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Aim: The intraglomerular mesangial cells are located between the glomerular capillaries. Here we hypothesized that mesangial cells regulate the single nephron glomerular filtration rate (snGFR) and that mesangial cells support the integrity of the glomerular filtration barrier. Methods: We assessed the function of mesangial cells in vivo by multiphoton microscopy. Mesangial cells were depleted in Munich Wistar Froemter rats using the Thy1.1 antibody model. Results: The Thy1.1 antibody caused the cell‐specific loss of 82 ± 3% of mesangial cells. After mesangial cell depletion, the baseline snGFR was reduced to 12.0 ± 1.2 vs 32.4 ± 3.2 nL/min in controls. In control rats, the snGFR decreased after angiotensin II infusion by 61 ± 3% (P =.004), whereas it remained unchanged in Thy1.1‐treated rats. The changes in the snGFR after angiotensin II infusion in control rats were accompanied by the marked rotation of the capillary loops within Bowman's space. This phenomenon was absent in anti‐Thy1.1‐treated rats. The glomerular sieving coefficient (GSCA) for albumin, used as a measure of the integrity of the glomerular filtration barrier, was low in control rats (0.00061 ± 0.00004) and increased after angiotensin II infusion (0.00121 ± 0.00015). In Thy1.1‐treated rats, the GSC was elevated (0.0032 ± 0.00059) and did not change in response to angiotensin II. Electron microscopy revealed the increased thickness of the glomerular basement membrane after mesangial cell depletion. Conclusion: Our data suggest that mesangial cells actively contribute to the regulation of the snGFR. Furthermore, mesangial cells are crucially involved in maintaining the integrity of the glomerular filtration barrier, in part by modulating the thickness of the glomerular basement membrane. [ABSTRACT FROM AUTHOR]

Adenosine coordinates organ metabolism and blood supply, and it modulates immune responses. In the kidney it mediates the vascular response elicited by changes in NaCl concentration in the macula densa region of the nephron, thereby serving as an important regulator of GFR. To determine whether adenosine formation depends on extracellular nucleotide hydrolysis, we studied NaCl-dependent GFR regulation (tubuloglomerular feedback) in mice with targeted deletion of ecto-5'-nucleotidase/CD73 (e-5'NT/CD73), the enzyme responsible for adenosine formation from AMP. e-5' NT/CD73-/- mice were viable and showed no gross anatomical abnormalities. Blood pressure, blood and urine chemistry, and renal blood flow were not different between e-5' NT/CD73-/- and e-5' NT/CD73-/- mice. e-5' NT/CD73-/- mice had a significantly reduced fall in stop flow pressure and superficial nephron glomerular filtration rate in response to a saturating increase of tubular perfusion flow. Furthermore, whereas tubuloglomerular feedback responses did not change significantly during prolonged loop of Henle perfusion in e-5' NT/CD73+/+ mice, a complete disappearance of the residual feedback response was noted in e-5' NT/CD73-/- mice over 10 minutes of perfusion. The contractile response of isolated afferent arterioles to adenosine was normal in e-5' NT/CD73-/- mice. We conclude that the generation of adenosine at the glomerular pole depends to a major extent on e-5' NT/CD73-mediated dephosphorylation of 5'-AMP, presumably generated from released ATP. [ABSTRACT FROM AUTHOR]

In the past few years the pivotal role of kidney Cl-channels (ClC-K) channels in maintaining salt and water homeostasis in the kidney has been established. The aim of the present study was to investigate the influence of the loop diuretic furosemide on the gene expression of the kidney chloride channel ClC-K1 and its recently described functional subunit barttin. Male Sprague Dawley rats received the loop diuretic furosemide (12 mg/kg/day) for 6 days. Rats had free access to 0.9% NaCl, 0.1%KCl solution to prevent volume depletion. Localisation and regulation of ClC-K1 and barttin mRNA was analysed by RNase protection and in situ hybridisation. Nephron-specific regulation was investigated by microdissection and real-time PCR quantification. In furosemide-treated rats ClC-K1 mRNA decreased to half in the inner medulla. In the renal cortex and outer medulla ClC-K1 mRNA levels were weak and did not change. Under furosemide treatment barttin mRNA was regulated in parallel with ClC-K1 mRNA. A significant mRNA decrease occurred after furosemide treatment in inner medulla (0.50 fold), whereas cortical and outer medulla levels remained unaffected. 35S in situ hybridisation confirmed the regulation and distribution seen in the RNase protection assay experiments. Microdissection of the inner medullary collecting duct and thin limb of Henle's loop followed by real-time PCR revealed that CLC-K1 and barttin mRNA regulation in inner medulla was limited to the thin limb; mRNA levels in collecting ducts were not affected by furosemide treatment. Our findings imply that during furosemide treatment selective down-regulation of ClC-K1 and barttin mRNAs in thin limb plays a role in maintaining salt and water homeostasis. [ABSTRACT FROM AUTHOR]

The aim of the present study was to determine the molecular responses of the main salt-reabsorbing systems in the distal nephron to changes of salt load of the organism. For this purpose we analysed messenger ribonucleic acid (mRNA) levels for the bumetanide-sensitive Na+K+2Cl– cotransporter (BSC1), the thiazide-sensitive Na+Cl– cotransporter (TSC), the kidney-specific inwards rectifier K+ channel (ROMK), the amiloride-sensitive epithelial Na+ channel (ENaC) and the kidney-specific Cl– channel ClC-K2, in the cortex and inner and outer medulla of kidneys from male Sprague-Dawley rats fed a high- (8% w/w), normal- (0.6%) or low- (0.02%) salt diet or treated chronically with subcutaneous infusions of furosemide (12 mg/kg per day). BSC1 and ROMK mRNA levels did not differ between the four treatment groups. TSC mRNA increased during furosemide treatment 1.75-fold versus control but was not affected by a high- or a low-salt diet. The mRNA for the α-subunit of ENaC increased with the low-salt diet (about 1.5-fold) and with furosemide (about 2.1-fold) in all kidney zones, but did not change with the high-salt diet. Dietary salt loading down-regulated ClC-K2 mRNA in the outer medulla 0.6-fold versus control whilst furosemide treatment, but not the low-salt diet, increased ClC-K2 mRNA in the outer (1.6-fold) and inner medulla (2.0-fold). These findings suggest that gene expression of Na+ and Cl– entry pathways in the distal nephron are at least partly regulated by the salt load of the organism, such that salt-reabsorbing systems are stimulated by salt deficiency and suppressed by salt overload. [ABSTRACT FROM AUTHOR]

ROP Os+/- mice, generated by a radiation-induced 10 Mb DNA inversion of chromosome 8 and resulting in an oligosyndactyly (Os) allele, have a 50% reduction in nephron number and develop glomerulosclerosis without altered survival. In contrast, Os+/- mice on an fvb background (fvb.ROP Os/+) die around 3 months of age of rapidly progressing renal failure. Here we assessed renal function in 4-5 wk old Os/+ and wild type (WT) mice. Kidneys of Os/+ were smaller than WT (135 ± 8 mg vs. 295 ± 20 mg; p<.001) whereas body weight did not differ. GFR determined by FITC-inulin clearance in conscious mice was reduced in Os/+ vs. WT (80 ± 10 vs. 258 ± 21 µl/min, p<.001). Glomerular counts per kidney averaged 1461 ± 163 in Os/+ and 10021 ± 480 in WT (p<.001). Calculated SNGFR was 26 ± 4.6 in Os/+ and 12.6 ± 1.4 nl/min in WT (p=.006), and this correlated with increased stop flow (49.8 ± 1.7 vs. 39.6 ± 1.5 mm Hg; p<0.001) and free flow pressures (17.2 ± 1.1 vs.14.5 ± 0.7 mm Hg, p<0.05). Proximal and distal tubular flow rates were elevated in Os/+ mice while distal Cl concentration was normal. BUN was elevated in Os/+ mice compared to WT (93 ± 8.8 vs. 29 ± 3.5 mg/dl; p<0.01). Ambient urinary osmolarity averaged 748 ± 29 mOsm in Os/+ and 1179 ± 118 mOsm in WT (p=.015). Plasma renin was reduced in Os/+ vs. WT (318 ± 59 vs. 807 ± 86 ng Ang I/ml/hr; p=.001) as was renin mRNA (21 ± 3% of WT normalized by 18s RNA; p<.001). In summary, Os/+ mice have an 85% reduction of nephron number, and this is associated with glomerular hypertension, hyperfiltration and hypertrophy of existing nephrons, likely causes of the progressive deterioration of renal function. [ABSTRACT FROM AUTHOR]

Abstract: Little information is available concerning the generation of renal tubules, but this information is urgently needed in regenerative medicine for the future treatment of acute and chronic renal failures. Of major interests are the integration of stem/progenitor cells, the cellular development and the tubular growth in a spatial environment. In this regard, we investigated the basal aspect of renal tubules generated at the interphase of an artificial interstitium. Stem/progenitor cells derived from neonatal rabbit kidney were mounted inside a specific tissue holder and covered by layers of polyester fleece. The tissue was then kept in a perfusion culture container for 13 days in chemically defined IMDM containing aldosterone (1×10−7 m) as a tubulogenic factor. The spatial development of tubules was registered on whole-mount specimens and on cryo-sections labeled with soybean agglutinin (SBA) and tissue-specific antibodies indicating that collecting duct tubules were developed. Scanning electron microscopy (SEM) revealed that the generated tubules were completely covered by a basal lamina. Most interestingly, the matrix was not consistently composed, but exhibited three categories of pores. The most frequently found pore type had an apparent diameter of 133±26nm followed by a medium-sized pore type of 317±35nm. Another category of pores with a diameter of 605±101nm was rather rarely found. All of the pores were evenly distributed and not restricted to particular sites. The newly detected pores are not related to culture artifacts, since they were also detected in collecting duct tubules of the neonatal rabbit kidney. It remains to be evaluated whether these pores support physiological transport functions or if they indicate the site where extracellular matrix proteins are inserted into newly synthesized basal lamina. [Copyright &y& Elsevier]