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.

Knockout of P2rx7 purinergic receptor attenuates cyst growth in a rat model of ARPKD.

The severity of polycystic kidney diseases (PKD) depends on the counterbalancing of genetic predisposition and environmental factors exerting permissive or protective influence on cyst development. One poorly characterized phenomenon in the cystic epithelium is abnormal purinergic signaling. Earlier experimental studies revealed the high importance of the ionotropic P2X receptors (particularly, P2X7) in the pathophysiology of the cyst wall. To study mechanisms of P2X7 involvement in cyst growth and aspects of targeting these receptors in PKD treatment we performed a CRISPR/SpCas9-mediated global knockout of the P2rx7 gene in PCK rats, a model of autosomal recessive PKD (ARPKD). A single base insertion in exon 2 of the P2rx7 gene in the renal tissues of homozygous mutant animals leads to lack of P2X7 protein that did not affect their viability or renal excretory function. However, PCK.P2rx7 rats demonstrated slower cyst growth (but not formation of new cysts) compared with heterozygous and PCK.P2rx7+ littermates. P2X7 receptors are known to activate pannexin-1, a plasma channel capable of releasing ATP, and we found here that pannexin-1 expression in the cystic epithelium is significantly higher than in nondilated tubules. P2X7 deficiency reduces renal pannexin-1 protein expression and daily urinary ATP excretion. Patch-clamp analysis revealed that lack of P2X7 increases epithelial sodium channel activity in renal tissues and restores impaired channel activity in cysts. Interpretation of our current data in the context of earlier studies strongly suggests that P2X7 contributes to cyst growth by increasing pannexin-1-dependent pathogenic ATP release into the lumen and reduction of sodium reabsorption across the cyst walls.

Dissociation of Hypertension and Renal Damage After Cessation of High-Salt Diet in Dahl Rats.

BACKGROUND: Every year, thousands of patients with hypertension reduce salt consumption in an effort to control their blood pressure. However, hypertension has a self-sustaining character in a significant part of the population. We hypothesized that chronic hypertension leads to irreversible renal damage that remains after removing the trigger, causing an elevation of the initial blood pressure. METHODS: Dahl salt-sensitive rat model was used for chronic, continuous observation of blood pressure. Rats were fed a high salt diet to induce hypertension, and then the diet was switched back to normal sodium content. RESULTS: We found that developed hypertension was irreversible by salt cessation: after a short period of reduction, blood pressure grew even higher than in the high-salt phase. Notably, the self-sustaining phase of hypertension was sensitive to benzamil treatment due to sustaining epithelial sodium channel hyperactivity, as shown with patch-clamp analysis. Glomerular damage and proteinuria were also irreversible. In contrast, some mechanisms, contributing to the development of salt-sensitive hypertension, normalized after salt restriction. Thus, flow cytometry demonstrated that dietary salt reduction in hypertensive animals decreased the number of total CD45+, CD3+CD4+, and CD3+CD8+ cells in renal tissues. Also, we found tubular recovery and improvement of glomerular filtration rate in the postsalt period versus a high-salt diet. CONCLUSIONS: Based on earlier publications and current data, poor response to salt restriction is due to the differential contribution of the factors recognized in the developmental phase of hypertension. We suggest that proteinuria or electrolyte transport can be prioritized over therapeutic targets of inflammatory response.

Probenecid slows disease progression in a murine model of autosomal dominant polycystic kidney disease.

Development of autosomal dominant polycystic kidney disease (ADPKD) involves renal epithelial cell abnormalities. Cystic fluid contains a high level of ATP that, among other effects, leads to a reduced reabsorption of electrolytes in cyst-lining cells, and thus results in cystic fluid accumulation. Earlier, we demonstrated that Pkd1RC/RC mice, a hypomorphic model of ADPKD, exhibit increased expression of pannexin-1, a membrane channel capable of ATP release. In the current study, we found that human ADPKD cystic epithelia have higher pannexin-1 abundance than normal collecting ducts. We hypothesized that inhibition of pannexin-1 function with probenecid can be used to attenuate ADPKD development. Renal function in male and female Pkd1RC/RC and control mice was monitored between 9 and 20 months of age. To test the therapeutic effects of probenecid (a uricosuric agent and a pannexin-1 blocker), osmotic minipumps were implanted in male and female Pkd1RC/RC mice, and probenecid or vehicle was administered for 42 days until 1 year of age. Probenecid treatment improved glomerular filtration rates and slowed renal cyst formation in male mice (as shown in histopathology). The mechanistic effects of probenecid on sodium reabsorption and fluid transport were tested on polarized mpkCCDcl4 cells subjected to short-circuit current measurements, and in 3D cysts grown in Matrigel. In the mpkCCDcl4 epithelial cell line, probenecid elicited higher ENaC currents and attenuated in vitro cyst formation, indicating lower sodium and less fluid retention in the cysts. Our studies open new avenues of research into targeting pannexin-1 in ADPKD pathology.

Renal baroreceptor-stimulated renin in the eNOS knockout mouse.

The role of endothelium-derived nitric oxide (NO) in renal baroreceptor stimulation of renin was tested comparing endothelial nitric oxide synthase (eNOS)-deficient mice with C57BL/6J (C57) controls. We measured blood pressure, renal blood flow (RBF), and plasma renin concentration (PRC) in Inactin-anesthetized mice. Blood pressure in eNOS knockout mice was higher than in controls (100 +/- 3 vs. 86 +/- 1 mmHg, respectively; P < 0.001), but RBF was similar (1.71 +/- 0.06 vs. 1.66 +/- 0.09 ml. min(-1). 100 mg kidney wt(-1), respectively), so that renal vascular resistance was also higher in the knockouts (59.81 +/- 2.07 vs. 51.81 +/- 2.66 resistance units, respectively; P < 0.025). PRC was similar (8.24 +/- 1.57 in eNOS knockouts vs. 7.10 +/- 1.19 ng ANG I. ml(-1). h(-1) in C57). NOS inhibition with nitro-L-arginine methyl ester (L-NAME) in C57 controls increased blood pressure (from 85 +/- 2 to 106 +/- 1 mmHg, P < 0.001) and decreased RBF (from 1.66 +/- 0.09 to 1.08 +/- 0.02; P < 0.005), but L-NAME had no effect in eNOS knockout mice. When renal perfusion pressure was reduced in C57 controls to 55 mmHg, PRC increased from 6.6 +/- 0.9 to 14.5 +/- 1.9 microg. ml(-1). h(-1) (P < 0.025), but this response was blocked by L-NAME. However, in eNOS knockouts, reduced renal perfusion pressure increased PRC from 7.6 +/- 1.4 to 15.0 +/- 2.8 microg. ml(-1). h(-1) (P < 0.001). Thus in the chronic absence of eNOS, blood pressure was elevated, but RBF was normal. Additionally, the absence of eNOS did not modify baroreceptor-stimulated renin, suggesting that eNOS-derived NO does not directly mediate this renin-regulating pathway.

Cardiovascular and renal phenotype in mice with one or two renin genes.

We compared the phenotype of two common mouse models, C-57BL/6J (C57), which carries only the Ren-1c gene, and 129/SvJ (Sv-129), with both Ren 1d and Ren-2. We hypothesized two renin gene Sv-129 would have increased blood pressure and the renin-angiotensin system would be more influential in regulating renal function compared with one renin gene mice. Sv-129 consistently had higher blood pressure than C-57, whether conscious (128 versus 108 mm Hg, P<0.001) or anesthetized (102 versus 88 mm Hg, P<0.001). Plasma renin concentration in both conscious and anesthetized C-57 mice was 3- to 4-fold higher than in Sv-129 (P<0.05), whereas renal cortical renin content was 2.5-fold higher (P<0.005). Renal blood flow and renal vascular resistance were the same in C-57 and Sv-129. Exogenous angiotensinogen produced identical pressor and renal vasoconstrictor responses in both strains. Blocking AT1 receptors with losartan reduced blood pressure by 19 mm Hg in both strains. Nitric oxide synthase inhibition by l-NAME increased blood pressure by 29 mm Hg in C-57 and 35 mm Hg in Sv-129 mice, but the decrease in renal blood flow was 30% less in C-57 (P<0.025). We conclude that Sv-129 mice with two renin genes have higher blood pressure but lower plasma and renal renin than C-57 mice with one renin gene. Renin substrate may limit angiotensin II production in the mouse. In Sv-129, the influence of nitric oxide on renal but not systemic resistance may be exaggerated. Renin from Ren-2 may act independently of normal renin control mechanisms.

Role of nitric oxide in the renal hemodynamic response to unilateral nephrectomy.

Reduction of renal mass by unilateral nephrectomy results in an immediate increase in renal blood flow (RBF) to the remnant kidney, followed by compensatory renal hypertrophy. Whether the increase in RBF after unilateral nephrectomy is mediated by nitric oxide (NO) was tested. It was found that immediately after nephrectomy, blood flow to the remaining kidney increased by 8% (P < 0.01), and inhibition of NO synthesis with Nomega-nitro-L-arginine methyl ester (L-NAME) blocked the increase in RBF. In addition, 2 d after nephrectomy, there was a 49% increase in RBF (corrected per gram of kidney weight), a 25% increase at 7 and 14 d, and a 16% increase after 28 d. Acute inhibition of NO synthesis with L-NAME in uninephrectomized rats caused a greater decrease in RBF on days 2 and 7 compared with controls, whereas by 14 and 28 d, the response to L-NAME was similar to controls. Urinary excretion of cyclic guanosine monophosphate, a marker for renal NO production, increased 2.5-fold by 2 d after uninephrectomy (P < 0.005) and remained at this level through 28 d. Pretreating rats chronically with a subpressor dose of L-NAME beginning 2 d before nephrectomy blocked the increase in RBF seen at 2 and 7 d and retarded the renal hypertrophy that should have developed by 7 d. It is concluded that after unilateral nephrectomy, immediate and sustained increases in RBF are mediated at least in part by NO. The hypertrophic response to unilateral nephrectomy may be partially initiated by the signal of hemodynamic changes.