIa, the early distal tubule, and the collecting tubule. Thus Leucoraja, in contrast to the mammalian kidney, employs only SGLT1 to reabsorb d-glucose in the early, as well as in the late segments of the proximal tubule and probably also in the late distal tubule (LDT). Thereby, it differs also partly from the kidney of the close relative Squalus acanthias, which uses SGLT2 in more distal proximal tubule segments but shows also expression in the later nephron parts.