Role Of A1AR And Peritubular Capillary Loss In Hypertension Background

Hypertension, especially malignant hypertension (MHT), can cause kidney injury and bad prognosis. Peritubular capillary (PTC), whose perfusion comes from glomerular efferent arterioles, can determine the blood supply of renal tubulo-interstitial area. In essential MHT (EMHT), our group has observed severe PTC loss, which correlated with kidney function decline and renal tubulo-intertitial lesions. We also detected highly activation of renin-angiotensin-aldosterone system (RAAS). Whether these phenomena appear in benign nephrosclerosis (BHN) or other secondary MHT such as IgA nephropathy associated MHT (IgAN-MHT), remains unclear. A fine tone in hemodynamic regulation can be achieved by RAAS which mainly contracts efferent arterioles and adenosine which usually contracts afferent arterioles through A1adenosine receptor (A1AR) activation. On the other hand, inflammation such as renal macrophage (MΦ) infiltration itself can result in imbalance of angiogenic and anti-angiogenic factors, which may lead to PTC injury. A1AR was observed to have some anti-inflammatory effects in ischemic-reperfusion kidney injury mice. However, the anti-inflammatory role of A1AR in hypertensive PTC injury has not been studied. Therefore, it might be hypothesized that in hypertensive PTC injury, A1AR may protect the kidney through whether the classic hemodynamic regulation role or the relatively new anti-inflammatory mechanism.Purpose1. Compare the differences of benign and malignant hypertensive kidney injury patients in PTC loss, renal MΦ infiltration and local renin expression, and analyze their correlation with proteinuria, kidney function and pathological lesions.2. Establish the DOCA-salt hypertensive mice model to observe the association between PTC injury and other indicators of kidney injury, and analyze the role of RAAS and renal interstitial MΦ infiltration in PTC injury.3. Establish the DOCA-salt hypertensive model in A1AR knock-out mice to observe the effects and mechanism of Al AR in hypertensive PTC injury and MΦ infiltration.Method and Results1. PTC loss was uncorrelated with local inflammation or high renin expression among hypertensive patientsFrom January2003to March2012,103hypertensive kidney injury patients (HTN group), including17BHN,34IgAN-MHT, and52EMHT, with diagnosis confirmed by kidney biopsy in Peking Union Medical College Hospital, were involved. Nineteen patients with glomerular minimal lesions (GML) of the same period were involved as the control group. Renal pathological lesions were semi-quantitively evaluated. The PTC was evaluated by immunohistochemical (IHC) staining of CD34, a specific marker of arteriolar endothelium. IHC staining of CD68and renin were also performed to evaluate the pattern of renal MΦ infiltration and renin-positive juxtaglomerular apparatus (JGA). The results are:1) These HTN patients showed PTC loss and its severity correlated with kidney function impairment and tubulo-interstitial injuries.2) These HTN patients presented renal interstitial MΦ infiltration and its degree correlated with kidney function decline, proteinuria, and tubulo-interstitial lesions, but not with PTC loss.3) The ratio of renin-positive JGA was similar in BHN and GML patients, but was higher in MHT patients. Its degree correlated with neither clinico-pathological lesions nor PTC loss.4) Compared with BHN patients, the MHT patients had more severe kidney function impairment, PTC loss, and renal interstitial MΦ infiltration.2. Renal MΦ infiltration and PTC injury in salt-sensitive hypertensive mice2.1Establishment of DOCA-salt hypertensive miceDOCA-salt hypertensive mouse model, a model with high mineralocorticoids but low renin level, was established by removing left kidney, implantation of a DOCA pellet (200mg,60-day release), and feeding with high salt diet (8%NaCl). Blood pressure, heart rate, and24h urine biochemical indicators were evaluated.The systolic blood pressure (SBP) of DOCA-salt mice increased progressively from week1([122.3±9.5]mmHg) to week8([141.5±10.4]mmHg). They also showed decreased heart rate, polyuria, lower urine osmolarity with response to acute argipressin (AVP) stimulation, and more urinary excretion of sodium as well as microalbuminuria.2.2Renal MΦ infiltration and PTC loss in DOCA-salt hypertensive miceThe DOCA-salt hypertensive model was established as above. Blood and kidney sample were collected by sacrifice at week4or8. The plasma RAAS level and renal pathological lesions were evaluated. IHC staining o f CD34and CD68were used to estimate PTC loss and renal MΦ infiltration. The mRNA expression level of adenosine synthetase (CD39, CD73) and A1AR were measured by realtime-PCR. We observed in DOCA-salt hypertensive mice:1) The kidney weight index was increased and renal tubular atrophy and interstitial fibrosis appeared at week8(not at week4).2) PTC area ratio was lower than the control (4w:[2.62±0.34]%vs.[4.57±0.79]%, P=0.004;8w:[2.57±0.20]%vs.[3.41±0.48]%,P=0.018) and its loss was correlated with polyuria and microalbuminuria.3) MΦ infiltration was more severe than the control (4w:[7.28±3.46]/mm2vs.[0.46±0.29]/mm2,P=0.008;8w:[24.87±12.13]/mm2vs.[0.54±0.74]/mm2, P=0.028), with more MΦs at week8. Its degree did not correlate with PTC loss.4) Plasma renin activity declined progressively and correlated with polyuria. It did not have a correlation with PTC loss. The levels of AngⅡ and aldosterone remained unchanged.5) The level of CD73mRNA expression was2.22times higher than the control at week4while both CD39and A1AR mRNA levels increased at week8.3. A1AR protected hypertensive PTC injury in DOCA-salt miceDOCA-salt model was established in A1AR knockout (A1AR-/-) mice. All the mice were sacrificed at week4. The other methods were the same as above.A1AR-/-control mice had higher SBP than wildtype control mice, while A1AR-/-DOCA-salt mice did not present higher SBP than A1AR-/-control or wildtype DOCA-salt mice. Compared with wildtype DOCA-salt mice, A1AR-/-DOCA-salt mice had more serious polyuria ([7003±3742]μl vs.[3606±2359]μl, P=0.031), earlier tubulo-interstitial lesions (4w), and more severe PTC loss ([2.13±0.17]%vs.[2.62±0.34]%, P=0.042), without difference in renal MΦ infiltration or plasma renin activity.Conclusion1. PTC loss and renal interstitial MΦ infiltration were popular in hypertensive patients, which correlated well with kidney function decline and tubulo-interstitial lesions. The ratios of renin-positive JGA were not different in BHN and GML, which did not correlate with clinico-pahtological indicators. PTC loss did not correlate with MΦ infiltration or high renin expression.2. In DOCA-salt hypertensive mice, a model with high mineralocorticoids but low renin expression, after3weeks of elevation in blood pressure, obvious PTC loss appeared with higher expression of CD73(adenosine synthetase) mRNA, followed by prominent renal interstitial MΦ infiltration and interstitial fibrosis4weeks later.3. Compared with wildtype DOCA-salt mice, establishing DOCA-salt model in A1AR-/- mice did not make the hypertension progress, but could result more severe PTC loss and earlier interstitial fibrosis without exacerbation of renal MΦ infiltration.In conclusion, hypertensive PTC loss was popular and may correlate with microalbuminuria and tubulo-interstitial lesions. The PTC loss may result from direct effects of aldosterone or glomerular hyperperfusion and hyperfiltration, among which adenosine and A1AR may show protective roles through tubuloglomerular feedback, instead of local inflammation or high renin expression.