Role of Alström syndrome 1 in the regulation of glomerular hemodynamics.

Inactivating mutations in the ALMS1 gene in humans cause Alström syndrome, characterized by the early onset of obesity, insulin resistance, and renal dysfunction. However, the role of ALMS1 in renal function and hemodynamics is unclear. We previously found that ALMS1 is expressed in thick ascending limbs, where it binds and decreases Na+-K+-2Cl- cotransporter activity. We hypothesized that ALMS1 is expressed in macula densa cells and that its deletion enhances tubuloglomerular feedback (TGF) and reduces glomerular filtration rate (GFR) in rats. To test this, homozygous ALMS1 knockout (KO) and littermate wild-type Dahl salt-sensitive rats were studied. TGF sensitivity was higher in ALMS1 KO rats as measured by in vivo renal micropuncture. Using confocal microscopy, we confirmed immunolabeling of ALMS1 in macula densa cells (nitric oxide synthase 1 positive), supporting a role for ALMS1 in TGF regulation. Baseline glomerular capillary pressure was higher in ALMS1 KO rats, as was mean arterial pressure. Renal interstitial hydrostatic pressure was lower in ALMS1 KO rats, which is linked to increased Na+ reabsorption and hypertension. GFR was reduced in ALMS1 KO rats. Seven-week-old ALMS1 KO rats were not proteinuric, but proteinuria was present in 18- to 22-wk-old ALMS1 KO rats. The glomerulosclerosis index was higher in 18-wk-old ALMS1 KO rats. In conclusion, ALMS1 is involved in the control of glomerular hemodynamics in part by enhancing TGF sensitivity, and this may contribute to decreased GFR. Increased TGF sensitivity, enhanced glomerular capillary pressure, and hypertension may lead to glomerular damage in ALMS1 KO rats. These are the first data supporting the role of ALMS1 in TGF and glomerular hemodynamics.NEW & NOTEWORTHY ALMS1 is a novel protein involved in regulating tubuloglomerular feedback (TGF) sensitivity, glomerular capillary pressure, and blood pressure, and its dysfunction may reduce renal function and cause glomerular damage.

Decreased tubuloglomerular feedback response in high-fat diet-induced obesity.

Obesity increases the risk of renal damage, but the mechanisms are not clear. Normally, kidneys autoregulate to keep the glomerular capillary pressure (PGC), renal blood flow, and glomerular filtration rate in a steady state. However, in obesity, higher PGC, renal blood flow, and glomerular filtration rate are noted. Together, these may lead to glomerular damage. PGC is controlled mainly by afferent arteriole resistance, which, in turn, is regulated by tubuloglomerular feedback (TGF), a vasoconstrictor mechanism. High fat-induced obesity causes renal damage, and this may be related to increased PGC. However, there are no studies as to whether high-fat diet (HFD)-induced obesity affects TGF. We hypothesized that TGF would be attenuated in obesity caused by HFD feeding (60% fat) in Sprague-Dawley rats. Sprague-Dawley rats fed a normal-fat diet (NFD; 12% fat) served as the control. We studied 4 and 16 wk of HFD feeding using in vivo renal micropuncture of individual rat nephrons. We did not observe significant differences in body weight, TGF response, and mean arterial pressure at 4 wk of HFD feeding, but after 16 wk of HFD, rats were heavier and hypertensive. The maximal TGF response was smaller in HFD-fed rats than in NFD-fed rats, indicating an attenuation of TGF in HFD-induced obesity. Baseline PGC was higher in HFD-fed rats than in NFD-fed rats and was associated with higher glomerulosclerosis. We conclude that attenuated TGF and higher PGC along with hypertension in HFD-fed obese Sprague-Dawley rats could explain the higher propensity of glomerular damage observed in obesity.NEW & NOTEWORTHY Reduced tubuloglomerular feedback, higher glomerular capillary pressure, and hypertension in combination may explain the higher glomerular damage observed in high-fat diet-induced obesity.

Role of connecting tubule glomerular feedback in obesity related renal damage.

Zucker obese rats (ZOR) have higher glomerular capillary pressure (PGC) that can cause renal damage. PGC is controlled by afferent (Af-Art) and efferent arteriole (Ef-Art) resistance. Af-Art resistance is regulated by factors that regulate other arterioles, such as myogenic response. In addition, it is also regulated by 2 intrinsic feedback mechanisms: 1) tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to increased NaCl in the macula densa and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation in response to an increase in NaCl transport in the connecting tubule via the epithelial sodium channel. Since CTGF is an Af-Art dilatory mechanism, we hypothesized that increased CTGF contributes to TGF attenuation, which in turn increases PGC in ZOR. We performed a renal micropuncture experiment and measured stop-flow pressure (PSF), which is an indirect measurement of PGC in ZOR. Maximal TGF response at 40 nl/min was attenuated in ZOR (4.47 ± 0.60 mmHg) in comparison to the Zucker lean rats (ZLR; 8.54 ± 0.73 mmHg, P < 0.05), and CTGF was elevated in ZOR (5.34 ± 0.87 mmHg) compared with ZLR (1.12 ± 1.28 mmHg, P < 0.05). CTGF inhibition with epithelial sodium channel blocker normalized the maximum PSF change in ZOR indicating that CTGF plays a significant role in TGF attenuation (ZOR, 10.67 ± 1.07 mmHg vs. ZLR, 9.5 ± 1.53 mmHg). We conclude that enhanced CTGF contributes to TGF attenuation in ZOR and potentially contribute to progressive renal damage.

Renal Protective Effects of N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) in Obese Rats on a High-Salt Diet.

BACKGROUND: Obesity is a public health problem, associated with salt sensitive hypertension, kidney inflammation, and fibrosis. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a tetra peptide with anti-inflammatory and anti-fibrotic properties. However, its effect on preventing kidney damage in obesity is unknown. We hypothesized that Zucker obese (ZO) rats on a high-salt (HS) diet develop renal damage, inflammation, fibrosis, and this is prevented with Ac-SDKP treatment. METHODS: Zucker lean (ZL) and ZO rats (8 weeks old) were treated with Ac-SDKP (1.6 mg/kg/day) while maintained on either a normal-salt (NS; 0.4%) or HS (4%) diet for 8 weeks. Systolic blood pressure (SBP), albuminuria, renal inflammation, and fibrosis were evaluated. RESULTS: HS diet increased macrophage infiltration in the kidneys of both ZL and ZO rats but was significantly higher in ZO rats receiving the HS diet (ZL + NS, 13.9 ± 1.3 vs. ZL + HS, 19.14 ± 1.5 and ZO + NS, 25.5 ± 1.4 vs. ZO + HS, 87.8 ± 10.8 cells/mm2; P < 0.05). Ac-SDKP prevented macrophage infiltration in ZO rats (ZO + HS + Ac-SDKP, 32.18 ± 2.4 cells/mm2; P < 0.05). Similarly, glomerulosclerosis, cortical, and medullary interstitial fibrosis were increased in ZO rats fed the HS diet, and Ac-SDKP attenuated these alterations (P < 0.05). SBP was increased in ZO rats fed the HS diet (ZO + NS, 121.3 ± 8.9 vs. ZO + HS, 164 ± 6.9 mm Hg; P < 0.05), and it was significantly decreased with Ac-SDKP treatment (ZO + HS + Ac-SDKP, 144.05 ± 14.1 mm Hg; P = 0.004). Albuminuria was higher in ZO rats than in ZL rats; however, neither HS nor Ac-SDKP treatment affected it. CONCLUSIONS: Ac-SDKP treatment in ZO rats fed a HS diet prevented renal damage by reducing inflammation, fibrosis, and SBP.

Connecting tubule glomerular feedback mediates tubuloglomerular feedback resetting after unilateral nephrectomy.

Unilaterally nephrectomized rats (UNx) have higher glomerular capillary pressure (PGC) that can cause significant glomerular injury in the remnant kidney. PGC is controlled by the ratio of afferent (Af-Art) and efferent arteriole resistance. Af-Art resistance in turn is regulated by two intrinsic feedback mechanisms: 1) tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to increased NaCl in the macula densa; and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation in response to an increase in NaCl transport in the connecting tubule via the epithelial sodium channel (ENaC). Resetting of TGF post-UNx can allow systemic pressure to be transmitted to the glomerulus and cause renal damage, but the mechanism behind this resetting is unclear. Since CTGF is an Af-Art dilatory mechanism, we hypothesized that CTGF is increased after UNx and contributes to TGF resetting. To test this hypothesis, we performed UNx in Sprague-Dawley (8) rats. Twenty-four hours after surgery, we performed micropuncture of individual nephrons and measured stop-flow pressure (PSF). PSF is an indirect measurement of PGC. Maximal TGF response at 40 nl/min was 8.9 ± 1.24 mmHg in sham-UNx rats and 1.39 ± 1.02 mmHg in UNx rats, indicating TGF resetting after UNx. When CTGF was inhibited with the ENaC blocker benzamil (1 µM/l), the TGF response was 12.29 ± 2.01 mmHg in UNx rats and 13.03 ± 1.25 mmHg in sham-UNx rats, indicating restoration of the TGF responses in UNx. We conclude that enhanced CTGF contributes to TGF resetting after UNx.

Effect of salt intake on afferent arteriolar dilatation: role of connecting tubule glomerular feedback (CTGF).

Afferent arteriole (Af-Art) resistance is modulated by two intrinsic nephron feedbacks: 1) the vasoconstrictor tubuloglomerular feedback (TGF) mediated by Na+-K+-2Cl- cotransporters (NKCC2) in the macula densa and blocked by furosemide and 2) the vasodilator connecting tubule glomerular feedback (CTGF), mediated by epithelial Na+ channels (ENaC) in the connecting tubule and blocked by benzamil. High salt intake reduces Af-Art vasoconstrictor ability in Dahl salt-sensitive rats (Dahl SS). Previously, we measured CTGF indirectly, by differences between TGF responses with and without CTGF inhibition. We recently developed a new method to measure CTGF more directly by simultaneously inhibiting NKCC2 and the Na+/H+ exchanger (NHE). We hypothesize that in vivo during simultaneous inhibition of NKCC2 and NHE, CTGF causes an Af-Art dilatation revealed by an increase in stop-flow pressure (PSF) in Dahl SS and that is enhanced with a high salt intake. In the presence of furosemide alone, increasing nephron perfusion did not change the PSF in either Dahl salt-resistant rats (Dahl SR) or Dahl SS. When furosemide and an NHE inhibitor, dimethylamiloride, were perfused simultaneously, an increase in tubular flow caused Af-Art dilatation that was demonstrated by an increase in PSF. This increase was greater in Dahl SS [4.5 ± 0.4 (SE) mmHg] than in Dahl SR (2.5 ± 0.3 mmHg; P < 0.01). We confirmed that CTGF causes this vasodilation, since benzamil completely blocked this effect. However, a high salt intake did not augment the Af-Art dilatation. We conclude that during simultaneous inhibition of NKCC2 and NHE in the nephron, CTGF induces Af-Art dilatation and a high salt intake failed to enhance this effect.

The anti-inflammatory peptide Ac-SDKP is released from thymosin-ß4 by renal meprin-α and prolyl oligopeptidase.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide with anti-inflammatory and antifibrotic properties. Previously, we have shown that prolyl oligopeptidase (POP) is involved in the Ac-SDKP release from thymosin-ß4 (Tß4). However, POP can only hydrolyze peptides shorter than 30 amino acids, and Tß4 is 43 amino acids long. This indicates that before POP hydrolysis takes place, Tß4 is hydrolyzed by another peptidase that releases NH2-terminal intermediate peptide(s) with fewer than 30 amino acids. Our peptidase database search pointed out meprin-α metalloprotease as a potential candidate. Therefore, we hypothesized that, prior to POP hydrolysis, Tß4 is hydrolyzed by meprin-α. In vitro, we found that the incubation of Tß4 with both meprin-α and POP released Ac-SDKP, whereas no Ac-SDKP was released when Tß4 was incubated with either meprin-α or POP alone. Incubation of Tß4 with rat kidney homogenates significantly released Ac-SDKP, which was blocked by the meprin-α inhibitor actinonin. In addition, kidneys from meprin-α knockout (KO) mice showed significantly lower basal Ac-SDKP amount, compared with wild-type mice. Kidney homogenates from meprin-α KO mice failed to release Ac-SDKP from Tß4. In vivo, we observed that rats treated with the ACE inhibitor captopril increased plasma concentrations of Ac-SDKP, which was inhibited by the coadministration of actinonin (vehicle, 3.1 ± 0.2 nmol/l; captopril, 15.1 ± 0.7 nmol/l; captopril + actinonin, 6.1 ± 0.3 nmol/l; P < 0.005). Similar results were obtained with urinary Ac-SDKP after actinonin treatment. We conclude that release of Ac-SDKP from Tß4 is mediated by successive hydrolysis involving meprin-α and POP.

Tubuloglomerular and connecting tubuloglomerular feedback during inhibition of various Na transporters in the nephron.

Afferent (Af-Art) and efferent arterioles resistance regulate glomerular capillary pressure. The nephron regulates Af-Art resistance via: 1) vasoconstrictor tubuloglomerular feedback (TGF), initiated in the macula densa via Na-K-2Cl cotransporters (NKCC2) and 2) vasodilator connecting tubuloglomerular feedback (CTGF), initiated in connecting tubules via epithelial Na channels (ENaC). Furosemide inhibits NKCC2 and TGF. Benzamil inhibits ENaC and CTGF. In vitro, CTGF dilates preconstricted Af-Arts. In vivo, benzamil decreases stop-flow pressure (PSF), suggesting that CTGF antagonizes TGF; however, even when TGF is blocked, CTGF does not increase PSF, suggesting there is another mechanism antagonizing CTGF. We hypothesize that in addition to NKCC2, activation of Na/H exchanger (NHE) antagonizes CTGF, and when both are blocked CTGF dilates Af-Arts and this effect is blocked by a CTGF inhibitor benzamil. Using micropuncture, we studied the effects of transport inhibitors on TGF responses by measuring PSF while increasing nephron perfusion from 0 to 40 nl/min. Control TGF response (-7.9 ± 0.2 mmHg) was blocked by furosemide (-0.4 ± 0.2 mmHg; P < 0.001). Benzamil restored TGF in the presence of furosemide (furosemide: -0.2 ± 0.1 vs. furosemide+benzamil: -4.3 ± 0.3 mmHg; P < 0.001). With furosemide and NHE inhibitor, dimethylamiloride (DMA), increase in tubular flow increased PSF (furosemide+DMA: 2.7 ± 0.5 mmHg, n = 6), and benzamil blocked this (furosemide+DMA+benzamil: -1.1 ± 0.2 mmHg; P < 0.01, n = 6). We conclude that NHE in the nephron decreases PSF (Af-Art constriction) when NKCC2 and ENaC are inhibited, suggesting that in the absence of NKCC2, NHE causes a TGF response and that CTGF dilates the Af-Art when TGF is blocked with NKCC2 and NHE inhibitors.

HO-1 induction improves the type-1 cardiorenal syndrome in mice with impaired angiotensin II-induced lymphocyte activation.

Type-1 cardiorenal syndrome, characterized by acute kidney dysfunction secondary to cardiac failure and renal arteriolar vasoconstriction, is mediated by the renin-angiotensin-aldosterone axis and sympathetic nervous system activation. Previous reports indicate that angiotensin II modulates immune function and causes recruitment and activation of T-lymphocytes. The goal of this study was to evaluate the effects of postischemic heart failure on renal morphology and circulation and the beneficial effects of heme oxygenase-1 (HO-1) induction in T-lymphocyte-suppressed severe combined immune deficiency (SCID) mice. Mice were divided into 4 groups: sham, myocardial infarction (MI), MI treated with an HO-1 inducer, cobalt protoporphyrin, and with or without stannous mesoporphyrin, an inhibitor of HO activity. Heart and kidney function were studied 30 days after surgery. Fractional area change was reduced 30 days after surgery in both the C57 and SCID MI-groups as compared with their respective controls (P<0.01). Renal Pulsatility Index and renal injury were increased in C57 and SCID MI-groups compared with the sham group. HO-1 induction improved renal vasoconstriction as well as ameliorated renal injury in both the SCID and C57 MI-groups (P<0.01). However, improvement was more evident in SCID mice. In addition, our results showed that plasma creatinine, angiotensin II, and renin were significantly increased in the C57 and SCID MI-groups as compared with their respective controls. HO-1 induction decreased these parameters in both MI groups. Stannous mesoporphyrin reversed the beneficial effect of cobalt protoporphyrin in both mouse strains. The study demonstrates that T-lymphocyte suppression facilitated the HO-1-dependent improvement in the attenuation of type-1 cardiorenal syndrome.

Antioxidants condition pleiotropic vascular responses to exogenous H(2)O(2): role of modulation of vascular TP receptors and the heme oxygenase system.

AIMS: Hydrogen peroxide (H(2)O(2)), a nonradical oxidant, is employed to ascertain the role of redox mechanisms in regulation of vascular tone. Where both dilation and constriction have been reported, we examined the hypothesis that the ability of H(2)O(2) to effect vasoconstriction or dilation is conditioned by redox mechanisms and may be modulated by antioxidants. RESULTS: Exogenous H(2)O(2) (0.1-10.0 µM), dose-dependently reduced the internal diameter of rat renal interlobular and 3rd-order mesenteric arteries (p<0.05). This response was obliterated in arteries pretreated with antioxidants, including tempol, pegylated superoxide dismutase (PEG-SOD), butylated hydroxytoluene (BHT), and biliverdin (BV). However, as opposed to tempol or PEG-SOD, BHT & BV, antioxidants targeting radicals downstream of H(2)O(2), also uncovered vasodilation. INNOVATIONS: Redox-dependent vasoconstriction to H(2)O(2) was blocked by inhibitors of cyclooxygenase (COX) (indomethacin-10 µM), thromboxane (TP) synthase (CGS13080-10 µM), and TP receptor antagonist (SQ29548-1 µM). However, H(2)O(2) did not increase vascular thromboxane B(2) release; instead, it sensitized the vasculature to a TP agonist, U46619, an effect reversed by PEG-SOD. Antioxidant-conditioned dilatory response to H(2)O(2) was accompanied by enhanced vascular heme oxygenase (HO)-dependent carbon monoxide generation and was abolished by HO inhibitors or by HO-1 & 2 antisense oligodeoxynucleotides treatment of SD rats. CONCLUSION: These results demonstrate that H(2)O(2) has antioxidant-modifiable pleiotropic vascular effects, where constriction and dilation are brought about in the same vascular segment. H(2)O(2)-induced oxidative stress increases vascular TP sensitivity and predisposes these arterial segments to constrictor prostanoids. Conversely, vasodilation is reliant upon HO-derived products whose synthesis is stimulated only in the presence of antioxidants targeting radicals downstream of H(2)O(2).

High fat diet enhances cardiac abnormalities in SHR rats: Protective role of heme oxygenase-adiponectin axis.

BACKGROUND: High dietary fat intake is a major risk factor for development of cardiovascular and metabolic dysfunction including obesity, cardiomyopathy and hypertension. METHODS: The present study was designed to examine effect of high fat (HF) diet on cardio-vascular structure and function in spontaneously hypertensive rats (SHR), fed HF diet for 15 weeks, a phenotype designed to mimic metabolic syndrome. RESULTS: Development of metabolic syndrome like phenotype was confirmed using parameters, including body weight, total cholesterol and blood pressure levels. High fat diet impaired vascular relaxation by acetylcholine and exacerbated cardiac dysfunction in SHRs as evidenced by lower left ventricular function, and higher coronary resistance (CR) as compared to controls (p < 0.05). The histological examination revealed significant myocardial and peri-vascular fibrosis in hearts from SHRs on HF diet. This cardiac dysfunction was associated with increased levels of inflammatory cytokines, COX-2, NOX-2, TxB2 expression and increase in superoxide (O2-) levels in SHR fed a HF diet (p < 0.05). HO-1 induction via cobalt-protoporphyrin (CoPP,3 mg/kg), in HF fed rats, not only improved cardiac performance parameters, but also prevented myocardial and perivascular fibrosis. These effects of CoPP were accompanied by enhanced levels of cardiac adiponectin levels, pAMPK, peNOS and iNOS expression; otherwise significantly attenuated (p < 0.05) in HF fed SHRs. Prevention of such beneficial effects of CoPP by the concurrent administration of the HO inhibitor stannic mesoporphyrin (SnMP) corroborates the role of HO system in mediating such effects. CONCLUSION: In conclusion, this novel study demonstrates that up-regulation of HO-1 improves cardiac and vascular dysfunction by blunting oxidative stress, COX-2 levels and increasing adiponectin levels in hypertensive rats on HF diet.